Irene Sanchez-Martin

Pedro Magalhães

Parisa Ranjzad

Ahmed Fatmi

Fabrice Richard

Thien Phong

Vu Manh

Andrew J Saurin

Guylène Feuillet

Colette Denis

Adrian S Woolf

Joost P Schanstra

Petra Zürbig

Xavier Caubit

Laurent Fasano
email: laurent.fasano@univ-amu.fr

Affiliation

Haploinsufficiency of the mouse Tshz3 gene leads to kidney defects

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of renal failure in children [START_REF] Schedl | Renal abnormalities and their developmental origin[END_REF]. Ureteropelvic junction obstruction (UPJO), the most common paediatric renal obstructive disorder, has an incidence of 1 in 1000-1500 live births screened by antenatal ultrasound [START_REF] Chang | Calcineurin is required in urinary tract mesenchyme for the development of the pyeloureteral peristaltic machinery[END_REF]. Congenital UPJO is usually caused by the presence of an aperistaltic segment of the ureter, preventing the efficient transport of the urine from the kidney to the bladder. UPJO can result from the decrease in the number of smooth muscle cells, interstitial Cajal-like cells and nerve fibers in the ureteropelvic junction. Therefore, impaired transport of urine can lead to an increase in back-pressure on the kidney, hydronephrosis, and progressive damage to the kidney function [START_REF] Klein | Congenital ureteropelvic junction obstruction: human disease and animal models[END_REF].

The TSHZ3 gene (Teashirt zinc-finger homeobox family member 3; also known as ZNF537), which encodes a zinc-finger transcription factor, was recently identified as the critical region for a 19q12 deletion syndrome (19q12DS): patients with TSHZ3 heterozygous deletion show lower (i.e. vesicoureteral reflux grade 2) and upper (i.e. UPJO) urinary tract defects as well as kidney (i.e. hydronephrosis and nephrolithiasis) defects [START_REF] Caubit | TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons[END_REF].

Tshz3 homozygous mutant mice (Tshz3 lacZ/lacZ ) have been used to explore the pathogenesis of Tshz3 in UPJO [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF][START_REF] Martin | TSHZ3 and SOX9 regulate the timing of smooth muscle cell differentiation in the ureter by reducing myocardin activity[END_REF]. Studies have shown that from embryonic day (E) 12.5 onwards TSHZ3 positive cells are detected in the mouse ureter and the kidney [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF].

In the embryo, Tshz3 plays a key role in smooth muscle differentiation by regulating Myocardin (myocd) expression and MYOCD activity [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF][START_REF] Martin | TSHZ3 and SOX9 regulate the timing of smooth muscle cell differentiation in the ureter by reducing myocardin activity[END_REF]. Tshz3 lacZ/lacZ mutant mice die perinatally because of their inability to breathe and display bilateral UPJO and hydronephrosis [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF][START_REF] Caubit | Teashirt 3 regulates development of neurons involved in both respiratory rhythm and airflow control[END_REF]. In comparison, one-fourth of Tshz3 +/lacZ heterozygous embryos display a unilateral UPJO and hydronephrosis and about 50% die at birth [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF][START_REF] Martin | TSHZ3 and SOX9 regulate the timing of smooth muscle cell differentiation in the ureter by reducing myocardin activity[END_REF][START_REF] Caubit | Teashirt 3 regulates development of neurons involved in both respiratory rhythm and airflow control[END_REF]. However, the impact of Tshz3 haploinsufficiency on the postnatal kidneys has never been investigated so far.

Therefore, the main aim of this study was to determine whether Tshz3 heterozygote mice exhibit kidney defects in order to gain insights on whether haploinsufficiency may help explaining kidney diseases reported in patients with heterozygous deletion of TSHZ3.

Results

TSHZ3 expression in adult kidneys.

Previous analyses performed during development indicate that the temporal and spatial distribution of β-galactosidase (ß-gal) protein in Tshz3 +/lacZ mice faithfully reproduces the expression of Tshz3/TSHZ3 [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF][START_REF] Martin | TSHZ3 and SOX9 regulate the timing of smooth muscle cell differentiation in the ureter by reducing myocardin activity[END_REF]. Here we used the same approach to characterize the expression of Tshz3/TSHZ3 in sections of Tshz3 +/lacZ adult kidneys. X-Gal staining performed on Tshz3 +/lacZ adult kidney revealed that Tshz3 is expressed in the pelvic region and ureter, the papilla, perivascular region and the glomeruli (Fig. 1A-F). To further characterize the expression of TSHZ3 in the glomeruli, we performed immunostaining, using glomerular cells markers. These analyses showed that -gal positive cells were endothelial cell (CD31-positive) but not podocytes (Dachshund 1 positive) or mesangial cells (NG2 positive) (Fig. 1G-I). In one-week old postnatal Tshz3 +/lacZ mouse kidneys, TSHZ3 was detected in the tufts of maturing glomeruli and in nuclei of thin tubule-like structures in the kidney cortex (Fig. 2A). Subsets of tubule-like profiles also immunostained for TSHZ3 in the outer medulla and the inner medulla (Fig. 2A-C).

Comparison with immunostaining patterns for aquaporin-1, uromodulin and calbindin-28 (Fig. 2D-L), suggest that thin descending limbs of loops of Henle express TSHZ3 because patterns for this protein and aquaporin-1 are similar in the thin tubules within the inner medulla. In this location, calbinin-28 positive collecting ducts are wider diameter and uromodulin is not detected. The TSHZ3 positive tubules in the cortex and outer medulla may also include the pars recta, or straight section, of the aquaporin-1 positive proximal tubules. The tubule signals for TSHZ3 appeared attenuated in adult kidneys into which we did not observe any major morphological changes (data not shown). Double immunostaining for β-gal and Smooth muscle alpha-actin (SMAA) confirmed the expression of TSHZ3 in smooth muscle cells in the pelvic region and in SMA-negative Dachshund 1-positive stromal cells lining the urothelium (Fig. 3).

Tshz3 +/lacZ mice showed glomerular defects.

