Ilaria Giovanna Luigi

Eleonora Matteo

Pamela

Federica Martin

Valentina Oscar

Loris Annalisa Antonio

Stefano Pierluigi

Rocco C Letizia

Giorgio Giulia

Carlo Marco

Simone D Michela

Valentina P Giovanni

Daniel Giulia

Keywords: HNF1B, RCAD, gene-dosage sensitivity, kidney development, transcriptomics

Aussi, merci aux membres de ma famille française préférée: Sidonie, Fabrice et Malo. Votre authenticité et votre complicité m'enrichissent à chaque réunion; vous êtes un exemple.

Et maintenant, passons aux remerciements sur le

s'incontreranno di nuovo. "Testa!" my friend. E non tornare indietro neanche per prendere la rincorsa.

Grazie anche al mio amico Girisaran Gangatharan, ci siamo conosciuti quasi alla fine del mio percorso di dottorato ma i nostri pranzi e le nostre chiacchierate sulla vita e sul mondo lavorativo hanno avuto un grande effetto sulla mia immaginazione. Sogna sempre in grande.

Inoltre, grazie di cuore ai membri della mia famiglia francese preferita: Sidonie, Fabrice e Malo. La vostra genuinità e complicità mi arricchiscono a ogni incontro; siete un esempio.

E ora, passiamo ai ringraziamenti sul fronte bellico italiano. Vivere in terra straniera non è mai stato facile per nessuno. Ma voi tutti siete stati il ponte emotivo che mi ha tenuto coi piedi per terra, motivandomi e incoraggiandomi costantemente. Siete voi, famigliari e amici, la mia vera forza. Anche il 24 settembre so che guardandomi intorno incrocerò molti vostri sguardi e spero che voi incrociando il mio penserete "Non lo sta facendo da solo, lo stiamo facendo insieme".

Grazie ai miei amici inossidabili: Luigi, Ilaria, Giovanna, Eleonora, Matteo, Pamela, Martin, Federica, Oscar, Valentina, Marica, Annalisa, Antonio, Loris, Stefano, Pierluigi, Valentina D, Simone R, Serena, Rocco C, Letizia, Giulia, Giorgio, Marco, Carlo, Michela, Simone D, Rocco F, Valentina P, Giovanni, Francesca, Marzia, Giuseppe, Paolo e tutti gli altri! I giochi, le confidenze e le risate non basteranno mai tra di noi. Siete forti, siete belli, siete partecipi, in alto i calici, questa festa è anche per voi.

Grazie a tutti i miei famigliari, gli zii straordinari, i cugini amorevoli, i nonni imbattibili. Siete le radici del mio essere e per voi sarà sempre il mio primo pensiero.

Grazie a Mauro, che sotto uno sguardo umile nasconde una grande saggezza. Hai una bella anima e so di poter imparare sempre da te. "Lean on me, brother".

Grazie a Giampaolo, ciò che ci unisce è tanto più forte quanto invisibile agli occhi. Continua a segnare e fare magie coi numeri per me, e io continuerò a stupirti.

Grazie a Maria Irene, mia preferita. In te giacciono la purezza di una bambina e la fame di una leonessa. Ti stimo ancor prima di volerti bene, illumina il paesaggio come sai fare solo tu.

Grazie a papà Francesco, da cui ho ereditato la bontà e il desiderio del quasi impossibile. Da grande voglio diventare come te.

Grazie a mamma Marisa da cui ho ereditato pazienza e tenacia. Se esistessero i supereroi tu saresti il loro capo.

Grazie a Giulia, Daniel, Patrizia e Luciano. Siete la mia seconda famiglia e una mia certezza primaria. La vita ha ancora così tanto da mostrarci, e so che lo scopriremo insieme.

Grazie a Mariagrazia, mia sposa, mia metà più emozionante e temeraria. Ogni mio successo è per te, ogni gesto è per noi. Mi hai seguito in questa folle vita lontano da casa e ti sarò sempre grato per questo. Tra tutte le luci sei la più intensa e durevole. Abbiamo fatto vedere al mondo cosa possono fare due ragazzi che si amano, e siamo ancora in cammino. I nomi elencati qui sopra rappresentano davvero ciò che s'intende all'estero per grande Famiglia Italiana. Ci saremo sempre gli uni per gli altri, è questo ciò che conta.

Infine voglio ringraziare il Consorzio RENALTRACT e l'Unione Europea. Senza di voi nulla di tutto questo sarebbe stato possibile.

GRAZIE A TUTTI VOI! SUMMARY

Heterozygous mutations in the gene encoding the transcription factor HNF1B are the cause of a complex multisystem syndrome known as Renal Cysts And Diabetes (RCAD). A novel mouse model, previously generated by introducing a human splicing mutation, was shown to reproduce several features of the human disease, including bilateral cystic embryonic kidneys, delayed proximal tubule differentiation, genital tract abnormalities and postnatal pancreas dysfunction. To further understand how miRNAs, mRNAs and the HNF1B transcriptional factor are integrated into gene regulatory networks underlying the disease phenotype, we performed high-throughput mRNA and microRNA sequencing at different developmental stages (E14.5, E15.5, E17.5). We showed that the most down-regulated genes are expressed in proximal tubules and to a lesser extent in the loop of Henle and collecting ducts and are involved in transport, lipid and organic acid metabolic processes. Consistent with the disease phenotype of our model de-regulated genes are related to abnormal renal/urinary system physiology and kidney diseases.

microRNAs sequencing showed that, depending of the stage, 11 to 25 microRNAs were significantly de-regulated. The higher number of differentially expressed miRNAs was observed at E15.5 and the majority was down-regulated. Moreover, different microRNAs were predicted to be involved in kidney development and maintenance. Taking advantage of a HNF1B ChIP sequencing data generated from E14.5 kidneys we selected two microRNAs (mir-802 and 192), which were down-regulated at all the three developmental stages analyzed, mir-194-2 down-regulated at E15.5 and at E17.5 and mir-30a down-regulated only at E15.5. Luciferase assays in HEK-293 cells showed that HNF1B was able to specifically transactivate in a dose response mode these microRNAs through the binding to consensus binding sites in their regulatory promoter/enhancer upstream sequences. We subsequently showed using vectors luciferase containing the 3'UTR of Hnf1b and miRNA MIMICS, were able to inhibit luciferase activity. These results together show that are linked by an autoregulatory-loop. Moreover, HNF1B is able to transactivate mir-30a gene through a far upstream enhancer element.

In parallel, proteomic analysis on urines of RCAD adult mice carried out by capillary electrophoresis coupled to mass spectrometry (CE-MS) uncovered 40 differentially excreted peptides including an increase of collagen fragments and a strong decrease of EGF and uromodulin fragments. Because of such characterized signature of the urinary proteome of the RCAD mice, we decided to investigate the urinary proteome of a pediatric cohort of RCAD patients searching for a similar urinary proteome signature. Analysis of urine samples from 22 RCAD patients and 22 healthy controls led to the identification of 146 peptides differentially excreted and associated with RCAD including a similarity regarding collagen and uromodulin fragments with the RCAD mouse model. Combining the peptides into a mathematical model we used independent cohorts of patients exhibiting different renal diseases to validate the prediction of the RCAD syndrome. Our classifier efficiently predicted RCAD syndrome with 91.7% sensitivity and 91.1% specificity on a wide population of control patients (including patients with unrelated cystic diseases and nephrotic syndromes).

In conclusion, the combined analysis of a unique RCAD mouse model and urinary proteomic approaches allowing the identification of a unique urinary proteomic signature of RCAD children, our work deepens different aspects of this complex and heterogeneous syndrome and provides new insights on the molecular basis underlying this disease. "Now is the time to understand more, so that we may fear less" stated Maria Skłodowska-Curie more than 80 years ago. This quote can be attributed to any phenomenon that scare us and we do not fully comprehend; nowadays among the most imperative mysteries to disclose there is the understanding of diseases.

The etiologies of diseases are often complex and unclear, but almost all have a genetic component. This thesis work was aimed to bring novel knowledge on a rare disease named Renal Cysts and Diabetes syndrome (RCAD), which is caused by mutations of a single gene.

Heterozygous mutations in the gene Hepatocyte Nuclear Factor-1-Beta (HNF1B) are responsible for the broad spectrum of symptoms that characterize the disease. Consistent with HNF1B role during development of different organs, RCAD clinical features are extremely heterogeneous and associates with morphological or functional anomalies of the kidneys, pancreas, liver and genital tract. This autosomal dominant disorder is not yet been comprehensively understood as shown by a lack of unequivocal prevalence or diagnostic tools and the difficulty in establishing its pathophysiological basis.

We studied this disease in two closely related mammals, the mouse and the human.

Regarding the murine approach a first mouse model that reproduces different phenotype of the RCAD disease was created in the laboratory. Using this model, we investigated transcriptional profiling, renal anomalies, and kidney functionalities at different time either during development or post-natal, uncovering significant data on the disease origination.

Moreover, we reveal in this mouse model a tight regulation between HNF1B and three microRNAs.

Regarding the human approach, within a collaborative project involving three European centers, we characterized the first urinary proteome of children affected by RCAD syndrome, providing new insights on the main elements that differentiate this disease from other renal disorders.

INTRODUCTION _________________

CHAPTER 1. Hepatocyte Nuclear Factor 1B

1.1 The HNF1B transcriptional factor: genomic organization and functional domains

Hepatocyte nuclear factor-1-beta (HNF1B), also known as LF-B3, HNF-1-β, vHNF1, and TCF2 (Transcription Factor 2), belong to a class of POU-like homeodomain of transcription factors, composed by two genes: HNF1A and HNF1B. HNF1B is located on Chr 17q12 in humans (position 17: 37,686,745,247;Ensembl); Chr11 in mice (11: 83,850,063-83,905,819).

The Hnf1b mRNA is approximately 2.8 kb split into 9 exons (Figure 1). Alternative splicing generates two main isoforms, named Hnf1b-A and Hnf1b-B, which are expressed at the same level in different tissues. Studies on human cells and mouse showed that Hnf1b-A transcript has an additional exon of 78 bp inserted between exon two and exon three generating 26 amino acids more at the end of the POU S domain [START_REF] Rey-Campos | vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1[END_REF] and is the predominant form both in embryonic and adult tissues compared with Hnf1b-B [START_REF] Cereghini | Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles[END_REF]. They display distinct activation potential depending on the context of the promoter and appear to complement each other during development [START_REF] Haumaitre | Functions of HNF1 family members in differentiation of the visceral endoderm cell lineage[END_REF]. A third isoform named Hnf1b-C was identified in human liver; it is shorter than the transcript -A and -B, and generated by the use of alternative polyadenylation site [START_REF] Bach | More potent transcriptional activators or a transdominant inhibitor of the HNF1 homeoprotein family are generated by alternative RNA processing[END_REF]. In vitro analyzes have shown that this isoform act as a dominant negative, repressing the expression of certain targets [START_REF] Bach | More potent transcriptional activators or a transdominant inhibitor of the HNF1 homeoprotein family are generated by alternative RNA processing[END_REF]. Comparative expression analysis in rat, mouse and human tissues showed controversial results concerning the relative expression levels of the three isoforms in different species [START_REF] Cereghini | Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles[END_REF]Harries L et al., 2009;[START_REF] Haumaitre | Severe pancreas hypoplasia and multicystic renal dysplasia in two human fetuses carrying novel HNF1beta/MODY5 mutations[END_REF]. The amino acid sequences of the different HNF1B isoforms are highlighted in Figure 2. The transcription factor HNF1B was identified after HNF1A in hepatic cells where it binds to the same promoter sequences of Hnf1A [START_REF] Baumhueter | A variant nuclear protein in dedifferentiated hepatoma cells binds to the same functional sequences in the beta fibrinogen gene promoter as HNF-1[END_REF]Cereghini S et al., 1988;Cereghini S et al., 1990). Subsequently, it has been confirmed that these two transcription factors were encoded by two different genes [START_REF] Bach | Two members of an HNF1 homeoprotein family are expressed in human liver[END_REF][START_REF] Rey-Campos | vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1[END_REF][START_REF] Simone | LFB3, a heterodimerforming homeoprotein of the LFB1 family, is expressed in specialized epithelia[END_REF].

HNF1B-A/B/C isoforms encode respectively 557-, 531-, and 457-proteins; the isoform A is the longest, has a molecular weight of 61,324 Da and showed a stronger transactivation activity than HNF1B-B (Ringeisen F 1993[START_REF] Ringeisen | The transactivation potential of variant hepatocyte nuclear factor 1 is modified by alternative splicing[END_REF]. With the exception of the isoform C which corresponds to a truncated protein, the isoforms A and B present three distinct functional domains: an N-terminus dimerization domain, a conserved DNA binding domain, and a C-terminus transactivation domain (Figure 3).

The dimerization domain of HNF1B is a structure composed by 32 amino acids forming four α-helices [START_REF] Pastore | The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?[END_REF]Rose RB et al., 2000 bis;Narayana N et al., 2001) and through binding this domain HNF1B can act as a homodimer or as a heterodimer with HNF1A to regulate gene expression (Chouard T et al., 1990;Rey-Campos J 1991;[START_REF] Frain | The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain[END_REF][START_REF] Nicosia | A myosin-like dimerization helix and an extra-large homeodomain are essential elements of the tripartite DNA binding structure of LFB1[END_REF]. This domain presents 72% of homology between the two transcription factors. When dimerization domain is mutated, less ability to form dimers and less affinity for the DNA binding site were described (Chouard T et al., 1990;[START_REF] Tomei | A POU-A related region dictates DNA binding specificity of LFB1/HNF1 by orienting the two XL-homeodomains in the dimer[END_REF].

The DNA binding domain is divided into a Pit-1/Oct-1/Unc-86 specific domain (POU S ) and a POU homeodomain (POU H ). POU S confers to the protein its DNA binding specificity [START_REF] Tomei | A POU-A related region dictates DNA binding specificity of LFB1/HNF1 by orienting the two XL-homeodomains in the dimer[END_REF]. It is atypical as it is composed of at least one α-helice more than the canonical POU S domains; HNF1A possess five α-helices and HNF1B six α-helices (Figure 4 A). Crystallography analyses suggest that the supplementary N-terminal α-helix could lead to a decrease in DNA binding (Chi YI et al., 2002;[START_REF] Lu | Structural basis of disease-causing mutations in hepatocyte nuclear factor 1beta[END_REF]. POU H forms three α-helices, the last of which is involved in the interaction with the nucleobases at the level of the major groove of DNA (Ceska TA et al., 1993;[START_REF] Leiting | The three-dimensional NMR-solution structure of the polypeptide fragment 195-286 of the LFB1/HNF1 transcription factor from rat liver comprises a nonclassical homeodomain[END_REF][START_REF] Schott | The NMR solution structure of the non-classical homeodomain from the rat liver LFB1/HNF1 transcription factor[END_REF]. Unlike the other characteristic POU domains, the fifth helix of the POU S domain, the region between the two POU domains and the second helix of the POU H domain are longer. These characteristics would lead to an increase in the interaction between the POUs and POU H domains and a greater rigidity in the DNA interaction. Between the POU S and POU H domains (amino acids 186-229) there is the nuclear localization signal (NLS) [START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF] During kidney development, its expression is detected in the mesonephric tubules, the entire Wolffian duct (WD) from E9.0 and at E10.5 at the level of the emerging ureteric bud (UB) (Figure 7). Its expression is maintained in the epithelium of the UB during its arborization. Then, Hnf1b is found throughout the nephrogenesis in the distal part of the renal vescicles (RV), and comma-shaped and S-shaped bodies in a proximal-distal pattern at E13.5.

At E16.5, the expression pattern is similar to the adult [START_REF] Cereghini | Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles[END_REF] with both proximal and distal tubules staining positive [START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF][START_REF] Simone | LFB3, a heterodimerforming homeoprotein of the LFB1 family, is expressed in specialized epithelia[END_REF], Ott MO et al., 1991). Hnf1b is expressed in the endoderm of the primitive gut and the multipotent pancreatic precursors of the pancreatic bud. From E14.5 until birth, its expression is restricted in the pancreatic duct network [START_REF] Haumaitre | Lack of TCF2/vHNF1 in mice leads to pancreas agenesis[END_REF][START_REF] Maestro | Hnf6 and Tcf2 (MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas[END_REF]De Vas MG et al., 2015) (Figure 8 A, B, C).

Hnf1b is also expressed during the development of the gallbladder, bile ducts and lungs. A subpopulation of hepatocytes, the periportal hepatocytes, located at the periphery of the hepatic lobules, also expresses Hnf1b at the end of embryogenesis and in adults [START_REF] Coffinier | Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta[END_REF]. Finally, as early as E16.5, the bronchi and bronchioles of the lungs express Hnf1b (Coffinier C et al., 1999b) (Figure 8 D, E, F). In adult mice, Hnf1b is strongly expressed in the polarized epithelial tubes of the kidney (all segments of the nephron and in renal collecting ducts), and at lover levels in the lungs, gonads, biliary and pancreatic ducts, stomach and intestine epithelia [START_REF] Cereghini | Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles[END_REF][START_REF] Lazzaro | LFB1 and LFB3 homeoproteins are sequentially expressed during kidney development[END_REF][START_REF] Simone | LFB3, a heterodimerforming homeoprotein of the LFB1 family, is expressed in specialized epithelia[END_REF][START_REF] Rey-Campos | vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1[END_REF]).

( 1 mm; (F) 400 µm. (Adapted from Coffinier C et al., 1999b and[START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF] 1.3 HNF1B during mouse development

Early post-implantation stages

In normal embryos, Hnf1b is initially expressed in extraembryonic tissues, and subsequently in the primitive endoderm (the precursor of both visceral and parietal endoderm) [START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF].

Constitutive inactivation of Hnf1b causes a severe disorganization of all embryonic and extraembryonic tissues, leading to embryonic lethality around E6.5 [START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF]Coffinier et al., 1999a). This early lethality is due to the absence of the visceral endoderm, an extraembryonic tissue essential for the nutrition and the hemopoietic function of the embryo.

Chimera tetraploid morulae aggregation strategy, obtained by fusing blastomers at the 2cells stage, has been used to overcome the early lethality linked to its germline inactivation and further examine the role of Hnf1b during early organogenesis. This technique consists in the aggregation of Hnf1b-deficient embryonic stem (ES) cells with wild-type tetraploid embryos, which contribute exclusively to the formation of extraembryonic tissues, while the ES Hnf1-/will contribute to the embryo (epiblast). These studies confirmed that the early lethality is due to the absence of visceral endoderm formation and highlight the crucial role of Hnf1b in the establishment of this tissue and its importance for proper growth and differentiation of pregastrulating embryos [START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF].

Role of HNF1B during organogenesis

Pancreas

Hnf1b deficient embryos rescued by tetraploid aggregation show an absence of ventral pancreas and a significant reduction of the dorsal pancreas, which degenerates causing pancreatic agenesis at E13.5 [START_REF] Haumaitre | Lack of TCF2/vHNF1 in mice leads to pancreas agenesis[END_REF]. At E11.5 the residual dorsal pancreatic bud of the mutant embryos expresses Pdx1, a very early pancreatic marker [START_REF] Offield | PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum[END_REF], and Hlxb9, which is a transcription factor involved in the dorsal pancreas specification [START_REF] Harrison | Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice[END_REF][START_REF] Li | Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9[END_REF]. On the other hand, the transcription factor Ptf1a, which is involved in the pancreatic fate acquisition, is not detected [START_REF] Lokmane | Crucial role of vHNF1 in vertebrate hepatic specification[END_REF].

These results show that Hnf1b is necessary for the specification of the ventral pancreas and the differentiation and proliferation of the dorsal pancreas. The conditional inactivation of Hnf1b in pancreatic progenitors with the Pdx1-Cre line highlighted the major role of this transcription factor in the morphogenesis of the pancreas. These mutants show significant pancreatic hypoplasia associated with a decrease in the number of acinar cells, the formation of cysts at the level of the ducts and a defect in morphogenesis and generation of endocrine progenitors via the direct regulation of Ngn3. Interestingly ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, including the known HNF1B targets Pkhd1, Kif12, Bicc1 as well as the novel identified Hnf1b targets, Cys1 and (De Vas et al., 2015). Conditional inactivation of Hnf1b in the β-pancreatic cells uncovered a later role of Hnf1b in glycolytic signaling using cells [START_REF] Wang | Selective deletion of the Hnf1beta (MODY5) gene in beta-cells leads to altered gene expression and defective insulin release[END_REF].

Glis3

These mice present a normal level of insulin and glucose but their insulin secretion after glucose injection is impaired. The analysis of different markers revealed a deregulation of transcription factors involved in the differentiation of β-pancreatic cells including Pdx1 and Hnf4α.

Liver

Hnf1b is expressed very early in the liver primordium, in the hepatoblast and subsequently is restricted to the bile ducts. Rescued embryos by tetraploid aggregation show a lack of hepatic bud formation associated with the deregulation of the expression of genes encoding hepatic key transcription factors such as Hnf4α, Prox1, Foxa1, Foxa2 and Foxa3, thus revealing a critical role at the earliest steps of liver induction: the acquisition of endoderm competence and the hepatic specification [START_REF] Lokmane | Crucial role of vHNF1 in vertebrate hepatic specification[END_REF]. Interestingly analysis of liver induction in zebrafish indicated a conserved role of HNF1B in vertebrates [START_REF] Lokmane | Crucial role of vHNF1 in vertebrate hepatic specification[END_REF] (see also chapter 1.7).

Conditional invalidation of Hnf1b in hepatocytes and the biliary system by using an Alfp-Cre mouse strain [START_REF] Kellendonk | Hepatocyte-specific expression of Cre recombinase[END_REF] leads to a severe defect in intrahepatic biliary ducts development and metabolic abnormalities at E15-E18. Mutant mice also suffer from severe jaundice and hepatic metabolic effects [START_REF] Coffinier | Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta[END_REF][START_REF] Kellendonk | Hepatocyte-specific expression of Cre recombinase[END_REF]. A recent study has shown that in the absence of Hnf1b or Hnf6, the polarity of the bile duct cells is defective and is accompanied by a decrease in the expression of the cystic gene associated with the eyelash: Cystin1 (Cys1) (Raynaud et al. al., 2011). These studies highlight the role of HNF1B in liver morphogenesis, lipid metabolism by hepatocytes and in the maintenance of the polarity of the bile duct epithelium.

Intestine

In mice, the specific inhibition of Hnf1b in the adult intestine lead to a lack of differentiation of the epithelial cells of the intestine followed by early lethality. This study demonstrates a redundant role of Hnf1b and Hnf1a for the regulation of cell fate and function of the intestinal epithelium [START_REF]Hepatocyte nuclear factor 1alpha and beta control terminal differentiation and cell fate commitment in the gut epithelium[END_REF]. The combination of germline inactivation of Hnf1a associated to a intestinal specific inactivation of Hnf1b, leads to an impaired terminal differentiation of the secretory and absorptive lineage, due to a defective Notch signaling [START_REF]Hepatocyte nuclear factor 1alpha and beta control terminal differentiation and cell fate commitment in the gut epithelium[END_REF]. Moreover, studies on embryos rescued by tetraploid aggregation show a defect in the regionalization of the primary intestine in these mutants since they present an ectopic expression of Sonic hedgehog (Shh) associated with the absence of Indian hedgehog (Ihh) and Pancreatic and duodenal homeobox 1 (Pdx1) in the posterior stomach and the duodenum [START_REF] Haumaitre | Lack of TCF2/vHNF1 in mice leads to pancreas agenesis[END_REF][START_REF] Lokmane | Crucial role of vHNF1 in vertebrate hepatic specification[END_REF]. .

HNF1B during kidney development

Overview of kidney development and function

Despite the crucial role of Hnf1b in mouse endoderm derived organs (pancreas, liver, intestine), its major site of expression is the kidney both during development and in adults.

Consistently with the mouse studies described in the next chapter, the most common pathological features related to HNF1B heterozygous mutations in humans affect the kidneys.

Since the thesis studies were focused essentially on the role of HNF1B in kidney development and disease, the development of this organ will be described more in-depth.

Kidney development in mice begins at embryonic day E10.5 and depends on an appropriate interaction between the ureteric bud (UB), which originally derives from the mesonephric duct (also called Wolffian duct, WD) and the adjacent metanephric mesenchyme (MM). The population of metanephric mesenchyme cells capping each branch encompass two lineage compartments: (A) nephron progenitors that produce all cell types of the nephron (from proximal podocytes to distal connecting segments) [START_REF] Kobayashi | Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development[END_REF][START_REF] Boyle | Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia[END_REF], and (B) interstitial progenitors that give rise to all interstitial cell types, mesangial cells within the glomerulus, vascular associated pericytes, and interstitial fibroblasts [START_REF] Humphreys | Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis[END_REF][START_REF] Little | A high-resolution anatomical ontology of the developing murine genitourinary tract[END_REF]. On the other hand, ureteric bud cells give rise to collecting ducts and the number of ureteric branches determines nephron number [START_REF] Schedl | Renal abnormalities and their developmental origin[END_REF].

A process of reciprocal signaling between the MM and the UB is the basis for renal development. UB undergoes branching morphogenesis to give rise to the renal collecting system composed of the ureter, renal pelvis and collecting duct. In parallel, groups of MM cells proximal to the ureteric bud tips, are induced to form the pretubular aggregates, which start epithelial transitions to form renal vesicles (RV) [START_REF] Mcmahon | Development of the Mammalian Kidney[END_REF]. RV differentiates into the comma-shaped and then S-shaped bodies. S-shaped bodies finally elongate and differentiate into the various segments of the nephron [START_REF] Dressler | The cellular basis of kidney development[END_REF][START_REF] Dressler | Advances in early kidney specification, development and patterning[END_REF]Kato N et al., 2009 bis) (Figure 9). As a consequence of this continuous process, primitive nephrons are found in regions close to the outer cortex, whereas more mature structures reside next to the medulla [START_REF] Schedl | Renal abnormalities and their developmental origin[END_REF]. At the first postnatal week, nephrogenesis ceases and the number of mature nephrons present in each kidney is definitive. At this point, the mouse kidney (the mammalian kidney in general) looks like a complex organ comprising over 25 distinct cell types organized in tissues that accomplish a large amount of tasks including blood filtration, regulation of blood pressure and pH, reabsorption of water and compounds required by the body. Proximal tubule (PT): a segment located between the glomerulus and the loop of Henle. It is characterized by a brush-border that consists of densed packed microvilli that increase the surface area to reabsorb small molecules from the filtrate to the interstitium. It is responsible for passive and active resorption of solutes from the preurine. The four vital functions performed by kidneys thanks to its different structures are:

Loop of

(1) filtration of the blood and reabsorbtion of water, glucose, and amino acids from the filtrate;

(2) excretion of wastes such as urea and ammonium;

(3) regulation of the balance of electrolytes and acide base;

(4) production of hormones, including calcitriol, erythropoietin, and the enzyme rennin, which modulates blood pressure.

A comprehensive picture of a mouse kidney and its structures is depicted in Figure 11. MH et al., 2007) Macroanatomic features are conserved in the human and mouse, although with distinct organization, temporal/spatial dynamics and differences in associated gene expression [START_REF] Lindström | Conserved and Divergent Features of Human and Mouse Kidney Organogenesis[END_REF]. 3D architecture and composition of human and mouse nephrogenic niches follow a similar developmental progression in their reduction of progenitor cells but proceed at different time-scales [START_REF] Lindström | Conserved and Divergent Features of Human and Mouse Kidney Organogenesis[END_REF]. In the end, the human kidney generates approximately 1,000,000 nephrons [START_REF] Bertram | Human nephron number: Implications for health and disease[END_REF] whereas mouse around 16,000 (Short KM et al., 2014).

Interestingly and unlike the mouse, the human kidney is made up of multiple lobes [START_REF] Oliver | Nephrons and Kidneys: A Quantitative Study of Development and Evolutionary Mammalian Renal Architectonics[END_REF] and in human is possible to observe the formation of rosette-like clusters of ureteric tips, where nephrogenic niches face outwards away from the center (Lindström NO et al., 2018). Another major difference is related to the anchor genes that identify discrete structures in kidney; only three of 26 anchor genes conserved what appeared to be a "mouselike" expression pattern in the human kidney [START_REF] Little | Defining kidney biology to understand renal disease[END_REF].

HNF1B requirement for ureteric bud branching morphogenesis and nephrogenesis induction

Hnf1b transcripts and protein are detected in the WD epithelium and cranial mesonephric tubules at E9.5; at E10.5 Hnf1b is expressed in the epithelium of the emerging UB and UB branches and nascent nephrons (renal vesicles, Comma-and S-shaped bodies) (Figure 12). However, Hnf1b is not expressed in the MM. From E17.5 to the adult it is expressed in all tubular epithelial cells of the nephrons (but not in the glomeruli) and the collecting duct system.

Consistent with the early expression pattern, constitutive inactivation of Hnf1b in the epiblast leads to severe defects in UB branching and WD and Mullerian duct differentiation, in addition, the MM covering the extremities of the UB is unable to condense, to achieve the mesenchymal-epithelial transition (MET) and thus to initiate nephrogenesis. Transcriptional and ChIP-PCR studies suggest that HNF1B exert these functions, at least in part via the direct control of key regulators, including Pax2, Lhx1 and Wnt9. In particular, acting directly upstream of Wnt9b control non-cell-autonomous Wnt-signaling and the initiation of nephrogenesis (Lokmane L et al., 2010) (Figure 13).

Collecting duct morphogenesis and cystic medullar tubules

Specific Hnf1b kidney inactivation was obtained using a Ksp-cadherin-Cre mouse line at a later time point when tubules have already formed but are still elongating [START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF]. Adult transgenic mice from this line expressed Cre recombinase in medullar collecting ducts and thick ascending limbs of Henle's loops [START_REF] Shao | Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract[END_REF]. These mutants present a polycystic kidney phenotype associated with death by postnatal day 28 [START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF] (Figure 15). The involvement of HNF1B in the differentiation of tubular cells and cyst formation was also demonstrated using a transgenic mouse line expressing a dominant negative form of HNF1B under control of the Ksp-Cadherin promoter. [START_REF] Hiesberger | Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis[END_REF]. Hnf1b inactivation in medullar renal tubules is associated with the downregulation of genes involved in cystogenesis, including Pkd2, Pkhd1, Nphp1, Tg737/polaris (Ift88) and

Umod [START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF], thus potentially linking the HNF1B transcriptional regulatory cascade to ciliogenesis.

By preforming inducible inactivation of Hnf1b at different postnatal stages, Verdeguer et al. showed that inactivation just until P10, at a time when proliferative morphogenetic elongation is ongoing, cause cystic tubular dilations, [START_REF] Verdeguer | A mitotic transcriptional switch in polycystic kidney disease[END_REF]. They propose a time window of action for HNF1B for the maintenance of renal tubule morphology.

Moreover, they studied the role of the transcription factor after P10 forcing Hnf1b deficient quiescent cells to proliferate following induced lesions; in this conditions, cysts appeared showing a potential role of Hnf1b in cell proliferation for tissue reparation. This has been related to the role proposed for HNF1B as a "bookmarking factor" thus remaining attached to chromatin during mitosis and then reopening the chromatin of its target genes immediately after completion of cell division and activating transcription [START_REF] Verdeguer | A mitotic transcriptional switch in polycystic kidney disease[END_REF]. It should be noted that similar findings were observed with other cystic disease genes as Pkd1 [START_REF] Piontek | A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1[END_REF]. Although the molecular mechanism remains poorly understood, it appears in this case not to be related to cellular proliferation. Recently, Desgrange A et al.

found the requirement of Hnf1b for the formation of a specialized UB tip domain through conditionally inactivation in the UB and collecting duct system. Using the HoxB7-Cre line allowing the conditional inactivation of Hnf1b in the entire WD and UB derivatives, they observed that HNF1B is required in cellular organization and epithelial polarity during UB branching and collecting duct (CD) morphogenesis. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating the Ret-co-receptor Gfra1 and the Ret direct target Etv5.

At later stages, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct maturation and disrupted tissue architecture, leading to cystogenesis and perinatal death. Late-stage embryos or P0 mutants exhibit severe urogenital abnormalities reminiscent of CAKUT [START_REF] Desgrange | HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis[END_REF].

Expression of dominant-negative mutant HNF1B or kidney-specific knock-out of Hnf1b in mice reduces the levels of Pkhd1 mRNA transcripts in the kidney via direct downregulation of Pkhd1 promoter activity in renal collecting ducts (CDs) (Hiesberger T et al., 2004;[START_REF] Hiesberger | Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis[END_REF][START_REF] Williams | Tissuespecific regulation of the mouse Pkhd1 (ARPKD) gene promoter[END_REF].

Aboudehen et al.

showed that mice where Hnf1b was specifically deleted in renal collecting ducts at rather later stages using the Pkhd1/Cre reporter, survived long term and developed slowly progressive cystic kidney disease, renal fibrosis, and hydronephrosis.

Moreover, they observed that treatment of HNF1B mutant mIMCD3 cells with hypertonic NaCl inhibited the induction of osmoregulated genes, including Nr1h4, which encodes the transcription factor FXR that is required for maximal urinary concentration (Aboudehen K et al., 2017). Notably, a direct transcriptional regulatory cascade has also linked HNF1B with several cystic disease genes; studies in postnatal cystic kidneys, upon Hnf1b inactivation in medullar renal tubules, reported reduced expression of genes including Umod, Pkhd1, Pkd2, Nphp1 and Tg377/Polaris (Ift88) [START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF]. Further in vivo ChiP experiments in adult kidneys confirmed that HNF1B is recruited on the regulatory regions of these genes.

In addition to control nephron patterning and renal tubular morphogenesis, HNF1B

regulates different genes associated with ion transport as FXYD2, KCNJ10, and UMOD.

Whereas a deregulation of FXYD2, encoding for the γ subunit of Na + /K + -ATPase, is associated with hypomagnesemia in human patients [START_REF] Adalat | HNF1B mutations associate with hypomagnesemia and renal magnesium wasting[END_REF][START_REF] Ferrè | HNF-1B specifically regulates the transcription of the γa-subunit of the Na+/K+-ATPase[END_REF], impairment of KCNJ10 function, that encodes for the K + channel Kir5.1, cause a saltwasting syndrome characterized by hypomagnesemia and hypokalemia [START_REF] Kompatscher | Loss of transcriptional activation of the potassium channel Kir5.1 by HNF1β drives autosomal dominant tubulointerstitial kidney disease[END_REF][START_REF] Simon | Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2[END_REF]. HNF1B can directly regulate the transcription of UMOD, which is involved in K + handling [START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF]. Moreover, the calcium-sensing receptor CaSR present in the TAL and contributing potentially to hypocalciuria in some RCAD patients [START_REF] Kompatscher | Transcription factor HNF1β regulates expression of the calcium-sensing receptor in the thick ascending limb of the kidney[END_REF] and the osmosensitive gene FXR, which is related to polyuria [START_REF] Aboudehen | Hepatocyte Nuclear Factor-1beta Regulates Urinary Concentration and Response to Hypertonicity[END_REF] were recently found as novel targets of HNF1B. Moreover, HNF1B directly regulates the proximal tubule urate transporter I (URAT1/SLC22A12), which has been related hyperuricemia [START_REF] Kikuchi | Regulation of tissuespecific expression of the human and mouse urate transporter 1 gene by hepatocyte nuclear factor 1 alpha/beta and DNA methylation[END_REF] and the trans-membrane transporter TMEM27 gene, encoding collectrin, and whose disruption in mice result in a severe defect in renal amino acid uptake [START_REF] Zhang | The role for HNF-1beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells[END_REF][START_REF] Desgrange | HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis[END_REF] (Figure 17).

The epithelial cell differentiation and the cell polarity are other domains handled by HNF1B as it has been shown to regulate Crb3 (encoding crumbs homolog-3), Bicc1

(encoding bicaudal C homolog 1) AP-2β (Tcfap2b) [START_REF] Verdeguer | A mitotic transcriptional switch in polycystic kidney disease[END_REF], Atp6v1b1, Slc26a4, Pdzk1, Aqp5, Hpn and Tmem27 [START_REF] Desgrange | HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis[END_REF].

Recent findings correlated HNF1B with the intrarenal cholesterol metabolism as well as mitochondrial respiration of proximal tubule renal cells. Kidney-specific inactivation of HNF1B or the expression of dominant negative mutant HNF1B decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and HMG-CoA reductase (Hmgcr) (Aboudehen K et al., 2015) (Figure 18).

HNF1B transcriptional homologs, cofactors and coactivators

HNF1B functions as a homodimer or heterodimer with the structurally related protein HNF1A [START_REF] Frain | The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain[END_REF]Chouard T et al., 1990), with which it shares more than 50% of the amino acid sequence [START_REF] Power | Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1[END_REF] (Figure 20). The two heterologous proteins appeared presumably by duplication of a common ancestral gene as they map to parts of the genome known to have duplicated in early vertebrate evolution, namely 12q24.31 (HNF1A, near LHX5 and on the same arm as the HOXC cluster) and 17q12 (HNF1B between LHX1 and the HOXB cluster). HNF1A, also known as HNF1 or TCF1, is encoded by a gene of 10 exons whose locus in the chromosomes 12 and 5 in human and mouse respectively [START_REF] Holland | Classification and nomenclature of all human homeobox genes[END_REF]. Overexpression experiments in different cell types showed that HNF1A and HNF1B can regulate gene transcription via binding the same consensus sequence [START_REF] Liu | The mouse alpha-albumin (afamin) promoter is differentially regulated by hepatocyte nuclear factor 1α and hepatocyte nuclear factor 1β[END_REF]. However, it seems that HNF1B has lower transcriptional activation efficiency than HNF1A [START_REF] Bach | More potent transcriptional activators or a transdominant inhibitor of the HNF1 homeoprotein family are generated by alternative RNA processing[END_REF][START_REF] Rey-Campos | vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1[END_REF]. Mendel DB et al. demonstrated that the 12kDa cofactor DCoH (Dimerisation Cofactor of HNF1, also known as pterin-4 alpha-carbinolamine dehydratase, PCDB1) interact with HNF1A-HNF1B dimers forming a tetrameric structure that stabilize the complex (Mendel DB et al., 1991 bis;Rose RB et al., 2000). PCDB1 is also important for renal Mg 2+ imbalances as it regulates the HNF1B-mediated FXYD2 transcription, influencing active renal Mg 2+ reabsorption in the distal convolute tubule [START_REF] Ferrè | Mutations in PCBD1 cause hypomagnesemia and renal magnesium wasting[END_REF].

An increase in the transcriptional activity of HNF1B has been described in the case of interactions in the C-terminal transactivation domain with histone acetyltransferases such as CBP [cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB)

binding protein], and P/CAF [p300/CBP-associated factor], SRC-1 and RAC3 [START_REF] Soutoglou | Acetylation regulates transcription factor activity at multiple levels[END_REF][START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF]. HNF1B is also able to interact in vitro and in vivo with HDAC1 histone deacetylase [START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF]. Zyxin is another HNF1B cofactor, which form a transcriptional complex that include CBP, binding to the POU domain and the C-terminal transactivation domain of HNFB (Choi YH et al., 2013).

A functional association of the N-terminal domain of HNF1B with two other regulatory proteins, ZFP36L1 (predominant in the cytoplasm, activator) and E4F1

transcription factor (predominant in the nucleus, inhibitory), has been shown. Their overexpression in Xenopus embryos leads to defects in pronephros formation suggesting their control of HNF1B during kidney development [START_REF] Dudziak | Transcription factor HNF1beta and novel partners affect nephrogenesis[END_REF]. --------------------- -------------------

Regulation of HNF1B

In order to better understand the regulation of Hnf1b, the promoter of this gene has been cloned and analyzed [START_REF] Power | Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1[END_REF]. This study reveals five sites of interaction between DNA and proteins at the first 260 base pairs upstream of the transcription initiation site involving at least three major families of transcription factors. Two of these sites are particularly important for the activity of the promoter. Gel delay and transient transfection experiments show that several members of the steroid hormone receptor family such as HNF4α, COUP-TF1/Ear3, COUPTFII/Arp1 as well as retinoic acid receptors RXR and RAR may play a role in the regulation of Hnf1b expression. In addition, a proximal Octamer protein binding site (OCT) is required for transactivation of Hnf1b by COUP-TF1/Ear3 a nd COUP-TFII/Arp1. Several studies suggest that HNF1B is a mediator downstream of retinoic acid signaling. Indeed, Hnf1b is induced in the embryonic carcinoma cells F9 after treatment with retinoic acid. In addition, retinoic acid appears necessary for the induction of Hnf1b and Hoxb1 for the establishment of the identity of the rhombomers and the r4/r5 border, but also during the development of the endocrine pancreas of the zebrafish [START_REF] Hernandez | vhnf1 integrates global RA patterning and local FGF signals to direct posterior hindbrain development in zebrafish[END_REF][START_REF] Sirbu | Shifting boundaries of retinoic acid activity control hindbrain segmental gene expression[END_REF][START_REF] Song | Vhnf1 acts downstream of Bmp, Fgf, and RA signals to regulate endocrine beta cell development in zebrafish[END_REF]. A highly conserved 800bp enhancer element located in the fourth intron of Hnf1b integrating direct inputs from the retinoic acid signaling cascade and MAF-related factors was identified [START_REF] Pouilhe | Direct regulation of vHnf1 by retinoic acid signaling and MAF-related factors in the neural tube[END_REF].

The recruitment of HNF4α on the Hnf1b promoter was confirmed by chromatin immunoprecipitation (ChIP) in human hepatocytes and pancreatic islets [START_REF] Boj | A transcription factor regulatory circuit in differentiated pancreatic cells[END_REF][START_REF] Odom | Control of pancreas and liver gene expression by HNF transcription factors[END_REF]. However, it does not appear that this factor is essential to the expression of Hnf1b. It has been shown that PDX1 is able to bind to the Hnf1b promoter in the embryonic pancreas and that mice deficient for Pdx1 show a decrease in Hnf1b suggesting that PDX1 participate in the transcriptional regulation of Hnf1b in pancreatic progenitors [START_REF]The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice[END_REF]. In addition, HNF6 transactivates the Hnf1b promoter in vitro and also during the development of bile ducts and pancreatic ducts (Clotman F et al., 2002;[START_REF] Pierreux | The transcription factor hepatocyte nuclear factor-6 controls the development of pancreatic ducts in the mouse[END_REF][START_REF] Poll | A vHNF1/TCF2-HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells[END_REF]. Despite a number of studies, it remains to determine which regulatory elements are involved in the induction of Hnf1b expression during kidney and endoderm derived tissues during development as well as in adult tissues.

The conservation of HNF1B in other species

Formation of the basic nephron with its segments took place probably more than 360 million years ago in a common ancestor of mammals and amphibians [START_REF] Schedl | Renal abnormalities and their developmental origin[END_REF]. Since then, many evolutionary events succeeded until the arrival of the transcription factor HNF1B. No orthologue of the HNF1 factors exists in the invertebrates (Figure 21). On the other hand, different studies on vertebrates such as Danio rerio [START_REF] Gong | Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish[END_REF][START_REF] Song | Vhnf1 acts downstream of Bmp, Fgf, and RA signals to regulate endocrine beta cell development in zebrafish[END_REF], Salmo salar [START_REF] Deryckere | Salmon HNF1: cDNA sequence, evolution, tissue specificity and binding to the salmon serum albumin promoter[END_REF], and Xenopus laevis [START_REF] Bartkowski | Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis[END_REF][START_REF] Zapp | Genomic structure of the Xenopus laevis liver transcription factor LFB1[END_REF][START_REF] Demartis | Cloning and developmental expression of LFB3/HNF1 beta transcription factor in Xenopus laevis[END_REF][START_REF] Heliot | HNF1B controls proximal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2[END_REF] showed similarity with mice in the expression pattern and its corresponding role in their organogenesis.

In zebrafish, hnf1b expression pattern is remarkable relative to mice and begins early during embryogenesis between stage 256 and 1000 cells (Sun and Hopkins 2001). Hnf1b expression was shown in gut, liver, pancreas (Sun and Hopkins 2001;[START_REF] Gong | Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish[END_REF][START_REF] Lokmane | Crucial role of vHNF1 in vertebrate hepatic specification[END_REF], hindbrain [START_REF] Wiellette | Wiellette EL, Sive H. vhnf1 and Fgf signals synergize to specify rhombomere identity in the zebrafish hindbrain[END_REF] and pronephric duct (Naylor RW et al., 2013). Tubular cysts have been observed in the pronephric tubules of hnf1bmutated zebrafish (Sun and Hopkins 2001).

In Xenopus embryos, hnf1b start to be expressed in the pronephros, in liver and pancreas [START_REF] Raciti | Organization of the pronephric kidney revealed by large-scale gene expression mapping[END_REF] and transgenic expression of HNF1B carrying human mutations in the DNA binding domain leads to pronephros abnormal development (Bohn S et al., 2003). Wu et al. showed that three domains must be present in the mutated protein to obtain a reduction in the pronephros; these include the dimerization domain, the 26 amino acid segment specific for splice variant A as well as the POU H domain. [START_REF] Wu | The HNF1beta transcription factor has several domains involved in nephrogenesis and partially rescues Pax8/lim1-induced kidney malformations[END_REF].

Moreover, the role of Hnf1b in morphogenesis of all nephron segments was confirmed in Xenopus [START_REF] Heliot | HNF1B controls proximal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2[END_REF] as well in Zebrafishi (Naylor RW et al., 2012).

Overview of the RCAD syndrome

Consistent with multiorgan expression pattern of HNF1B, heterozygous mutations of hepatocyte nuclear factor 1B gene (HNF1B), were reported associated with the Renal Cysts and Diabetes Syndrome (RCAD, OMIM #137920) (Bingham C et al., 2001), originally referred Maturity Onset Diabetes of the Young type 5 (MODY5) [START_REF] Horikawa | Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY[END_REF].

The disease is inherited in an autosomal dominant fashion (Bingham C et al., 2004) and is characterized by a wide range of phenotypes encompassing multiple organs (reviewed by [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF][START_REF] Clissold | HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum[END_REF]. The most prominent clinical features in HNF1B-associated syndrome are developmental kidney disease and diabetes mellitus.

Besides, other manifestations include genital tract malformations, pancreas and liver structural anomalies and dysfunctions, and electrolytes imbalances. HNF1B mutations are also associated with two autosomal dominant disorders: the Hypoplastic Glomerulocystic Kidney Disease (GCKD) and the Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD).

The phenotype of HNF1B mutant carriers is highly variable both within and between families and no correlation was observed between the type or position of a given mutation and the severity of the phenotype. These observations led to the hypothesis that non-allelic factors, as well as stochastic variation in temporal HNF1B gene expression and environmental factors, could bring the strong intrafamilial variability of RCAD patients [START_REF] Edghill | Mutations in hepatocyte nuclear factor-1beta and their related phenotypes[END_REF][START_REF] Clissold | HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum[END_REF]. The origin of the pathology can, therefore, be associated with a reduction of the HNF1B activity with haploinsufficiency being the main disease mechanism. In the following part of the chapter, the various phenotypes observed in RCAD patients are described individually (Figure 23). 25)

RCAD clinical features (Figure

Renal abnormalities

HNF1B mutations are found in 20-30% of fetuses with renal abnormalities representing the most frequent prenatal cause of hyperechogenic kidneys (Bingham C et al., 2004;Decramer S et al., 2007) and represent the most frequent genetic cause of renal malformation during childhood [START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF][START_REF] Ulinski | Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort[END_REF]. HNF1B-related abnormalities have been recognized to cause congenital anomalies of the kidney and urinary tract (CAKUT) (Nakayama M et al., 2010;[START_REF] Madariaga | Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes[END_REF][START_REF] Raaijmakers | Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract[END_REF], predominantly affecting bilateral renal malformations.

The most common kidney abnormality implicates the formation of cysts in the renal tubules, and the acronym RCAD (Renal Cysts And Diabetes) has been created to highlight this main feature together with diabetes. The cystic spectrum, observed in the majority of RCAD patients ( 62% Faguer S et al., 2011;∼75% Bingham C et al., 2002 and[START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]83% Edghill EL et al., 2006), includes simple cysts, multicystic renal dysplasia, and glomerulocystic kidneys. The cysts may appear on a renal ultrasound (mostly in the renal cortex), they are usually small in size [START_REF] Ulinski | Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort[END_REF] and were not reported to progressively increase in number over time [START_REF] Faguer | Diagnosis, management, and prognosis of HNF1B nephropathy in adulthood[END_REF].

In addition to cysts, other renal morphological anomalies have been reported as renal hypoplasia, renal agenesis, oligomeganephronia, solitary kidneys, horseshoe kidneys, and hydronephrosis [START_REF] Ulinski | Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort[END_REF][START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF][START_REF] Clissold | HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum[END_REF].

Prenatal renal anomalies encompass renal failure, oligohydramnios and pulmonary hypoplasia [START_REF] Madariaga | Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes[END_REF] and genetic variants in HNF1B are predicted to be the most common cause of isolated renal hypodysplasia in fetuses and children [START_REF] Weber | Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study[END_REF][START_REF] Thomas | HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort[END_REF]. Renal interstitial fibrosis can be also present in RCAD patients and has been associated with a highly variable rate of decline of estimated glomerular filtration rate (eGFR) [START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF][START_REF] Bellanné-Chantelot | Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations[END_REF]Edghill EL 2006).

Kidney disease can get worse progressing from chronic kidney disease (CKD) to kidney failure, also called end-stage renal disease (ESRD) [START_REF] Edghill | Hepatocyte nuclear factor-1beta gene deletions--a common cause of renal disease[END_REF] and between 13 and 20% of RCAD adult patients develop ESRD [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF][START_REF] Faguer | Diagnosis, management, and prognosis of HNF1B nephropathy in adulthood[END_REF][START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]. CKD has been reported in children (1-5%) (Verbitsky M et al., 2015;[START_REF] Thomas | HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort[END_REF] as well as in adults (15-40%) [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF][START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]Musetti C et al., 2014).

Renal physiological imbalances

Physiological imbalances in plasma and urine include hypocalciuria, hypermagnesuria, hypomagnesemia [START_REF] Adalat | HNF1B mutations associate with hypomagnesemia and renal magnesium wasting[END_REF][START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF][START_REF] Faguer | Diagnosis, management, and prognosis of HNF1B nephropathy in adulthood[END_REF][START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF][START_REF] Van Der Made | Hypomagnesemia as First Clinical Manifestation of ADTKD-HNF1B: A Case Series and Literature Review[END_REF], hyperuricemia (Bingham C et al., 2003;[START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF] and hypokalemia [START_REF] Faguer | Diagnosis, management, and prognosis of HNF1B nephropathy in adulthood[END_REF]. The prevalence of hypomagnesemia ranges drastically in different studies: 25% [START_REF] Raaijmakers | Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract[END_REF], 44% [START_REF] Adalat | HNF1B mutations associate with hypomagnesemia and renal magnesium wasting[END_REF], 45-60% (Faguer S et al., 2014), 72% (Ferrè S et al., 2013), 75% [START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]. Urinary magnesium wasting could be related to HNF1B transactivation role on FXYD2 gene, which encodes the γ subunit of the Na + -K + -ATPase involved in the reabsorption of Mg 2+ in the distal convoluted tubule [START_REF] Adalat | HNF1B mutations associate with hypomagnesemia and renal magnesium wasting[END_REF][START_REF] Ferrè | HNF-1B specifically regulates the transcription of the γa-subunit of the Na+/K+-ATPase[END_REF]. Potassium depletion may be related to magnesium depletion as a decrease in intracellular Mg 2+ concentrations may lead to the release of inhibition of renal outer medullary potassium channel (ROMK) causing K + wasting [START_REF] Yang | Magnesium modulates ROMK channel-mediated potassium secretion[END_REF].

Patients with HNF1B mutations display hyperuricemia and some present also early onset gout (Bingham C et al., 2003); also in these cases, the bibliography does not show the same values, attesting hyperuricemia between 11,6% and 63% [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF][START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF][START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF]Bingham C et al., 2003). Gout is predicted between 20% and 27% [START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF], Bingham C et al., 2003). It has been shown that a decrease in uric acid excretion was associated with mutations in the gene UMOD, encoding uromodulin (Bingham C et al., 2003;[START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF]; abnormal urate transport followed by elevated serum acid concentration, which leads to hyperuricemic nephropathy in some RCAD patients, could be related to UMOD gene misregulation since Hnf1b-conditional inactivation in mouse renal tubules results in reduced transcriptional activation of Umod [START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF].

Proteinuria and microalbuminuria have also been reported in ∼25% and ∼30% of subjects of HNF1B mutant carriers respectively (Dubois-Laforgue D et al., 2017).

Diabetes

Mutations in HNF1B could lead to the monogenic form of diabetes called maturity-onset diabetes of the young type 5 (MODY5). However, HNF1B mutations account for only ∼1-5%

of MODYs [START_REF] Beards | Mutations in hepatocyte nuclear factor 1beta are not a common cause of maturity-onset diabetes of the young in the U[END_REF][START_REF] Sadler | Genitourinary system[END_REF][START_REF] Edghill | HNF1B deletions in patients with youngonset diabetes but no known renal disease[END_REF]. Diabetes mellitus is the most frequent extra-renal feature present in HNF1B-associated patients and it is diagnosed at ∼24 years old [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF]. A diagnosis can be done sometimes also during the neonatal period [START_REF] Yorifuji | Neonatal diabetes mellitus and neonatal polycystic, dysplastic kidneys: Phenotypically discordant recurrence of a mutation in the hepatocyte nuclear factor-1beta gene due to germline mosaicism[END_REF][START_REF] Edghill | Hepatocyte nuclear factor-1 beta mutations cause neonatal diabetes and intrauterine growth retardation: support for a critical role of HNF-1beta in human pancreatic development[END_REF]. Different studies evaluated the prevalence of diabetes in RCAD patients showing that it is still an ambiguous value among HNF1B-associated patients: 45% [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF], 48% [START_REF] Faguer | Diagnosis, management, and prognosis of HNF1B nephropathy in adulthood[END_REF], 56% [START_REF] Bingham | Solitary functioning kidney and diverse genital tract malformations associated with hepatocyte nuclear factor-1beta mutations[END_REF], 60% [START_REF] Edghill | Hepatocyte nuclear factor-1beta gene deletions--a common cause of renal disease[END_REF]), 77% (Bellanné-Chantelot C et al., 2004), 82% (Dubois-Laforgue D et al., 2017).

The pathophysiology of diabetes in HNF1B mutations carriers is related to β-cell dysfunction and insulin resistance. The first results in reduced insulin secretion probably due to pancreatic hypoplasia and/or to a decrease in the number of β-cell available at birth [START_REF] Haldorsen | Lack of pancreatic body and tail in HNF1B mutation carriers[END_REF][START_REF] El-Khairi | The role of hepatocyte nuclear factor 1β in disease and development[END_REF], the second is correlated with reduced insulin sensitivity to endogenous glucose production in RCAD patients [START_REF] Pearson | Contrasting diabetes phenotypes associated with hepatocyte nuclear factor-1alpha and -1beta mutations[END_REF][START_REF] Brackenridge | Contrasting insulin sensitivity of endogenous glucose production rate in subjects with hepatocyte nuclear factor-1beta and -1alpha mutations[END_REF].

Pancreas morphological abnormalities and exocrine dysfunctions

Pancreas disorders could be divided into morphological abnormalities and exocrine dysfunctions. Pancreas structural abnormalities have been found in ∼10% of RCAD patients (reviewed in [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF][START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF] and encompass pancreatic hypoplasia [START_REF] Haumaitre | Severe pancreas hypoplasia and multicystic renal dysplasia in two human fetuses carrying novel HNF1beta/MODY5 mutations[END_REF][START_REF] Haldorsen | Lack of pancreatic body and tail in HNF1B mutation carriers[END_REF], atrophy, calcifications, pancreas divisum, ring pancreas, malrotation, intraductal papillary mucinous tumor and rarely cysts [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF]. Pancreas exocrine dysfunctions are often subclinical as reduced Vitamin D and E concentration, or fecal elastase concentration [START_REF] Bellanné-Chantelot | Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations[END_REF][START_REF] Haldorsen | Lack of pancreatic body and tail in HNF1B mutation carriers[END_REF].

Liver dysfunctions

Liver dysfunctions manifest as asymptomatic increase in levels of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), and γ-glutamyl transferase (GGT) in ∼15% of RCAD patients [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF] but the percentage can increase drastically according to the different studies (71% [START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF], 84% Bellanné-Chantelot C et al., 2004). On the other hand, liver structure anomalies as cholestasis [START_REF] Beckers | Neonatal cholestatic jaundice as the first symptom of a mutation in the hepatocyte nuclear factor-1beta gene (HNF-1beta)[END_REF], fibrosis, steatosis, and hepatomegaly account for less than ∼5% [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF].

Imaging evaluations highlighted abnormal biliary duct and biliary cysts in patients (32% [START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF] and electron microscopy has shown a reduction of primary cilia on the epithelial cells of the bile duct [START_REF] Roelandt | HNF1B deficiency causes ciliary defects in human cholangiocytes[END_REF].

Genital tract abnormalities

Genital malformations are found in almost 14% of HNF1B-associated patients and are detected preferentially in females (∼18%) than in males (∼10%) (reviewed by [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF]. The prominent feature in female patients is uterus development abnormalities, which cover uterus agenesis, bicornuate uterus, and didelphic uterus [START_REF] Avni | Renal cystic diseases in children: new concepts[END_REF][START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF]. In a cohort of females with congenital uterine abnormalities, HNF1B

heterozygous mutations were detected in nine of 108 patients (8.3%) [START_REF] Oram | Mutations in the hepatocyte nuclear factor-1β (HNF1B) gene are common with combined uterine and renal malformations but are not found with isolated uterine malformations[END_REF].

In male patients, genital tract abnormalities could be presented as cryptorchidism, testicular hypoplasia, vas deferens agenesis, epididymal cysts, enlarged seminal vesicles, hypospadias/phimosis, and asthenospermia [START_REF] Bingham | Solitary functioning kidney and diverse genital tract malformations associated with hepatocyte nuclear factor-1beta mutations[END_REF][START_REF] Bellanné-Chantelot | Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations[END_REF][START_REF] Faguer | Diagnosis, management, and prognosis of HNF1B nephropathy in adulthood[END_REF][START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF].

HNF1B mutations could also lead to failure of the fusion of the Müllerian ducts producing rare congenital uterine and upper vaginal abnormalities or aplasia; this indicates HNF1B as a candidate gene for Mayer-Rokitansky-Küster syndrome [START_REF] Bernardini | Recurrent microdeletion at 17q12 as a cause of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome: two case reports[END_REF][START_REF] Ledig | Recurrent aberrations identified by array-CGH in patients with Mayer-Rokitansky-Küster-Hauser syndrome[END_REF]. Moreover, HNF1B mutations were found in two of 34 individuals

(5,8%) with prune-belly syndrome, which is characterized by bilateral undescended testes, a triad of dilatation of the urinary tract and deficiency or absence of the abdominal wall musculature [START_REF] Murray | Whole gene deletion of the hepatocyte nuclear factor-1beta gene in a patient with the prune-belly syndrome[END_REF]Haeri S 2010;Granberg CF 2011).

Other clinical features

Neurodevelopmental disorders have been associated with a 1.4 Mb deletion at chromosome 17q12, counting 15 genes including HNF1B. In a wide cohort of 15,749 patients with autism spectrum disorders or cognitive impairment, this deletion was found in 18 subjects (1.1%) [START_REF] Moreno-De-Luca 2010) Moreno-De-Luca | Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia[END_REF] suggesting that one or more of these genes in the deleted interval is dosage sensitive and essential for normal brain development and function. Findings suggested that prevalence of autistic spectrum disorders recorded in the general pediatric population (1:150-1:300) is lower than in cases of patients with whole gene deletions of HNF1B and cystic kidney disease (3:53). For this reason. nephrologists should be aware of this potential association and refer to psychiatric counseling in uncertain situations [START_REF] Loirat | Autism in three patients with cystic or hyperechogenic kidneys and chromosome 17q12 deletion[END_REF].

Different studies suggest that lack of HNF1B expression is related to chromophobe RCC [START_REF] Rebouissou | Germline hepatocyte nuclear factor 1alpha and 1beta mutations in renal cell carcinomas[END_REF][START_REF] Lebrun | Cystic kidney disease, chromophobe renal cell carcinoma and TCF2 (HNF1 beta) mutations[END_REF][START_REF] Terasawa | Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines[END_REF]Wang CC et al., 2012). Moreover, overexpression of HNF1B has been found frequently in clear cell ovarian cancer [START_REF] Tsuchiya | Expression profiling in ovarian clear cell carcinoma: identification of hepatocyte nuclear factor-1 beta as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma[END_REF][START_REF] Kato | Expression of hepatocyte nuclear factor-1beta (HNF-1beta) in clear cell tumors and endometriosis of the ovary[END_REF][START_REF] Kato | Hypomethylation of hepatocyte nuclear factor-1beta (HNF-1beta) CpG island in clear cell carcinoma of the ovary[END_REF] and prostate

RCAD diagnosis and treatment

There are presently no clear directives on which patients should be genetically tested to confirm the diagnosis. In general, nephrologists might request a genetic test to confirm HNF1B heterozygous mutations in patients referred with a suspected diagnosis of autosomal dominant polycystic kidney disease, medullary cystic kidney disease, diabetic nephropathy, or CKD of unknown cause (Clissold RL et al., 2014;[START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF]. Other clinical features except for renal and glucose metabolic disorders as genital tract malformations, gout and elevated liver enzymes [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF], should provide extra clues to genetic testing analyses and early clinical intervention. Proteinuria is also a feature of RCAD syndrome as it has been found in almost one on three patients with HNF1B-associated disease [START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]. Importantly, mutations can occur spontaneously so family history may be absent. Because of this lack of familiarity in about 50% of patients and the intrafamilial variability, the prevalence of HNF1B gene mutations in the general population is unknown and it is probable that a large portion of RCAD patients was not recognized [START_REF] Beards | Mutations in hepatocyte nuclear factor 1beta are not a common cause of maturity-onset diabetes of the young in the U[END_REF][START_REF] Raaijmakers | Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract[END_REF].

Even if no casual therapy exists, an early diagnosis of RCAD syndrome is paramount for multiple reasons: to prevent unnecessary examination reducing costs to the healthcare system, to make (family) screening for diabetes, renal function decline, hypomagnesemia, to follow the degree of renal impairment and the presence of diabetes, to prevent diabetes complications and cardiovascular risk factors, to avoid the manifestation of gout adapting a diet and to prepare patients psychologically to potential limitation in their life (like a lack of a reproductive apparatus) [START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF][START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]. Faguer S et al. have developed the most efficient tool to select patients for genetic analysis based on 17 clinical, imaging, and biological variables related to RCAD syndrome like renal hyperechogenicity, cystic kidney, MODY, pancreatic hypoplasia etc. (sensitivity 98,2%, specificity 41,1%) (Faguer S et al., 2014). The observations about the difficulty in recognizing the syndrome highlight the requirement for an improved method of patient selection for genetic testing. In fact, RCAD patients could be misdiagnosed with other diseases for similar features in common. First, the most frequently observed phenotype in fetuses with the HNF1B-gene mutation is isolated bilateral hyperechogenic kidneys of normal or slightly increased size [START_REF] Ulinski | Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort[END_REF]Decramer S et al., 2007); this phenotype is common with ARPKD, ADPKD, maternal induced diseases, infection, ischemia, metabolic diseases, dysplasia, and nephrotic syndromes. After birth, some patients with HNF1B mutations would meet the diagnostic criteria for familial juvenile hyperuricemic nephropathy [START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF] as different patients with HNF1B mutations present early-onset gout and kidney disease. The RCAD disease can mimic also polycystic kidney disease especially in the prenatal setting and early childhood (Bergmann C et al., 2014). HNF1B mutations could even mimic Gitelman syndrome because of the concomitant hypomagnesemia and hypocalciuria [START_REF] Verhave | Hepatocyte Nuclear Factor 1β-Associated Kidney Disease: More than Renal Cysts and Diabetes[END_REF]. Dubois-Laforgue D et al hypothesized that some patients might be misdiagnosed as having type 1 diabetes at onset [START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF]. RCAD patients with CKD in combination with renal cysts are easily falsely diagnosed as autosomal dominant polycystic kidney disease (ADPKD) or medullary cystic kidney disease (MCKD). HNF1B-associated disease in CKD patients with diabetes is frequently misdiagnosed as diabetic nephropathy, CKD with gout and/or hyperparathyroidism is easily misdiagnosed as complications secondary to CKD itself. In all the cases the presence of multiorgan symptoms should help in being alert for the RCAD syndrome.

Three main methods have been developed to analyze the HNF1B gene:

1) direct sequencing of proximal promoter and intron exon-junctions

2) QMPSF (Quantitative Multiplex PCR of Short Fragments)

3) MLPA (Multiplex Ligation-dependent Probe Amplification)

The first method is used to detect mutations primarily within the coding sequences as well as the splicing mutations, the two following methods are used to detect deletions or exonic duplications. As whole gene deletions are responsible for the most of cases (Decramer et al., 2007;Bellane-Chantelot et al., 2005;[START_REF] Alvelos | A novel mutation of the HNF1B gene associated with hypoplastic glomerulocystic kidney disease and neonatal renal failure: a case report and mutation update[END_REF][START_REF] Dubois-Laforgue | Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects[END_REF] the search for a complete deletion by QMPSF is now the first molecular analysis followed by a direct sequencing in case of deletion absence.

The treatment is dependent on the degree of renal impairment and the presence of other features such as diabetes. Most of patients with HNF1B-associated diabetes mellitus need treatment with insulin as they respond poorly to sulphonylureas in contrast to HNF1Aassociated diabetes (MODY3) [START_REF] El-Khairi | The role of hepatocyte nuclear factor 1β in disease and development[END_REF]. Renal transplantation should be considered for patients who require renal replacement therapy. [START_REF] Pearson | Contrasting diabetes phenotypes associated with hepatocyte nuclear factor-1alpha and -1beta mutations[END_REF][START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF].

Molecular mechanisms underlying the RCAD disease

The molecular mechanism by which heterozygous mutations in HNF1B produce the disease has been debated especially for the lacking of genotype-phenotype correlation even between members of the same family with identical mutations, suggesting haploinsufficiency as the underlying disease mechanism as mentioned in the chapter 2.3 (Clissold RL et al., 2014).

The production of dominant-negative mutant products has been proposed as a possible mechanism on the basis of in vitro functional analysis of several mutations either affecting DNA binding or within the C-terminal transactivation domain and preventing the recruitment of coactivators [START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF][START_REF] Igarashi | Roles of HNF-1beta in kidney development and congenital cystic diseases[END_REF]. An apparent gain-of-function mutation has been also reported [START_REF] Wild | The mutated human gene encoding hepatocyte nuclear factor 1beta inhibits kidney formation in developing Xenopus embryos[END_REF] leaving still open the molecular mechanisms by which HNF1B heterozygous mutations are responsible for the syndrome. It is worth noting that transgenic mouse overexexpressing in the developing kidney a truncated version of Hnf1b functions in vivo as a dominant negative mutation [START_REF] Igarashi | Roles of HNF-1beta in kidney development and congenital cystic diseases[END_REF]).

Yet, it is unknown whether the putative truncated mutant proteins encoded by the mutated HNF1B allele are produced in vivo or are unstable and degraded. Note also that not patients with the whole gene deletion were leaner than those with HNF1B point mutations and present a more severe diabetes phenotype. On the other hand, patients with HNF1B mutations had a poorer renal prognosis than those with a whole gene deletion (lower eGFR and higher frequency of CKD3-4 or ESRD), a more frequent need of renal transplantation, and a less frequent normal function at follow-up. The authors suggested that some intragenic HNF1B mutations might exert a dominant negative effect that results in a more severe phenotype, which as mentioned above still do not explain the non correlation genotype-phenotype.