Comparison of 8-week-old adult kidney histology revealed a decrease of 28.8% in glomerular density in Tshz3 +/lacZ compared to WT (Fig. 4A, B and supplementary Fig. 4). The same analysis performed with 40-week-old adult kidneys revealed a 44.2% reduction of glomerular density in Tshz3 +/lacZ vs. WT. In order to identify glomerular morphological changes in Tshz3 +/lacZ mice, we conducted an ultrastructural analysis using transmission electron microscopy (TEM). While this analysis did not identify a significant variation of the proportion of the size of the fenestration and endothelial layer (Fig. 4C,D), it revealed a significant reduction of the thickness of the glomerular basement membrane (GBM) in glomeruli of Tshz3 +/lacZ mutants (144.2 ± 4.2 nm) compared to WT (155.1 ± 3.32 nm) (Fig. 4E,F). TEM analysis also showed that in the Tshz3 +/lacZ kidneys the foot processes were effaced, reflected by a foot process width (FPW) of 351.7 ± 5.03 nm, ≈14% larger (P<0.0001) than in the WT kidneys (303.4 ± 6.57 nm) (Fig. 4G,H). Blood electrolytes are modified in Tshz3 +/lacZ mice.

To characterize the effects of Tshz3 haploinsufficiency on kidney filtration, we performed biochemical measurements on blood samples and generated biochemical profiles for Tshz3 +/lacZ and WT adult mice tested at 58-64 days-of-age. Blood analysis demonstrated that Tshz3 +/lacZ mice have significant reduction of the urea (P<0.013), phosphates (P<0.011), magnesium (P<0.014), potassium (P<0.0034) as well as an increased concentration of sodium (P <0.012) (Fig. 5; Table 1a). We found no significant variation in creatinine concentration in Tshz3 +/lacZ compared to WT (15.91 ± 1.51 vs. 12.36 ± 2.07 µM/l; P<0.09). The Tshz3 +/lacZ adult kidney showed differential expression of genes involved in inflammatory processes.

To identify Tshz3-regulated genes in the adult kidney, RNA sequencing analysis was performed with samples extracted from Tshz3 +/lacZ (n=6) and wild type (WT; controls, n=6) adult mouse kidneys. 97 differentially expressed genes (DEGs) were identified, among which 55 were up-regulated and 42 were down-regulated (adjusted p-value < 0.01) (Table 1b). Thereafter, we sought to take advantage of the single-cell transcriptome of mouse glomeruli [START_REF] Karaiskos | A Single-Cell Transcriptome Atlas of the Mouse Glomerulus[END_REF] and adult kidney [START_REF] Park | Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease[END_REF] to identify the cell types in wild type kidneys that expressed the genes differentially expressed in Tshz3 +/lacZ kidney. This analysis identified 58 genes expressed in podocytes, 31 in mesangial cells, 27 in immune cells, 40 in tubules and 43 in GECs, which might be direct targets of TSHZ3 (Table 1c). We also found that some DEGs were shown to be markers of proximal tubules (Aspdh, Snhg11 and D630029K05Rik, a long non-coding RNA) or myeloid lineage (Cd74, Gusb, H2-Aa, H2-Ab1, Lyz2, Mpeg1 and Vcam1), including macrophages and dendritic cells [START_REF] Liu | Cell-specific translational profiling in acute kidney injury[END_REF].

To characterize the differential expression data and to identify biological changes that occur in Tshz3 +/lacZ kidneys, we performed different enrichment analyses. As only a few genes showed very strong differential expression, we applied gene set enrichment analysis (GSEA [START_REF] Mootha | PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[END_REF][START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genomewide expression profiles[END_REF]) using a ranked gene list. We chose the updated public genesets available on MSigDB [START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genomewide expression profiles[END_REF] and identified positive enrichment in the kidneys of Tshz3 +/lacZ adult mice for gene sets related to "interferon-gamma response", "epithelial to mesenchymal transition", "inflammatory response" and negative enrichment for genes related to "Oxidative Phosphorylation" and "Xenobiotic Metabolism" (Table 1d; Supplementary Fig. 1). Furthermore, ingenuity pathway analysis (IPA) of the DEGs revealed enrichment of cellular processes centered on inflammatory and kidney diseases (Supplementary Fig. 2A,B). This analysis showed that the relevant toxicity phenotypes and pathology endpoints associated with the DEGs in Tshz3 +/lacZ mice were centered on the kidney and identified interferon-gamma (IFNG) as an upstream regulator (Supplementary Fig. 2B,C). Moreover, enrichment analysis of pathways and transcription factor using enrichR (13) also identified the "interferon gamma" pathway but also "collagen formation" and "extracellular matrix organization" as significantly enriched in Tshz3 +/lacZ adult kidneys (Table 1e). Interestingly, the ChIP enrichment analysis (ChEA) and a database search revealed that 22 DEGs (21.68%) are direct targets of the transcription factor interferon regulatory factor 8 (IRF8) (Table 1f).

Using RT-qPCR we confirmed the DEG status observed by RNA-seq analysis for Ciita, Pld4 (two targets of IRF8), Tlr7 (IRF8 target that promotes IFNG production (14)) and the proinflammatory Npy (Supplementary Fig. 3).

Genes differentially expressed in Tshz3 +/lacZ adult kidney are associated with ASD

To gain insights into TSHZ3 function, we studied the disease association of the 80 nonambiguous human orthologs of the mouse DEGs (Table 1g). This analysis identified 35/80 genes (43.75%), which were established or putative causes for kidney disorders. Interestingly, 8 of these genes have been also associated with ASD (Table 1h). Identification of Tshz3 +/LacZ -related urinary peptides Since Tshz3 +/lacZ mice exhibit dysregulated kidney expression of several genes and altered hematological parameters, we sought to analyze their urinary peptidome. Urine samples derived from Tshz3 +/LacZ (n=15) and WT (n=12) were analyzed by capillary electrophoresis coupled to mass spectrometry (CE-MS). Comparison of urinary peptidome profiles led to the identification of 33 peptides that were significantly (adjusted p-value < 0.05) associated with Tshz3 +/lacZ (Fig. 6). Protein fragments from clusterin (CLUS), complement factor D (CFAD), histone H2B type 1-F/J/L (H2B1F), major urinary protein 17 (MUP17), tripeptidyl-peptidase 1 (TPP1) and uromodulin (UMOD), as well as a large number [START_REF] Liu | How fibroblast growth factor 23 works[END_REF] of collagen fragments, were identified (Table 1i).