Alternatively,, the cause of this difference could be the lost of the protective effect from genes in the 17q12 deletion and the lost of other genes predisposing to renal disease. In fact, deletions in the 17q12 can encompass 15 genes (AATF, ACACA, C17orf78, DDX52, DHRS11, DUSP14, GGNBP2, HNF1B, LHX1, MRM1, MYO19, PIGW, SYNRG, TADA2A, and ZNHIT3). Interestingly no genotype/phenotype correlations in the two groups were observed for kidney morphology, pancreas morphology and exocrine function, plasma Cpeptide concentrations, liver tests, and genital tract abnormalities. Similar results concerning renal impairment were observed in patients with HNF1B deletions or mutations, but in smaller cohorts of patients. Other two publications from 2010 and 2016 [START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF]Clissold RL et al., 2016) showed similar results concerning renal impairment in patients with HNF1B deletions or mutations, but in smaller cohorts of patients.

There are several additional potential mechanisms by which heterozygous mutations in HNF1B lead to variable pathogenic phenotypes, including:

-Single nucleotide polymorphisms (SNPs) in non-coding regions may affect the stability of HNF1B mRNA and/or HNF1B gene expression; -Additional mutations in other genes that share the same biological pathways as HNF1B may either compensate or worsen HNF1B haploinsufficiency; -Gene modifiers that either aggravate or increase the severity of phenotype as it has been identified for several diseases (Cutting GR 2010); -Additionally, proteins interacting with HNF1B as cofactors or coactivators may present own variations depending on the patients; so, lack of interaction between mutated HNF1B and its partners may contribute to the misregulation of HNF1B functionality [START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF][START_REF] Hiesberger | Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis[END_REF][START_REF] Soutoglou | Acetylation regulates transcription factor activity at multiple levels[END_REF] as observed for PCBD1 mutations [START_REF] Ferrè | Mutations in PCBD1 cause hypomagnesemia and renal magnesium wasting[END_REF]. Finally and related to this, the dysregulation of micro RNAs (miRNAs) targeting HNF1B or controlled by HNF1B, may also be involved in the disease phenotype.

Until now, the lack of mouse models that reproduce the RCAD syndrome at the heterozygous state has been a major limitation. Similarly, heterozygous mutations in HNF1A, HNF4A, and GATA6 lead to diabetes in humans, but have no effect in mice. It is known that diseases prompted by haploinsufficiency are more common in humans than in mice but molecular studies are still necessary to uncover the reasons. This variation in phenotype may exist because of species-specific differences in the dosage of the transcription factor that is needed for full function [START_REF] El-Khairi | The role of hepatocyte nuclear factor 1β in disease and development[END_REF]. Alternatively as in the case of Hnf1 mutant mouse models, the mutations introduced do not replicate the human mutations and this could be another major lack in reproducing molecular events leading to a disease state.

The laboratory has generated a mouse model of RCAD syndrome introducing a human splicing mutation in Hnf1b gene. Our phenotypic analysis of this model show that the heterozygous mutants for the splicing mutation exhibit several features of the human RCAD pathology. Although not replicating entirely the RCAD disease to our knowledge this mouse line represent the first Hnf1b model that manifest a RCAD like phenotype at the heterozygous state, thus allowing to examine the consequences of the Hnf1b mutation in the context of the whole organism. In the Results section (Topic 1), a manuscript will be presented describing the development and the features of this model in detail with a major interest on the renal abnormalities, the main feature of the RCAD syndrome.

Furthermore, recent studies have demonstrated that HNF1B is recruited on the regulatory sequences of the miR-200 gene and directly controls the transcription of the miR-200b~429 cluster via a long non-coding RNA [START_REF] Hajarnis | Transcription factor hepatocyte nu-clear factor-1beta (HNF-1beta) regulates microRNA-200 expression through a long noncoding RNA[END_REF]. At the same time

Hnf1b was reported to be controlled by several microRNAs (Kornfeld JW et al., 2013;[START_REF] Patel | miR-17~92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease[END_REF]. Taking in consideration this new tight molecular control, which may shed more light on HNF1B gene network and regulation, we decided to search for miRNAs potentially regulated by this transcription factor as well as microRNAs targeting HNF1B. This study will be described in detail in a separate section of Results (Topic 2).

CHAPTER 3. microRNAs and renal diseases

Overview of microRNAs

A microRNA is a small non-coding RNA molecule of ∼22 nucleotides that negatively regulates gene expression at post-transcriptional level of numerous organisms including fungi, algae, plants, insects and animals (Bartel DP 2004;[START_REF] Griffiths-Jones | miRBase: tools for microRNA genomics[END_REF]. The first microRNA (abbreviated miRNA), lin-4, was discovered in 1993 in Caenorhabditis elegans [START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF] but only in the early 2000s miRNAs were recognized as a real class of biological regulators able to silence gene products [START_REF] Reinhart | The 21nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[END_REF]Pasquinelli AE et al., 2000;[START_REF]Identification of novel genes coding for small expressed RNAs[END_REF]. Since then, hundreds of miRNAs were characterized and together with siRNAs (small inhibitory RNAs) and piRNAs (PIWI interacting RNAs) they constitute the small noncoding RNA world [START_REF] Ghildiyal | Small silencing RNAs: an expanding universe[END_REF]. miRNAs induce gene silencing via base pairing to complementary sequences present on target molecules (mRNA or lncRNA) [START_REF] Ameres | Diversifying microRNA sequence and function[END_REF].

Considering that a single microRNA may regulate more than a hundred mRNAs [START_REF] Rajewsky | microRNA target predictions in animals[END_REF]Bartel DP 2009) and that microRNAs appear to target about 60% of all mammal genes [START_REF] Friedman | Most mammalian mRNAs are conserved targets of microRNAs[END_REF], the size of this regulation appear extremely elaborated. Recent analysis indicates the number of functional microRNAs in human at around 600 [START_REF] Fromm | A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome[END_REF]. Most of miRNAs are located within the cell, but some miRNAs (commonly known as circulating miRNAs), have been found in extracellular environment, including various biological fluids and cell culture media [START_REF] Weber | The microRNA spectrum in 12 body fluids[END_REF][START_REF] Boeckel | From heart to toe: heart's contribution on peripheral microRNA levels[END_REF], thus potentially serving as powerful tools for diagnosis and treatment [START_REF] Kaucsár | Post-transcriptional gene-expression regulation by micro RNA (miRNA) network in renal disease[END_REF].

microRNA biogenesis

miRNAs are the final product of a multi-step biogenesis process (Bartel DP 2009) and their biogenesis may be resumed in four steps: i. transcription;

ii. nuclear processing;

iii. nuclear exportation; iv. cytoplasmic processing

microRNAs in renal development and diseases

miRNAs are essential for virtually all aspects of mammalian biology, including development of key organs such the brain, the heart, and the kidney. This is demonstrated by the incompatibility with life in mouse models where factors involved in the maturation and functionality of microRNA are knocked-out (Wang Y et al., 2007;[START_REF] Bernstein | Dicer is essential for mouse development[END_REF]Morita S et al., 2007). The striking conservation of microRNAs during evolution is frequently associated with the conservation of their gene targets, which further denotes additional evidences about their biological importance [START_REF] Friedman | Most mammalian mRNAs are conserved targets of microRNAs[END_REF].

This chapter is mainly constructed to highlight the role of microRNAs in the kidney, as we performed almost all our analysis in renal tissues or renal cells. microRNAs act at different levels in this organ: (i) during development, (ii) for maintenance of renal functions, and (iii) for the progression of kidney diseases.

(i) Starting from nephrogenesis to renal senescence, different studies demonstrated the role of microRNAs in kidney development. Conditionally ablating the function of Dicer using Six2Cre in cells of the nephron lineage promoted apoptosis, induced premature interruption of nephrogenesis, and disrupted branching morphogenesis [START_REF] Nagalakshmi | Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney[END_REF]. Moreover, HoxB7Cre-mediated removal of Dicer from the ureteric bud and collecting system led to hydronephrosis and hydroureter, whereas some mutants with milder phenotypes retained most of the renal parenchyma but developed cysts in the collecting ducts [START_REF] Nagalakshmi | Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney[END_REF]. This phenotype was accompanied by disrupted ciliogenesis within the ureteric bud epithelium and the development of renal cysts [START_REF] Nagalakshmi | Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney[END_REF].

Characterization of three mouse models where key miRNA pathway genes Dicer, Dgcr8, and the entire Argonaute gene family (Ago1, 2, 3, and 4) were inactivated, revealed that inhibition of miRNAs in CDs spontaneously evokes a renal tubule injury-like response, which culminates in progressive tubulointerstitial fibrosis (TIF) and renal failure [START_REF] Hajarnis | Suppression of microRNA Activity in Kidney Collecting Ducts Induces Partial Loss of Epithelial Phenotype and Renal Fibrosis[END_REF].

Inactivation of Drosha or Dicer in podocytes produced dysplasia in early developing glomeruli and caused proteinuria and glomerulosclerosis [START_REF] Harvey | Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease[END_REF][START_REF] Ho | Dicer cuts the kidney[END_REF][START_REF] Shi | Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis[END_REF][START_REF] Zhdanova | The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy[END_REF]. Bai et al.

combining the expression profiles of microRNAs in young (3-month) and old (24month) rat kidneys together with senescence study in mesangial cells confirmed the regulatory importance of microRNAs in renal aging by inhibiting intracellular pathways such as those involving the mitochondrial antioxidative enzymes SOD2 and Txnrd2 (Bai XY et al., 2011).

(ii) miRNAs' are predicted to act in kidney homeostasis and maintenance by modulating the expression of different proteins. They regulate water re-absorption thorough the control of aquaporin (AQPs) channels expression [START_REF] Sepramaniam | MicroRNA 320a functions as a novel endogenous modulator of aquaporins 1 and 4 as well as a potential therapeutic target in cerebral ischemia[END_REF]2011), and they modulate osmotic response targeting Sodium-hydrogen antiporter 3 regulator 1 (Nherf1) [START_REF] Flynt | miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos[END_REF] or the osmotic response element binding protein (OREBP) [START_REF] Huang | Tonicity-responsive microRNAs contribute to the maximal induction of osmoregulatory transcription factor OREBP in response to high-NaCl hypertonicity[END_REF]. microRNAs maintain also the renin-producing juxtaglomerular cells and the morphologic integrity and function of the kidney (Sequeira- [START_REF]The microRNAprocessing enzyme dicer maintains juxtaglomerular cells[END_REF].

(iii) miRNAs' involvement have been identified also in a variety of renal diseases (reviewed by [START_REF] Ichii | Ichii O, Horino T1. MicroRNAs associated with the development of kidney diseases in humans and animals[END_REF][START_REF] Ma | The function of microRNAs in renal development and pathophysiology[END_REF], including diabetic nephropathy [START_REF] Dewanjee | MicroRNA: A new generation therapeutic target in diabetic nephropathy[END_REF], acute kidney injury (AKI) [START_REF] Jones | MicroRNAs in Acute Kidney Injury[END_REF], chronic kidney disease (CKD) [START_REF] Lv | Therapeutic potential of microRNAs for the treatment of renal fibrosis and CKD[END_REF], polycystic kidney disease [START_REF] Tran | The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity[END_REF][START_REF] Duan | PKHD1 post-transcriptionally modulated by miR-365-1 inhibits cell-cell adhesion[END_REF][START_REF] Phua | MicroRNAs in the pathogenesis of cystic kidney disease[END_REF], cancer [START_REF] Juan | Identification of a microRNA panel for clear-cell kidney cancer[END_REF]Catto JW et al., 2011) and others. A schematic representation of the renal pathogenic role of microRNAs is illustrated below (Figure 31).

HNF1B and microRNAs

Different studies performed in cell lines and mouse models suggest a fine regulation between HNF1B and some microRNAs. First, HNF1B was recenlty shown to be able to regulate the expression of different microRNAs as the long noncoding RNA (lncRNA) containing the miR-200 cluster [START_REF] Hajarnis | Transcription factor hepatocyte nu-clear factor-1beta (HNF-1beta) regulates microRNA-200 expression through a long noncoding RNA[END_REF] or miR-194-2/192 cluster [START_REF] Jenkins | Transforming growth factor β1 represses proximal tubular cell microRNA-192 expression through decreased hepatocyte nuclear factor DNA binding[END_REF]. A decreased expression of miR-200 has been observed in kidney tubule-specific deletion of Hnf1b, and in turn, knockdown of miR-200 in cultured renal epithelial cells inhibits tubulogenesis and produces cyst-like structures [START_REF] Hajarnis | Transcription factor hepatocyte nu-clear factor-1beta (HNF-1beta) regulates microRNA-200 expression through a long noncoding RNA[END_REF].

Knockdown of Hnf1b in HK-2 proximal tubule cell line inhibited mature miR-192 and miR-194 expression and the transcriptional factor was found to bind their promoter sequences [START_REF] Jenkins | Transforming growth factor β1 represses proximal tubular cell microRNA-192 expression through decreased hepatocyte nuclear factor DNA binding[END_REF].

Different microRNAs (miRNAs) have been found to control Hnf1b mRNA abundance in mouse kidneys as miR-17∼92, miR-92a or miR-802. Patel et al. reported that the miR-17∼92 miRNA cluster regulates the posttranscriptional expression of PKD genes, including Pkd1, Pkd2, and Hnf1b [START_REF] Patel | miR-17~92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease[END_REF]: kidney-specific transgenic overexpression of this oncogenic miRNA cluster seems to produce kidney cysts in mice, while a kidneyinactivation in a mouse model of PKD retards kidney cyst growth, and improve renal function. Within this cluster, miR-92a repressed the 3′ UTR of Hnf1b. Moreover, Hnf1b was shown to be a target of miR-802-dependent silencing in hepatocytes potentially reducing its transcript and causing glucose intolerance, and impairing insulin signalling in humans and mice (Kornfeld JW et al., 2013).

In the result section (Topic II) we present our last findings about a potential autoregulatory loop regulation between HNF1B and three microRNAs in the RCAD mouse model (miR-802, miR-192/194-2 and miR-30a), which may be essential during the last stages of kidney development.

CHAPTER 4. The urinary proteome as research tool

The urinary proteome

Proteomics is one of the main challenges of the post-genomic era consisting in the identification, expression, and characterization of proteins. Derived from the fusion of the words "protein" and "genome", proteomic evaluates the complete set of proteins encoded in the genome of a specific cell or organism. The researched information in a proteome corresponds to the sequence, structure, and localization of each protein, as well as the interaction with other proteins [START_REF] Ideker | A new approach to decoding life: systems biology[END_REF]. In doing so, proteomic analysis provides informative data related to the biological mechanisms, which can be useful for a better readout of the physiological and pathological processes affecting the life of organisms [START_REF] Latterich | Proteomics: new technologies and clinical applications[END_REF]Wright PC et al., 2012). Biological specimens, such as tissue, blood (plasma and serum) or urine are valuable sources of information. In particular, urinary proteomics represents an enlarging field of interest for biologists and physicians and proteomic analysis have been employed for the discovery and the validation of biomarkers of several kidney diseases [START_REF] Mischak | Proteomic biomarkers in kidney disease: issues in development and implementation[END_REF][START_REF] Magalhães | Urinary biomarkers for renal tract malformations[END_REF]. Urine represents an easy accessible biological fluid, which can be obtained in large quantities via a noninvasive procedure and may provides insights about different organs because it results from glomerular filtration via blood [START_REF] Thongboonkerd | Renal and urinary proteomics: current applications and challenges[END_REF]. Moreover, its proteome is originated 70% from the kidney and the urinary tract along with 30% from the circulation, which allows the study of different renal diseases and consequently associated events such as extracellular matrix (ECM) remodelling or fibrosis [START_REF] Schaub | Urine protein profiling with surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry[END_REF][START_REF] Klein | Comparison of CE-MS/MS and LC-MS/MS sequencing demonstrates significant complementarity in natural peptide identification in human urine[END_REF]Magalhães P et al., 2017). Oppositely, its limitation is due to daily variability caused by circadian rhythms, physical activity, diet and metabolic or catabolic processes (Weissinger E et al., 2004;[START_REF] Decramer | Urine in clinical proteomics[END_REF], which can be corrected by different adjustment or calibration/normalization methods (Jantos- [START_REF]Quantitative Urinary Proteome Analysis for Biomarker Evaluation in Chronic Kidney Disease[END_REF]. In the following manuscript we performed the first clinical proteomic analysis of the RCAD syndrome in a pediatric cohort of patients to provide new insights of this multisystem disorder identifying novel urinary biomarkers.

Analytic approaches

Proteomic analysis may require complex analytical steps to separate the mixture of all the proteins in accordance with their mass/charge ratio (m/z). Subsequently, these molecules are determined by mass spectrometry [START_REF] Resing | Proteomics strategies for protein identification[END_REF][START_REF] Zürbig | Peptidomics: methods and applications[END_REF]. While in a bottom-up approach proteins are cleaved into short peptides by the usage of a synthetic enzyme (usually trypsin) [START_REF] Resing | Proteomics strategies for protein identification[END_REF][START_REF] Zürbig | Peptidomics: methods and applications[END_REF], top-down methods allow the identification of naturally occurring peptides, which does not require any prior proteolytic procedure. These peptides are stable and soluble biomolecules ranging from 0.5 to 20 kDa, not requiring any protein digestion before MS analysis, and being usually derived by the activity of endogenous proteases/peptidases [START_REF] Zürbig | Peptidomics: methods and applications[END_REF][START_REF] Finoulst | Sample preparation techniques for the untargeted LC-MS-based discovery of peptides in complex biological matrices[END_REF].

For the analysis of pediatric RCAD urinary proteome we performed capillary electrophoresis coupled to mass spectrometry (CE-MS), which is one of the most relevant top-down approach that can be clinically applicable.

CE-MS workflow

Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has been employed in body fluids for the detection and identification of biomarkers for many diseases (Coon JJ et al., 2008;Mischak H et al., 2011;Klein J et al., 2016). The workflow of CE-MS normally comprises different steps. First, a step of protein separation is performed based on the electrophoretic mobility (dependent on size and charge of molecules) by CE, followed by a detection step using a MS (Figure CE-MS). Subsequently, after CE-MS run, data are processed through the use of a software that translates the mass spectral ion peaks representing molecules at different charge states into singles masses. Following calibration and normalization step, values of molecular masses and CE-migration time are assigned to each peptide (Coon JJ et al., 2008). Afterwards, all detected peptides are deposited, matched, and annotated in a database, enabling digital data compilation. In our study, after biomarker discovery, we combined the significant peptides of RCAD children in a mathematical classifier via support vector machine (SVM), an algorithm that allows a separation of features via multiple independent parameters in a high-dimensional hyperplane [START_REF] Mischak | Technical aspects and inter-laboratory variability in native peptide profiling: The CE-MS experience[END_REF]. This mathematical classifier was further validated using a blinded cohort, encompassing RCAD patients along with patients suffering from cystic kidney disease, RESULTS _________________ There is no clear genotype-phenotype correlation with haploinsufficiency being the main disease mechanism. The pathogenesis underlying this disease remained unclear since mice with heterozygous mutations in Hnf1b have no phenotype, while constitutive or conditional Hnf1b-ablation led to more severe phenotypes without reflecting the human disease.

To better understand the pathogenesis of RCAD, the lab has previously developed a mouse model carrying an identified human mutation at the intron 2 splice donor site, referred as splicing mutation intron-2 (Sp2). At the time I arrived at the lab the embryonic phenotype of this mouse model was already characterized and I was initially involved in the embryonic transcriptional profiling, which I subsequently used also for the microRNA study presented in Topic II. I was also involved into postnatal analyses in particular basal urinary/plasma analyses and into the urinary proteome analyses at postnatal stages as well as immunostaining evaluations.

I.2. Results and discussion

Heterozygous Hnf1b Sp2/+ mutant embryos exhibited bilateral renal cysts from E15.5, originated primarily from proximal tubules (PT) and intermediate nephron segments and glomeruli. They also exhibited apico-basal polarity defects in different renal tubules as well as delayed differentiation and unequal distribution of PT. Hydronephrosis and genital tract anomalies were also observed at later stages and postnatal life.

Combined mRNA-seq and ChIP-sequencing analyses at different embryonic stages (E14.5, E15.5, E17.5 and P1) identified more than 200 genes down-regulated at both E17.5 and P1, primarily expressed in early proximal tubules and to a lesser extent in loops of Henle and collecting ducts and involved in transport, lipid and organic acid metabolic processes.

Several of these genes were putative HNF1B targets. Notably, among the different regulators, Hnf4a expressed in PT, and an HNF1B target, was the strongest down-regulated, while the expression of other previously identified targets, including the cystic disease genes (Pkh1, Pdk2, Bicc1) or Pax2, Wnt9b were slightly or not affected.

Postnatal analyses revealed a range of renal abnormalities ranged from few clusters of glomerular cysts, micro cysts, hydronephrosis and rare cases of multi cystic dysplasia.

Partially urinary concentration defects were observed and investigating the urinary proteome of this mice we found a particular signature composed of several differentially excreted peptides in Hnf1b Sp2/+ versus WT mice including down-excretion of Uromodulin (Umod) and Epidermal Growth Factor (EGF) and up-excretion of collagen fragments.

I.3. Conclusions

Together these data suggest that reduced expression of HNF1B below a critical level results in kidney abnormalities such as cyst initiation and hydronephrosis, associated with the deregulation of a subset of target genes. HNF1B impairment leads to progressive urinary system physiology dysfunctions consistent with a pathological state that mirror RCAD syndrome in different features.

This work is presented in the following pages as a manuscript.

SCIENTIFIC ARTICLE 1 A novel mouse model of the Renal Cysts and Diabetes (RCAD) syndrome uncovers functional HNF1B dosage-sensitive genes during kidney development contributing to the disease

Leticia L Niborski1# , Mélanie Paces-Fessy 1# , Pierbruno Ricci 1# , Adeline Bourgeois 1 , Pedro Magalhães These observations together led to the suggestion that mice are either less sensitive to haploinsufficiency or alternatively the mouse mutant models so far generated do not correctly represent the mutations identified in humans.

To explore these possibilities and obtain a more comprehensive view of HNF1B function in organ development and disease in the context of the whole animal, we generated a novel mouse RCAD model by introducing a previously identified human point mutation at the intron-2 splice donor site (<IVS2nt+1G>T, {Bingham, 2003#36}{Harries, 2004 #37}). Five different mutations have already been identified at this splice site indicating that it is a putative hotspot mutation. Moreover, patients with these mutations exhibited the typical features HNF1B mutations (renal cysts, diabetes, hyperuricaemia) {Bingham, 2003 #36} (Adalat et al, 2008) and could therefore be representative of a large number of HNF1B intragenic disease causing mutations.

We present here a comprehensive analysis of the renal phenotype of heterozygous mutants for the splicing mutation at different developmental stages and a preliminary characterization of the progression of the renal disease phenotype in adulthood. Heterozygous mutants (referred as splicing mutation intron-2 (Sp2) Hnf1b Sp2/-exhibited bilateral cysts and tubular dilatations as well as glomerular cysts from E15, along with rare cases of genital tract abnormalities and other extra renal manifestations that have been described in human HNF1B mutant carriers.

Unlike heterozygous mouse mutants for a null allele, in which the HNF1B protein levels remained essentially unchanged {Kornfeld, 2013 #39}, in the Hnf1b Sp2/+ heterozygous mutants the HNF1B protein levels were strongly reduced, in particular during development, without correlating with the more modest reduction of Hnf1b transcript levels.

Further molecular and global transcriptional profiling indicate that only a subset of target genes was sensitive to reduced levels of HNF1B protein at different embryonic stages, whereas the previously identified targets strongly downregulated in the absence of Hnf1b, including the cystic disease genes, were relatively insensitive to reduced levels of HNF1B

RESULTS

Generation of a mouse model reproducing a clinical HNF1B splicing mutation

The mouse model carrying a point mutation at the intron-2 splice donor site, was generated by homologous recombination by introducing a G to T point mutation (Fig. 1 A) thus mimicking the c.544+1G>T (<IVS2nt+1G>T) mutation identified in humans {Bingham, 2003 #36}. The LoxP-flanked neomycin-resistance cassette located within intron-1 was subsequently excised by breeding heterozygous mutant mice with a "Cre deleter" mouse line (Materials and Methods). Thus, the Hnf1b mutant allele encompassed the human splicing point mutation and a unique LoxP site within the intron-1.

To precisely define the consequences of this mutation, we performed semiquantitative RT-PCR using RNA from heterozygotes for the splicing mutation (Hnf1b sp2/+ ) and wild type (WT) kidneys using primers located in exon-1 and exon-3. Sequence of the PCR products obtained indicated the expected production in heterozygous mutants of the two Hnf1b spliced isoforms A and B. Four additional novel transcripts were detected, corresponding to isoforms A and B in which exon 2 was deleted and the isoforms A and B deleting respectively the last 32 bp of exon 2 through the use of a novel cryptic splice donor site (Fig. 1 B). This pattern of alternative splicing from the mutant allele was observed at different stages and in other tissues expressing Hnf1b (not shown). The detection of HNF1B variants lacking the exon 2 was consistent with previous analysis in human patients {Harries, 2004 #37}. Yet the variants lacking the last 32 bp of exon 2 has not been described in humans.

The predicted consequences of deleting either the entire or part of exon 2 is the production of putative truncated HNF1B proteins encompassing the N-terminal dimerisation domain, but lacking the DNA binding domain and the C-terminal transactivation domain.

Moreover and consistent with the absence of the NLS, upon transient expression in different cell-lines, these truncated proteins remained mainly in the cytoplasm (Supplementary Fig. S1).

Therefore, if these putative truncated proteins are expressed they would be non functional (see below). In agreement with these observations, analysis of embryos from heterozygous intercrosses at different developmental stages indicated that the homozygous Hnf1b Sp2/Sp2 mutation was lethal soon after implantation similarly to the Hnf1b homozygous mutants for a null allele previously described {Barbacci, 1999 #26}, thus confirming that the splice mutation generate nonfunctional Hnf1b transcripts (data not shown).

Heterozygous embryos for the Hnf1b Sp2/+ allele exhibit bilateral tubular dilatations, glomerular cysts and hydronephroses

To evaluate the onset and progression of the mutant phenotype, histological analyses of heterozygous mutants and WT littermates were performed at different embryonic stages in a mixed background C57BL/6N x 129/sv. Up to E14.5, heterozygous and WT embryos developed normally (Supplementary Fig. S2). However from E15, we reproducibly observed bilateral cystic kidneys, including glomerular and tubular cysts predominantly in the cortical and medullar regions, respectively (Fig. 2). A similar phenotype was observed at E16.5 (not shown). As kidney growth progressed, by E17.5 there was an apparent reduction of the volume of cystic structures relative to whole kidney volume, while glomerular cyst exhibited similar distribution all throughout development (Fig. 2).

To examine the influence of mouse strain susceptibility into the disease phenotype we backcrossed the Hnf1b sp2/+ mutant mice into two inbreed backgrounds: C57BL/6N and 129/sv. We observed similar tubular dilatations and glomerular cysts from E15 in the two inbreed backgrounds (Fig. 2). However, in the C57BL/6N background the Hnf1b sp2/+ embryonic kidneys exhibited a more severe and variable renal phenotype, in particular at later stages and newborn pups with higher numbers of both medullar and glomerular cysts frequent pelvic dilatations and hydronephroses, which in some cases occurred bilaterally as well as duplicated kidneys (Fig. 2 and Supplementary Fig. S3: E-F C57BL/6N).

Heterozygous mutant embryos or newborns were obtained at the expected Mendelian ratio in different background; however in the C57BL/6N background 10-15% of heterozygous mutants die between P1-25 days of age (Supplementary Table S1), in general without being able to define the causes due to cannibalism. Heterozygous mutants in both backgrounds were able to reproduce, but they were less fertile than WT littermates. Further postnatal analysis revealed rare cases of genital tract abnormalities (agenesis of an uterine horn, epididymis cysts, abnormal branched and dilated seminal vesicles) in either C57BL/6N or 129/sv backgrounds. Heterozygous mutants exhibited also pancreatic dysfunction with glucose intolerance, a phenotype that will be described elsewhere by C. Haumaitre's laboratory (IBPS, UMR7622). Other associated phenotypes, but only in the C57BL/6N background were unilateral or bilateral absence of eyes (30% of n: 36 Hnf1b Sp2/+ males and n=20 Hnf1b Sp2/+ females), which is manifested from embryo stages along with a higher susceptibility to eye infections. This ocular phenotype could eventually be linked to the mutation Rd8 mutation of the Crb1 gene present in the strain C57BL/6N {Mattapallil, 2012 #70}. Unless otherwise indicated the following analysis were mainly performed in the C57BL/6N background.

Altogether, these data show the heterozygous mutants exhibited several of the urogenital phenotypes described in RCAD patients and further suggest that genetic modifiers may either aggravate (C57BL/6N) or attenuate (129/sv) the phenotype.

Relative expression of normal Hnf1b transcript and protein levels in Hnf1b Sp2/+ mutant kidneys at different stages

To further understand the molecular mechanism by which the heterozygous mutants for the splicing mutation exhibited a RCAD like disease phenotype, a finding that contrasts with the absence of phenotype in heterozygous mutants for a null allele (Hnf1b lacZ/+ {Barbacci, 1999 #26}), we first examined the transcript levels produced by the normal allele in Hnf1 Sp2/+ heterozygous kidneys relative to WT. We used primers located in the ATG translational site and in the last 32bp of the exon 2, which encompassed sequences absent in the abnormal spliced transcripts produced by the mutant allele (Supplementary Table S2). Unexpectedly and similarly to heterozygous mutants for a null allele, in our heterozygous mutants, the Hnf1b transcripts were decreased by only 20-25% relative to WT, instead of the expected 50% reduction (Fig. 3 A). We therefore hypothesized that the HNF1B protein levels produced by the normal allele were either decreased under lower levels than those of transcripts or alternatively, the functional protein levels were decreased by heterodimerization with the putative truncated proteins produced by the mutant allele (depicted in Fig. 1B) HNF1B immunostainings at different embryonic stages revealed a global decrease in nuclear staining in Hnf1b Sp2/+ mutants, although dilated tubules did exhibit nuclear staining without any evidence of a further decrease or a focal lack of expression as reported in other autosomal dominant polycystic kidney diseases (Fig. 3 B).

To obtain a quantitative evaluation of the levels of HNF1B protein and of the putative truncated variants, Western blot analyses were performed using whole cell extracts from at least 4 independent mice/embryo kidneys microdissected at different stages. In contrast to the moderate decrease of transcript levels, in heterozygous mutants we observed a stronger decrease in the HNF1B protein levels, in particular at earlier stages (Fig. 3 C,D). This finding provides further evidence of a posttranscriptional and/or translational control of HNF1B protein by a not yet identified mechanism. We also confirmed that in Hnf1b Lacz/+ mice, the HNF1B protein levels were actually increased (Fig. 3 D) consistent with the absence of a disease phenotype as previously reported {Kornfeld, 2013 #39}). More importantly, we were unable to detect any truncated protein encoded by abnormally spliced Hnf1b transcripts, under conditions the endogenous WT HNF1B protein was easily detected, thus suggesting that they were either very unstable or not produced.

Altogether these results suggest that decreased levels in the HNF1B functional protein are likely responsible of the cystic disease phenotype of our mutants, rather than through a dominant negative effect via the formation of nonfunctional heterodimers between HNF1B

and the truncated proteins encoded by the abnormal spliced transcripts.

Embryos heterozygous for the splicing mutation display glomerular cysts, early proximal tubule dilatations and delayed differentiation of proximal tubules and collecting ducts

We previously reported that Hnf1b, expressed in the ureteric bud (UB), the entire collecting system and nascent nephrons, is required for early ureteric bud branching and initiation of nephrogenesis {Lokmane, 2010 #12}. We therefore began the phenotypic analysis by examining the expression of Calbindin-D-28K, a protein first expressed in the emerging UB branches of the metanephric kidney and found that it showed a similar expression profile in E14.5 heterozygous mutants and WT (Fig. 4 We next examined the origin of cystic structures by staining with different nephron and collecting ducts markers at different stages (E14.5-16.5 in Fig. 4; E17.5 and P1 in Fig. 5). For these analyses we selected mutant kidneys without overt hydronephrosis in order to visualize the collecting duct network and medullar nephron tubules. Interestingly, using the marker of early proximal tubules (PTs) HNF4A we observed not only decreased expression of this transcription factor, but also a decrease in the number of labeled structures (Fig. 4 E,E').

Notably, the mature PT marker LTA was not expressed up to the E16.5 stage (Fig. 4 F') and began to be expressed by E17.5 (Fig. 5 A'). However, the Hnf1b Sp2/+ PT clusters were consistently reduced in size and exhibited an unequal distribution both at E17.5 and P1 (Fig. 5 B'). Interestingly, most of the Hnf1b Sp2/+ medullar cysts and tubular dilatations observed, both at E17.5 and P1, were stained by the Na-K-Cl cotransporter (NKCC2), which is expressed in the thick ascending Loop of Henle (Fig. 5 In summary, in addition to cortical glomerular cysts, cysts and tubular dilatations in Hnfb1 Sp2/+ appear to predominate initially in early proximal tubules and subsequently from E17.5 in the thick Loop of Henle, while neither nascent nor mature collecting ducts appeared significantly affected (see below).

Hnf1b Sp2/+ tubular dilatations of developing nephrons are associated with apical polarity defects Cystogenesis in embryonic kidneys has been reported to be associated with several cellular defects, including abnormal cell-polarity changes in cell-cell and cell-matrix interactions as well as increased proliferation and apoptosis {Wilson, 2011 #46}.

We found that the PT brush border marker villin was normally expressed in Hnf1b Sp2/+ nondilated tubules, but it was interrupted in cystic proximal tubules (Fig. 6). PTs also exhibited lower expression of HNF4A with a far stronger decrease in cystic PTs (compare Interestingly, the basal membrane marker Laminin (Lam) exhibited a global decrease and partial disorganization in E15.5 heterozygous kidney tubules (both nondilated and dilated) (Fig. 6 F, H). In medullar cystic tubules, the NKCC2 apical staining of epithelial cells of the thick ascending limb of the Loop of Henle (TAL) was also decreased in a proximo-distal gradient and disorganized (Fig. 6 J). At later stages most of the tubular dilations were found in the TAL, while PTs exhibited rare dilatations.

Consistent with the lack of dilatations/cysts in Hnf1 Sp2/+ collecting system, the apical markers of collecting ducts Muc1 and CK, were both correctly expressed (Fig. 6 E, F, K, L).

As previously reported in other Hnf1b-deficient kidneys {Gresh, 2004 #10}{Desgrange, 2017 #5}, staining with α-acetylated tubulin, a component of cilia, showed a reduction of cells with cilia in cystic tubules, but a rather similar distribution in non-dilated heterozygous mutants.

Further analysis of proliferation and apoptosis using respectively, the mitosis marker phosphorylated histone H3 and Tunel assay, did not detect significant changes in the tubular structures of heterozygous mutants as compared with WT at E15.5 (data not shown). Together these results show that decreased levels of HNF1B appear to affect basal membrane organization without affecting apical cell polarity markers in nondilated tubules. The decreased expression of apical and brush border markers as well as the defects in cilia integrity in cystic tubules of Hnf1 Sp2/+ mutants appear to be secondary to tubular dilations.

These observations contrast our previous results showing that lack of Hnf1b results in disrupted apico-basal cell-polarity and epithelial organization of both non dilated and dilated collecting duct tubules {Desgrange, 2017 #5}.

Transcriptomic analyses at different stages uncover common genes sensitive to reduced levels of HNF1B protein

We initially focused our expression analysis on cystic disease genes previously identified as HNF1B targets {Gresh, 2004 #10}{Verdeguer, 2010 #45} as well as additional putative target genes involved in cystogenesis (see {De Vas, 2015 #8}). The expression of most of these genes was however, only slightly or not affected (Supplementary Fig. S4),

suggesting that a more complex process is involved in the disease phenotype. To further elucidate the cellular and molecular components sensitive to HNF1B levels, we performed RNA-sequencing on WT and heterozygous mutant kidneys at E14.5 (considered as predisease kidneys) and at disease stages (E17.5 and P1). Microdissected kidneys from embryos/newborns of the same litter were used. We also included in these analysis deep RNA-sequencing at E15.5 on pools of the two kidneys from n=3 WT and n=3 heterozygous embryos (6 kidneys/sample) (Supplementary Material and Methods).

Since WT mice were compared with heterozygous mutants still expressing HNF1B we did not expected differentially expressed genes at very high levels and therefore considered an absolute fold-change cutoff value > or < 0.70 log, instead of 1 with an adjusted p-value < 0.05. At E17.5 an additional list of genes with a cutoff value of < -0,6 log was included since contained many known HNF1B target genes that were significantly downregulated (Supplementary Table S3 provide the lists of up-and down-regulated at each stage).

Remarkably, the RNA-seq data identified several genes differentially expressed with foldchanges > 1 at all stages. From E14.5 the number of differentially downregulated genes was increased (51, 37, 241 and 127 genes at E14.5, E15.5, E17.5 and P1, respectively consistent with the growth and further morphogenesis of the kidney, Only a reduced number of UP-regulated genes differentially expressed were observed with no common genes between stages neither putative HNF1B targets among them. In contrast, the number of shared downregulated genes increased from 10 at E14.5 and E15.5 to reach 91 genes at E17.5 and P1 (Supplementary Table S3, File 5). The down regulated genes with greater fold-changes were expressed predominantly in developing PTs and to lesser extent in primitive Loops of Henle and S-shaped bodies and in the cortical and medullary collecting ducts. GO-term analysis {Chen, 2009 #71} highlighted transporter activity, active trans-membrane transporter activity, organic acid and lipid metabolic processes and metabolism terms as well as an association with abnormal renal/urinary system physiology, renal reabsorption, aminoaciduria, decreased urine osmolality and nephrocalcinosis (Table 1, Supplementary Table S4). From E17.5 and consistent with the onset of mature cell-types in the developing nephrons, the pathways enriched included SLC-active transmembrane transport, transport of glucose and small molecules and metabolism (Supplementary Table S4).

It is of interest that among the 25 identified anchor genes of early proximal tubule (EPT) and predicted to be controlled by HNF1B and HNF4A {Thiagarajan, 2011 #72}, 18 genes were found down-regulated at E17.5 and 15 genes at P1 (Supplementary Table S3, File 6). While nine EPT-anchor genes were downregulated at both stages, others were specific to either E17.5 or P1 suggesting a dynamic temporal control of these genes. It is worth noting that the expression of other EPT anchor genes was either not affected, such as Cml1, AI317395, Ugt2b37 or only modestly downregulated (Mogat2, Bdha2, Aqp11, C2, Sord, Cryl1) thus indicating that the observed down regulations are not simple due to a reduction in proximal tubule structures.

Similarly, both at E17 and P1, the downregulated genes included drug-metabolizing enzymes (Abcc2, Ggt1, Fmo2, Akr1c1) and more than fifty SLC-transmembrane-transporter genes (organic cation (the Slc22a family) and sodium glucose transporters gene, see Table S3, File 7) that have been shown to be transcriptionally controlled in postnatal proximal tubules by HNF4A along with HNF1A {Martovetsky, 2013 #73}. Unexpectedly, Hnf1a expression, which is restricted to developing and adult PTs was only moderately decreased at E17.5 but remained comparable to WT levels at P1 and postnatal mutant kidneys (not shown), further indicating that during development HNF1A was unable to compensate for the decreased protein levels of HNF1B.

It is worth noting that ChIP-PCR/ChIP-seq assays in kidney cell-lines and adult kidneys (reviewed by {Ferre, 2018 #69} or E14.5 kidney ChIP-seq ({Desgrange, 2017 #5}; C. Heliot and S.C. unpublished data) detected specific recruitment of HNF1B to most of the consensus binding sites found in the regulatory sequences of these downregulated genes. The mRNAseq data showed however that the expression of these targets was not similarly affected.

Amongst those HNF1B targets strongly downregulated at most of the stages in our mutants we can mention Hnf4a, Pdzk1, Pah, Tmem27, Cubn, Spp1, Spp2, Kcnj as well as many SLCtransmembrane transporters, thus indicating that these genes represent a subset of targets that appeared to be the most sensitive to reduced levels of HNF1B protein (Supplementary Table S3, see below Fig. 7). Intriguingly, Pdzk1, a HNF1A target in adult kidneys, encodes a either not affected or modestly and not significantly decreased (Supplementary Table S3).

We further confirmed the mRNA-seq data by in situ hybridization (ISH), immunostaining and additional Q-RTPCR analyses. Consistent with the mRNA-seq data, ISH of Fbp1 and Spp2, two early proximal tubule anchor genes {Thiagarajan, 2011 #72}, showed a strong downregulation of Fbp1 transcript levels and to a lesser degree of Spp2 both at E17.5 and P0 Hnf1b Sp2/+ kidneys (Supplementary Fig. S5), while as expected ISH for Wnt9b, Pax2

and Pkhd1 showed similar expression to WT littermates (not shown). Similarly, immunofluorescence analysis for SPP1 (osteopontin), CUBN and LRP2 in E17.5 and P0

Hnf1b Sp2/+ kidneys, showed a strong downregulation of SPP1 and CUBN, and a more modest decrease of LRP2 (Fig. 7A).

While in the mRNA-seq analysis the known targets Umod, Tmem27 and Pkhd1 were significantly downregulated at E17.5, but not at P1 (Supplementary Table S3), further validations by Q-RTPCR at different stages and adult kidneys showed a significant downregulation of Umod and Tmem27 from E16.5 to adults in heterozygous mutants (Fig. 7 E;

Fig. S7). The additional targets examined Hnf4a, KcnJ1 (RomK), Lrp2, Ihh, exhibited a preferential downregulation during development up to P0, whereas Umod and Tmem27

remained significantly downregulated in adult mice (Fig. 7 E). In addition, we examined the expression of Aqp2, a gene found downregulated during development in collecting ducts lacking Hnf1b {Desgrange, 2017 #5} and upregulated in postnatal kidneys of Hnf1b conditional inactivated medullar renal tubules {Gresh, 2004 #10}. We found that Aqp2 expression was indeed downregulated throughout development, whereas it was only modestly and not significantly increased in adult mutant kidneys (Fig. 7 E).

Together these results show differential dosage sensitivity among the HNF1B-activated genes during metanephric kidney development. Our data reveal that among the various metanephric kidney developmental processes known to be controlled by HNF1B, the genes participating to proximal tubule differentiation and the onset of nephron cellular maturation are those exhibiting a unique response to the levels of HNF1B protein.

Preliminary postnatal characterization of heterozygous Hnf1b sp2/+ mutant mice

As mentioned above (Supplementary TableS1), a fraction of heterozygous died between P1 and P25, yet the renal phenotype in these cases could not be studied owing cannibalism.

Otherwise, mice lived more than one year, beginning to manifest disease symptoms approximately after 12 months. In few cases, disease symptoms were manifested already in 5 month-old mice and required euthanasia (Fig. 8 D).

Body weight curve analyses showed approximately 20% reduction in weight of heterozygous mice relative to WT, with males and females behaving rather similarly (Supplementary Fig S6). Analysis of kidney weight/body weight ratio of males at different ages did not show significant differences as compared to WT littermates (Supplementary Fig. S6). Yet, one month-old heterozygous mutants exhibited lower kidney weight/body weight ratios than WT reflecting mild hypoplastic kidneys.

Further histological analysis of adult Hnf1b sp2/+ mice revealed variability in the severity of the renal phenotype with increased abnormalities observed both in aged males and females.

They usually exhibited unilateral hydronephrosis while the other kidney less affected, displayed mainly cortical or medullar glomerular cysts with collapsed or rudimentary capillary tufts (Fig. 8, B, C, H, I) as well as microcysts (Fig. 8 B,I). Dilated of Bowmann's spaces were often filled with finely granular proteinaceous material (Fig. 8 B', E', I'). A rare case (1/48 heterozygous mutants) exhibited severely bilateral affected kidneys: one kidney was highly multicystic/hypoplastic and nonfunctional and the other was severely hydronephrotic (Supplementary Fig. S6 H,H'). This mouse exhibited obvious symptoms of severe renal dysfunction, including bloody urines, at the time of dissection (12-month-old), but at 6 months its renal functions were similar heterozygous littermates indicative of progressive hydronephrosis with age. Accordingly, some old mice (males or females) developed giant hydronephrosis.

We then performed preliminary urinary analyses at different ages under basal conditions. Three month-old Hnf1b sp2/+ mutant mice exhibited normal physiological parameters. However, by 6 month-old they exhibited defective urine concentrating ability with polyuria, and reduced urine osmolality (Table 2). Reduced urine osmolality was also observed after 22hs of water deprivation (data not shown). By 12 months, although urine volumes remained higher than WT, urine osmolality was more modestly and non-significantly decreased (Table 2). A similar increase in 24-hour urine output and daily water consumed was observed in three independent groups of mice in 5-6 month-old mutant mice, followed by an attenuation of these parameters in 12 month-old mice (Supplementary Table S6).

Furthermore, impaired urinary concentration ability was also observed in 8-months Hnf1b sp2/+ mice in a mixed genetic background, but not in 6-month-old in the 129/sv background (not shown).

In agreement with the lower urine osmolality, 6-month-old Hnf1b sp2/+ mice had significantly lower urinary magnesium, sodium and potassium concentrations. However, the total excretion of these solutes was increased as compared to WT (Table 2); this tendency was maintained in 12-month-od mice, although did not reach significance. Interestingly, urinary calcium concentration was significantly increased despite decreased osmolality. Further, plasma analysis in a separate group of mice revealed an increase of creatinine levels, although non-significant, while the levels of Mg were similar to WT. We have aso observed a significant increase in the levels of the alanine aminotransferase ALT and a tendency of higher levels aspartate aminotransferase (AST), reflecting potential liver dysfunctions in our heterozygous mutant mice as previously reported in some RCAD mutant carriers {Iwasaki, 1998 #77}.

To gain further insight into the pathophysiology of the renal disease we also performed urinary proteome analysis. Urinary low molecular weight proteomes were investigated from urine samples of 17 Hnf1b sp2/+ mutant and 18 WT mice with an age ranging from 3 months to 17 months, using Capillary electrophoresis-mass spectrometry (CE-MS) and tandem mass spectrometry (MS/MS). We identified 40 significant differentially excreted peptides (Supplementary Table S7A). The most prominent findings associated to our mouse model were a substantial decrease of Epidermal Growth Factor (EGF), uromodulin (UMOD)

and the kidney androgen-regulated protein (KAP) and a tendency of an increase of collagen fragment urinary excretion. Amongst the collagen fragments (e.g. collagen type I alpha-1, type I alpha-2 and type III), 23 were increased and only 4 displayed a decrease in the urine of heterozygous mutant mice (Supplementary Table S7A). Interestingly, no significant differences were detected in the peptides excreted by younger and older Hnf1b sp2/+ mice (data are not shown). Subsequently, we performed a peptide-based in silico protease analysis based on the N-and C-terminal and on the mean intensities (peptide amplitudes) of the 40 sequenced naturally occurring peptides and found both downregulated proteases, such as cathepsins and matrix-metalloproteinases and upregulated proteases, such as granzymes and collagenases potentially responsible for generation of the urinary peptides in our mouse model (Supplementary Table S7B).

The urinary proteomic profile of Hnf1b sp2/+ is consistent with the HNF1B-dependent regulation of both Umod {Gresh, 2004 #10; Fig. 7) and of the Kap gene, found strongly downregulated at all stages examined (Supplementary Table S3). In this context, mutations in the Umod gene lead to autosomal dominant tubule interstitial diseases, a rare disease characterized by progressive tubulointerstitial damage, impaired urinary concentrating ability, hyperuricemia, renal cysts, and progressive renal failure [START_REF] Rampoldi | The rediscovery of uromodulin (Tamm-Horsfall protein): from tubulointerstitial nephropathy to chronic kidney disease[END_REF], thus sharing some of the features exhibited by our mouse model as well as RCAD patients. The function of the Kap gene, although encoding one of the most abundant proteins in the mouse PT, remains mainly unknown and no orthologous was found in humans. Of note, a decreased EGF urinary concentration has been reported in several kidney injury rodent models (reviewed {Klein, 2016 #80}{Kok, 2014 #81}) and low urinary excretion of EGF was found in ADPKD patients at an early stage in the disease {Weinstein, 1997 #82}.

DISCUSSION

We report here the generation of a novel mouse model of the RCAD disease by replicating a previously identified human splicing mutation and show that at the heterozygous state reproduces several of the urogenital defects described in HNF1B mutant carriers. These observations show that not only humans but also mice are sensitive to haploinsufficiency for HNF1B.

Our findings highlight the importance to precisely replicate the human disease mutations within the Hnf1b locus. Although previous mouse Hnf1b mutations ({Barbacci, 1999 #26};Coffinier, 1999) generated a loss of function alleles as our new model, the levels of the HNF1B protein at the heterozygous state did not decrease and were even increased (Kornfeld, 2013 #39}, Fig. 3 C, D). In these previous knock out models one of the major difference with our model was the presence of the neomycin resistance cassette with its regulatory elements, which may impact severely the genomic structure of the locus.

An additional important observation is that the levels of HNF1B protein in heterozygous mutants were strongly reduced, in particular during development, without correlating with the corresponding transcript levels. Thus, HNF1B activity appears to be highly regulated at the post-transcriptional and/or translational level. Previous analyses have also reported the apparent non-correlation between the transcript levels and HNF1B binding activity during mouse development [START_REF] Cereghini | Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles[END_REF]. Further studies of our mouse model are required to fully define the underlying molecular mechanisms involved and eventually uncover how the HNF1B protein levels could be manipulated. The ubiquitin proteasome pathway and miRNA-mediated regulation of HNF1B are, amongst others, possible mechanisms mediating tightly regulation of HNF1B protein levels and potentially deregulated in our model. This knowledge will certainly have potential implications for disease therapy.

The high variability in the phenotype of HNF1B mutant carriers has been explained by diverse mechanisms ranging from modifier genes, environmental factors, epigenetic influences, interacting cofactors and potential dominant negative function of certain mutations. We show that heterozygous mouse mutants, similar to RCAD patients develop heterogeneous urogenital abnormalities. Moreover, they exhibited a more severe phenotype in the C56BL/6N background than in the 129/sv background with 10% of C57BL/6N Hnf1b Sp2/+ mutants dying during the first month of life. Thus, genetic modifiers may indeed either aggravate or attenuate the disease phenotype. Interestingly, even in these two inbreed backgrounds we still observed variability in the phenotype presentation of heterozygous Hnf1b Sp2/+ notably at later stages of development and postnatal life. In particular, in the C57BL/6N background very often one kidney was more severely affected and hydronephrotic while the other was moderately affected mainly with glomerular cysts and microcysts. This variability was also observed in Hnf1b Sp2/+ mutants from the same litter and raised together reducing different environmental exposition. One possible explanation of this variability could be the known inherent stochasticity in transcription and translation processes (Bar-Even A et al, 2006) or epigenetic modifications that may also underlie the observed variability in HNF1B mutant carriers.

Our results also show that although transcripts from the mutated allele are transcribed at relative lower levels that those from the WT allele, there were no evidences of nonsense mediated mRNA decay (Fig. 1 B), thus confirming previous observations of a differential susceptibility to mRNA decay of naturally occurring HNF-1B mutations [START_REF] Harries | The position of premature termination codons in the hepatocyte nuclear factor -1 beta gene determines susceptibility to nonsense-mediated decay[END_REF]).

However, we did not detect any of the potential truncated proteins encoded by the alternative spliced transcripts, thus excluding a proposed dominant negative action. It is possible that these truncated proteins are misfolded and degraded by ubiquitin-mediated protein quality control system (Rousseau A, Bertolotti 2018). These observations further suggest that intragenic HNF1B mutations leading to premature stop codons do not necessarily encode the respective truncated proteins thus excluding, at least in these cases, the hypothesis that function as dominant negative via heterodimerisation with the WT protein.

Owing the developmental character of the RCAD disease, we focused our analysis throughout embryogenesis. Global transcriptional profiling at different embryonic stages indicate that only a subset of the established target genes was sensitive to reduced levels of HNF1B protein. Unexpectedly, most of the targets strongly decreased in the absence of Hnf1b, most notably the cystic genes considered the major downstream effectors of the HNF1B-associated cystic renal disease, were relatively insensitive to HNF1B dosage.

Remarkably, the most downregulated genes were primarily involved in proximal tubule unpublished data), is the unique transcriptional regulator strongly reduced in our mutants at all developmental stages, further suggesting that the transcription factor HNF4A cooperate with HNF1B in the observed developmental renal phenotype. Such a functional interaction between HNF1A and HNF4A has already been described in pancreatic islets [START_REF] Boj | Epistasis of transcriptomes reveals synergism between transcriptional activators Hnf1alpha and Hnf4alpha[END_REF].

Despite a global increase in the number of downregulated genes from E14.5 to E17.5, we also observed dynamic and temporal regulation patterns (i.e. some genes were strongly downregulated at an early stage but not at a later stage) suggesting an increasing complexity in the regulatory network involved in the differentiation and maturation of nephron segments.

It is tempting to speculate that similar changes in promoter occupancy patterns during development involving new recruitments, release, and exchange of specific factors as described during hepatocyte differentiation (Kyrmizi et al, 2006), take also place during renal development.

Not unexpectedly since HNF1B and the structurally related transcription factor HNF1A bind to the same sequences either as dimers or heterodimers, several of the proximal tubule genes strongly downregulated during development in our mutants have also been shown to be targets of HNF1A or HNF1A/HNF1B heterodimers in adult kidneys [START_REF] Saji | Transcriptional regulation of human and mouse organic anion transporter 1 by hepatocyte nuclear factor 1 alpha/beta[END_REF]Kikuchi et al.2007;[START_REF] David-Silva | Hepatocyte nuclear factors 1α/4α and forkhead box A2 regulate the solute carrier 2A2 (Slc2a2) gene expression in the liver and kidney of diabetic rats[END_REF]. In our mutants, the expression of Hnf1a in the PT was only moderately decreased at E17.5 and not affected at P1 and P21 (Supplementary Table S3 and data not shown), indicating that HNF1A was unable to compensate for HNF1B decreased levels during development. However, it appears, at least partially, to compensate in postnatal life as indicated the observed restoration of the expression of some proximal tubule markers such as Lrp2 and Spp2 in Hnf1b Sp2/+ adult kidneys. In this context, heterozygous Hnf1b Sp2/+ did not show glycosuria, suggesting that HNF1A replaced HNF1B in the control of Sglt2 (Slc5a2), a gene strongly downregulated in our mutants at both E17.5 and P1. Other targets however were found strongly downregulated at all stages in both our mutants and Hnf1a-/-mice, such as the phenylalanine hydroxylase (Pah) and the PDZ Domain-Containing Protein 1 (Pdzk1), suggesting their transcriptional dependence on HNF1A/HNF1B heterodimers. Unexpectedly, the renal expression of other known HNF1 targets, including Tmem27 and the cationic amino acid exchanger Slc7a9 were not affected in Hnf1a-deficient mice [START_REF] Bonzo | Metabolomics identifies novel Hnf1alpha-dependent physiological pathways in vivo[END_REF] and remained downregulated in our heterozygous mutants (Fig. 7), highlighting the complexity in the regulatory networks of these two transcription factors in the cells they coexpress.

Adult Hnf1b sp2/+ mice often exhibited unilateral hydronephrosis (40% n: 45). One the most common cause of hydronephrosis is ureter obstruction. Although we did not observed ureter obstruction in our mutants, occasionally we observed dilated ureters at the ureteropelvic junction (Sup. Fig. 3-C57BL/6N). Of note, hydronephrosis and hydroureter have been already described in some RCAD patients (Adalat et al, 2008) conditions along with transcriptomics and urinary metabolomics, we have not observed misexpression of AQP2 in our mutant mice (data not shown). It is worth noting that the defects observed in Hnf1b sp2/+ mutant mice are not restricted to the collecting ducts. Indeed, the combined decreased expression of Tmem27 and Umod found in heterozygous mutant mice (Fig. 7 E) may at least in part, explain the observed urinary concentration defects since reduced urinary concentrating ability has been described in both Umod-and Tmem27deficient mice [START_REF] Bachmann | Renal effects of Tamm-Horsfall protein (uromodulin) deficiency in mice[END_REF][START_REF] Mutig | Activation of the bumetanide-sensitive Na+,K+,2Cl-cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner[END_REF][START_REF] Malakauskas | Hepatocyte nuclear factors 4alpha and 1alpha regulate kidney developmental expression of drug-metabolizing enzymes and drug transporters[END_REF] Interestingly, Tmem27-deficiency leads also to defective renal amino acid uptake in mice and generalized aminoaciduria [START_REF] Malakauskas | Hepatocyte nuclear factors 4alpha and 1alpha regulate kidney developmental expression of drug-metabolizing enzymes and drug transporters[END_REF], which it would be important to further explore in our model.

We recently reported a metabolic profiling of different organs, including kidney, pancreas and liver as well as plasma of adult Hnf1b sp2/+ mice (8-months, mixed genetic background). Although urinary metabolomics was not performed, we found evidences of impaired amino acid renal metabolism in kidneys and reduced levels of total free amino acids and increased myo-inositol in plasma samples that are metabolic parameters reflecting impaired renal function (Torrel et al, 2018). The metabolomic analyses also suggested that Hnf1b sp2/+ mice exhibited disturbed hepatic lipid metabolism and endocrine pancreas alterations. Likewise, and as previously been reported in RCAD mutant carriers, we also observed in our mutants a significant increase in the plasma levels of the alanine aminotransferase (ALT) and a tendency of higher levels aspartate aminotransferase (AST)

reflecting liver dysfunctions (Table 2). Unlike humans, however adult mice did not exhibit hypo-magnesemia and/or hyper-magnesuria (Table 2) reported in more than 40% of HNF1B mutant patients. We do not have a mechanistic explanation for the normal urine and plasma Mg levels in our mutants, which may reflect differences between mouse and humans in ion transport regulation and/or adaptive mechanisms. For instance, the lack of hyperuricaemia observed in Umod transgenic mice despite the defective transport in the TAL, is likely due to the uricase activity that catalyses the conversion of uric acid to allantoin and is present in rodents but not in humans [START_REF] Rampoldi | The rediscovery of uromodulin (Tamm-Horsfall protein): from tubulointerstitial nephropathy to chronic kidney disease[END_REF].

More importantly, our urinary proteome analysis uncovered a particular profile in our heterozygous mutants, with a substantial decrease of uromodulin (UMOD) and epidermal growth factor (EGF) peptides, predictive of progressive decline in kidney function and kidney injury, as well as UP-excreted collagen fragments, which are consistent with an excessive extracellular matrix (ECM) turnover. Interestingly, a subsequent analysis in a pediatric cohort of RCAD patients revealed a similar signature with a majority of peptides collagen type I or type III fragments enriched in the urine of RCAD patients and a down-excretion of uromodulin fragments, together with other additional de-regulated peptides (Ricci P, Magalhães P, et al., Submitted (#footnote) In summary, the Hnf1b sp2/+ heterozygous mutant mouse model represents a unique clinical/pathological viable model of the human disease and promises to be important in the integrative evaluation of the broad facets of this disease ranging from various developmental abnormalities to kidney, pancreas and liver dysfunctions in the context of the whole animal.

# Ricci P 1 *, Magalhães P 2,3 *, Krochmal M 2 , Pejchinovski M 2 , Daina E 4 , Caruso MR 4 , Goea L 1 , Iwona Belczacka I

MATERIAL AND METHODS

Generation of a mouse model carrying a point mutation at the intron 2 splice donor site

The knock-in mouse carrying a human splicing point mutation was generated according to a project presented by S. Cereghini with the support of the GIS Maladies Rares and the Mouse Clinical Institute. The splice mutation into the Hnf1b locus was introduced by homologous recombination. The WT sequence GAC/g taagtgttttaacctt was mutated to GAC/t taagtgttttaagctt sequence (Capital letters show end Exon2 bases, lower letters indicates the intron2-Exon2 junction sequence, in bold the point mutations). The LoxP-flanked neomycinresistance cassette was located within intron-1 and subsequently excised by breeding heterozygous mutant mice with a "Cre deleter" mouse line, in this way the mutated allele encompassed the point mutation at the splice donor site in addition to a single LoxP site and HindIII restriction site aagctt (underlined) within intron 2. Mice heterozygous for the Hnf1b null allele (Hnf1b

RNA extraction and real-time PCR analysis

Both metanephroi of each embryo were dissected in ice cold Dulbecco Modified Medium (DMEM), washed in PBS. Total RNA was extracted using the miRNase Mini Kit (Qiagen), Dnase1 treated on the columns and 250 ng was reverse-transcribed using the high capacity cDNA reverse transcription kit (Applied Biosystems). Real-time PCR was performed using Fast SYBR Green master mix (Applied Biosystems) and the Step-One Plus system (Applied Biosystems) as described (Paces-Fessy, 2012 #1). The method of relative quantification (2 -ΔΔCT ) was used to calculate the expression levels of each target gene, normalized to cyclophilin A [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[END_REF], and relative to WT cDNA from E15.5 embryonic kidney. The primers used are listed in Supplementary Table S2. All PCR reactions were run in duplicate. Number of kidney samples is indicated in the figures. The mean and SEM were calculated by genotypes and the statistical significance was determined using Student's t-test (significance at *P < 0.05, **P < 0.01, ***P < 0.001).

Semiquantitative RT-PCR

Total RNA from microdissected kidneys was extracted and subjected to semiquantitative RT-PCR as described {Lokmane, 2008 #2} with the following modifications. The conditions were chosen so the RNAs analyzed were in the exponential phase of amplification by performing different PCR cycles indicated in Fig. 1. PCR products were resolved in 2% agarose/TBE ethidium bromide gels and photographed using GELDOC documentation system. Densitometry quantification was performed in ImageJ software. Primer sequences used were Gapdh for normalization and for Hnf1b, vATG and v695 (Supplementary Table S2)

Western blots

The two kidneys of each embryo from at least three (n=3) different embryos were pooled, snap-frozen in liquid N2 and reduced to a fine powder under Liquid N2. They were lysed in ice-cold buffer (10% glycerol, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% NonidetP-40 with complete protease inhibitor cocktail ROCHE) using a dounce-homogenizer. Samples were centrifuged for 15 minutes at 13,000 rpm 4°C, and the supernatant kept in Liquid N2 until use. Whole cell extracts containing 30µg of protein were prepared in SDS sample buffer and subjected to SDS-PAGE (4-15% Mini-PROTEAN® TGX™ Precast Protein Gels, Biorad). After transfer to PVDF membrane and blocking with TBST (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1 % Tween), 5% skim milk, immunostaining was performed by overnight incubation in TBST 1% skim milk buffer with a rabbit polyclonal antibody against HNF1B (1:500) raised in the laboratory against residues 39-89 of the mouse HNF1b protein {Haumaitre, 2005 #3}; α-Actin was subsequently used as a loading control (monoclonal antibody A4700, 1:1,000; Sigma-Aldrich). Secondary antibodies are listed in Supplementary Table S8. Positive bands were detected by chemiluminescence (ECL, Pierce; SuperSignal West Dura, Thermo Scientific). Images were captured with a Fusion Fx7 and quantified with Bio-1D software (Vilbert-Lourmat).

Plasma and urine analyses

Urine and plasma were obtained on age-and gender-matched heterozygous and WT mice (males). They were housed in light-and temperature-controlled room with ad libitum access to tap water and standard chow (Diet AO4, SAFE, France). 24-h urine samples collected under mineral oil to avoid evaporation were obtained at baseline in individual metabolic cages, after 2-3 days habituation. Each 24-h, animals were weighed and the food, water intake, urine volume and fecal weight recorded. Blood was sampled by retro-ocular puncture in general 2 weeks after being in the metabolic cages and plasma samples were kept at -80°C.

The urinary concentrating ability was tested after 22h water deprivation. Urinary creatinine, urea and electrolytes, plasma urea, creatinine, magnesium, ASAT, ALAT and were measured on Olympus AU400 Chemistry Analyzer (ICB-IFR2, Laboratoire de Biochimie UFR de Médecine Paris 7, Bichat. Osmolality was measured using a vapor pressure osmometer (Wescor 5500, USA).

Statistical

Data are represented as mean ± standard error of mean (SEM). Comparisons between groups were performed using with a two-tailed test of significance. P < 0.05 was considered to be significant with *P < 0.05, **P < 0.01, ***P < 0.001.

Immunofluorescence on transfected cells, In situ hybridization (ISH) and immunohistochemistry

The human epithelial C33 cell line was transiently transfected with reporter constructs of WT-HNF1B and truncated HNF1B proteins lacking the exon 2 and immunofluorescence analysis on glass-slides was carried out as described {Barbacci, 2004 #25}, using rabbit anti-HNF1B (Supplementary Table S2),

In situ hybridization (ISH) on paraffin sections was performed as described {Lokmane, 2008 #2}. The Fbp, Spp2 cRNA probes were generated by PCR (GUDMAP database). Embryos and postnatal kidneys up to 2 month-old were fixed with 60% ethanol/11% formaldehyde and 10% acetic acid. Adult kidneys (> 2months) were fixed in alcoholic Bouin (Duboscq-Brasil) solution and paraffin sections were used for H&E histological analysis and immunohistochemistry. Antibody staining on paraffin sections was performed as described {Lokmane, 2010 #12}{Desgrange, 2017 #5}. For each probe (ISH) or antibody sections from at least three different embryos were used. The primary and secondary antibodies are listed in supplementary Table S7. Terminal deoxynucleotidyl transferase, mediated digoxigenindeoxyuridine nick-end labelling (TUNEL) was performed on E15.5 kidney sections as described {Paces-Fessy, 2012 #1}{Heliot, 2013 #7}.

SUPPLEMENTARY MATERIAL AND METHODS

Transcriptional profiling at different stages by mRNA-sequencing

The two kidneys from heterozygous for the Hnf1b s p l i c i n g m u t a t i o n a n d W T e m b r y o s w e r e microdissected from the same litter. This requirement limited the number of samples used in general to 2 controls and 2 mutants, independently of the sex. Note that we found a similar phenotype in males and females. The stages analyzed included E14.5 (when morphologically the mutant kidneys were morphological normal and considered as pre-disease and at disease stages E15.5, E17.5 and postnatal day 1 (P1). Note that at E15.5 we performed deep sequencing from pooled samples of 3 WT and 3 Hnf1b Sp2/+ (6 kidneys each sample) RNA from microdissected kidneys was extracted by Tryzol, using the miRNA mini kit Qiagen for the extraction of total and miRNAs. The quality of the RNA samples was assessed on the Agilent Bioanalyzer system using the Agilent RNA 6000 Nano Kit (Agilent Technologies) and RNA with a Ring higher than 8 was used. 1-2 µg of total RNA were used for mRNA isolation using the Dynabeads® mRNA DIRECT™ Micro Kit (ThermoFisher Scientific). mRNA was digested with RNase III, purified, hybridized and ligated to Ion Adaptors, reverse transcribed, barcoded and amplified, using the Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific). RNA sequencing was performed on an Ion Proton™ System, according to the manufacturer's instructions. The prepared libraries were quantified and pooled together in duplicates at the required concentration. The pools were then processed on an OneTouch 2 instrument and enriched on a One Touch ES station. Templating was performed using the Ion PI™ Template OT2 200 Kit (ThermoFisher Scientific) and sequencing with the Ion PI™Sequencing 200 Kit on Ion Proton PI™ chips (ThermoFisher Scientific) according to commercial protocols.

The resulting RNA-Seq BAM files were analyzed with the Bioconductor package metaseqR {Moulos, 2015 #60} a n d a p p l y i n g the edgeR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) methodology for differential expression analysis with default settings.