Tshz3 +/lacZ mice showed a decreased concentration of uromodulin and an increased concentration of peptides from CLUS, CFAD, H2B1F, MUP17 as well as TPP1 fragments. Twelve collagen peptides were in higher abundance and fifteen in lower abundance. As UPJO can lead to significant kidney damage, we compared the identified 33 urinary peptides from mice with those of the CKD273 classifier, a predictive marker of chronic kidney disease (CKD) progression in humans [START_REF] Good | Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease[END_REF]. This analysis identified 14 similar (orthologs) human peptides in the CKD273 classifier, most of which were collagen fragments (10 from collagen type I alpha-1 chain and 3 from type III alpha-1 chain). One peptide fragment was from uromodulin.

Discussion

Previously, we reported that in mice and humans, haploinsufficiency of Tshz3/TSHZ3 results in hydroureter [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF][START_REF] Caubit | TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons[END_REF] and that individuals with TSHZ3 heterozygous deletion are at risk to develop kidney diseases [START_REF] Caubit | TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons[END_REF]. However, the expression of Tshz3 in adult kidney and the morphology of Tshz3 heterozygous adult kidney have not been investigated before. Herein, we characterized TSHZ3 expression in the adult mouse kidney, in particular in glomeruli where TSHZ3 is specifically expressed in endothelial cells (GEnCs), which is supported by single-cell RNA sequencing analysis [START_REF] Karaiskos | A Single-Cell Transcriptome Atlas of the Mouse Glomerulus[END_REF]. Using Tshz3 +/lacZ heterozygous mice, we have also uncovered a key role for the TSHZ3 transcription factor in controlling the glomerular density and morphology. Notably, we showed that Tshz3 +/lacZ mice have abnormal blood electrolytes and identified Tshz3 +/LacZrelated urinary peptides. In addition, by coupling transcriptomics with in silico analysis, we showed that in kidneys from Tshz3 +/LacZ mice, the expression of genes associated with inflammatory processes and of renal-and ASD-associated was different from that in WT mice. Combined with our previous reports, our data suggest that the Tshz3 heterozygous mice constitute a model that replicates many of the corresponding human disease phenotypes.

The GEnCs, together with the glomerular basement membrane (GBM) and the podocytes, constitute the glomerular filtration barrier (GFB), which selectively filtrates the plasma.

The GBM derives from the fusion of the basement membranes of both GEnCs and podocytes, and in adult kidney, the GEnCs may contribute to the renewed biosynthesis of the GBM. Because abnormalities in each of the three constituents of the GFB can lead to proteinuria and kidney disease, TEM was used to assess fine structures of the GFB.

While TEM analysis revealed no obvious structural phenotype in TSHZ3-expressing GEnCs, it provided evidence that Tshz3 haploinsufficiency leads to GBM thinning and change in podocyte morphology evidenced by effaced foot process. Interestingly, hematuria has been reported in individuals with a thin GBM (17) but we did not observe blood staining (macrohematuria) in the urine samples from Tshz3 heterozygous mice. In the future, it would be interesting to investigate whether these mice have microhematuria.

At present, the primary cause of the structural defects observed in the GBM and the podocytes remains unknown. However, since communication between intraglomerular cells is required for proper development and maintenance of the GFB [START_REF] Dimke | Crosstalk in glomerular injury and repair[END_REF], GBM thinning and larger FPW might be the direct or indirect consequences of an abnormal cross-talk between the TSHZ3-positive GEnCs and other glomerular cells. Furthermore, Tshz3 may be transiently expressed in glomerular cell lineages. Nevertheless, the comparison of the urinary peptidome of Tshz3 +/lacZ and WT adult led to the identification of 27 collagen and 6 non-collagen peptides associated with Tshz3 +/lacZ mice. Among the collagen fragments displaying a deregulation (e.g. 12 increased or 15 decreased), we identified thirteen collagen fragments that overlap the CKD273 classifier that might be indicative of the development of fibrosis [START_REF] Magalhaes | Association of kidney fibrosis with urinary peptides: a path towards non-invasive liquid biopsies?[END_REF][START_REF] Wei | Alterations in urinary collagen peptides in lupus nephritis subjects correlate with renal dysfunction and renal histopathology[END_REF]. As previously suggested [START_REF] Klein | Urinary peptidomics provides a noninvasive humanized readout of diabetic nephropathy in mice[END_REF], the fragments overlapping the human CKD273 classifier could be used as biomarkers to assess renal function in patients with TSHZ3 heterozygous deletion. Interestingly, our analysis of urinary peptides also revealed an under-representation of a MUP17 peptide in Tshz3 +/lacZ mice. The MUP17 protein is predominantly expressed in males and dominant males significantly increased the secretion of MUP17 in social conditions [START_REF] Mudge | Dynamic instability of the major urinary protein gene family revealed by genomic and phenotypic comparisons between C57 and 129 strain mice[END_REF][START_REF] Thoss | Regulation of volatile and non-volatile pheromone attractants depends upon male social status[END_REF]. In the future, it would be interesting to study the relationship between variations in the level of MUP17 and the severity of the social behavior deficit observed in Tshz3 +/lacZ mice. To further evaluate renal function, we performed biochemical measurements on blood samples and generated biochemistry profiles. This analysis identified a significantly reduced concentration for urea, phosphates, magnesium and potassium in Tshz3 +/lacZ mice.