Urinary proteomic analysis

Urines from wild-type (WT) and Hnf1 Sp2/+ mutant mice (males) were collected by spontaneous voiding and kept frozen at -80°C or after being placed individually in metabolic cages as described in Material and Methods. The total volume of 24-hs urines was aliquoted and frozen at -80°C. A 150 µl sample of mouse urine was diluted with the same volume of urea buffer (2 M urea, 10 mM NH4OH, 0.2% sodium dodecylsulfate). The total volume of urines was aliquoted and frozen at -80°C. 150 µl of urine samples of were ultrafiltrated, desalted, lyophilized and resuspended for proteomics analyisis as described {von zur Muhlen, 2012 #61}.

CE-MS analysis and data processing.

Capillary electrophoresis-mass spectrometry (CE-MS) analysis was performed using a Beckman Coulter Proteome Lab PA800 capillary electrophoresis system (Fullerton, CA) online coupled to a micrOTOF II MS (Bruker Daltonic, Bremen, Germany) as described {Coon, 2008 #59}{Mischak, 2013 #58}. For normalization caused by analytical variances and differences in urine dilution, MS signal intensities were normalized relative to 41 internal standard peptides generally present in at least 90% of all urine samples, with small relative standard deviation (SD) {von zur Muhlen, 2012 #61}. The peak lists obtained characterized each peptide by its molecular mass (in daltons), normalized CE migration time (in minutes) and normalized signal intensity. The data of all detected peptides were deposited, matched, and annotated in a Microsoft SQL database as previously described allowing further analysis and comparison of multiple samples {Siwy, 2011 #62}.

Peptide sequencing and In silico protease prediction.

For sequencing peptides, tandem mass spectrometry was performed as described {Klein, 2014 #66}. Concisely, MS/MS analysis were performed using a Dionex Ultimate 3000 RSLC nano flow system (Dionex, Camberly, UK) coupled to an Orbitrap Velos MS instrument (Thermo Fisher Scientific). Data files were analyzed using Proteome Discoverer 1.2 and searched against the Swiss-Prot Mus Musculus database {Nkuipou-Kenfack, 2017 #63}. To link the urinary peptide fragments to the proteases involved, in silico protease mapping was generated using Proteasix software as described by {Klein, 2013 #65}.

Statistical analysis.

The main peptidomic differences between the WT and Hnf1 Sp2/+ urines were obtained using the P-values based on Wilcoxon rank-sum test. Statistical adjustment of P-values due to multiple testing was performed by Benjamini and Hochberg method. Peptides that were detectable in >90% of mice and reached an adjusted P value of <0.05 were further considered as relevant. A-Q-RT PCR of normal Hnf1b transcripts at the indicated stages from WT and Hnf1b Sp2/+ kidneys. Number of samples were: at E14.5: 2 WT and 5 Hnf1b Sp2/+ ; at E15.5: 3 WT and 6 Hnf1b Sp2/+ (2 litters), at E16.5: 3 WT and 8 Hnf1b Sp2/+ (2 litters); at E17.5: 3 WT, 4 Hnf1b Sp2/+ ; at P0: 5 WT, 10 Hnf1 Sp2/+ (2 litters) and adults: 3 WT and 3 Hnf1b Sp2/+ . The 2 kidneys of each embryo/mice were pooled. Hnf1b WT transcripts were amplified by primers located in the ATG translational site and in the last 32bp of the Exon 2. Asterisk show significant values p<0,05 (*) and p<0,001 (**). Error bars represent standard error of the mean (SEM). B-HNF1B immunostaining of E15.5 embryo sections show a partial decrease in nuclear staining in Hnf1b Sp2/+ . Note HNF1B nuclear staining in both nondilated and dilated Hnf1b Sp2/+ tubules. C-Representative western blot of WT and Hnf1b Sp2/+ kidney extracts at the indicated stages form WT and Hnf1b Sp2/+ . The two HNF1b variant A and B are visible on blot. α-actin was used to normalize for protein amount. D-Quantification of western blots show more than 50% reduction in HNF1B protein levels in Hnf1b Sp2/+ relative to WT particularly at earlier stages. A decrease of 55% and 67% is observed respectively at E15.5 and E17.5, while at P0 is decreased by 30% and in adults by 60%, with some variability. In adult heterozygous for the null allele of Hnf1b (Hnf1b Lac/+Z ) we observe an increase of 98% in HNF1B levels. N= 4 to 6 WT and Hnf1b Sp2/+ embryo /mouse kidneys were used per stage and 2 independent experiments were performed. Significant differences between mutants and WT (P < 0.05) are indicated (*).

LEGENDS TO FIGURES

Figure 4: Hnf1b Sp2/+ exhibit normal branching of the ureteric bud but delayed differentiation of proximal tubules and collecting duct maturation. Immunohistochemical analyses of E14.5 to E16.5 WT and Hnf1b Sp2/+ kidney embryos from the same litter, with Calbindin-D-28K (A, A') and PAX2 (B, B') and (C, C'), WT1 (D, D'), the early proximal tubule marker HNF4A (E, E'), the proximal tubule LTA (F, F'), the collecting duct markers pancytokeratin CK (G, G') and DBA H, H') at the indicated embryo stages. Note decreased expression of HNF4A (E'), and the absence of LTA (F') and DBA (H') in Hnf1b Sp2/+ sections, while the other markers express similar to WT. Sections (except C, C') are co-stained with DAPI.

Figure 5: Immunohistochemical analyses of nephron segment and collecting duct markers of E17.5 -E18.5 and P0 kidney sections In Hnf1b Sp2/+ kidneys LTA staining shows decreased and unequal clusters of proximal tubules both at E17.5 (compare A and A') and P0 (compare B and B'), NKCC2 a marker of the thick ascending Loop of Henle, stains predominantly medullar-cortical cystic structures (compare C and C'; D and D') and DBA is absent in Hnf1b Sp2/+ collecting ducts at E17.5 (I'), P0 (L'). ). Sections (except C, C', E, E') are co-stained with DAPI.

Figure 6: Cystic and tubular dilations of proximal tubule and thick Loop of Henle cells exhibit abnormal apico-basal polarity and reduced number of cells with cilia

Confocal microscopy of co-stained sections with Villin (Vil1) and HNF4A (A, B, B' magnification in B') and Vil1 and HNF1B (C, D, magnification D') revealed that the expression of Villin, specifically localized on the brush border of proximal tubules, is interrupted in several regions of cystic structures of Hnf1b Sp2/+ indicative of brush border loss, as highlighted in B' and D' magnifications. Confocal images of Muc1 and Lam1 (E, F)) and Lam1 and ZO1 ((G, H) co-immunohistochemistry show that the collecting duct apical marker Muc1 in E15.5 Hnf1b Sp2/+ (F) is expressed similar to WT (E), while the basement membrane Lam1 exhibit a partial disorganized pattern in Hnf1b Sp2/+ (F and H) compared to WT kidneys (E, G). The tight junction ZO1 is normally expressed (compare G and H). Co-stained P0 kidney sections with NKCC2 and pancytokeratin (CK) with only NKCC2 in I, J and merged NKCC2 and CK in K, L, show strong decreased staining of NKCC2 in cystic medullar Loops of Henle of Hnf1b Sp2/+ (compare WT 1, K with J, L), while the apical expression of collecting ducts with CK (K, L) is not affected in Hnf1b Sp2/+ kidneys. Acetylated tubulin cilium staining (M, N, N' magnification) shows partial loss of primary cilia in cystic structures in E15.5 mutant cysts, while non-dilated tubules have apparent normal cilia distribution of cilia (compare M (WT) with N, N' (Hnf1b Sp2/+ ).

Figure 7: Reduced expression of a subset of HNF1B targets in Hnf1b Sp2/+ kidneys Representative Immunostainings of E17.5 WT and Hnf1b Sp2/+ kidneys with TMEM27 (Collectrin) (A, A') and CUBN (B, B'), SPP1 (Osteopontin) (C, C') and LRP2 (Megalin) (D, D') showing strongly reduced expression of TMEM27, CUBN and SPP1 (A', B', C'), in Hnf1b Sp2/+ mutant kidneys while the expression of LRP2 is only moderately reduced (compare D with D'). E: QRTPCR analysis of selected HNF1B targets at different stages show significant and strong downregulation of Hnf4a, Ihh, Lrp2, Kcnj1, Tmem27 and Umod during embryo stages up to P0, consistent with mRNA-seq data (Sup Table S3). Also included Aqp2, which is not a HNF1B target. Aqp2, Lrp2 and Ihh while downregulated during development are either slightly up upregulated or expressed similarly to WT in adult mice. In adults, Umod and Tmem 27 remained downregulated.

Figure 8: Representative histological sections of the renal phenotypes of adult kidneys from Hnf1 Sp2/+ males and females (A-F) H&E stained sections of male kidneys of WT 12 months (A) and Hnf1b Sp2/+ (B-F) showing kidney sections of 2month (B), 11 month-(C) , 5 month-(D), 17 month-(E), 12 month-old (F). H&E stained sections (G, J, K, L) and trichrome Masson (H, I) stained sections of female kidneys. G: WT; H to L of Hnf1b Sp2/+ of 2months (H), 12 months (I), 17 months (J), 15 months (K, L). Note clusters of glomerular cysts (magnifications shown in B', E', F', I', I"), microcysts (B, H, I), severe hydronephrosis both in old males (E) and females (L) and rare cases of a severe dysplastic kidney (F; see also supplementary Figure S6). Note no evidences of fibrosis in trichrome Masson stained sections (H, I). TABLE 1: Selected Gene ontology (GO) terms of differentially expressed genes at different embryonic stages TABLE 2: Urine and plasma parameters of WT and Hnf1b Sp2/+ mice Values are the mean ± SEM and correspond to the average of measurements from 2 or 3 days of urine sample collection under basal conditions at the indicated ages. Urine samples at 3 and 6-months were from the same series of mice, while at 12-months as well as the plasma analysis were from separate groups of mice. Left Kidney Weight (KW) (g) 0,14 ± 0,01 0,11 ± 0,02* 0,2 ± 0,02 0,19 ± 0,03 0,22 ± 0,04 0,22 ± 0,04

Right Kidney Weight (KW) (g) 0,14 ± 0,01 0,11 ± 0,02* 0,22 ± 0,01 0,23 ± 0,04 0,24 ± 0,04 0,21 ± 0,04 %KW (left) / BW 0,71 ± 0,05 0,58 ± 0,07* 0,6 ± 0,09 0,66 ± 0,24 0,72 ± 0,07 0,74 ± 0,15 %KW (right) / BW 0,69 ± 0,05 0,59 ± 0,05* 0,65 ± 0,07 0,79 ± 0,03 0,78 ± 0,11 0,70 ± 0,15 Left Kidney Weight (KW) (g) 0,29 ± 0,05 0,17 ± 0,09 0,25 ± 0,06 0,22 ± 0,07

%KW (left+right) / BW 1,40 ± 0,1 1,16 ± 0,
Right Kidney Weight (KW) (g) 0,26 ± 0,02 0,19 ± 0,001 0,27 ± 0,08 0,22 ± 0,06 %KW (left) / BW 0,77 ± 0,08 0,59 ± 0,13 0,75 ± 0,07 0,72 ± 0,19 %KW (right) / BW 0,72 ± 0,08 0,66 ± 0,05 0,82 ± 0,15 0,72 ± 0,13 %KW (left+right) / BW 1,49 ± 0,14 1,15 ± 0,18 1,58 ± 0,19 1,44 ± 0,23

n=3 n=2 n=5 n=11
Supplementary Table S6 (Bartel DP 2004;[START_REF] Griffiths-Jones | miRBase: tools for microRNA genomics[END_REF]. miRNAs induce gene silencing via base pairing to complementary sequences present on target molecules (mRNA or lncRNA) [START_REF] Ameres | Diversifying microRNA sequence and function[END_REF] and recent analysis indicates the number of functional microRNAs in human at around 600 [START_REF] Fromm | A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome[END_REF]. Most of miRNAs are located within the cell, but some miRNAs (commonly known as circulating miRNAs), have been found in extracellular environment, including various biological fluids and cell culture media [START_REF] Weber | The microRNA spectrum in 12 body fluids[END_REF][START_REF] Boeckel | From heart to toe: heart's contribution on peripheral microRNA levels[END_REF], thus potentially serving as powerful tools for diagnosis and treatment [START_REF] Kaucsár | Post-transcriptional gene-expression regulation by micro RNA (miRNA) network in renal disease[END_REF].

As different studies performed in cell lines and mouse models suggest a fine regulation between HNF1B and some microRNAs [START_REF] Hajarnis | Transcription factor hepatocyte nu-clear factor-1beta (HNF-1beta) regulates microRNA-200 expression through a long noncoding RNA[END_REF][START_REF] Jenkins | Transforming growth factor β1 represses proximal tubular cell microRNA-192 expression through decreased hepatocyte nuclear factor DNA binding[END_REF][START_REF] Patel | miR-17~92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease[END_REF]Kornfeld JW et al., 2013) and the post-transcriptional regulation of HNF1B may be an undervalued factor related to RCAD syndrome, we performed genome wide transcriptomic analyses at different developmental stages from our RCAD mouse model and investigate the possible roles of miRNAs that may contribute to post-transcriptional regulation of Hnf1b and to the disease phenotype.

II.2. Summary of results

Performing mRNA and microRNA sequencing analysis made at three developmental stages (E14.5, E15.5 and E17.5) from kidney samples of Hnf1b Sp2/+ mutant mice and taking advantage from ChIP seq HNF1B (E14.5) analysis made by Heliot C. and Cereghini S.

(unpublished data), we performed a transcriptomic reverse correlation analysis presenting a list of genes potentially controlled by microRNAs in the RCAD phenotype during development.

Subsequently, we focalized the analysis in cell culture testing the capacity of HNF1B in transactivating three regulatory sequences coding for four microRNAs (miR-194-2, miR-192, miR-30a and miR-802). Luciferase assays in HEK293 cells demonstrated that HNF1B was able to transactivate the regulatory sequences upstream these microRNAs through specific binding. Moreover, luciferase assays showed also that miR-194-2, miR-192, and miR-802 were able to inhibit HNF1B transcript via binding its 3'UTR.

When I arrived in the lab, mRNA and microRNA sequencing at different developmental stages were just performed. I began all the analyses on microRNAs of interest and potential regulations with other mRNAs taking advantage of HNF1B ChIP sequencing data available in the lab, updated bibliography, and from the different databases available online. I performed all the bioinformatic analysis and with the help of my supervisors, we designed the project to analyze potential HNF1B-microRNAs regulatory networks. I subsequently performed the main experiments concerning this project including construction of vectors, cell-transfections, luciferase assays, site-directed mutagenesis, and q-RT-PCRs.

Results interpretation was made with the help of Dr. Silvia Cereghini, who also suggested completing this study with additional data of ChIP seq at later embryonic stages done in collaboration with Dr. JS Park and Dr. Chung E.

II.3. Discussion and conclusions

Here we show that the transcriptional factor HNF1B is integrated into a microRNA regulatory network during kidney development. Our analyses suggest that Hnf1b de-regulation in the RCAD mouse model compromised the direct transcription of different microRNAs such as miR-802, the cluster miR-194-2/192 and miR-30a potentially altering the pathways involved in normal renal tubule differentiation and morphogenesis.

As most microRNAs negatively regulate the levels of expression of their targets, miRNA-mRNA target pair should be inversely correlated for their expression levels. So, if the expression of a miRNA is UP-regulated, levels of its target genes should be DOWN-regulated and vice-versa. The predicted mRNAs target of each significant microRNA listed above were found from the major Internet databases (TargetScan, miRSystem, and miRDB) and crossed with the mRNAs from our mRNA seq at the selected stage. At E14.5, the expression of 11 microRNAs was inversely related to expression of 6 potential target genes and at the following stage the expression of 25 microRNAs was inversely related to expression of 22 potential target genes (Table 1). The number of mRNA potential targets increased to 67 at E17.5 linking their control to 14 microRNAs de-regulated in the RCAD mouse model at this stage. Following this double study approach "ChIP seq/miRNA seq", together with the previous and recent studies on miRNA expression and function revision, we shorten the list of microRNAs focusing our attention on four microRNAs: 802, 192, 194, and 30a.

E15.5

miR-802 express in the same tissues of HNF1B (kidney, pancreas, liver) [START_REF] Higuchi | Identification of circulating miR-101, miR-375 and miR-802 as biomarkers for type 2 diabetes[END_REF] and it was the most strongly DOWN-regulated microRNA at all the 3 stages (E14.5, E15.5, E17.5). We found that HNF1B is recruited to the upstream sequence of its genomic locus within 1 kb of distance, thus potentially participating in its transactivation. Surprisingly, Kornfeld et al. have previously described a direct down-regulation of Hnf1b by miR-802 in Hepa1-6 cells (Kornfeld JW et al., 2013) and therefore we did not expect that miR802 be the most strongly downregulated miRNA in our mode.

As miR-802, miR-192 was DOWN-regulated at all the 3 stages (E14.5, E15.5, E17.5).

HNF1B is recruited to the upstream sequence of its genomic locus within 1 kb of distance. It is co-transcribed with miR-194-2 and it is found DOWN-regulated at both E15.5 and E17.5

together with its isoform miR-194-1. miR-192 and miR-194 are among the most expressed miRNAs in kidney (preferentially in the renal cortex) [START_REF] Bhatt | microRNAs in kidneys: biogenesis, regulation, and pathophysiological roles[END_REF][START_REF] Sun | Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs[END_REF][START_REF] Tian | MicroRNA-target pairs in the rat kidney identified by microRNA microarray, proteomic, and bioinformatic analysis[END_REF]. Interestingly, Jenkins et al. have previously demonstrated that HNF1A

was able to bind the miR-194-2/192 promoter in proximal tubule epithelial cell line (HK-2), whereas knockdown of HNF1α and HNF1β with siRNAs resulted in a decrease expression of miR-192 and miR-194 of -40% and -50% respectively [START_REF] Jenkins | Transforming growth factor β1 represses proximal tubular cell microRNA-192 expression through decreased hepatocyte nuclear factor DNA binding[END_REF]. It has also been shown a correlation between low miR-192 expression and structural (tubulointerstitial fibrosis) and functional (reduced estimated glomerular filtration rate) indicators of renal damage [START_REF] Krupa | Loss of MicroRNA-192 promotes fibrogenesis in diabetic nephropathy[END_REF].

miR-30a was DOWN-regulated at E15.5 and HNF1B is recruited 10 kb upstream to its sequences in a region without any other coding or non coding genes; miR-30 family showed a prominent kidney-restricted expression [START_REF] Shi | Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis[END_REF][START_REF] Wu | Downregulation of microRNA-30 facilitates podocyte injury and is prevented by glucocorticoids[END_REF] and research on pronephros development in Xenopus demonstrated that miR-30 regulated normal renal development by targeting Xlim/Lhx1 (Agrawal R et al., 2009). Microarrays in a PKD mouse model (PKD/Mhm(cy/+) at P36 identified miR-30a as DOWN-regulated [START_REF] Pandey | Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease[END_REF] and together with miR-194, and miR-802 it was reported in the published list of DOWN-regulated microRNAs in another PKD mouse model (Kif3a-KO) at P28 (Patel V et al., 2013).

Moreover, a loss of regulation of miR-192, miR-194, miR-802, and miR-30a could regulate epithelial to mesenchymal transition (EMT) (Khella et al. 2013, Kato et al. 2007[START_REF] Dong | MicroRNA-194 inhibits epithelial to mesenchymal transition of endometrial cancer cells by targeting oncogene BMI-1[END_REF][START_REF] Wang | microRNA-802 inhibits epithelial-mesenchymal transition through targeting flotillin-2 in human prostate cancer[END_REF][START_REF] Peng | MiR-30a Inhibits the Epithelial--Mesenchymal Transition of Podocytes through Downregulation of NFATc3[END_REF], which is predicted to play a role in epithelial tubular morphogenesis and polycystic kidney disease (Togawa et al. 2011) HNF1B stimulates the luciferase activity of all the three constructs in a dose response mode (Figure 4). Moreover and supporting the specificity of HNF1B binding to the miRNA regulatory sequences coexpression of a mutant Q136E that abolish binding or of a HNF1B mutant encompassing a selective in phase deletion of exon 7 that abolish transcriptional activation [START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF] fails to transactivate the regulatory sequences of miR802, 194 or 30a. Importantly, these regulatory sequences are conserved between mouse and human. promoter/enhancer elements and either a human Hnf1b expression vector, or "Hnf1b ΔEx7" vector containing a deletion in the Exon7 in the transactivation domain or "Hnf1b Q136E" vector containing a human missense mutation that abolish DNA binding [START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF]. HNF1B lacking binding or transactivation activities fails in transactivate the regulatory sequences efficiently. The normalized firefly luciferase activity was obtained by measuring firefly luciferase activity/Renilla luciferase activity. In each condition the amount DNA transfected was 600 ng (500 ng pGL4.10 vector + different quantities of Hnf1b vectors adjusted with pCB6 empty vector). All experiments were performed at least three times. Statistical significance was analyzed by oneway ANOVA for multiple comparisons.

Subsequently, to further confirm the specificity of HNF1B binding we mutagenized HNF1B Interestingly, also miR-192 is also able to inhibit luciferase activity by specific binding to the Hnf1b 3'UTR but the mutation of its MRE is lacking to confirm its specificity.

Discussion and conclusions

We have attempted to reveal microRNAs playing a role in the RCAD mouse model during kidney development. The results obtained so far demonstrated that HNF1B and the microRNAs -802 and -194-2 are linked by an autoregulatory-loop. Potentially, miR-192 also participates in this molecular autoregulation. We established an unknown transactivation capacity of HNF1B on miR-802 and miR-30a and we revealed for the first time a role of miR-802 in the kidney, thus showing a multi-organ regulation of this microRNA (Figure 7). We discovered that miR-194-2 and miR-192 are able to inhibit HNF1B transcripts through binding its 3'UTR, and confirmed Jenkins et al. studies on HNF1B transactivation activity on the cluster miR-194-2/192 [START_REF] Jenkins | Transforming growth factor β1 represses proximal tubular cell microRNA-192 expression through decreased hepatocyte nuclear factor DNA binding[END_REF] using a different cellular system.

miRNA Isolation and Quantitative reverse transcription PCR (qRT-PCR) miRNA sequencing results were confirmed by reverse transcription quantitative real-time PCR analysis for four different microRNAs. Total RNA was extracted from entire kidneys and reverse transcribed using miScript II RT kit (QIAGEN). The cDNA (diluted 10 times) was used for a 40-cycle Q-PCR reaction with miScript specific Primers (forward primer) and miScript Universal Primer (reverse primer, sequences copyright of QIAGEN). All reactions were performed using the Applied Biosystems StepONEPlus Real-Time system (ThermoFisher Scientific). All the amplifications were done using SYBR Green PCR Master Mix (Applied Biosystems). The control target SNORD61 (sequence copyright of QIAGEN) was used to normalize and the expression values of the respective miRNA were compared using ΔΔC T method. The thermal cycling conditions included an initial holding stage of 20s, followed by the cycling stage composed by 40 cycles (95°C for 3sec, 60°C for 30s), followed by the melt curve stage (95°C for 15s, 60°C for 60 sec, temp increase of 0.3°C until 95°C for 15 sec). Melting curve analysis of every qPCR was conducted at the end of the cycling stage. [START_REF] Mischak | Proteomic biomarkers in kidney disease: issues in development and implementation[END_REF][START_REF] Magalhães | Urinary biomarkers for renal tract malformations[END_REF]. Urine represents an easy accessible biological fluid, which can be obtained in large quantities via a noninvasive procedure and may provides insights about different organs because it results from glomerular filtration via blood [START_REF] Thongboonkerd | Renal and urinary proteomics: current applications and challenges[END_REF]. Moreover, its proteome is originated 70% from the kidney and the urinary tract along with 30% from the circulation, which allows the study of different renal diseases and consequently associated events such as extracellular matrix (ECM) remodelling or fibrosis [START_REF] Schaub | Urine protein profiling with surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry[END_REF][START_REF] Klein | Comparison of CE-MS/MS and LC-MS/MS sequencing demonstrates significant complementarity in natural peptide identification in human urine[END_REF]Magalhães P et al., 2017). Oppositely, its limitation is due to daily variability caused by circadian rhythms, physical activity, diet and metabolic or catabolic processes (Weissinger E et al., 2004;[START_REF] Decramer | Urine in clinical proteomics[END_REF], which can be corrected by different adjustment or calibration/normalization methods (Jantos- [START_REF]Quantitative Urinary Proteome Analysis for Biomarker Evaluation in Chronic Kidney Disease[END_REF].

Primers

Top-down methods, as Capillary electrophoresis-mass spectrometry (CE-MS), allow the identification of naturally occurring peptides, which does not require any prior proteolytic procedure. These peptides are stable and soluble biomolecules ranging from 0.5 to 20 kDa, not requiring any protein digestion before MS analysis, and being usually derived by the activity of endogenous proteases/peptidases [START_REF] Zürbig | Peptidomics: methods and applications[END_REF][START_REF] Finoulst | Sample preparation techniques for the untargeted LC-MS-based discovery of peptides in complex biological matrices[END_REF].

Following the promising results on the urinary proteome of Hnf1b Sp2/+ mutant mice described in the Topic II, we decided to perform the first clinical proteomic analysis of the RCAD syndrome in a pediatric cohort of patients to provide new insights of this multisystem disorder identifying novel urinary biomarkers. For this analysis we performed capillary electrophoresis coupled to mass spectrometry (CE-MS).

Regarding my personal role in this study, in the context of the RENALTRACT

III.2. Results and discussion

Performing CE-MS we found 146 peptides associated with RCAD urines in 22 pediatric patients compared to 22 healthy age-matched controls. Interestingly, comparable results regarding collagen fragments (generally UP-excreted) and uromodulin fragments (DOWNexcreted) were obtained between RCAD human and RCAD mice proteome. Following the identification of RCAD urinary proteome signature, a classifier based on these peptides was generated and further tested on an independent blinded cohort, clearly discriminating RCAD patients from different groups of controls (ADPKD, nephrotic and CKD patients).

III.3. Conclusions

Our study demonstrates that the urinary proteome in children with RCAD syndrome differs from autosomal dominant polycystic kidney disease (PKD1, PKD2), congenital nephrotic syndrome (NPHS1, NPHS2, NPHS4, NPHS9), and also from chronic kidney disease conditions, suggesting differences between the pathophysiology behind these disorders.

Future studies will further evaluate the performance of the RCAD panel in additional pediatric cohorts of disorders more closely related to RCAD such as autosomal recessive polycystic kidney disease (ARPKD), medullary cystic kidney disease (MCKD), autosomal dominant tubulointerstitial kidney disease (ADTKD) or diabetic patients. Moreover, and along with the evaluation of the performance of this classifier to predict the progression of RCAD, follow-up clinical data of the patients described in this study will be addressed.

Introduction

Renal Cysts and Diabetes (RCAD) syndrome is caused by heterozygous mutations in the HNF1B gene, encoding the transcriptional factor hepatocyte nuclear factor-1B. RCAD syndrome (RCAD, OMIM #137920) 1 can also be referred as MODY5 (Maturity Onset Diabetes of the Young type 5) 2 . The wide spectrum of clinical features in RCAD patients is due to the multisystem role of HNF1B, which is involved in normal morphogenesis of several organs, including kidneys, pancreas, liver, and genitourinary tract 3 . Consistent with its broad developmental expression pattern 4,5 , studies on fetuses carrying HNF1B mutations revealed a fundamental function during kidney, urogenital tract and pancreas development 4,6-8 .

RCAD disease is inherited in an autosomal dominant pattern 9 . To date, more than 150 mutations have been described in the HNF1B gene, including missenses, nonsenses, frameshifts, splice site mutations, insertions/deletions as well as whole gene deletions that represent approximately 50% of all mutations found in patients 10 .

The most prominent clinical feature in HNF1B-associated syndrome is the renal disease, usually characterized by congenital anomalies of the kidney and urinary tract (CAKUT) including such as renal cysts, renal dysplasia, solitary or horseshoe kidney, hydronephrosis, and hyperuricaemic nephropathy 11 . In addition, cases of otherwise unexplained chronic kidney disease (CKD) have been reported both in children 12 and adults 13,14 . Tubular dysfunction manifesting by hypomagnesemia, hypocalciuria 15-17 , and hyperuricemia 13,18 has also been described. Extrarenal features comprise maturity-onset diabetes of the young, pancreatic hypoplasia, abnormal liver function, and genital tract malformations. The phenotype of HNF1B mutant carriers is indeed highly variable within and between families 19 . These observations led to the hypothesis that non-allelic factors, as well as stochastic variation in temporal HNF1B gene expression and environmental factors, could cause the strong intrafamilial variability of RCAD patients 3,20 .

Urinary proteomics is increasingly being employed in kidney disease research. Several studies have demonstrated that capillary electrophoresis coupled to mass spectrometry (CE-MS) enables the identification and validation of several biomarkers or peptide signatures classifying the diagnosis and prognosis of various kidney diseases 21-24 . In addition to their diagnostic and prognostic usefulness, proteomics derived biomarkers may advance the understanding of the molecular pathways involved in the pathogenesis of a specific condition.

In this study, we aimed to obtain more insights of the RCAD syndrome by applying urinary proteomics to discover peptides that can be used to characterize RCAD patients' proteome.

Results

Identification of RCAD-related urinary peptides and development of a urinary peptidebased classifier

Urine samples from RCAD patients obtained from three European centers were analyzed with CE-MS. Furthermore, data from previously measured samples were extracted from the Human Urinary Proteome Database 25-27 . The data of those samples were separated in two different groups: a discovery cohort (n=44) and an independent cohort for validation (n=230) (Figure 1A). For the identification of significant urinary peptides related to RCAD syndrome, we compared the urinary proteome profiles of 22 RCAD patients with a group of 22 age-and gender-matched healthy patients (discovery cohort) (Table 2A and Supplementary Table 1).

The selection of the peptides was adjusted for multiple testing following the concept described by Benjamini and Hochberg 28 and following the clinical proteomics guidelines 29 .

This led to the identification of 294 differentially excreted peptides (corrected p<0.05) between these two groups. For 146 out of the 294, high-confidence sequence information could be assigned. Fragments of uromodulin, osteopontin and mucin, as well as a large number of collagen fragments were identified. Moreover, peptides associated to cystic conditions as annexin A1 and unc-119 homolog A were de-regulated (details such as the sequence information as well as the fold-change are described in detail in Supplementary Table 2). The difference in abundance of these 146 peptides between RCAD patients and healthy controls is shown in Figure 1B. These 146 peptides were combined into a classifier termed "RCAD146" using a support vector machine (SVM), defining a cut-off score based on the classification of the patients in the discovery cohort. Based on the cut-off score 0.30, the RCAD classifier discriminated RCAD from healthy controls with 90.9% sensitivity and 100%

specificity and an AUC of 0.99 in the discovery cohort (Figure 1C).

Validation of RCAD146 in an independent group

The mathematical RCAD146 model was validated in an independent group of samples (Table 2B), consisting of 24 RCAD and 20 healthy controls. The analysis revealed an AUC of 1.00 [0.92 to 1.00 (95% CI); p<0.0001]. At the pre-defined cut-off level of 0.30 based on the discovery cohort, the classifier displayed a sensitivity of 91.67% and specificity of 100%. To obtain confirmation about the performance of the RCAD146 in differentiating pediatric RCAD patients from patients suffering from other kidney diseases, we further selected a group of patients with chronic kidney disease (CKD) (n=85). This control group of children was particularly interesting as i) 40% of adults with HNF1B mutations develop CKD 13 , ii) it represents a condition with severe chronic kidney damage and, iii) to confirm that the performance of the RCAD146 classifier is independent of proteinuria in RCAD patients. This analysis showed a specificity of 97.7% and an AUC of 0.99 [0.949 to 1.00 (95% CI); p<0.0001] for the classification of children with CKD as non RCAD. To further evaluate the specificity and validity of the pediatric RCAD urinary proteomic pattern, the classifier was subsequently tested in patients with different monogenic kidney diseases, including ADPKD and nephrotic syndrome subjects. In the case of ADPKD, 53 out of 55 patients were scored as non RCAD corresponding to a specificity of 96.4% [AUC: 0.974] (Figure 2A). On the other hand, in the group of patients with nephrotic syndrome, 39 of 46 patients were scored by RCAD146 as non-RCAD displaying a specificity of 84.8 [AUC: 0.952] (Figure 2A).

When evaluating all data sets combined the overall sensitivity and specificity were 91.7% and 91.1%, respectively. Furthermore, this receiver operating characteristic (ROC) analysis revealed an AUC of 0.978 [0.950 to 0.993 (95% CI); p<0.0001] (Figure 2B).

RCAD patients from children affected by CKD was evaluated. The RCAD146 classifier correctly identified the majority of CKD patients as non-RCAD (97.6%) demonstrating that the RCAD signature is clearly different from the CKD background peptide excretion, which is conversely characterized by a down excretion of collagen peptides 22 . The increased excretion of collagen fragments in the RCAD urinary samples may reflect the cystic phenotype and the still non-fibrotic status of patients' kidneys, whereas tubulointerstitial fibrosis determines the peptide excretion pattern in CKD 38 . Since CKD is a rare condition in RCAD children 12 , it would be of interest testing a cohort of adult RCAD patients suffering from CKD, in order to investigate the performance of the RCAD146 classifier.

The other disorders used as diseased controls in this study were biologically related (e.g ADPKD) or non-related (e.g. nephrotic syndrome) to the RCAD syndrome. In particular, a group of patients affected by autosomal dominant polycystic kidney disease (ADPKD) appears relevant as there are genotypic and phenotypic correlations with the RCAD syndrome. HNF1B was shown to regulate Pkd2 in mouse 39 and mutations in HNF1B can mimic polycystic kidney disease especially in prenatal setting and early childhood 40,41 .

Notably, the RCAD146 classifier precisely discriminated RCAD from ADPKD.

We then selected other patients with monogenic mutations sharing a common nephrotic syndrome phenotype. In this case too, RCAD146 classifier identified subjects carrying mutations in NPHS1, NPHS2, WT1 (NPHS4), and ADCK4 (NPHS9) as non-RCAD.

This result may highlight that RCAD146 is not just reflecting proteinuria, which has been found in almost one of three RCAD patients 13 , but reflecting a specific pathophysiological state.

Interestingly, in a parallel test, the urinary proteome of a patient with Pax2 mutation was misrecognized by RCAD146 (data not shown). This observation suggests that the RCAD pediatric proteome can potentially be closer to patients with mutations in the gene encoding the transcription factor PAX2, known to cooperate with HNF1B in kidney morphogenesis and ureter differentiation 42 , than patients with either polycystic or nephrotic syndrome. Additional samples are required to further validate this common feature.

A limitation of the current study is related to the ADPKD cohort, which were not children, but adults. This is due to the difficulty in recruiting children with ADPKD because the average age at the diagnosis is approximately 30 years old 43 . Another issue that could be a limitation is that this study included post hoc analysis, due to the selection of the diseased control population from previous studies. Overall, the study, performed in agreement with all guidelines for clinical proteomics demonstrates a significant value of the urinary proteome analysis in the detection of RCAD, highlighting some proteins that potentially participate in the development of cysts and may be useful for early diagnosis.

In conclusion, the urinary proteome of pediatric patients with HNF1B mutations was investigated here, providing novel insights into the pathophysiology of the RCAD phenotype.

The urinary peptide signature of pediatric RCAD patients is mainly characterized by the upregulation of collagens peptides (especially type I o type III fragments), and osteopontin along with the downregulation of uromodulin. Including the 146 peptides differentially excreted between RCAD and healthy patients in a diagnostic biomarker panel, we demonstrated that RCAD pediatric urinary proteome is different from the proteome of patients with Pkd1-2 and Nphs1-2-4-9 mutations, and also from the urinary proteome of CKD patients.

Future studies will further evaluate the performance of the RCAD panel in additional pediatric cohorts of disorders more closely related to RCAD such as autosomal recessive polycystic kidney disease (ARPKD), medullary cystic kidney disease (MCKD), autosomal dominant tubulointerstitial kidney disease (ADTKD) or diabetic patients. Moreover, and along with the evaluation of the performance of this classifier to predict the progression of RCAD, follow-up clinical data of the patients described in this study will be addressed. Collagen alpha-1 (I) chain COL1A1 52 4.97E-02 1.6 (± 3.44) *Information described are the number of significant protein fragments for each protein origin, the highest observed P-value as well as the estimated mean fold change (± standard deviation).

CONCLUSION AND DISCUSSION _________________

Conclusions and discussion

As shown in the introduction, HNF1B has a multiorgan role during mammalian development and heterozygous mutations in this gene are at the basis of a rare human syndrome called Renal Cysts and Diabetes (RCAD). The understanding of the molecular misregulation of HNF1B in patients carrying these mutations is essential for an early diagnosis and a potential cure.

One of the main limits to study the RCAD syndrome was the lacking of a mouse model, as heterozygous mutations in Hnf1b did not present phenotype. For the first time the lab developed a mouse model able to mimic different features of RCAD syndrome allowing to perform functional and molecular examinations. In sharp contrast with previous studies we introduced a human mutation, which was able to overcome the mouse strengthness in resist to diseases prompted by haploinsufficiency. Mirroring the human disease, heterozygous Hnf1b Sp2/+ mutant mice mainly exhibited bilateral renal cysts (originated primarily from proximal tubules (PT) and intermediate nephron segments and glomeruli), renal hydronephrosis and genital tract anomalies. Moreover, a partial urinary concentration defect was observed, thus further validating this model as a valuable instrument to investigate the RCAD disease.

Interestingly, the canonical cystic diseases genes (Pkh1, Pdk2, Bicc1) and branching genes (Pax2, Wnt9b) were slightly or not affected, and mRNA-seq analysis at different embryonic stages (E14.5, E15.5, E17.5 and P1) revealed a de-regulation of more than 300 hundred genes. These results demonstrate that i) cysts and morphological anomalies do not derived from the misregulation of classical cystic/branching genes and that ii) HNF1B deregulation impairs several other genes expressed primarily in proximal tubules and mostly involved in transport, and lipid and organic acid metabolic processes.

In this context we strongly believe that the severity of de-regulated gene network together with the other causes characterizing RCAD features both in human and mouse is the failure to reach a minimal threshold value of HNF1B protein during development and potentially postnatally. The etiologies of the strong intrafamilial variability, found also in our RCAD model littermates, have been previously attributed to stochastic variation in temporal gene expression as well as environmental factors. We do not distance from this assumption, but we insisted on the fact that the only reason at the basis of the RCAD pathology is a failure to reach a minimum level of HNF1B protein, which can be also organ-specific.

Related to this, it is tempting to speculate that a significant number of patients with HNF1B heterozygous mutations do not show pathological RCAD features because, somehow, HNF1B minimal level for correct development and maintenance was achieved during their life (at least in the most of organs where HNF1B accomplish its functions). In this perspective, a dominant negative role of HNF1B-truncated mutant proteins at the basis of RCAD syndrome is unlikely.

If our theory is correct, the modulation of HNF1B protein during development may be at the basis of a treatment to ensure a proper development of kidney, pancreas, liver and genital tract in HNF1B heterozygous patients.

Taking into consideration that miRNAs regulation during kidney development, homeostasis and disease is dependent on transcription factors, the understanding of microRNAs involved in RCAD syndrome and controlled by HNF1B is a priority for molecular biology and medicine. In fact, microRNAs may be a tool to modulate transcription factor expression as HNF1B or at least to early predict diseases as RCAD (Christopher AF et al., 2016;[START_REF] Meuth | The expanding roles of microRNAs in kidney pathophysiology[END_REF]. We demonstrated that HNF1B and the microRNAs -802 and -194-2 and very likely miR-192 are linked by an autoregulatory-loop, and that HNF1B transactivates in a dose response mode an upstream enhancer likely controlling miR-30a expression. As these microRNAs are de-regulated in the RCAD mouse model, it is conceivable that they have an important role in RCAD pathogenesis but further studies on their other potential targets are required. Moreover, to add further value to this discovery, analyses in human kidney tissues may be done to show the conservation of this regulation.

Another analysis reinforces the value of the RCAD mouse model as consistent with the human disease: the urinary proteomic study. The first results obtained comparing wt and Hnf1b Sp2/+ mutant mice urinary proteome revealed a particular signature of the RCAD urinary proteome consisting mainly of UP-excreted collagen fragments, and DOWN-excreted uromodulin (UMOD) and epidermal growth factor (EGF) peptides. The results were coherent with an excessive extracellular matrix (ECM) turnover. Interestingly, a subsequent analysis in a pediatric cohort of RCAD patients revealed a similar signature with a majority of peptides collagen type I or type III fragments enriched in the urine of RCAD patients and a downexcretion of uromodulin fragments, together with other de-regulated peptides. Following studies in human, we hypothesize that this active extracellular matrix (ECM) remodeling may BIBLIOGRAPHY _________________ (Brunskill EW et al., 2008) Brunskill EW, Aronow BJ, Georgas K, Rumballe B, Valerius MT, Aronow J, Kaimal V, Jegga AG, Yu J, Grimmond S, McMahon AP, Patterson LT, Little MH, Potter SS. Atlas of gene expression in the developing kidney at microanatomic resolution. Dev Cell. 2008 Nov;15(5):781-91. doi: 10.101615(5):781-91. doi: 10. /j.devcel.200815(5):781-91. doi: 10. .09.007. (Cai X et al., 2004) ) Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA. 2004 Dec;10(12):1957-66. Epub 2004Nov 3. (Calabrese JM et al., 2007) Calabrese JM, Seila AC, Yeo GW, Sharp PA. RNA sequence analysis defines Dicer's role in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18097-102. Epub 2007Nov 7. (Cao G et al., 2003) Cao G, Yang G, Timme TL, Saika T, Truong LD, Satoh T, Goltsov A, Park SH, Men T, Kusaka N, Tian W, Ren C, Wang H, Kadmon D, Cai WW, Chinault AC, Boone TB, Bradley A, Thompson TC. Disruption of the caveolin-1 gene impairs renal calcium reabsorption and leads to hypercalciuria and urolithiasis. Am J Pathol 162: 1241-1248, 2003. (Carroll TJ et al., 2005) Carroll TJ, Park JS, Hayashi S, Majumdar A, McMahon AP. Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev Cell. 2005 Aug;9(2):283-92. (Cereghini S et al., 1987) Cereghini S, Raymondjean M, Carranca AG, Herbomel P, Yaniv M. Factors involved in control of tissue-specific expression of albumin gene. Cell. 1987 Aug 14;50(4):627-38. (Cereghini S et al., 1988) Cereghini S, Blumenfeld M, Yaniv M. A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes Dev. 1988 Aug;2(8):957-74. (Cereghini S et al., 1990) Cereghini S, Yaniv M, Cortese R. Hepatocyte dedifferentiation and extinction is accompanied by a block in the synthesis of mRNA coding for the transcription factor HNF1/LFB1. EMBO J. 1990 Jul;9(7):2257-63. [START_REF] Cereghini | Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles[END_REF] [START_REF] Chen | Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5[END_REF] Chen YZ, Gao Q, Zhao XZ, Chen YZ, Bennett CL, Xiong XS, Mei CL, Shi YQ, Chen XM. Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5. Chin Med J (Engl). 2010 [START_REF] Kato | Hepatocyte nuclear factor-1beta (HNF-1beta) in human urogenital organs: its expression and role in embryogenesis and tumorigenesis[END_REF]123(22):3326-33. (Chi YI et al., 2002) Chi YI, Frantz JD, Oh BC, Hansen L, Dhe-Paganon S, Shoelson SE. Diabetes mutations delineate an atypical POU domain in HNF-1alpha. Mol Cell. 2002;10:1129-1137. doi: 10.1016/S1097-2765(02) 00704-9. (Chouard T et al., 1990) Chouard T, Blumenfeld M, Bach I, Vandekerckhove J, Cereghini S, Yaniv M. A distal dimerization domain is essential for DNA-binding by the atypical HNF1 homeodomain. Nucleic Acids Res. 1990 Oct 11;18(19) Nat Rev Nephrol. 2015 Feb;11(2):102-12. doi: 10.1038/nrneph.2014.232. Epub 2014Dec 23. (Clissold RL et al., 2016) Clissold RL, Shaw-Smith C, Turnpenny P, Bunce B, Bockenhauer D, Kerecuk L, Waller S, Bowman P, Ford T, Ellard S, Hattersley AT, Bingham C. Chromosome 17q12 microdeletions but not intragenic HNF1B mutations link developmental kidney disease and psychiatric disorder. Kidney Int. 2016 Jul;90(1):203-11. doi: 10.1016/j.kint.2016.03.027. Epub 2016May 24. (Clotman F et al., 2002) Clotman F, Lannoy VJ, Reber M, Cereghini S, Cassiman D, Jacquemin P, Roskams T, Rousseau GG, Lemaigre FP. The onecut transcription factor HNF6 is required for normal development of the biliary tract. Development. 2002 Apr;129(8):1819-28.

  Analysis of HNF1B regulating microRNAs in the renal pathogenesis of a mouse model of

Figure 1 .

 1 Figure 1. Genomic organization of the Hnf1b locus illustrating the 9 coding exons and main functional domains (N-terminal Dimerization, the POU-specific (POU S ) and POU-homeodomain (POU H ) and the C-terminal transactivation domains). Exons, but not introns, are depicted in nucleotides scale size.

Figure 2 .

 2 Figure 2. NCBI protein blast of HNF1B isoforms highlighting conservation in amino acidic sequences

Figure 4 .

 4 Figure 4. A. Crystallographic structure in ribbon comparison between HNF1A and HNF1B binding a DNA double helix (source RCSB protein data bank). B. HNF1B binding motif to DNA from JASPAR (source http://jaspar.binf.ku.dk/ Khan A et al., 2018) (Adapted from Lu P et al., 2007).

Figure 6 .

 6 Figure 6. Analysis of the β-galactosidase expression pattern in Hnf1b heterozygous mouse embryos. Expression in the gut epithelium (g), neural tube (nt), mesonephric duct (m), pancreatic bud (pb), otic vescicle (ov). Scale bar (1-2-3): 200 µm. (Adapted from[START_REF] Barbacci | Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification[END_REF]

Figure 7 .

 7 Figure 7. Analysis of the β-galactosidase expression pattern in Hnf1b heterozygous mouse embryos and microdissected urogenital tract at the indicated developmental stage showing mesonephric tubules, Wolffian ducts (WD), and ureteric bud (Adapted from Lokmane L et al., 2010).

8 )

 8 Analysis of the β-galactosidase expression pattern in developing pancreas and other endoderm derived organs in Hnf1b lac/+ heterozygous mouse embryos. A. Pancreas buds at E12.5: black arrow, dorsal pancreatic bud; white arrow, ventral pancreatic bud; arrowhead, duodenum. B. Dissected pancreas at E14: arrowhead, duodenum. Note the restriction of the staining to the ductular part of the pancreas. C. Sagittal section of the pancreas; D. Biliary duct network surrounding a vessel in a E18.5 liver; E. Hnf1b expression in the lung: arrows, bronchiole; F. Adult polmonary lobe: arrows, bronchiole. Scale bar (A-B): 100 µm; (C): 200 µm; (D-E):

Figure 9 .

 9 Figure 9. Ureteric bud formation, brunching and nephrogenesis. Above: renal morphogensis in mouse; in red the mesenchymal structures, blue corresponds to epithelial structures. Below: Nephron formation in the kidney is simultaneous with ureteric tree branching. Schematic representation of the major stages of kidney development in mice. All nephrons in the kidney develop from cap mesenchyme (CM) cells (purple). To develop into a nephron, CM cells form a pretubular aggregate (PA), with each aggregate developing in a specific region of the kidney, underneath the tips (UT) of the growing ureteric tree. Subsequently, these mesenchymal aggregates differentiate into nephrons by undergoing a mesenchyme to epithelial transition (MET). The formed renal vesicles (RV) develop into comma-shaped bodies (CSB), S-shaped bodies (SSB) and capillary loop stage nephrons, which finally differentiate into mature nephrons. Each mature nephron is comprised of a glomerulus connected via distinct tubules to the collecting ducts, which drain into the pelvis of the kidney. The pelvis is connected to the bladder via the ureters. The collecting ducts develop from the ureteric tree epithelium (orange).

  Henle: intermediate portion of the nephon between the proximal and the distal tubule. This tubular segment enters the medulla of the kidney and returns back to the cortex where it contacts its own glomerulus. Two functionally distinct limbs form the Henle's loop segment: the thin descending (TDL) limb and the thick ascending (TAL) limb of Henle. It is responsible for the resorption of water and ions from the pre-urine. Distal convolute tubule (DCT): a segment located after the Loop of Henle and connected to the collecting ducts. It continues to reabsorb useful solutes from the filtrate to the peritubular capillaries, actively pumping small molecules out of the tubule lumen into the interstitial space. It regulates the pH and salt concentration of calcium, potassium and sodium. Collecting Duct (CD): structure formed in the renal cortex by the connection of several nephrons. Collecting ducts enter in the cortex, penetrate the outer and the inner medulla where they successively fuse together to bring the urine produced by the nephrons into the ureter and finally in the bladder.

Figure 11 .

 11 Figure 11. Diagram and histological sections of the complete murine metanephros at E15. The histology sections (haematoxylin and eosin) show sagittal sections through the metanephros and ureter (left) and just beyond the midline of the metanephros (right). Below (left): diagrammatic representation of a wedge of the E15 metanephros, from renal capsule to pelvis next to a maturing nephron structure. Below (right): nephron structure and vascularization (Adapted from gudmap.org and LittleMH et al., 2007)

Figure 12 .

 12 Figure 12. Expression pattern of Hnf1b during kidney development. Whole-mount in situ hybridization of Hnf1b in WT embryos at E9.5 (A), E10.5 (B) and E11.5 (C). Immunostaining of HNF1B on sagittal sections of the kidney at E13 (D), E17.5 (E) and in the adult (F). g, glomeruli; cs, comma-shaped bodies; mt, mesonephric tubules; sb, S-shaped bodies; ub, ureteric bud; wd, Wolffian duct (Adapted from Lokmane L et al., 2010).

Figure 13 .

 13 Figure 13. X-gal staining of WT Hnf1b lacZ/-(A,C) and Hnf1b -/-(B,D) urogenital tracts dissected at E13.5 (A, B) and E14.5 (C, D). At E14.5 homozygous mutants exhibited arrested UB branching, severe hypoplastic kidneys and hypertrophic andrenal glands. Note interrupted WTs and absence of mesonephric tubules. The white arrowhead in B shows the strong reduction in UB branching in mutants relative to heterozygous (A) (Adapted fromLokmane L et al., 2010)

  Aboudehen K et al., further confirmed by chromatin immunoprecipitation and sequencing exp eriments that HNF1B binds to the Nr1h4 promoter, suggesting an additional role of HNF1B in osmoregulation.

Figure 15 .

 15 Figure 15. Inactivation of HNF1B in kidney cells leads to polycystic kidney disease. Kidney sections from control and mutant mice at P1 (A, B), P8 (C, D) and P12 (E, F) stained with hematoxylin-eosin. At P1 tubular dilations were visible in t h e medullary region of mutants, with an increased number of nuclei per tubule section compared to controls (B versus A). In mutants, the medulla was completely disrupted by large cysts by P8 (D). The size of cysts increased with age (F). A few glomerular cysts were observed in the deep cortex (F, black arrowheads). Scale bars: A, B: 100 mm, C-F: 400 mm (Adapted from[START_REF] Gresh | A transcriptional network in polycystic kidney disease[END_REF].

Figure 20 .

 20 Figure 20. NCBI protein blast of human HNF1A and HNF1B show 54% of amino acid identity (314 on 577)

Figure 21 .

 21 Figure 21. Comparative alignments of Hnf1b genomic sequences from different species reconstructs its phylogenetic history and show its conservation during evolution

  all intragenic mutations are predicted to function as dominant mutations and the potential dominant negative effect therefore does not explain the non-correlation between the type and position of the mutation and the phenotype A recent study made by Dubois-Laforgue D et al. on 201 patients with HNF1B heterozygous mutations, showed marked differences in the phenotypes of the patients with HNF1B deletion compared with those with mutations (Dubois-Laforgue D et al., 2017):

  A, A'). Likewise, the expression of the key regulator PAX2, whose transcription in the collecting system has been shown to depend on Hnf1b {Lokmane, 2010 #12}{Desgrange, 2017 #5}{Paces-Fessy, 2012 #1} was not affected neither at E14.5 and E15.5 (compare Fig 4 B, C with B', C') nor at later stages (not shown). Thus, in Hnf1 Sp2/+ mutants decreased HNF1B protein levels did not affect branching of the UB, nor the expression of the early HNF1B targets previously identified. WT1 was also normally expressed in heterozygous both in the condensed mesenchyme around the ureteric buds and in the podocytes. Moreover, nephrogenesis appeared normally induced as indicated by the glomeruli stained by WT1 and the presence of normally shaped Sshaped bodies, in contrast to the abnormal S-shaped bodies observed upon Hnf1b-conditional inactivation in nephron progenitors {Heliot, 2013 #7} (Fig. 4 D, D').

  C'; D'). Distal tubules labeled by SLC12a3 (NCC) showed only rare and mild dilatations (data not shown). Surprisingly, Hnf1b Sp2/+ collecting ducts were normally stained by either AQP2 (Fig. 5 E', F') or pancytokeratin (CK) (Fig. 4 G'; Fig. 5 G'; H') and mainly devoid of dilatations. However, they were not stained by the lectin DBA, neither during development nor in postnatal life suggesting defective maturation of collecting duct cells (Fig. 4 H'; Fig. 5 I'; L').

Fig. 6 B

 6 Fig. 6 B, insert B' and A). The expression of HNF1B was moderately decreased in these dilated PTs as compared to its expression in other renal tubules (Fig. 6 C, D, insert D').

  scaffolding protein involved in the normal localization and function to the brush border of the PT of several transporters {Thomson, 2005 #74}, further suggesting additional indirect roles of HNF1B/HNF1A in renal tubule transport via its transcriptional regulation. Remarkably and confirming our initial Q-RTPCRs (Supplementary Fig S7) the mRNAseq data showed that the expression of several known HNF1B targets strongly reduced in the absence of Hnf1b, including Wnt9b and Pax2 {Lokmane, 2010 #12}, Pkd2, Cdh16, Crb3, Kif12 {Verdeguer, 2010 #45}, Cys1, Glis2, Glis3, Fgfr4 {De Vas, 2015 #8}, were indeed

  differentiation and onset of nephron tubule mature functions. Consistent with these findings, analyses of human fetuses carrying heterozygous mutations in HNF1B showed decreased numbers of nascent nephron structures {Haumaitre, 2006 #4}. Accordingly, the HNF1B target gene Hnf4a {Heliot, 2013 #7}, involved in proximal tubule differentiation and function {Martovetsky, 2013 #73} (A. Desgrange and S.C.

  as well as in different mouse models of Hnf1b-inactivated in the collecting ducts{Desgrange, 2017 #5}{Aboudehen, 2017 #75}. Interestingly, compound heterozygotes for Pax2 and Hnf1b null alleles displayed hydronephroses and megaureters, which appeared to result from a functional defect in the ureter smooth muscle differentiation {Paces-Fessy, 2012 #1}. Further analyses of Hnf1b sp2/+ mutant are required to define whether hydronephrosis is due to similar perturbations of smooth muscle differentiation of the ureter and/or through other proposed mechanisms, such as polyuria (see below, {Aboudehen, 2017 #75}. In this context, we have also shown that heterozygous Hnf1b sp2/+ exhibited progressive renal abnormalities associated with defective urinary concentration ability under basal conditions and hypercalcuria by 6 month-old followed by a partial restoration by 12-month-old. Similar defects in urine concentration have recently been described in mice with specific Hnf1b inactivation in the collecting ducts at later stages {Aboudehen, 2017 #75}, which were associated with increased expression and abnormal apical localization of AQP2, indirect downregulation of the PT urea transporter UT-A1 (Slc22a12) and the direct control of the HNF1B target Fxr. Although additional analyses are required, included urinary concentration ability under different

Figure 1 :

 1 Figure1: Structure and generation of the Hnf1b intron-2 spliced mutant mice A-Genomic organization of the Hnf1b locus, illustrating the 9 coding exons and the main functional domains (N-terminal Dimerization, POU-specific (POU S ) and POU-homeodomain (POU H ) and C-terminal transactivation domain). Also indicated are the mutation G to T introduced at the intron-2 splice donor site, the cryptic splicing site within Exon 2 (ss); the Nuclear Localization Signal (NLS) and the location of the alternative exon of 78 bp present in the Hnf1b isoform A. B-Characterization of abnormal spliced Hnf1b transcripts generated by the HNF1b Sp2/+ mutant allele. Representative semiquantitative RT-PCR from P1-kidney RNA of WT (lanes a, b) and HNF1b Sp2/+ (lanes c, d), using primers located in exon 1 and exon 3 (Material and Methods). Amplifications were performed for different cycles ranging 27 to 36 for Hnf1b (lanes a, c and lanes b, d correspond respectively to 30 and 33 cycles) and for Gapdh, used to normalyze for RNA amount for 24 to 30 cycles, (e, f, show 28 cycles). Sequence of PCR products indicated that WT express Hnf1b variants A and B, while Hnf1b Sp2/+ mice express four additional abnormal spliced transcripts corresponding to variants A and B lacking either exon 2 or the last 32bp of exon2. The structure of these spliced products and putative encoded proteins are depicted in the middle and right panels. Right panel: grey shows the dimerization and DNA-binding domains, checkerboard box represent the new protein sequences encoded by abnormal spliced transcripts.

Figure 2 :

 2 Figure 2: Hnf1b heterozygous for the splicing mutation Hnf1b Sp2/+ exhibit renal cysts and hydronephrosis during embryogenesis (A-F) Representative Hematoxylin and Eosin (H&E) staining sections of embryo kidneys show glomerular cysts and tubular dilatations in the medulla at E15.5 and E17.5 (arrows in E' higher magnification of E). Cystic glomeruli are shown in F' (arrows in F' higher magnification of F). (G-N) Backcrosses to inbreed C57BL/6N and 129/Sv backgrounds show different severity of phenotype. Note in C57BL/6N background pelvic dilatations at E17.5 (H) and hydronephrosis and duplicated kidney at P0 (J, see also supplementary Fig S3), in addition to cystic glomeruli and medullar tubules dilatations also observed in 129/sv background (L, N). Images are representative of n = 6 for each genotype. See also Supplementary Fig.S3. Scale bar: 200µm.

Figure 3 :

 3 Figure3: Expression levels of Hnf1b transcripts and protein from WT and heterozygous mutants Hnf1 Sp2/+ A-Q-RT PCR of normal Hnf1b transcripts at the indicated stages from WT and Hnf1b Sp2/+ kidneys. Number of samples were: at E14.5: 2 WT and 5 Hnf1b Sp2/+ ; at E15.5: 3 WT and 6 Hnf1b Sp2/+ (2 litters), at E16.5: 3 WT and 8 Hnf1b Sp2/+ (2 litters); at E17.5: 3 WT, 4 Hnf1b Sp2/+ ; at P0: 5 WT, 10 Hnf1 Sp2/+ (2 litters) and adults: 3 WT and 3 Hnf1b Sp2/+ . The 2 kidneys of each embryo/mice were pooled. Hnf1b WT transcripts were amplified by primers located in the ATG translational site and in the last 32bp of the Exon 2. Asterisk show significant values p<0,05 (*) and p<0,001 (**). Error bars represent standard error of the mean (SEM). B-HNF1B immunostaining of E15.5 embryo sections show a partial decrease in nuclear staining in Hnf1b Sp2/+ . Note HNF1B nuclear staining in both nondilated and dilated Hnf1b Sp2/+ tubules. C-Representative western blot of WT and Hnf1b Sp2/+ kidney extracts at the indicated stages form WT and Hnf1b Sp2/+ . The two HNF1b variant A and B are visible on blot. α-actin was used to normalize for protein amount. D-Quantification of western blots show more than 50% reduction in HNF1B protein levels in Hnf1b Sp2/+ relative to WT particularly at earlier stages. A decrease of 55% and 67% is observed respectively at E15.5 and E17.5, while at P0

Figure 4 :

 4 Figure4: HEK293 cells were co-transfected with pGL4.10[luc2]vectors containing the indicated promoter/enhancer elements and either a human Hnf1b expression vector, or "Hnf1b ΔEx7" vector containing a deletion in the Exon7 in the transactivation domain or "Hnf1b Q136E" vector containing a human missense mutation that abolish DNA binding[START_REF] Barbacci | HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment[END_REF]. HNF1B lacking binding or transactivation activities fails in transactivate the regulatory sequences efficiently. The normalized firefly luciferase activity was obtained by measuring firefly luciferase activity/Renilla luciferase activity. In each condition the amount DNA

  binding sites in miR-802 and miR-30a regulatory sequences (not in miR-194-2/192 promoter because already demonstrated by Jenkins et al.) (Supplementary Figure 1, 2 and 3). As a result of mutations, HNF1B was not anymore able to transactivate miR-802 promoter and miR-30a enhancer sequences. A summary of these results is shown in Figure 5.

Figure 5 .

 5 Figure 5. pGL4.10[luc2]-802 or -30a vectors containing single or double HNF1B binding site mutations in the promoter and enhancer elements. The normalized firefly luciferase activity was obtained by measuring firefly luciferase activity/Renilla luciferase activity. In each condition the amount of DNA reporter transfected was 500 ng and the amount of DNA expression vector was 100 ng. All experiments were performed at least three times. Statistical significance was analyzed by one-way ANOVA for multiple comparisons.

  miRNA MIMICS with luciferase vectors containing wt versions of 3'UTR of Hnf1b, we have shown that miR-802 and miR-194-2 mimics are able to inhibit luciferase activity. Furthermore, mutating the miRNA regulatory elements (MRE) for miR-802 and miR-194-2 on HNF1B 3'UTR, did not lead to a down-regulation of the transcript, demonstrating the specificity of the regulation (Figure6and Supplementary Figure4).

Figure 6 .

 6 Figure 6. Luciferase assays were made co-transfecting 250 ng of mirGLO-Hnf1b-3'UTR vectors and 100 nM of the indicated miRNA mimics for each condition. mirGLO vectors was either WT or specifically mutated for mir-802 and mir-194-2 recognition elements (MRE) as shown in Supplementary Figure 4.

  consortium and in collaboration with the Mosaiques Diagnostic and Therapeutics AG (Hannover) company, I performed with the help of my colleague Pedro Magalhães the first proteomic analysis on mouse urines. Following these results I thought with him to apply the same scientific approach to RCAD human patients and after a discussion with our supervisors we decided to contact nephrologists in different European Center. I was involved in the collection of samples and during my secondments in the Mosaiques Diagnostic and Therapeutics AG (Hannover) I performed urine preparation (ultrafiltration process) under the supervision of Pedro Magalhães and Dr Zürbig. Subsequently, my role has been focused in experimental design deciding the different cohorts to use as discovery and validation along with data interpretation and writing the manuscript.

Figure 1 .

 1 Figure 1. Study design and CE-MS analysis in urine of patients with RCAD. A. This study was performed in two phases: a discovery phase, where the urinary proteome of 44 pediatric subjects (22 healthy, 22 RCAD) was analyzed leading to the identification of 146 sequenced urinary peptides that were further modelled in a SVM-classifier, termed RCAD146. In the next step, the validation phase, we evaluated the discriminatory ability of the panel RCAD146 panel in a new cohort of RCAD patients (n=24) as well as individuals with CKD or patients carrying monogenic mutations associated with different renal diseases. B. Representation of the 146 urinary peptides significantly modified between RCAD and healthy controls. Normalized molecular mass (kDa) was plotted against normalized capillary electrophoresis (CE)-migration time (min). Mean signal intensity was given in 3-dimensional depiction. C. Box-and-Whisker plot of cross-validation scores of the RCAD146 model from the analysis of the discovery cohort along with the definition of the cut-off 0.3 (dashed line).

Figure 2 .

 2 Figure 2. Independent validation of urinary peptide classifier RCAD146 in a separate RCAD patient population together with healthy controls and patients suffering from other kidney diseases. A. Box-and-Whisker plot for the classification of all patient cohorts (RCAD subjects, non-RCAD groups) of the validation set according to the RCAD146 scores. B. ROC curve for the RCAD146 classifier based on all samples used in the validation cohort.

Figure 3 .

 3 Figure 3. HNF1B gene transcript with known mutations. The mutations shown in the graph are registered in the Human Gene Mutation Database (Stenson PD 2003, accessed on 20 February 2018). They are grouped by three mutation type within the nine exons and splice sites of the transcript: missense/nonsense mutations, insertions/deletions, and splice mutations. The numbered white boxes correspond to the exons, and the different functional domains are shown above the gene transcript. The colored boxes correspond to the number of registered mutations up to date. The whole gene deletions are not depicted. Abbreviations: POU S , POU specific domain; POU H , POU homeodomain.

  Cereghini S, Ott MO, Power S, Maury M. Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles. Development. 1992 Nov;116(3):783-97. (Cereghini S 1996) Cereghini S. Liver-enriched transcription factors and hepatocyte differentiation. FASEB J. 1996 Feb;10(2):267-82. (Casemayou A et al., 2017) Casemayou A, Fournel A, Bagattin A, Schanstra J, Belliere J, Decramer S, Marsal D, Gillet M, Chassaing N, Huart A, Pontoglio M, Knauf C, Bascands JL, Chauveau D, Faguer S. Hepatocyte Nuclear Factor-1β Controls Mitochondrial Respiration in Renal Tubular Cells. J Am Soc Nephrol. 2017 Nov;28(11):3205-3217. doi: 10.1681/ASN.2016050508. Epub 2017 Jul 24. (Catto JW et al., 2011) Catto JW, Alcaraz A, Bjartell AS et al. MicroRNA in prostate,bladder, and kidney cancer: a systematic review. Eur Urol 2011; 59: 671-681 (Ceska TA et al., 1993) Ceska TA, Lamers M, Monaci P, Nicosia A, Cortese R, Suck D. The X-ray structure of an atypical homeodomain present in the rat liver transcription factor LFB1/HNF1 and implications for DNA binding. EMBO J. 1993 May;12(5):1805-10. (Chandrasekaran K et al., 2012) Chandrasekaran K, Karolina DS, Sepramaniam S, Armugam A, Wintour EM, Bertram JF, Jeyaseelan K. Role of microRNAs in kidney homeostasis and disease. Kidney Int. 2012 Apr;81(7):617-27. doi: 10.1038/ki.2011.448. Epub 2012 Jan 11. (Cheng HT et al., 2007) Cheng HT, Kim M, Valerius MT, Surendran K, Schuster-Gossler K, Gossler A, McMahon AP, Kopan R. Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron. Development. 2007 Feb;134(4):801-11. Epub 2007 Jan 17.

  :5853-63. (Choi YH et al., 2013) Choi YH, McNally BT, Igarashi P. Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β. Am J Physiol Renal Physiol. 2013 Jul 1;305(1):F100-10. doi: 10.1152/ajprenal.00582.2012. Epub 2013 May 8. (Christopher AF et al., 2016) Christopher AF, Kaur RP, Kaur G, Kaur A, Gupta V, Bansal P. MicroRNA therapeutics: Discovering novel targets and developing specific therapy. Perspect Clin Res. 2016 Apr-Jun;7(2):68-74. doi: 10.4103/2229-3485.179431. (Chung AC et al., 2010) Chung AC, Huang XR, Meng X, Lan HY. miR-192 mediates TGF-beta/Smad3-driven renal fibrosis. J Am Soc Nephrol. 2010 Aug;21(8):1317-25. doi: 10.1681/ASN.2010020134. Epub 2010 May 20. (Clissold RL et al., 2014) Clissold RL, Hamilton AJ, Hattersley AT, Ellard S, Bingham C. HNF1B-associated renal and extra-renal diseasean expanding clinical spectrum.