Reduced serum urea is less frequent and usually of less clinical significance than increased serum urea [START_REF] Gallagher | Significance of abnormally low blood urea levels[END_REF][START_REF] Lum | Significance of low serum urea nitrogen concentrations[END_REF]. The lower plasma magnesium (hypomagnesaemia), phosphate (hypophosphatemia) and potassium (hypokalemia) serum concentration are associated with defective reabsorption/excretion process in the distal nephron [START_REF] De Baaij | Regulation of magnesium balance: lessons learned from human genetic disease[END_REF][START_REF] Liu | How fibroblast growth factor 23 works[END_REF][START_REF] Lederer | Regulation of serum phosphate[END_REF].

However, the decrease in potassium concentration in Tshz3 +/lacZ mice should be viewed with caution since the method of euthanasia had an effect on potassium level [START_REF] Traslavina | Euthanasia by CO(2) inhalation affects potassium levels in mice[END_REF]. In line with this, single-cell transcriptional profiling of the healthy kidney of 4-8 weeks old mice showed expression of Tshz3 in the proximal tubule, distal convoluted tubule as well as in collecting duct principal and intercalated cells [START_REF] Park | Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease[END_REF] and our immunostaining studies suggest that Tshz3 is indeed not only expressed in glomeruli but also, at least in the early post-natal period, in subsets of tubules, most likely the straight section of the proximal tubule and the thin descending limb of the loop of Henle. This segments are respectively implicated in reclaiming substances, including magnesium, from the glomerular ultrafiltrate, and in the mechanism required for urinary concentration in the medulla (30-32). Altogether, our data suggest that Tshz3 haploinsufficiency might impact different components of the nephron.

To identify pathways that might be altered, we performed RNA-seq analysis and detected 97 statistically significant changes in gene expression in adult kidneys of Tshz3 +/lacZ as compared to WT. Based on the results of enrichment analyses, we detected the involvement of inflammation-related pathways such as interferon-gamma. Of note, seven DEGs (Crym, Ctss, Fasn, Fcgrt, Gabrb3, Lyz2, and Vcam1) were found to be associated with kidney diseases, including renal inflammation, crescentic glomerulonephritis or end-stage renal failure. Interestingly, population-based studies support that autism spectrum disorder (ASD) and kidney disease coexist in several genetic disorders (for review see Table 2 in (33)), suggesting that the same genetic modification can affect neurodevelopment and nephrogenesis. However, because the genetic alterations (deletion or duplication) associated with these disorders often encompass several genes, it is still unclear how these genes contribute to the underlying molecular mechanisms. In this context, the TSHZ3 gene which associates ASD with a congenital kidney condition is quite unique (4) and our findings that heterozygous deletion of Tshz3 leads to kidneys defects might be relevant for patients with TSHZ3 heterozygous deletion.

For example, we found a decreased glomerular density that potentially might lead to a reduced glomerular filtration rate (GFR), a defect that was reported in a patient with heterozygous deletion of the TSHZ3 gene (Table1 in ( 4)) who required renal transplant (Table 2 in (34)). However, we did not find that creatinine level was significantly altered in Tshz3 heterozygous mice but this measurement is not very accurate to estimate the GFR. Therefore, we must be cautious about whether Tshz3 heterozygous mice have an altered GFR. It is possible to measure GFR in adult mice, although the technology to do so is technically challenging [START_REF] Scarfe | Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy[END_REF]. In future, this could be applied to our mouse model.

The reduced number of glomeruli also questions about the mechanism leading to this defect. The reduction of the glomerular density could be a secondary defect in a subtle branching defect but it would require a new study to formally validate this hypothesis.

We actually do not observe Tshz3 expression in the ureteric bud-collecting duct epithelial lineage (( 5) and this study). On the other hand, we do see Tshz3 in the interstitial stroma around this epithelial lineage (( 5) and this study) and we cannot exclude that there is a defect in the crosstalk between these two compartments [START_REF] Paroly | Stromal protein Ecm1 regulates ureteric bud patterning and branching[END_REF] in Tshz3 heterozygous mice.

Our results might also provide to be of interest for cognitive defects link to TSHZ3 haploinsufficiency. For now, the role of low plasma concentration of magnesium in ASD is still a matter of debate. While two studies did not find a statistically significant difference in levels of magnesium in children diagnosed ASD [START_REF] Wu | Evaluation of Whole Blood Trace Element Levels in Chinese Children with Autism Spectrum Disorder[END_REF][START_REF] Saldanha Tschinkel | Plasma concentrations of the trace elements copper, zinc and selenium in Brazilian children with autism spectrum disorder[END_REF], two other studies [START_REF] Strambi | Magnesium profile in autism[END_REF][START_REF] Guo | Vitamin and mineral status of children with autism spectrum disorder in Hainan Province of China: associations with symptoms[END_REF] demonstrated lower levels of magnesium in children diagnosed with ASD. So far, Tshz3 mouse models have been shown to be quite analogous to the clinical problems (i.e. ASD-associated deficits, hydroureter and hydronephrosis) reported in patients with TSHZ3 heterozygous conditions (4, 5). Our results generate new hypotheses that might lead to further understanding of the clinical problems and to a better diagnosis management of TSHZ3 patients.

Materials and methods

Mouse strain and genotype

The Tshz3 +/LacZ mouse line has been described previously [START_REF] Caubit | Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4[END_REF].

Samples collection and RNAseq

For RNA sequencing, Tshz3 +/LacZ and wild-type mice kidneys were collected at 60 daysof-age. The mean values for body weight of the Tshz3 +/LacZ and wild-type were 34.7± 3.9 g (n=6) and 34.6 ± 6.5 g (n=6), respectively. Dissected kidneys were stored in RNAlater solution (Qiagen) and kept frozen at -80 °C until RNA extraction.

Total individual kidney RNA was extracted using anRNeasy Maxi kit75162

Lot.260018727/ Lot.160012031 from Quiagen according to manufacturer's instructions.