TABLE OF CONTENTS INTRODUCTION CHAPTER 1. Hepatocyte Nuclear Factor 1B

 OF

	1.1 The HNF1B transcriptional factor: genomic organization and functional domains
	1.2 HNF1B expression pattern during mouse development
	1.3 HNF1B during mouse development
	1.3.1 Early post-implantation stages
	1.3.2 Role of HNF1B during organogenesis
	1.3.2.1 Pancreas
	1.3.2.2 Liver
	1.3.2.3 Intestine
	1.3.3 HNF1B during kidney development
	1.3.3.1 Overview of kidney development and function
	1.3.3.2 HNF1B requirement for ureteric bud branching morphogenesis and
	nephrogenesis induction
	1.3.3.3 HNF1B and patterning of early nephron structures
	1.3.3.4 Collecting duct morphogenesis and cystic medullar tubules	1.4
	HNF1B regulatory network and gene targets
	1.5 HNF1B transcriptional homologs, cofactors and coactivators
	1.6 Regulation of HNF1B
	1.7 The conservation of HNF1B in other species

CHAPTER 2. Renal Cysts and Diabetes syndrome

	2.1 HNF1B expression in human
	2.2 Overview of the RCAD syndrome
	2.3 HNF1B mutations and genetic analysis
	2.4 RCAD clinical features
	2.4.1 Renal abnormalities
	2.4.2 Renal physiological imbalances
	2.4.3 Diabetes
	2.4.4 Pancreas morphological abnormalities and exocrine dysfunctions
	2.4.5 Liver dysfunctions
	2.4.6 Genital tract abnormalities
	2.4.7 Other clinical features
	2.5 RCAD diagnosis and treatment
	2.6 Molecular mechanisms underlying the RCAD disease

  TOPIC I. A novel mouse model of the Renal Cysts and Diabetes disease uncovers HNF1B

	gene dosage targets during kidney development
	I.1. Context of the study
	I.2. Results and discussion
	I.3. Conclusions
	SCIENTIFIC ARTICLE 1
	I.1. Context of the study
	The transcription factor HNF1B is an important regulator of early liver, pancreas and kidney

development in vertebrates. In humans, heterozygous mutations in HNF1B cause a complex syndrome, known as Renal Cysts and Diabetes, characterized by developmental abnormalities of kidney, genital tracts and pancreas as well as several disorders, including hyperuricaemia, hypomagnesemia, early onset of diabetes and liver dysfunctions. Renal abnormalities are the most prominent features, including single kidneys, hypoplasia and glomerulocsytic dysplasia.

  2,3 , Maria Kuzma-Kuzniarska 1 , Celine Lesaulnier 1 , Martin Rezko 4 , Edwige Declercq 1 , Petra Zürbig 2 , Alain Doucet 5 and Silvia Cereghini 1* Hnf1b null allele have no apparent phenotype, while the homozygous deletion results in early embryonic death due to defective visceral endoderm formation {Barbacci, 1999 #26}, thus delaying our understanding of the pathogenic mechanisms of HNF1B-mutations. Likewise, constitutive inactivation of Hnf1b in the entire mouse epiblast {Haumaitre,2005 #3}{Lokmane, 2008

	INTRODUCTION
	The transcription factor HNF1B is an important regulator of kidney, liver and pancreas
	organogenesis either at the specification stage or during branching morphogenesis
	{Haumaitre, 2005 #3}{Coffinier, 2002 #9}{Lokmane, 2008 #2}{Lokmane, 2010 #2}{Lokmane, 2010 #12} or specific inactivation of Hnf1b either in renal tubules, liver or
	#12}{Gresh, 2004 #10}{Heliot, 2013 #7}{Massa, 2013 #11}{De Vas, 2015 #8}. In humans pancreas {Gresh, 2004 #10}{Coffinier, 2002 #9} {Heliot, 2013 #7}{Desgrange, 2017
	heterozygous germ-line mutations in the HNF1B gene cause a complex and heterogeneous #5}{De Vas, 2015 #8} results either in early embryonic or perinatal death along with far more
	syndrome, known as Renal Cysts and Diabetes (RCAD, OMIM #137920) and characterized severe phenotypes than those observed in humans. Regarding renal development, mouse
	by early onset of Diabetes and developmental abnormalities of the kidney, genital tract and genetic studies have shown that HNF1B acts at multiple and sequential steps during early
	pancreas as well as a variety of liver, biliary, pancreas and renal dysfunctions{Chen, 2010 metanephric development. Initially required for early ureteric bud branching and the induction
	#40} {Clissold, 2015 #68}. This pattern of clinical features is consistent with the broad of nephrogenesis {Lokmane, 2010 #12}, specific deletion in nephron progenitors uncovered a
	expression profile of HNF1B in the human developing metanephric kidney, urogenital tract crucial role in early nephron segmentation {Heliot, 2013 #7} {Massa, 2013 #11}.
	and endodermal derived tissues {Haumaitre, 2006 #4}{Kato, 2009 #14}, which is remarkably
	conserved in vertebrates {Sun, 2001 #15}{Lokmane, 2008 #2}{Naylor, 2017
	#16}{Kaminski, 2016 #17}.
	Heterozygous mutations in HNF1B are as well one the most common monogenic causes
	of developmental kidney disease. A large spectrum of renal abnormalities has been observed
	including unilateral or bilateral cysts, severe multicystic dysplasia, oligomeganonephronia,
	hypoplastic Glomerulocystic Kidney Disease (GCKD), solitary or horseshoe kidney
	(reviewed by {Bingham, 2004 #31}{Heidet, 2010 #20}{Chen, 2010 #40} {Clissold, 2015
	#68}.

To date, more than one hundred fifty heterozygous mutations in HNF1B have been described, including missense, nonsense, frameshift, splice site mutations and insertion/deletions as well as whole gene deletions

{Stenson, 2003 #18}{Edghill, 2006 #19}{Heidet, 2010 #20{Chen, 2010 #40}

. The phenotype of HNF1B carriers is highly variable both between and within families. No clear genotype-phenotype correlations were observed neither for the type or location of mutations, and haploinsufficiency has been the main underlying disease mechanism proposed.

Despite the increasing number of mutations identified, the molecular mechanisms by which heterozygous mutations in the HNF1B gene cause this broad spectrum of clinical symptoms remains poorly understood. Unlike humans, heterozygous mice for an Additionally, Hnf1b-ablation in the collecting ducts resulted into massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture leading to cystogenesis and perinatal death {Desgrange, 2017 #5}, while deletion at relatively later stages in medullar tubules led to cystic kidneys after birth associated with downregulation of several cystic disease genes, including Pkhd1, Pkd2 and Umod {Gresh, 2004 #10}{Hiesberger, 2005 #33}.

Molecular characterization of fetuses carrying different HNF1B heterozygous mutations demonstrated that HNF1B plays in humans, as in mice, an important role in both kidney and pancreas morphogenesis {Haumaitre, 2006 #4}. However, the cystic renal phenotype either in these fetuses {Haumaitre, 2006 #4} or in adult mutant carriers {Faguer, 2012 #34}{Casemayou, 2017 #35} was not associated with the decreased expression of the renal cystic disease genes previously identified as HNF1B targets in mice {Gresh, 2004 #10}.

  lacZ/+ ), with the lacZ gene replacing the first exon of Hnf1b {Barbacci, 1999 #26}, were maintained as heterozygotes. Animal care and the experimental protocols were approved by and conducted in accordance with French and European ethical legal guidelines and the local ethical committee for animal care (Comité d'éthique en Expérimentation Animale Charles Darwin N°5, approval number N° 04817.02), respecting the 3R rule.

Table 1 . Gene ontology

 1

	E15.5	E17.5	P1	TOTAL

Table 2 . Urine and plasma parameters of WT and Hnf1b Sp2/+ mice

 2

	Age	3 months	6 months	10-11 months
	Basal parameters	WT	Hnf1b Sp2/+	WT	Hnf1b Sp2/+	WT	Hnf1b Sp2/+
	Body weight (BW) (g)	26,1 ± 1,5	23,4 ± 2,4*	29,5 ± 2,8	26,5 ± 3,13*	36,5 ± 2,7	34,9 ± 3,12
	Water intake (ml/day)	4,75 ± 0,67	5,01 ± 0,64	3,90 ± 1,43	5,36 ± 1,81*	2,51 ± 1,1	3,4 ± 1,71
	Water intake day (20g BW)	3,63 ± 0,51	4,28 ± 0,54	2,64 ± 0,96	4,04 ±1,36*	1,37 ± 0,6	1,95 ± 0,98
	Urine volume (ml/day)	1,24 ± 0,41	1,23 ± 0,73	0,79 ± 0,50	1,61 ± 1,18*	0,72 ± 0,33	0,98 ± 0,38
	Urine volume day (20g BW)	0,951± 0,31	1,05± 0,63	0,535±0,33	1,21±0,89*	0, 394±0,18	0,561±0,217
	Aliments (g/day)	4,24 ± 0,16	4,23 ± 0,31	3,91 ± 1,46	3,39 ± 0,58	1,22 ± 0,96	1,44 ± 0,9
	Urine parameters
	U-Creat (mM)	4,48 ± 0,91	4,11 ± 0,40	5,94 ± 0,88	4,76 ± 1,53	4,29 ± 2,85	3,75 ± 1,84
	U-Prot (g/L)	5 ± 2,67	4,7 ± 3,06	7,09 ± 0,85	4,5 ± 2,07	5,07 ± 1,91	5,71 ± 3,67
	Mg 2+ (mM)	31,9 ± 7	29,4 ± 2,8	35,9 ± 7,2	24,2 ± 2,3*	18,6 ± 10,5	14,3 ± 4,64
	Total Mg2+ excreted day	30,33±2,17	30,8±1,76	19,2±2,43	29,2±2*	7,33±3,45	8,03±1
	U-Urea (mM)	1376,8 ± 169	1440 ± 135	1264,5 ± 291	1269,3 ± 173	822,7 ± 530	837,7 ± 136
	Total Urea excreted day	1309±52	1512±85,2	676,5±98,5	324,14±153	324,14±95,4	469,94±29,6
	Na + (mM)	189 ± 26,1	152,2 ± 10,8*	188,4 ± 29,1	132,3 ± 35,4*	85,2 ± 48,13	98,5 ± 26,2
	Total Na+ excreted day	179,73±8	159,8±6,8*	100,79±9,83	160,08±31*	33,56±8,66	55,25±5,6
	K + (mM)	311,3 ± 62,7	272,4 ± 38,6	329,4 ± 41,5	231,8 ± 47*	183,2 ± 88,3	170,2 ± 32,7
	Total K+ excreted day	296±19,43	286±24	176,23±14	280,47±41*	72,18±15,9	95,48±7,1
	Ca 2+ (mM)	1,26 ± 0,41	1,43 ± 0,42	0,94 ± 0,25	1,46 ± 0,18*	1,71 ± 0,57	1,6 ± 0,45
	Total Ca2+ excreted day	1,198±0,12	1,57±0,26	0,502±0,08	1,766±0,16*	0,673±0,1	0,897±0,09
	U-Pi (mM)	45,4 ± 12	47,9 ± 13,2	70,3 ± 14,2	52,5 ± 17,6	74,8 ± 43	52,5 ± 27,6
	Total U-PI excreted day	43,17±3,7	50,29±8,3	37,6±4,7	63,52±15,6	29,47±7,7	29,45±5,9
	Cl -(mM)	242,8 ± 36,0	221,5 ± 19,5	226,5 ± 42,4	224,3 ± 17,8	109,2 ± 73	150,2 ± 48
	Total Cl-excreted day	158±11	232,5±12,2	121,1±14,2	271,4±15,8	43±13	84,3±10,4
	Osmolality (mOsm/kg H 2 O)	2377 ± 445	2196,2 ± 76,3	2893,5 ± 166	2209 ± 271*	1738 ± 751	1583 ± 225
		n=4	n=4	n=4	n=4	n=4	n=4
	Plasma parameters (7months)	WT	Hnf1b Sp2/+
	Creat (µM)	30,3 ± 2,44	33,3 ± 4,67
	Mg 2+ (mM)	0,77 ± 0,08	0,89 ± 0,09*
	Urea (mM)	7,16 ± 0,84	8,2 ± 1,65
	Gluc (mM)	10,7 ± 1,78	11,7 ± 2,02
	Ala (U/l)	55,7 ± 14,5	136,3 ± 76,3*
	Asat (U/l)	155 ± 60,1	334 ± 282,9
		n=9	n=6

Table S1 : Mendelian inheritance in the offspring of Hnf1b Sp2/+ and WT intercrosses in different mouse backgrounds

 S1

			C57BL/6				129/Sv				F1
	Stage	Hnf1b Sp2/+	WT	TOT	%HET	Hnf1b Sp2/+	WT	TOT	%HET	Hnf1b Sp2/+	WT	TOT	%HET
	E14.5	25	32	57	43,8					18	11	29	62
	E15.5	13	15	28	46,4	9	9	18	50	9	10	19	47,3
	E17.5	13	12	25	52	9	12	21	42,8	16	19	35	45,7
	P0	10	12	22	45,4	10	8	18	55,5	20	21	41	48,7
	Adult	(125)	223	348	(35,9)	241	265	506	47,6	99	133	232	42,6
		(118*)	223		(33,9)
	Supplementary

Table S2 : Primers used for quantitative and semiquantitative RT-qPCRs

 S2

	Oligonucleotides used for semiquantitative PCR experiments
	Gene symbol		Gene name	RefSeq (NCBI)	Primers (5'-3')
	vATG	hepatocyte nuclear factor 1 (HNF-1), beta	NM_001291268.1	TGGTGTCACAGCTCACGTC
	V695	hepatocyte nuclear factor 1 (HNF-1), beta	NM_001291268.1	CAGTCGGAGGACACCTGCTC
	Gapdh	glyceraldehyde-3-phosphate dehydrogenase	NM_001289726.1	TCCAGTATGACTCCACTCAC ACCTTGCCCACAGCCTTG
	Supplementary Table S3 in excel. Transcriptional profiling at E14.5 -E15.5 -E17.5 -P1
	Gene symbol	Gene name		RefSeq (NCBI)	Primers (5'-3')
	Aqp2		aquaporin 2		NM_009699	CCATTGGTTTCTCTGTTACCCTG CGGTGAAATAGATCCCAAGGAG
	Bicc1		protein bicaudal C homolog 1 isoform 2	NM_001347189	TGAGGTCAGAACACCTACGA AGTGCAGTGAAGACTGTCCA
	Crb3		crumbs family member 3		NM_001347408	CCTGTCTTCAGGGGCTATTG TCGCATGAGCAGAAACAGTC
	Cyclo		cyclophilin A		NM_120034.2	CAGGTCCTGGCATCTTGTCC TTGCTGGTCTTGCCATTCCT
	Cys1		cystin 1		NM_001004455	AGAGGAGCTCATGGCGAGCATT CTGTGGCACAGATGCCAAGAGG
	Glis2		GLIS family zinc finger 2		NM_031184	GACGAGCCCCTCGACCTAA AGCTCTCGATGCAAAGCATGA
	Glis3		GLIS family zinc finger 3		NM_001305671	TGTGGCATGAATCTCCACCG TGATGGGAGGGATATGTTGACC
	Hnf1a		hepatocyte nuclear factor 1 (HNF-1), alpha	NM_009327.3	GTGTAACTGCACAGGAGGCAAA TTCTCACGTGTCCCAAGACCTA
	Hnf1b: WT transcript in Hnf1b sp2/+ samples	hepatocyte nuclear factor 1 (HNF-1), beta	NM_001291268.1	CGAGGTGGACCGGATGCTCA TCGGAGGATCTCCCGTTGCT
	Hnf4a		hepatocyte nuclear factor 4 (HNF-4), alpha	NM_001312906.1	CCATCATCTTCTTTGATCCAG CTCACTTGCACCTGTGACC
	Ihh		Indian hedgehog		NM_001313683	CCACGTGCATTGCTCTGTCA CACGCTCCCCGTTCTCTAGG
	Kcnj1		potassium inwardly-rectifying channel, subfamily J, member 1	NM_001168354	TGTCTCCACGTTTACCCCAGCA CGTTGCCGAGAACGCCCAAATA
	Kif12		kinesin family member 12		NM_001317352	GAGGCCAATAGCATCAACCG GGAGATGCAGTGGCCCAAT
	Lrp2		low density lipoprotein receptor-related protein 2	NM_001081088	AAAATGGAAACGGGGTGACTT GGCTGCATACATTGGGTTTTCA
	Nphp2		inversin		NM_010569	ACTTGTTACCCAGCATATGTGGTC AGGAGAAAACATTTGAACCTTGTCTT
	Nphp4		nephronophthisis 4 (juvenile) homolog (human)	NM_153424	TTGGCAATAAGCCAGAATCTCC TGATACAGCCTCAACCGTTTGTC
	Pkd1		polycystic kidney disease 1 homolog	NM_013630	GCTGCATGCCAGTTCTTTTG TTTTAAAGTGCAGAAGCCCCA
	Pkd2		polycystic kidney disease 2		NM_008861	CATGTCTCGATGTGCCAAAGA ATGGAGAACATTATGGTGAAGCC
	Pkhd1		polycystic kidney and hepatic disease 1	NM_153179	ATGGAGAACATTATGGTGAAGCC ATGTTTCTGGTCAACAGCCC
	Tg737		transgene insert site 737, insertional mutation, polycystic kidney disease	NM_009376	GCAGGCTTCAACCTCATCCTTA TTTCTCCCGATCTCCAATGG
	Tmem27		transmembrane protein 27		NM_001313719	TCTATCTGGAATCCGGCAAC CACTCCAGGTGGTCCTTTGT
	Wnt9B		wingless-type MMTV integration site family, member 9B	NM_011719	GTGAGAAGGAAGATGGTGAGC GCAGAATCTGGAGAACTTGGC
	Umod		uromodulin		NM_001278605.1	CTGCACCGATCCTAGTTCCG TCTACCCTGCATTCTTCGCAA

Supplementary Table S4 in excel. Gene ontology terms of downregulated genes at E14.5, E15.5, E17.5 and P1 Supplementary Table S5. Kidney-to-body weight ratios of WT and Hnf1b Sp2/+ males

	Age	1 month	5 months	10 months
	Parameter	WT	Hnf1b Sp2/+	WT	Hnf1b Sp2/+	WT	Hnf1b Sp2/+
	Body Weight (BW) (g)	20,2 ± 2,6	18,28 ± 0,83	35,1 ± 1,52	29,3 ± 6,46	31,2 ± 2,7	30,1 ± 1,5

. Basal Parameters of adult Hnf1b Sp2/+ and WT mice

		5-6 months	12 months
	RATIO	Hnf1b sp2/+ / WT	Hnf1b sp2/+ / WT
	Hnf1b /+ / WT Urine vol 20g BW	2,14*	1,354
	Hnf1b /+ / WT Water vol 20g BW	1,43*	1,45
	Supplementary
	AGE	5-6 months	12 months
	Basal parameters	WT	Hnf1b Sp2/+	WT	Hnf1b Sp2/+
	Body weight (BW, g)	31,137 ±5,622	29,683 ±3,5	36,5 ± 5,39	35,87 ± 4,9
	Water intake (ml/day)	3,909±1,43	5,361±1,81*	2,703±1,196	3,409±0,64
	Water intake (20g BW)	2,51±0,91	3,61±1,2*	1,47±0,65	1,9±0,35
	Urine volume (ml/day)	0,79±1,43	1,615±1,187*	0,79±1,43	0,79±1,43
	Urine volume day (20g BW)	0,507±0,9	1,088±0,79*	9±1,43	1±1,43
	Aliments (g/day)	3,91±1,46	3,39±0,58	1,15±0,7	1,58±0,9
		n=11	n=13	n=11	n=14

Table S7A : Differentially excreted urinary peptides between Hnf1b Sp2/+ and WT mice

 S7A

	TOPIC II. Analysis of HNF1B regulating microRNAs in the renal pathogenesis of a mouse
	model of the RCAD syndrome
	II.1. Introduction
	II.2. Summary of results
	II.3. Discussion and conclusions
	Sequence PROGRESS SCIENTIFIC REPORT (ongoing) Protein name Accession number	Start (aa position)	Stop	Adjusted p-value	Fold Change
		DOWN-REGULATED
	WTDVGMSPRIESASLQGSDRVL GRMAHASmGNRPYGPNMANMP PQVGS	Pro-epidermal growth factor containing protein 1A AT-rich interactive domain-II.1. Introduction P01132 A2BH40	620 857		0.0385 0.0009	-6,27 -5,02
	PAAPGPAGSPANDNGNGNGNGN GNGNGGKGKPA LSSLKHPSNIAVDPIERL microRNAs (miRNAs) are s m a l l n on-coding RNA molecule of ∼22 nucleotides that Striatin-interacting proteins Q8C9H6 4 36 0.002 -4,95 2 Pro-epidermal growth factor P01132 161 0.0028 -4,54
	SSLKHPSNIAVDPIERLM ALDYDPVESKIYFAQTA negatively regulates gene expression at post-transcriptional level of numerous organisms Pro-epidermal growth factor P01132 162 0.0063 Pro-epidermal growth factor P01132 522 0.0005	-3,7 -3,7
	SGNFIDQTRVLNLGPITR	Uromodulin	Q91X17	590		0.0385	-2,81
	SSLKHPSNIAVDPIERL GAGLEQEAAAG including fungi, algae, plants, insects and animals Pro-epidermal growth factor ribonucleoprotein U Heterogeneous nuclear	P01132 Q8VEK3	162 70	80	0.0114 0.0494	-2,78 -2,76
	SINKELQNSIIDL	Kidney androgen-regulated protein	P61110	26	38	0.0385	-2,36
	SPRIESASLQGSDRVL	Pro-epidermal growth factor	P01132	626		0.0304	-2,18
	LVSINKELQNSIIDLLNS	Kidney androgen-regulated protein	P61110	24	41	0.0448	-2,16
	PGApGAPGHPGPPGPV	Collagen alpha-1(III) chain	P08121	1039		0.002		-1,9
	GPpGPpGPpGPpG	Collagen alpha-1(XV) chain	O35206	704		0.0436	-1,71
	SINKELQNSIID	Kidney androgen-regulated protein	P61110	26	37	0.0132	-1,58
	DGQPGAKGEpGDTGVKGD	Collagen alpha-1(I) chain	P11087	809		0.0193	-1,54
	SpGPDGKTGPpGP	Collagen alpha-1(I) chain	P11087	535		0.0117	-1,18
		UP-REGULATED
	PGAKGEpGDTGVKGD	Collagen alpha-1(I) chain	P11087	812		0.0311		1,29
	pGpPGpRGPQGPNGADGPQGp	Collagen alpha-1(XI) chain	Q61245	1219		0.0145		1,38
	KpGERGLpGEF	Collagen alpha-2(I) chain	Q01149	575		0.0466		1,43
	GpGERGEHGPpGP	Collagen alpha-1(III) chain	P08121	795		0.0212		1,53
	GIpGTGGpPGENGKpGEpGP	Collagen alpha-1(III) chain	P08121	641		0.0032		1,62
	NIGFpGPKGPSGDpGKpGERGHpG	Collagen alpha-2(I) chain	Q01149	497		0.0311		1,68
	GPpGPTGPAGDKGD	Collagen alpha-1(III) chain	P08121	617		0.0348		1,69
	GLPGpAGPpGEAGKpGEQ	Collagen alpha-1(I) chain	P11087	633		0.0009		1,69
	GQPGAKGEpGDTGVKGDAGPpGP	Collagen alpha-1(I) chain	P11087	810		0.0348		1,74
	DGTpGGpGIRGMpG	Collagen alpha-1(III) chain	P08121	526		0.0464		1,8
	TGPIGpPGPAGApGDKGEA	Collagen alpha-1(I) chain	P11087	755		0.0287		1,87
	GPpGEAGKpGEQ	Collagen alpha-1(I) chain	P11087	639		0.0375		1,94
	GPIGPpGPAGQpGDKGEGGSpGLpG	Collagen alpha-1(III) chain	P08121	764		0.0132		2
	EAGKpGEQGVpGDLGApGP	Collagen alpha-1(I) chain	P11087	643		0.0348		2,04
	GQpGAKGEpGDTGVKGDAGppGP	Collagen alpha-1(I) chain	P11087	810		0.0348		2,16
	RDGApGAKGDRGETGP	Collagen alpha-1(I) chain	P11087	1015		0.0032		2,26
	GLpGPAGPpGEAGKpGEQGVpG	Collagen alpha-1(I) chain	P11087	633		0.0231		2,31
	TTGEVGKpGERGLpGEF	Collagen alpha-2(I) chain	Q01149	569		0.0375		2,48
	GEpGAKGERGApGEKGEG	Collagen alpha-1(III) chain	P08121	818		0.002		2,72
	AGQpGEKGPpGAQGPpGSpGPLG	Collagen alpha-1(III) chain	P08121	925		0.0054		3,06
	EVGKpGERGLpGEF	Collagen alpha-2(I) chain	Q01149	572		0.002		3,13
	pGPAGpPGEAGKPGEQGVpGDLG	Collagen alpha-1(I) chain	P11087	635		0.0005		6,9
	GpPGEAGKpGEQGVpGDLGApGP	Collagen alpha-1(I) chain	P11087	639		0.0008		7,07

Table 1 .

 1 Several genes are predicted as targets of microRNAs showing a reciprocal relationship at E15.5. In the table, values of log2FoldChange for mRNAs and microRNAs are denoted as well as the algorithm that predicted the regulation. microRNAs or mRNAs are colored in blue if UP-regulated, in red if DOWN-regulated. A complete table including also E14.5 and E17.5 inverse regulation is integrated in the Supplementary

	Genes	log2FoldChange (E15.5) on mRNA seq	Targe miRNA	log2FoldChange microRNA seq miRNA (E15.5) from	Prediction Algorithms
	IL31RA	1.59	mmu-miR-1199, mmu-miR-322	-1.38, -0.28	TargetScan, miRDB
	LARS2	1.62	mmu-miR-1199	-1.38	TargetScan
	REST	1.61	mmu-miR-1199	-1.38	TargetScan
	SCUBE3	1.54	mmu-miR-194-2	-0.76	TargetScan
	ZFP791	1.87	mmu-miR-1199	-1.38	TargetScan
	ASS1	-2.65	mmu-miR-488	0.47	TargetScan
	CLRN3	-2.32	mmu-let-7b	0.54	miRDB
	CPN1	-1.7	mmu-miR-488	0.47	TargetScan
	FBP1	-5.29	mmu-let-7c-1	0.45	TargetScan
	GPD1	-1.51	mmu-miR-379	1.32	miRSystem, miRDB
	HAO2	-2.65	mmu-miR-183	0.27	miRDB
	HNF4A	-1.63	mmu-miR-488, mmu-miR-5119	0.47, 2.53	TargetScan
			mmu-let-7b,
	KL	-1.8	mmu-miR-488,	0.54, 0.47, 1.72	TargetScan
			mmu-miR-382
	MT2	-2.24	mmu-let-7c-1	0.45	miRDB
	PCK1	-3.52	mmu-miR-5119, mmu-miR-6240	2.53, 2.17	TargetScan
	PDZK1	-2.17	mmu-miR-379	1.32	miRDB
	SLC5A8	-4.23	mmu-miR-183, mmu-let-7b	0.27, 0.54	TargetScan
	SULT1D1	-2.43	mmu-miR-183	0.27	miRDB
	TMEM229A	-1.61	mmu-miR-5119	2.53	TargetScan
	TMEM25	-1.49	mmu-let-7c-1	0.45	TargetScan, miRDB
	TMEM27	-1.64	mmu-let-7b, mmu-let-7c-1	0.54, 0.45	TargetScan
	UGT1A1	-1.5	mmu-miR-183, mmu-miR-5119	0.27, 2.53	TargetScan

Table 2 .

 2

  below all the microRNAs significantly de-regulated in the RCAD mouse model. Data were obtained from microRNA seq at three different developmental stages from kidney samples. In addition to foldChange and pvalues, the genomic sequence of each significantly de-regulated microRNA was examined through the use of HNF1B ChIP seq at E14.5; the nearest PEAK for each sequence was identified and evaluated for potential HNF1B binding sites. microRNAs are colored in blue if UP-regulated, in red if DOWN-regulated. microRNAs alignment with HNF1B 3'UTR wt and mutant. A. miR-802 alignment with HNF1B 3'UTR. B. miR-194 alignment with HNF1B 3'UTR. C. miR-30a alignment with HNF1B 3'UTR D. miR-192 alignment with HNF1B 3'UTR. While miR-802, miR-194 and miR-30a are sharing different common nucleotides in their miRNA regulatory elements (MRE) on 3'UTR of HNF1B, miR-192 is targeting another sequence.