The integrity of RNA was assessed using a chip-based capillary electrophoresis machine and RNA concentration determined using a full spectrum (220-750nm) spectrophotometer. The quality control of the RNA was additionally checked with RNA 6000 Pico de Agilent Technologies, according to the manufacturer's instructions. To obtain two independent total RNA preparations from the two different conditions (wild type: WT1 & WT2; Tshz3 +/LacZ : HET1 & HET2) we pooled RNA from 3 kidneys per group in the same proportion. The starting material (1µg Total mRNA, dissolved in RNase-, DNase-and protease-free molecular grade water) was sent to GATC (Eurofins) for sequencing (Genome Sequencer Illumina HiSeq).

Sequences (fastq format) were mapped to the mm10 version of the mouse genome to generate Sequence Alignment/Map (SAM/BAM) format. After normalization, analysis of differentially expressed genes (DEGs) was performed using both the Bioconductor (http://www.bioconductor.org) package DESeq/DESeq2 and the package edgeR. This analysis generated differential expression lists with False Discovery Rates (FDRs), which are derived from p-values corrected for multiple testing using the Benjamini-Hochberg method. 6 files in total were generated: FDR 1%, 10% for both UCSC (transcripts) and ENSEMBL (genes).

EnrichR tool [START_REF] Chen | Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool[END_REF] was used to performed enrichment analysis with "pathways", gene ontology biological processes (GOBP) and transcription factor (ChIP Enrichment Analysis, ChEA). Gene set enrichment analysis (GSEA) was performed using the software provided by the Broad Institute [START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genomewide expression profiles[END_REF][START_REF] Mootha | PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[END_REF] with default parameters and a preranked gene list calculated based on the10 negative log10 of the P-value from DESeq2 analysis multiplied by the sign of differential expression.

mRNA extraction, cDNA synthesis and quantitative real-time PCR (RT-qPCR)

Total RNA from adult kidneys of WT and Tshz3 +/lacZ mice was prepared using a RNeasy Maxi Kit (ref 75162 Qiagen™), and first-strand cDNA was synthesized using a Maxima 5 Gapdh

GTCTCCTGCGACTTCAACAGCA ACCACCCTGTTGCTGTAGCCGT

Expression data were normalized to controls and the variability in expression levels was analyzed using the 2 -ΔΔCT method described Livak and Schmittgen [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Variables which showed a p-value less than 0.05 in the resulting model were considered to have a significant effect. These statistical analyses were performed by unpaired t-tests with the qbase+ software version 2 (Biogazelle).

Immunological and in situ hybridization analysis

Adult mice were transcardially perfused with phosphate-buffered saline (PBS, 10 mM, pH 7.4), followed by 4% paraformaldehyde (PFA) in PBS under ketamine (150 mg/kg) and xylazine (20 mg/kg) anesthesia. Kidneys were post-fixed in 4% paraformaldehyde (PFA; EMS Lot.160401) for 2 h at 4 °C, cryoprotected with 30% sucrose solution in PBS and frozen (Iso-Pentane RPE524391 Carlo Erba at dry ice temperature). Immunostaining was performed on 12-µm cryosections from tissues embedded in OCT compound.

Cryosections were washed with 0.2% Tween/PBS for 15 min and then blocked in 10% goat serum 0.1%/0.1% Tween/PBS for 1 h. Sections were incubated with primary antibodies overnight at 4 °C. Secondary antibodies were incubated 2 h at room temperature, and after several washes, sections were counterstained with DAPI for 10 minutes.

The following primary antibodies were used: anti-β-galactosidase (chicken, 1/1000, ab9361, Abcam; rabbit, 1/1000, Cappel), mouse anti-NG2 (1/100, MAB5384 Millipore), rabbit anti-CD31/Pecam (1/100 MEC13.3, BD Pharmingen) and rabbit anti-DACH11

(1/100, Proteintech 10914-1-AP). Secondary antibodies: Alexa-Fluor-546 goat antimouse; Alexa-Fluor-546 donkey anti-rabbit, Alexa-Fluor 488 donkey anti-mouse, Alexa-Fluor-488 donkey anti-chicken. Slides were mounted with Fluoromount-G, Southern Biotech Lot: B0216-N156. Images were acquired using a laser scanning confocal microscope (LSM780; Carl Zeiss) and processed using Adobe Photoshop.

For X-gal staining, kidneys/ureters were dissected from non-perfused animals, kidneys were cut in two according to the sagittal plane, and tissues were fixed for 1 h in 1% PFA.

Detection of β-galactosidase activity was done on tissues or on 14-m cryostat sections incubated in the dark in staining solution at 37 °C. X-gal staining was performed as described [START_REF] Relaix | Divergent functions of murine Pax3 and Pax7 in limb muscle development[END_REF]. Samples were paraffinized after washing in increasing alcohol concentrations (70%, 80%, 90%, 97% and 100%) VWR chemicals for one day and finally with Xylene 33817 (Sigma-Aldrich) for 2 h, and immersed in paraffin Paraplast X-TRA from Sigma at 65 °C. Staining was performed with Hematoxylin (HHS32-1L Lot.

064K4354 Sigma-Aldrich), Eosin (Ht110230-1L Sigma-Aldrich), Trichrome Masson (Lot.17301-V04 Ral Diagnostics).

To characterize the expression of TSHZ3 in the tubules, immunostaining was performed on kidneys of Tshz3 +/lacZ mice, using renal tracts of normal external appearance (e.g. no hydronephrosis). Paraformaldehyde-fixed kidneys were embedded in paraffin and 4 µm sections were cut, dewaxed and rehydrated. These sections were incubated overnight at 4°C with antibodies raised in rabbits to define nephron segments and collecting ducts, as described (30-32, 45, 46): aquaporin-1 (1:500, Abcam, ab15080), a marker of proximal tubules and the thin descending limb of the loop of Henle; uromodulin (1:200, Santa Cruz, sc-20631), a marker of the thick ascending limb of the loop of Henle; and calbindin-28, a marker of distal tubules of the nephron and also of collecting ducts (1:500, Abcam, ab25085). For immunostaining of TSHZ3, we used an antibody (1:1000) raised in guinea pig, as described (5). Our preliminary experiments showed that this antibody did not work well on paraffin section but was effective on optimal cutting temperature compound (OCT)-embedded frozen sections; thus the latter technique was used for TSHZ3. OCTembedded frozen tissue sections were fixed in pre-cooled acetone for 15 min, then washed twice in PBS. Primary antibodies were detected using appropriate Biotin-conjugated secondary antibodies: 1:500, goat anti-guinea pig IgG (Abcam ab6970) and 1;200, goat polyclonal anti-rabbit IgG (Abcam ab6720) and a peroxidase-based system, generating a brown colour followed by haematoxylin counterstain (46). Negative controls omitted primary antibodies and these experiments showed no significant signal (data not shown).

Quantitation of glomeruli

The number of glomeruli was determined using kidney images acquire from multiple mid-sagittal longitudinal sections (12µm; Tshz3 +/lacZ , n=58; WT, n=52) of whole P60 adult kidneys (Tshz3 +/lacZ , n=4; WT, n=4) stained by hematoxylin/eosin. We used ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] to measure the renal cortex area into which the number of glomeruli was counted. The results are reported as the number of glomeruli per area (glomeruli/cm 2 ).

Transmission electronic microscopy and quantification

Kidneys were perfused as described above, rapidly dissected and postfixed in 2% PFA, 2.5% glutaraldehyde in cacodylate buffer (pH 7.2) overnight at 4 °C. The kidneys were put in 1% OsO4 solution in cacodylate buffer for 1 h on ice, then dehydrated on ice and embedded in resin (EPON 912). Sample were polymerized 48 h at 60 °C. Ultrathin sections (80-nm) performed on Leica UCT were poststained with 2% uranyl acetate, followed by Reynolds' lead citrate. Section were examined with a high-resolution transmission electron microscope (Tecnai G2 (FEI), Netherland) at 200 kV and images were acquired with a Veleta camera (Olympus). EM images were opened and analyzed with ImageJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]; the straight-line tool was used to measure GBM thickness and endothelial cell fenestration on randomly selected electron micrographs. The same approach was used for morphometric analysis of foot process width, as described previously [START_REF] Van Den Berg | Podocyte foot process effacement is not correlated with the level of proteinuria in human glomerulopathies[END_REF][START_REF] Deegens | Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis[END_REF].

Blood samples

Blood samples were collected from Tshz3

Urine samples

Urine samples were collected from Tshz3 +/LacZ (n= 7; 4 females and 3 males) and WT mice (n=7; 6 females and 1 males) at 60 days-of-age.

Animals were placed in a clean, dry, empty, and transparent individual cage. A nonabsorbable plastic, fully sanitizable material was laid on the floor of the cage. During urine collection, water bottles were provided but no food was given to limit contamination with faeces or animal feed. The mouse was monitored all time and removed as soon as it urinated. The voided urine was aspirated with a Gilson Pipetman and transfert to a 1.5 mL sterile microcentrifuge tube (kept on ice). Collected urine was stored at -80C prior to analysis. Due to the small amount of urine collected, the procedure was repeated during three consecutive days to obtain 300ul of urine for each mouse.

Urinary Proteomics

Sample Preparation: Urine samples were collected and frozen at -80°C as described above. Immediately before preparation, mice urine samples were thawed on ice. 150 µl of urine was mixed with a similar volume of a solution containing 2 M urea, 10 mM NH4OH, and 0.02% sodium dodecyl sulfate (SDS). To remove high molecular weight molecules, samples were ultrafiltrated (3,400 × g for 45 min at 4°C) using a Centrisart 20kDa cut-off centrifugal filter device (Satorius, Göttingen, Germany) until 200 µl of filtrate was obtained. Afterwards, the filtrate was desalted by a NAP5 gel filtration column (GE Healthcare BioSciences, Uppsala, Sweden) to eliminate electrolytes and urea, hence decreasing matrix effects. The samples were lyophilized in a Savant SpeedVac SVC100H connected to a Virtis 3L Sentry freeze dryer (Fischer Scientific, Illkirch, France), consequently stored at 4°C. Shortly before CE-MS analysis, the samples were resuspended in 10 µl high-performance liquid chromatography grade water (HPLC-H2O).

CE-MS analysis and data processing: CE-MS experiments were conducted as previously reported [START_REF] Von Zur Muhlen | Urine proteome analysis reflects atherosclerotic disease in an ApoE-/-mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis[END_REF]. Briefly, a Beckman Coulter Proteome Lab PA800 capillary electrophoresis system (Fullerton, CA) online coupled to a micrOTOF II MS (Bruker Daltonic, Bremen, Germany) was used. The electro-ionization (ESI) sprayer (Agilent Technologies, Palo Alto, CA) was grounded, and the ion spray interface potential was established to -4.5 kV.

Subsequently, data acquisition and MS acquisition methods were automatically measured through the CE by contact-close-relays. Spectra were accrued every 3 seconds, over a range of m/z 350 to 3000.

Mass spectral ion peaks signifying identical molecules at different charges were deconvoluted into singles masses using MosaiquesVisu [START_REF] Neuhoff | Mass spectrometry for the detection of differentially expressed proteins: a comparison of surface-enhanced laser desorption/ionization and capillary electrophoresis/mass spectrometry[END_REF]. The subsequent peak lists categorized each peptide according to its molecular mass (kDa), CE-migration time (min) and signal intensity (amplitude). Due to the analytical variances of urine samples, migration time and ion signal intensity (amplitude) were normalized using endogenous "housekeeping" peptides, normally displaying a small difference between at least 90% of all urine samples, as reported elsewhere [START_REF] Von Zur Muhlen | Urine proteome analysis reflects atherosclerotic disease in an ApoE-/-mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis[END_REF]. Lastly, all detected peptides were deposited, matched and annotated in a Microsoft SQL database [START_REF] Von Zur Muhlen | Urine proteome analysis reflects atherosclerotic disease in an ApoE-/-mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis[END_REF]. Thus, further comparisons and statistical analysis among both groups were performed.