	mmu-mir-135b-5p mmu-mir-194-1-5p TOPIC III. Urinary proteome signature of Renal Cysts and Diabetes syndrome in children 1,96 0,97 0,008733622 no 313 0,57 -0,81 2,41E-05 yes (3'UTR) 360 Supplementary Figure 4
	mmu-mir-194-2-5p mmu-mir-3968-3p III.1. Context of the study 0,64 0,67	-0,66 -0,57	0,000570755 0,001026361	yes (3'UTR) no	360 unknown
	mmu-mir-6236-5p mmu-mir-6240-5p III.2. Results and discussion 2,84 0,16 -2,65 1,51	2,78E-07 4,61E-13	no no	unknown unknown
	mmu-mir-6240-3p mmu-mir-6340-3p III.3. Conclusions 2,19 Inf	1,13 inf	5,79E-06 3,80E-06	no no	unknown unknown
	SCIENTIFIC ARTICLE 2
					E14.5
	id mmu-mir-192-5p mmu-mir-802-5p miR-802 alignment with HNF1B 3'UTR wt and mutant foldChange log2FoldChange pval 0,76 -0,40 0,005522256 0,15 -2,72 0,002401317 E14.5 Genes ____________________________________________________________ log2FoldChange Putative seed sequence in Hnf1b mRNA yes (CDS, 3'UTR) yes (3'UTR) III.1. Context of the study log2FoldChange Targe miRNA miRNA (E14.5) from Prediction Algorithms Nearest HNF1B Macs Peak from ChIP seq (kb) 1 Human WT 3'UTR 5' AAACUUGAAUC UGUUACUGAAAUAA 3' (E14.5) on mRNA seq microRNA seq WT 3'UTR 5' aaacuUGAAUC--UGUUACUGAAAUAA 3' 1 mmu-mir-342-5p 1,45 0,54 0,00066888 yes (CDS, 3'UTR) CLDN2 -1.92 mmu-mir-877 0.43 TargetScan |||||| ||||||||| 16 mmu-mir-30c-2-3p 0,75 -0,42 0,006256346 yes (3'UTR) 30 mmu-mir-24-1-3p 0,73 -0,46 0,002983014 yes (3'UTR) 32 mmu-mir-204-5p 0,70 -0,52 0,000728916 yes (CDS, 3'UTR) 48 mmu-mir-24-2-3p 0,74 -0,44 0,004202491 yes (3'UTR) GRHPR -1.68 mmu-mir-877, mmu-0.43, 0.54 TargetScan mmu-miR-802 3' uuccuACUUAGAAACAAUGACUAAUAA 5' Urinary proteomics represents an enlarging field of interest for biologists and physicians and mir-342 HCLS1 -1.48 mmu-mir-342 0.54 Mutant 3'UTR 5' AAACUguccga--guggcagucAAUAA 3' TargetScan HNF4A -1.61 mmu-mir-342 0.54 ____________________________________________________________ proteomic analysis have been employed for the discovery and the validation of biomarkers of TargetScan 58 mmu-mir-877-5p 1,35 0,43 0,00450893 yes (3'UTR) 144 mmu-mir-379-5p 0,68 -0,55 0,001397799 yes (3'UTR) 207 LGALS9 -1.51 mmu-mir-342 0.54 TargetScan PLCG2 -1.45 mmu-mir-342 0.54 miR-194 alignment with HNF1B 3'UTR wt and mutant TargetScan several kidney diseases
	Name mmu-mir-153-3p _______________________________________________________ 0,27 -1,90 0,000190631 Sequence mmu-mir-802 promoter FW mmu-mir-184-3p 0,18 -2,45 3,37E-20 Human WT 3'UTR 5' GAUGCGAAAACUUGAAUCUGUUACUG C CTCGAG GTACTGGGAAGGCAGAGACG no yes (3'UTR) 3' mmu-mir-802 promoter RV C AAGCTT CGTGGGCAGGCAGTATCCT mmu-mir-802 promoter FW 1st mutation CCGGGCCTTTAGCTTGCTAATCGGTTATGGATCCGGGAGTTAATAATTC 540 2000 mmu-mir-802 promoter RV 1st mutation GAATTATTAACTCCCGGATCCATAACCGATTAGCAAGCTAAAGGCCCGG mmu-mir-802 promoter FW 2nd mutation CGGTTATGGATCCGGGAGTTGGTAATGGATGTTAATTGGTGCTATAAGC mmu-mir-802 promoter RV 2nd mutation GCTTATAGCACCAATTAACATCCATTACCAACTCCCGGATCCATAACCG mmu-mir-194-2/192 promoter FW C CTCGAG TTAGCTGTGGTCGCTCCTCC mmu-mir-194-2/192 promoter RV CC AAGCTT ACTTGCTCAGAAGGGCTGATG mmu-mir-30A enhancer FW GA AGATCT CAGGGAGTAGCGACAAGC mmu-mir-30A enhancer RV CC AAGCTT TGCTAAGCACGACTGAACG mmu-mir-30A enhancer FW 1st mutation GCCCTGAAATTATAAGATGCTGTGGTCCAGGGACCAGACTTTAGGG mmu-mir-30A enhancer FW 2nd mutation CCCTAAAGTCTGGTCCCTGGACCACAGCATCTTATAATTTCAGGGC Hnf1b 3'UTR FW CG GCTAGC CACACCATCAGTCTCTGGG Hnf1b 3'UTR RV GC TCTAGA GAGTTCTGGCTGGCTTGC Primers used are represented with restriction sites in italic and supplementary nucleotides in bold. Bioinformatics Prediction of miRs targets was performed using TargetScan mouse 5.0 (http://targetscan.org), miRDB (http://www.mirdb.org/), mirSystem (http://mirsystem.cgm.ntu.edu.tw/) (which integrates seven well known miRNA target gene prediction programs) and mirWalk 2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/custom.html). Prediction of HNF1B binding sites in regulatory sequences was performed using the JASPAR database (http://jaspar.binf.ku.dk/) and the ECR Browser (https://ecrbrowser.dcode.org/). A limitation of the bioinformatic study may be that focusing on the predicted targets also excludes potential previously unknown mRNA targets that are not recognized by any of the well-known algorithms. Using a negative miRNA-mRNA correlation method excludes more complicated miRNA regulatory mechanisms, such as the interaction with transcription E15.5 id foldChange log2FoldChange pval Putative seed sequence in Hnf1b mRNA Nearest HNF1B Macs Peak from ChIP seq (kb) mmu-mir-192-5p 0,51 -0,96 2,11E-20 yes (CDS, 3'UTR) Mouse WT 3'UTR 5' gaUGCAAAAACUUGAAUCUGUUACUG 3' E15.5 Genes || | ||| ||||||| log2FoldChange mmu-miR-194 3' agGUGUACCUCAAC---GACAAUGu 5' log2FoldChange Targe miRNA miRNA (E15.5) from Prediction Algorithms || | | (E15.5) on mRNA seq Mutant 3'UTR 5' gaUGCAAAAACUguccgaguggcagu 3' microRNA seq IL31RA 1.59 _______________________________________________________ mmu-mir-1199, mmu-mir-322 -1.38, -0.28 TargetScan, miRDB 1 mmu-mir-194-2-5p 0,59 -0,76 1,77E-06 yes (3'UTR) LARS2 1.62 mmu-mir-1199 -1.38 TargetScan 1 mmu-mir-802-3p 0,14 -2,81 0,002994326 yes (3'UTR) REST 1.61 mmu-mir-1199 -1.38 TargetScan miR-30a alignment with HNF1B 3'UTR wt and mutant 1 mmu-mir-802-5p 0,13 -2,97 3,73E-07 yes (3'UTR) SCUBE3 1.54 mmu-mir-194-2 -0.76 TargetScan _____________________________________________________ 1 mmu-mir-1199-5p 0,38 -1,38 0,005306279 no 10 mmu-mir-30a-3p 0,82 -0,28 0,005701507 yes (3'UTR) 10 mmu-mir-187-3p 0,62 -0,70 0,00015646 no ZFP791 1.87 mmu-mir-1199 -1.38 TargetScan ASS1 -2.65 mmu-mir-488 0.47 Human WT 3'UTR 5' AACUUGAAUCUGUUACUGAAAUAA 3' TargetScan CLRN3 -2.32 mmu-let-7b 0.54 Mouse WT 3'UTR 5' aacuUGAAUCUGUUACUGAAAuAA 3' miRDB || | ||||||| 20 mmu-mir-204-5p 0,77 -0,38 0,000673598 yes (CDS, 3'UTR) CPN1 -1.7 mmu-mir-488 0.47 TargetScan mmu-mir-30a 3' cgacGUUUGUAGGCUGACUUUc 5' 48 mmu-let-7c-2-5p 1,37 0,46 2,49E-06 yes (3'UTR) 64 mmu-let-7b-5p 1,46 0,55 4,86E-07 yes (3'UTR) 65 mmu-mir-183-5p 1,21 0,27 0,005037814 no FBP1 -5.29 mmu-let-7c-1 0.45 | || TargetScan GPD1 -1.51 mmu-mir-379 1.32 Mutant 3'UTR 5' AACUguccgaguggcagucAAUAA 3' miRSystem, miRDB HAO2 -2.65 mmu-mir-183 0.27 _____________________________________________________ miRDB 120 mmu-mir-132-3p 0,74 -0,43 0,002172258 no 123 mmu-mir-379-5p 2,50 1,32 6,40E-07 yes (3'UTR) HNF4A -1.63 mmu-mir-488, mmu-0.47, 2.53 TargetScan mir-5119 207 mmu-mir-382-5p 3,30 1,72 3,25E-12 no 232 mmu-mir-194-1-5p 0,68 -0,55 0,001660338 yes (3'UTR) KL -1.8 mmu-let-7b, mmu-mir-488, mmu-mir-382 0.54, 0.47, 1.72 miR-192 alignment with HNF1B 3'UTR wt and mutant TargetScan ___________________________________________________ 360 mmu-mir-322-3p 0,82 -0,29 0,003910507 yes (3'UTR) 408 mmu-mir-488-3p 1,39 0,47 0,004607872 no 425 mmu-let-7c-1-5p 1,37 0,45 3,08E-06 yes (3'UTR) MT2 -2.24 mmu-let-7c-1 0.45 miRDB PCK1 -3.52 Human WT 3'UTR 5' ACUUUUCUUUGUAAAGGUCAG 3' mmu-mir-5119, mmu-mir-6240 2.53, 2.17 Mouse WT 3'UTR 5' acUUUUCUUUGUAAAGGUCAg 3' TargetScan | || | | |||||| 665 mmu-mir-674-3p 0,76 -0,40 0,001405383 no PDZK1 -2.17 mmu-mir-379 1.32 miRDB mmu-mir-192 3' ccGACAGUUAAGUAUCCAGUc 5' 1000 mmu-mir-3968-3p 0,74 -0,43 0,001363173 no unknown mmu-mir-5109-3p 4,92 2,30 9,40E-92 no unknown mmu-mir-5115-5p 5,17 2,37 7,47E-60 no mmu-mir-5119-3p 5,79 2,53 4,84E-06 no mmu-mir-6236-5p 3,69 1,89 6,49E-39 no mmu-mir-6240-3p 4,51 2,17 1,91E-47 no unknown mmu-mir-6340-3p Inf Inf 1,50E-30 no UGT1A1 -1.5 mir-5119 0.27, 2.53 TargetScan mmu-mir-183, mmu-unknown TMEM27 -1.64 7c-1 0.54, 0.45 TargetScan mmu-let-7b, mmu-let-unknown TMEM25 -1.49 mmu-let-7c-1 0.45 TargetScan, miRDB unknown TMEM229A -1.61 mmu-mir-5119 2.53 TargetScan unknown SLC5A8 -4.23 mmu-mir-183, mmu-let-0.27, 0.54 TargetScan No mutant form tested 7b SULT1D1 -2.43 mmu-mir-183 0.27 miRDB ___________________________________________________
	E17.5 factors, thus inducing or repressing the expression of multiple genes that are not a direct target Putative seed Nearest HNF1B of the respective miRNA. id foldChange log2FoldChange pval sequence in Hnf1b mRNA Macs Peak from ChIP seq (kb) E17.5
	Statistical Analyses mmu-mir-192-5p mmu-mir-802-3p mmu-mir-802-5p Genes (E17.5) on mRNA seq 0,49 0,33 0,35 log2FoldChange Data are shown as mean ± standard error. Unless indicated otherwise, data sets were analysed -1,01 6,74E-09 yes (CDS, 3'UTR) 1 -1,58 0,001939792 yes (3'UTR) 1 -1,52 4,67E-09 yes (3'UTR) 1 log2FoldChange Targe miRNA miRNA (E17.5) from Prediction Algorithms microRNA seq for statistical significance using a two-tailed unpaired Student t test. *=p<0.05, **=p<0.01, mmu-mir-218-1-5p 1,47 0,56 0,001566905 yes (3'UTR) 24 mmu-mir-146b-5p 1,40 0,49 0,005244074 no GABRP 0.88 mmu-mir-194-1, mmu-mir-194-2 -0.8, -0.65 TargetScan 63 mmu-mir-122-5p 0,19 -2,36 7,07E-06 yes (CDS, 3'UTR) 117 PMAIP1 0.65 mmu-mir-192 -1.01 TargetScan, miRSystem ***=p<0.001. mmu-mir-135a-2-3p 1,78 0,83 0,003657453 no 135 TCERG1L 0.59 mmu-mir-194-2 -0.8 TargetScan
	mmu-mir-218-2-5p TCHH	1,47	0.63	0,56	0,001539087 mmu-mir-192	yes (3'UTR) -1.01	218 TargetScan
	A1CF		-1.05		mmu-mir-218-1	0.55	TargetScan

Listed

Table 1 .

 1 Clinical characteristics of children with RCAD syndrome.

	All

Table 2 .

 2 Baseline characteristics of the subjects used in the A. discovery set and B. validation set.

			A. Discovery Set
	Group of subjects Mutation	Sample size (n)	Gender Male Female	Age (years)
	RCAD	HNF1B	22	17	5	9.4 ± 6.3
	Healthy	----	22	17	5	9.3 ± 4.1
			B. Validation Set
	Group of subjects Mutation	Sample size (n)	Gender	Age (years)
				Male	Female
	RCAD	HNF1B	24	20	4	7.5 ± 5.1
	Healthy	----	20	15	5	9.3 ± 2.4
	CKD	----	85	40	45	11.1 ± 5.7
	ADPKD	PKD1 PKD2	46 9	20 3	26 6	34.3 ± 6 40.2 ± 3.3
		NPHS1	2	2	-	6.5 ± 8.8
	Nephrotic Syndrome	NPHS2 NPSH4 (WT1)	35 6	14 1	21 5	10.5 ± 6.4 13.3 ± 3.7
		NPHS9 (ADCK4)	3	1	2	13.6 ± 3.1

Table 3 .

 3 Proteins origin of the 146 differentially excreted urinary peptides obtained by the comparison between RCAD patients and healthy controls.

	Protein name	Gene symbol	N° of protein fragments	P-value (adjusted)	Mean fold change
	Mucin-3a	MUC3A	1	1.10E-05	-56.9
	Gelsolin	GSN	1	1.66E-05	105.4
	Microfibrillar-associated protein 5	MFAP5	1	4.32E-05	308.3
	Interleukin-1 receptor-associated	IL-1R	1	1.27E-04	43.2
	Ig kappa chain C region	IGKC	1	1.88E-04	2.64
	Collagen alpha-6 (IV) chain	COL4A6	1	5.61E-04	3
	Collagen alpha-3 (V) chain	COL5A3	1	7.70E-04	-6.25
	Collagen alpha-4 (IV) chain	COL4A4	1	1.46E-03	4
	Collagen alpha-1 (XXVI) chain	COL26A1	1	1.72E-03	-11.2
	Collagen alpha-1 (XVII) chain	COL17A1	1	3.14E-03	-2.03
	Collagen alpha-1 (XI) chain	COL11A1	1	4.21E-03	1.19
	Protein unc-119 homolog A	UNC119	1	4.95E-03	-7.05
	Ephrin-A1	EFNA1	1	5.46E-03	5.55
	Fibrinogen alpha chain	FGA	2	5.87E-03	4.79 (± 0.48)
	Cystatin-A	CSTA	1	5.88E-03	-3.67
	Annexin A1	ANXA1	1	6.44E-03	-2.68
	Retinol binding protein 4	RBP4	1	7.78E-03	3.11
	Mucin-1 subunit alpha	MUC1	1	9.90E-03	2.4
	Beta-2-microglobulin	B2M	1	1.04E-02	-1.17
	Sarcalumenin	SRL	1	1.05E-02	-2.21
	Ig lambda-2 chain C regions	IGLC2	1	1.45E-02	-1.92
	Osteopontin	OPN	2	1.56E-02	3.85 (± 2.21)
	Collagen alpha-2 (V) chain	COL5A2	3	1.67E-02	0.22 (± 2.9)
	Kininogen-1	KNG1	1	1.69E-02	1.53
	Short transient receptor potential channel 4-associated protein	TRPC4AP	1	1.74E-02	1.98
	Immunoglobulin kappa variable 4-1	IGKV4-1	1	1.88E-02	56.7
	Collagen alpha-1 (XXVII) chain	COL27A1	1	2.04E-02	-5.66
	Collagen alpha-5 (IV) chain	COL4A5	1	2.88E-02	2.86
	Actin, cytoplasmic 1	ACTB	1	2.88E-02	3.3
	Collagen alpha-1 (II) chain	COL2A1	7	3.05E-02	4.45 (± 5.37)
	Protein scribble homolog	SCRIB	1	3.51E-02	3.15
	Collagen alpha-1 (XVI) chain	COL16A1	1	3.53E-02	2.24
	Collagen alpha-1 (VIII) chain	COL8A1	1	3.61E-02	1.29
	Hemoglobin subunit delta	HBD	1	3.65E-02	-3.38
	Neurosecretory protein VGF	VGF	1	3.80E-02	3.45
	Collagen alpha-1 (III) chain	COL3A1	21	3.89E-02	3.71 (± 4.37)
	Collagen alpha-1 (V) chain	COL5A1	2	4.36E-02	180.32 (± 253)
	Uromodulin	UMOD	4	4.82E-02	-5.7 (± 2.33)
	Collagen alpha-2 (I) chain	COL1A2	20	4.84E-02	3.28 (± 6.86)
	Serum amyloid A protein	SAA1	2	4.87E-02	-0.81 (± 2.95)

KKMRRNRFK 237[START_REF] Wu | The HNF1beta transcription factor has several domains involved in nephrogenesis and partially rescues Pax8/lim1-induced kidney malformations[END_REF]. HNF1B recognizes the consensus sequence

-Institut de Biologie Paris Seine (IBPS) -UMR7622 CNRS-Sorbonne Université -Université Pierre et Marie Curie. Paris-France

-Mosaiques Diagnostics Hannover, Germany

-Department of Pediatric Nephrology, Hannover Medical School, Hannover, Germany

-BSRC Al. Fleming, Division of Molecular Biology & Genetics Genomics Facility-Athens Greece

-Sorbonne Universités, Université Paris Descartes, UMRS 1138; CNRS, ERL 8228, Centre de Recherche des Cordeliers, Paris, France. # These authors contributed equally to the work * Silvia Cereghini corresponding author

In conclusion, heterozygous Hnf1b sp2/+ exhibit progressive renal abnormalities associated with a tendency to defective urinary concentration ability under basal conditions, associated with increased excretion of certain solutes and hypercalcuria, followed by a partial recovery in old animals. Interestingly, urinary proteome analysis uncovers a particular profile of our heterozygous mutants predictive of progressive decline in kidney function, kidney injury and fibrosis.

ACKNOWLEDGMENTS

We thank Daniel Darby and Adrian S. Woolf for critical reading of the manuscript, Eduard Manzoni for animal care. We also thank Pantelis Hatzis (Alexander Fleming Institute, Genomics Facility, Greece) for mRNA sequencing, Christophe Antoniewsky (IBPS UMR7622) for help in mRNA-seq statistical analyses, Mark Knepper (Bethesda USA), Renata Kozyraki (Institut Cordeliers, France) and Sylvie Robine (Intitut Curie, Paris) for kindly providing antibodies. FUNDINGS L.N and M. K were early researchers (ER) supported by the by the Biology of Liver and Pancreatic Development and Disease (BOLD) Marie Curie Initial Training Network (MCITN) EU-FP7 programme (No. 238821); P. R. and P.M. were recipients of PhD student fellowships from ITN RENALTRACT MSCA-ITN-2014-642937. C. L. was a postdoctoral researcher supported by Agence National de la Recherche (ANR) Blan06-2_139420. This work was supported by the GIS-Institut Maladies Rares Paris France & Institut Clinique de la Souris, Ilkirch, France, the Agence National de la Recherche (ANR) Blan06-2_139420 ; the Institut National de la Santé et de la Recherche Médicale (INSERM, France); the BOLD Marie Curie Initial Training Network (MCITN) EU-FP7 programme (No. 238821); by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement No. 642937 (RENALTRACT; MSCA-ITN-2014-642937); the by the Centre National de la Recherche Scientifique (CNRS, France) and the Université Pierre et Marie Curie (to S.C.). P.M. and P.Z. were supported by RENALTRACT MSCA-ITN-2014-642937 Acknowledgments The research presented in this manuscript was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement No. 642937 (RENALTRACT; MSCA-ITN-2014-642937). P.R., P.M. and L.G. were recipients of PhD student fellowships from ITN RENALTRACT MSCA-ITN-2014-642937. Furthermore, J.D. and L.P. were supported by the NEOCYST consortium, which is funded by the German Federal Ministry of Research and Education (BMBF, grant 01GM1515).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

SUPPLEMENTARY MATERIAL

Supplementary Figure S1: Truncated mutant proteins encoded by alternative spliced transcripts lacking the Exon2 are located primarily in the cytoplasm. Immunofluorescence of the human cell line C33 transiently expressing the WT HNF1B (isoform A) and the putative truncated proteins encoded by mutant alternative spliced isoforms A and B lacking the Exon2. Note the restricted nuclear expression of WT HNF1B in contrast to mutant truncated proteins detected both in the cytoplasm and to lesser degree into the nuclei. HNF1B (green), Dapi (nuclei, blue), middle panels (merged).

Supplementary Figure S2: Hematoxylin and Eosin (H&E) stained sections of E14.5 embryo kidneys. Upper panel F1 background (mixed genetic background C57BL/6Nx129/sv), middle panel C57BL/6N and Lower panel 129/sv background, showing in each case one E14.5 WT kidney and the two Hnf1b sp2/+ kidneys of the same litter. Scale bar: 200µm Supplementary Figure S3: Representative Hematoxylin and Eosin (H&E) stained sections of P0 kidneys in the 129/sv and C57BL/6N brackgrounds exhibiting increasing severity in the renal phenotype. Note frequent pelvic dilatations and /or hydronephrosis in the C57BL/6N background (lower panels). The last two C57BL/6N kidney sections correspond to the same Hnf1b sp2/+ embryo indicating bilateral severely affected kidneys (duplication, hydronephrosis) that would likely be incompatible with life.

Supplementary Figure S4: Comparison of mRNA levels of previously identified Hnf1b

target genes in WT and Hnf1b sp2/+ at different embryonic stages and at P0. mRNA levels of the indicated genes at E14.5, E15.5, E17.5 and P0 stages were determined by qRT-PCR and normalized by cyclophilin A expression. The indicated number of WT and Hnf1b sp2/+ samples were used with n being a pool of the 2 kidneys of each embryo. Values are represented as percentage of WT controls. Note the modest and non-significant downregulation of few genes. Significant differences between WT and Hnf1b sp2/+ (p<0.05 (*) and p <0.01 (**) are indicated. Error bars represent standard error of the mean (SEM) Supplementary Figure S5: In situ hybridization of early proximal tubule anchor genes Fbp and Spp2. In situ hybridization at E17.5 and P0 of WT and Hnf1b sp2/+ kidneys show a strong decrease of Fbp at both stages (Left panels), and a more modest decrease of Spp2 (Right panels).

Supplementary Figure S6: Representative histological sections of the Left and Right kidneys of Hnf1b Sp2/+ adult males and females, illustrating the variable severity and body weight curve analyses. H&E stained sections of the Left and Right kidneys of males (left panel ) and females (right panel). Male Age: A, A': 17-months ; B, B': 13-months, C, C': 10months ; D, D': 16-months; E, E': 9-months; F, F': 17-months; G, G': 17-months; H, H': 12 months. Female Age: A, A': 2 months; B, B': 13-months; C, C': 17-months. The right lower panel shows body weight curves of males (at the left) and females (at the right) at different ages. Note the tendency of 20% reduction in body weight of both Hnf1b Sp2/+ males and females, although lower numbers of females were examined.

Supplementary Figure S7: Representation of urinary peptides significantly altered between Hnf1b sp2/+ and WT male mice. Each peak corresponds to an identified peptide using capillary electrophoresis coupled to mass-spectrometry (CE-MS), and is characterized by specific mass (kDa), migration time (min) and abundance in the samples.

SUPPLEMENTARY TABLES

Table S1: Mendelian inheritance in the offspring of Hnf1b Sp2/+ and WT intercrosses in different mouse backgrounds. Are indicated the number of embryos and/or animals of WT and heterozygous Hnf1b sp2/+ mutant obtained from Hnf1b Sp2/+ males x WT females in the indicated genetic backgrounds, the total embryos or animals (TOT) and percentages of heterozygotes. Numbers with * in C57BL/6N background mice indicate that 7 Hnf1b Sp2/+ mice died from P1 and P25.

Table S2: Primers used for Q-RTPCR and semi-Q-RT-PCR

Table S3: Transcriptomic profiles at E14.5, E15.5, E17.5 and P1. Venn's diagram of shared differentially expressed genes between stages. Differentially anchor early proximal tubules and SLC transmenbrane differentially expressed Table S4: Gene ontology enrichment analysis of downregulated genes at E14.5, E15.5, E17.5 and P1 GO term enrichment analysis using using ToppGene informatics at different stages {Chen, 2009 #71}, showing the most important GO-terms at each stage Table S5: Kidney-to-body weight ratio of Hnf1b Sp2/+ and WT males in the C57BL/6N background Values represent the mean +/-SEM of the indicated numbers (n) of male mice. Animals with the 2 kidneys to total body weight ratio lower than 1.4 are considered as low. Note also that there is a bias in these ratios in cases of pelvic dilations of Hnf1b sp2/+ . One month-old Hnf1b Sp2/+ kidneys exhibit a moderate hypoplasia relative to the WT littermates as manifested by the low kidney-to-body ratio.

Table S6: Basal Parameters of adult Hnf1b Sp2/+ and WT mice Values are the mean ± SEM and are the average of measurements from 2 days of urine sample collection under basal conditions at the indicated ages.. Urine samples were from 3 different groups of mice at both 5-6 months and 12-months.

Table S7: Urinary proteomic analysis Adifferentially excreted urinary peptides between Hnf1b Sp2/+ and WT mice. Data were obtained from 17 Hnf1b Sp2/+ and 18 WT mice from 3, 8, 12, 17 months. 40 peptides with different abundance in urine between the two groups were selected after multiple testing (Supplementary Material and Methods). No differences were observed among different ages. P values were defined using Wilcoxon ranksum test followed by adjustment for multiple testing. Peptides are separated according to Hnf1b Sp2/+ mice urinary results (up-and down-excreted). B-Proteases potentially responsible for generation of the urinary peptides associated with RCAD mouse model. Using the 40 sequenced urinary peptides associated-proteases were predicted for N and C-terminal cleavage sites. Only proteases with ≥ 2 cleavage sites association and predicted as a high and medium prediction score were used for further analysis Mann-Whitney test with adjusted p-values <0.05 was applied to identify the proteases with a significant proteolytic activity responsible for protein/peptide fragmentation.

Cell Culture

Human embryonic kidney (HEK-293) cells were grown in DMEM (1X) + GlutaMAX medium supplemented with 10% BSA and maintained in 5% CO 2 atmosphere at 37°C.

Vector constructions

For Hnf1b transactivation analyses:

The regulatory sequences of each microRNA were amplified with PCR from mouse genome and inserted into pGEM®-T Easy vectors (PROMEGA). These sequences were confirmed by sequencing, removed from each plasmid with corresponding restriction sites and ligated into the pGL4.10[luc2]vector (PROMEGA) to form pGL4. and pGL4.10-miR-802 linked to luciferase cDNA. pGL4.10-miR-802 and pGL4.10-miR-30a point mutations variants were obtained by the use of a Site-Directed Mutagenesis using the QuikChange® II XL Site-Directed Mutagenesis Kit (Agilent Technologies) as depicted in Supplementary Figure 1 and2. Primers used are listed below.

For microRNA targeting analyses:

The 3'UTR of mouse Hnf1b was cloned into a p-mirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). A mirGLO-Hnf1b-3'UTR wt and a mutated version of these vectors were kindly provided by Dr. Jan-Wilhelm Kornfeld.

Chromatin immunoprecipitation (ChIP) assays

ChIP was performed on E14.5 kidneys using the rabbit HNF1B antibody (H85, Santa Cruz) as described by Heliot and Cereghini, 2012.

Dual Luciferase Reporter Assay

For Hnf1b transactivation analyses: HEK-293 cells were plated into twelve-well plates at a density of 2.5 x 10 5 cells/well. After 24 hours transfection mixtures containing Opti-MEM® Reduced Serum Media (ThermoFisher), 500 ng of each reporter vector (pGL4.10[luc2] + regulatory sequence wt or mutated), 5 ng of pCMV-renilla luciferase normalization vector and from 25 to 100 ng of transactivation vector (Hnf1b wt, the mutated versions ΔEx7 / Q136E or a pCB6 empty vector) were prepared. Subsequently, cells were transfected using 1.5 µL of X-tremeGENE HP DNA Transfection Reagent (Roche) for each well according to manufacturer's protocol. Cells were harvested after 48 hours and luciferase activity was calculated.

For microRNA targeting analyses: HEK-293 cells were plated into twenty-four-well plates at a density of 1.25 x 10 5 cells/well. After 24 hours, transfection mixtures containing Opti-MEM® Reduced Serum Media (ThermoFisher), 250 ng of reporter vector (mirGLO Hnf1B 3'UTR wt or mut) together with miRNA mimics (miRIDIAN microRNA Mimics Dharmacon) at the concentration of 100 nM for each condition were prepared. Subsequently, cells were transfected using 0.75 µL of X-tremeGENE™ Hp Dna Transfection Reagent (Roche) for each well according to manufacturer's protocol. Cells were harvested after 24 hours and luciferase activity was calculated. In both experiments luciferase expression was assayed using a Dual Luciferase Assay Kit (Promega) according to the manufacturer's protocol, and the luminescence was recorded on a microplate reader (Berthold Tech TriStar 2 LB 942). The normalized firefly luciferase activity was obtained by measuring firefly luciferase activity/Renilla luciferase activity. All experiments were performed at least three times.

Supplementary Table 1

Supplementary table 2

Inverse gene-(predicted)miRNA relations. Several genes are predicted as targets of microRNAs showing a reciprocal relationship during the three stages analyzed. In the

Abstract

Renal Cysts and Diabetes Syndrome (RCAD) is an autosomal dominant disorder caused by mutations in the HNF1B gene encoding for the transcriptional factor hepatocyte nuclear factor-1B. RCAD is characterized as a multi-organ disease, with a broad spectrum of symptoms including kidney abnormalities (renal cysts, renal hypodysplasia, single kidney, horseshoe kidneys, hydronephrosis), early-onset diabetes mellitus, abnormal liver function, pancreatic hypoplasia and genital tract malformations. In the present study, using capillary electrophoresis coupled to mass spectrometry (CE-MS), we investigated the urinary proteome of a pediatric cohort of RCAD patients and different controls to identify peptide biomarkers and obtain novel insights into the pathophysiology of this disorder. As a result, 146 peptides were found to be associated with RCAD in 22 pediatric patients compared to 22 healthy agematched controls. A classifier based on these peptides was generated and further tested on an independent blinded cohort, clearly discriminating RCAD patients from different groups of controls. This study demonstrates that the urinary proteome in children with RCAD syndrome differs from autosomal dominant polycystic kidney disease (PKD1, PKD2), congenital nephrotic syndrome (NPHS1, NPHS2, NPHS4, NPHS9), and also from chronic kidney disease conditions, suggesting differences between the pathophysiology behind these disorders.

Discussion

In this study, we aimed to identify urinary peptides related to RCAD syndrome with the goal of providing a non-invasive readout together with molecular changes that can be significant and specific of this disease. This is the first study showing a unique proteome profile, which distinguishes RCAD children from healthy controls and patients with different renal diseases.

To demonstrate the RCAD pediatric proteome specificity from genetic point-of-view, patients with more than ten different heterozygous mutations in the HNF1B gene were evaluated in our study. Considering that more than 150 heterozygous mutations in HNF1B have so far been shown to be pathogenic (Figure 3), this study covered a distinct range of exonic and intronic variations.

The most prominent finding of the study was the identification of 294 differentially excreted peptides potentially related to RCAD syndrome, where sequence information was obtained for 146 peptides. Due to the fact that changes in the kidney and genitourinary tract are reflected by modifications of the urinary proteome 30 , peptides may display a particular signature/feature, providing better insights of RCAD syndrome pathophysiology.

Interestingly, as previously shown in the ADPKD urinary proteome 31 , the majority of peptides enriched in the urine of RCAD patients were collagen type I or type III fragments. This may reflect active extracellular matrix (ECM) remodeling, which could be related to ECM modifications due to cyst expansion, which affects modifications in the ECM 32 .

In contrast, other peptides, previously associated with cystic conditions, were deregulated in RCAD. These included annexin A1 (ANXA1), a key protein in cystic fibrosis pathogenesis and a prognostic marker of glomerular injury 33,34 ; hemoglobin subunit delta (HBD), which was found downregulated in arachnoid cysts fluids compared with cerebrospinal fluids 35 ; the protein unc-119 homolog A (UNC119) that play a crucial role in proper ciliary targeting of the cystic gene nephrocystin-3.

In the RCAD urinary proteome we observed altered of several peptides with calcium binding or calcium regulating properties, such as sarcalumenin (SRL), and annexin A1 (ANXA1), gelsolin (GSN), short transient receptor potential channel 4-associated protein (TRPC4AP) and the direct target of HNF1B osteopontin (SPP1) 36 . This may be related to the role of HNF1B as a mediator of the urinary reabsorption of calcium 15,37 .

Following the identification of the proteomic signature, the urinary peptides were

Urine sample preparation and CE-MS analysis

Urine sample collection and CE-MS analysis were performed as reported previously 44,45 .

Briefly, immediately before preparation, urine samples aliquots stored at -20°C were thawed and 700 µl were diluted with the same volume of 2 M urea, 10 mM NH 4 OH comprising 0.02 % SDS. Then, samples were filtered via a Centristat 20-kDa cut-off centrifugal filter device (Sartorius, Goettingen, Germany) at 2,600 g for one hour at 4°C in order to remove high molecular weight compounds. The obtained filtrate was desalted using a PD-10 column (GE Healthcare, Sweden) equilibrated in 0.01% aqueous NH 4 OH to eliminate urea, electrolytes and salts. Finally, samples were lyophilized and stored at 4°C prior to CE-MS analysis. The samples were re-suspended in 10 µL of HPLC-grade H 2 O shortly before CE-MS analysis, as described 45 . CE-MS analyses were accomplished using a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, USA) online coupled to a MicroTOF MS (BrukerDaltonic, Bremen, Germany) 45 . The electro-ionization sprayer (Agilent Technologies) was grounded, and the ion spray interface potential was defined between -4 and -4.5 kV. Spectra were accumulated every 3s along with over a range of m/z to 350-3000.

Detailed information on accuracy, precision, selectivity, sensitivity, reproducibility and stability of the CE-MS method have been described previously 45 .

CE-MS data processing

A proprietary software (MosaiquesVisu) was used to deconvolute mass spectral ion peaks demonstrating identical molecules at different charge states into single masses 46 . The achieved peak list allows the characterization of each polypeptide according to its CEmigration time (in minutes), molecular mass (in Daltons), and ion signal intensity.

Subsequently, normalization of the amplitude of the urinary peptides was conducted on twenty-nine 'housekeeping' peptides (peptides varied slightly between samples, generally present in at least 90% of all urine), similarly to previous studies 47 . All detected peptides were deposited, clustered, matched and annotated in a Microsoft SQL database 25-27 , allowing further statistical analysis.

Peptide sequencing

Candidate peptides for the RCAD-classifier were identified and sequenced by the use of tandem mass spectrometry (MS/MS) analysis and searched against human entries in the UniProt database, as previously described previously 48,49 . Briefly, to acquire the sequence information, urine samples were separated on a Dionex Ultimate 3000 RSLC nano flow system (Dionex, Camberly, UK) or a Beckman CE systems (PACE MDQ) coupled to an Orbitrap Velos MS instrument (Thermo Fisher Scientific) 48,49 . Thereafter, data files were examined against the UniProt human non-redundant database using Proteome Discoverer 1.2 (Thermo) and the SEQUEST search engine. No fixed modifications were selected, hydroxylation of proline and lysine and oxidation of methionine were enabled as optional modification, nor enzyme specificity were specified in the settings 48 . The matching of the peptide sequence obtained by MS/MS analysis to the CE-MS peaks were based on molecular mass [Da] and theoretical migration time, calculated using the number of basic amino acids 50 .

Peptides were accepted only if they had a mass deviation below ±5 ppm and <50 mDa for the fragment ions.

Peptide identification and statistical analysis

For the identification of potential RCAD-related urinary peptide biomarkers, a comparison between RCAD cases and healthy controls was performed. Only peptides that were detected in at least 70% (frequency threshold) of the samples in at least one of the two groups were further considered for statistical analysis. Adjusted P-values were calculated based on the comparison between RCAD cases and healthy controls, using the Wilcoxon rank-sum test followed by adjustment for multiple testing with the false-discovery rate method presented by be also related to cyst expansion/presence, which affects modifications in the ECM [START_REF] Norman | Fibrosis and progression of Autosomal Dominant Polycystic Kidney Disease (ADPKD)[END_REF]. In addition, in the pediatric RCAD urinary proteome we observed altered of several peptides with calcium binding or calcium regulating properties as sarcalumenin (SRL), annexin A1 (ANXA1), gelsolin (GSN), short transient receptor potential channel 4-associated protein (TRPC4AP) and osteopontin (SPP1).

Furthermore, this proteomic analysis highlighted that RCAD syndrome has a unique urinary signature, which is different from the urinary proteome of patients from different groups of controls as Autosomal Dominant Polycystic Kidney Disease (ADPKD), nephrotic syndrome and chronic kidney disease CKD patients. This discovery is attractive as differentiates the biological events leading to the RCAD syndrome from other cystic diseases

and conditions and open the possibilities to an early tool of diagnosis as well as a better understanding of the molecular basis underlying HNF1B regulation and so the RCAD syndrome. Future studies will further evaluate the performance of the RCAD panel in additional pediatric cohorts of disorders more closely related to RCAD such as autosomal recessive polycystic kidney disease (ARPKD), medullary cystic kidney disease (MCKD), autosomal dominant tubulointerstitial kidney disease (ADTKD) or diabetic patients.

Moreover, and along with the evaluation of the performance of this classifier to predict the progression of RCAD, follow-up clinical data of the patients described in this study should be addressed.

The combined analysis of a unique RCAD mouse model together with urinary proteomic approaches provided new interesting means to comprehend the disease as well as revealed new insights on this disorder mainly related to the kidney phenotype, the urinary proteome and the post-transcriptional regulation of HNF1B transcriptional factor.