Sequencing of peptides: Tandem mass spectrometry (MS/MS) analysis were conducted to retrieve the sequence information of the peptides, as previously described [START_REF] Von Zur Muhlen | Urine proteome analysis reflects atherosclerotic disease in an ApoE-/-mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis[END_REF]. Briefly, MS/MS experiments were performed on a Dionex Ultimate 3000 RSLC nanoflow system (Dionex, Camberly, UK) coupled to an Orbitrap Velos MS instrument (Thermo Fisher Scientific). Thereafter, all resulting data files were evaluated by the use of SEQUEST (using Thermo Proteome Discoverer 1.2) without any enzyme specificity and searched beside the Swiss-Prot Mus Musculus database, as previously described [START_REF] Nkuipou-Kenfack | The use of urinary proteomics in the assessment of suitability of mouse models for ageing[END_REF].

Statistical analysis:

For the identification of potential significant different urinary peptides, urine samples from both groups (wild type and Tshz3 +/lacZ ) were compared. Pvalues were calculated according to the Wilcoxon Rank-Sum test. For multiple testing correction, the reported p-values were further adjusted via false discovery rate method described by Benjamini and Hochberg [START_REF] Benjamini | Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing[END_REF]. Only peptides with p-values less than 0.05 and detected in a frequency threshold of ≥70% in at least one of both groups were further considered as statistically significant. Statistical analysis was performed using Prism 7.05 (GraphPad Software, USA) and results considered significant at P < 0.05. Comparison with immunostaining patterns for aquaporin-1, uromodulin and calbindin-28, suggest that thin descending limbs of loops of Henle express TSHZ3 because patterns for this protein and aquaporin-1 are similar in the thin tubules within the inner medulla.

Legends to

In this location, calbinin-28 positive collecting ducts are wider diameter and uromodulin is not detected. The TSHZ3 positive tubules in the cortex and outer medulla may also include the pars recta, or straight section, of the aquaporin-1 positive proximal tubule.

Bars are 50 µm.

Gm5182 Predicted gene 5182 831,08 0,77

First

  Strand cDNA Synthesis Kit with dsDNase (ThermoFisher Scientific™ ref K1671 50). All samples from each experiment were reverse-transcribed at the same time, and real-time PCR was performed on a StepOne+ qPCR detection system (Applied Biosystems™) using Luminaris Color HiGreen High ROX qPCR Master Mix (Thermo Fisher Scientific™ ref K0362). RT-qPCR conditions were as follows: 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Reactions were run in triplicate in three independent experiments. The geometric mean of the housekeeping gene GAPDH was used as an internal control to normalize variability in expression levels, and samples were also normalized to their respective control group. Specificity of reactions was verified by melt curve analysis. Primer sequences used for Sybr qPCR are as follows:

Figures Figure 1 .

 1 Figure 1. Distribution of Xgal-positive cells in Tshz3 +/lacZ adult renal tract. (A, B) The two halves of the same adult kidney stained with X-Gal. (C) Cartoon showing the section sites and planes for D, E and F. (D-F) Xgal-Positive cells were found in papilla (p) and smooth muscle present in the pelvic region (arrow) (D), in the mesenchymal part of the ureter (E), in the glomeruli (g) and in close proximity to blood vessel (v) (F). (G-I) In glomeruli, TSHZ3 (ß-Gal) is detected in CD31+ glomerular endothelial cells (G) but not

Figure 2 .

 2 Figure 2. Immunohistochemistry for TSHZ3 and kidney segment markers. Immunohistochemistry (brown positive signal) of one week postnatal Tshz3 +/lacZ mouse kidneys for: A-C. Teashirt-3 (TSHZ3); D-F. Aquaporin-1 (marker of proximal tubules and thin descending limb of loop of Henle; G-I. Uromodulin (marker of the thick

Figure 3 .

 3 Figure 3. Characterization of β-galactosidase-positive cells in the pelvic region of Tshz3 +/lacZ adult kidney. (A-D) Comparative expression of ß-galactosidase (green) and Dachshund 1 (red) in the pelvic region. (F) Close up of the region boxed in D. Merge image (D, F) shows that a subset of βgal+ cells expresses DACH1 (arrowheads in C and F). These cells represent a cell layer in a sub urothelial position. (E) Double immunostaining for βgalactosidase (red) and smooth muscle actin (green) indicates that βgal expression is found in SMA expressing cells. Some βgal+ cells adjacent to the urothelium do not express SMA (arrowheads in E). P: papilla, u: urothelium. Scale bars, in A, 100µm; in E, 50µm.

Figure 4 :

 4 Figure 4: Reduced glomerular density, glomerular basement membrane thinning and larger foot process width in heterozygous Tshz3 lacZ/+ mice. (A) Representative images of haematoxylin and eosin-stained sections of WT and Tshz3 +/lacZ adult kidneys at post-natal day 60. Scale bar 1mm. (B) The graph shows the significant (P<0.0001) reduction of the glomerular density in heterozygous Tshz3 lacZ/+ (471 ± 0.29 /cm 2 , 48 sections from 3 kidneys) compare to wild-type kidney (695 ± 0.38 /cm 2 , 41 sections from 3 kidneys). (C, E, G) Representative TEM images of wild type and Tshz3 +/lacZ adult kidneys. (D, F, H) TEM morphometric analysis. (C) TEM: No abnormalities in endothelial cell fenestration are observe in Tshz3 +/lacZ mice. Scale bar, 500nm. (D)

Figure 5 .

 5 Figure 5. Differences in serum biochemical parameters between control and Tshz3 lacZ/+ mice. Plasma concentration of potassium (A), phosphates (B), urea (C), magnesium (D) and sodium (E). Data are represented as means ± SEM. Statistically significant difference from control at *p<0.02; **p<0.004. WT, wild type; +/-, Tshz3 +/lacZ

Figure 6 .

 6 Figure 6. Urinary peptides displaying differential abundance in Tshz3 +/LacZ mice. The volcano plot of quantified proteins revealed that 115 peptides (red and blue dots) show a significant change (p-value <0.05) in Tshz3 +/lacZ compared to control mice. However, for33 of these peptides we were able to find a sequence (labeled ones; see Table1i). The grey dots are peptides that have not a significant change (p-value >0.05).

  +/LacZ (n= 11; 5 females and 6 males) and WT mice (n=11; 6 females and 5 males) at 60 days-of-age. The mean values for body weight of Tshz3 +/LacZ and wild-type were 38.27± 7.8 (n=11) and 33.01 ± 4.9 (n=11). Mice were anaesthetized by an intraperitoneal injection of ketamine/xylazine (0.1 ml/10 g body weight) prior to manipulation. Anesthesia was maintained by using 1.7% to 2.5% isoflurane delivered in 600 ml/min oxygen and a closely fitting facemask. Blood was collected by cardiocentesis puncture in heparinize tubes with EDTA and also in tubes without anticoagulant, centrifuging at 4 ºC immediately after extraction. The total blood volume in 30-40 g mice is approximately 2 to 3 ml. The maximum volume that could be collected safely at a single survival time point was approximately 800-1000 μl. Blood

tests were outsourced to Laboklin G.m.b.H. and performed using a Siemens' high-volume hematology analyzer (ADVIA 2120i) and a Roche' chemistry analyzer (Cobas 8000 c701).

Table 1b . Significantly differentially expressed genes (DEGs) between WT and HET. FDR cutoff chosen was FDR <=0.01

 1b

	Ensembl ID ENSMUSG0000 0054676 ENSMUSG0000 0086474 ENSMUSG0000 0004668 ENSMUSG0000 0031659 ENSMUSG0000 0004730 ENSMUSG0000 0060470 ENSMUSG0000 0085180 ENSMUSG0000 0033715 ENSMUSG0000 0028356 2,1E-ENSMUSG0000 Gene symbol Gene Name baseM ean log2 Fold Change lfc SE sta t pval ue padj 1600014C 10Rik RIKEN cDNA 1600014C10 gene(1600014C10Rik) 7360,9 2 -0,63 0, 14 -4,4 1 1,0E -05 6,1E-03 9130204K 15Rik RIKEN cDNA 9130204K15 gene 181,94 -0,78 0, 21 -3,6 8 2,3E -04 6,9E-02 Abca13 ATP-binding cassette, sub-family A (ABC1), member 13 5366,7 5 0,63 0, 18 3,5 2 4,4E -04 9,6E-02 Adcy7 Adenylate cyclase 7 474,95 0,73 0, 20 3,6 2 2,9E -04 7,7E-02 Adgre1 Adhesion G protein-coupled receptor E1 387,86 0,69 0, 20 3,4 9 4,8E -04 9,6E-02 Adgrg3 Adhesion G protein-coupled receptor G3 347,56 0,65 0, 19 3,5 3 4,2E -04 9,6E-02 Ai838599 Expressed sequence AI838599 473,16 0,71 0, 17 4,0 6 4,8E -05 2,2E-02 Akr1c14 aldo-keto reductase family 1, member C14(Akr1c14) 30143, 69 -0,68 0, 19 -3,5 3 4,2E -04 9,6E-02 Ambp Alpha 1 microglobulin/bikunin precursor 105,82 0,89 0, 22 4,0 8 4,4E -05 0020609 Apob Apolipoprotein B 6556,7 9 -0,58 0, -3,4 7 5,1E -04 9,97E -02 ENSMUSG0000 0040694 Apobec2 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 175,14 1,27 0, 5,9 4 2,8E -09 1,1E-05 ENSMUSG0000 0020604 Arsg Arylsulfatase G 2625,2 3 -0,77 0, -4,5 9 4,5E -06 3,2E-03 ENSMUSG0000 0038704 Aspdh aspartate dehydrogenase domain containing(Aspdh) 5008,2 6 -0,59 0, -3,5 1 4,4E -04 9,6E-02 ENSMUSG0000 0017929 B4galt5 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5(B4galt5) 18312, 69 -0,65 0, -3,6 2 2,9E -04 7,7E-02 ENSMUSG0000 0090486 BC035947 CDNA sequence BC035947 4151,5 7 -0,60 0, -3,4 8 5,0E -04 9,96E -02 ENSMUSG0000 0030268 Bcat1 branched chain aminotransferase 1 6109,7 5 -0,92 0, -6,5 1 7,7E -11 3,6E-07 ENSMUSG0000 0058914 C1qtnf3 C1q and tumor necrosis factor related protein 3(C1qtnf3) 9805,3 8 -0,72 0, -3,5 9 3,3E -04 8,3E-02 ENSMUSG0000 0022416 Cacna1i calcium channel, voltage-dependent, alpha 1I subunit 126,26 0,91 0, 4,2 4 2,2E -05 1,1E-02 ENSMUSG0000 0038128 Camk4 Calcium/calmodulin-dependent protein kinase IV 274,72 0,84 0, 4,0 5 5,2E -05 2,2E-ENSMUSG0000 0024610 Cd74 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated)(Cd74) 12439, 99 0,62 0, 3,4 7 5,2E -04 9,97E -02 ENSMUSG0000 0022504 Ciita class II transactivator 431,87 1,10 0, 5,8 6 4,6E -09 1,4E-05 ENSMUSG0000 0056025 Clca3a1 Chloride channel accessory 3A1 875,04 0,74 0, 4,5 6 5,2E -06 3,6E-03 ENSMUSG0000 0040663 Clcf1 Cardiotrophin-like cytokine factor 1 246,91 0,74 0, 3,6 5 2,6E -04 7,2E-02 ENSMUSG0000 0024330 Col11a2 Collagen, type XI, alpha 2 191,72 1,00 0, 4,8 3 1,4E -06 1,4E-03 ENSMUSG0000 0030905 Crym Crystallin, mu 2030,9 9 -0,87 0, -4,4 2 9,7E -06 6,1E-03 ENSMUSG0000 0038642 Ctss Cathepsin S [Source:MGI Symbol;Acc:MGI:107341] 1091,2 7 0,74 0, 4,7 3 2,3E -06 1,9E-03 ENSMUSG0000 0027014 Cwc22 CWC22 spliceosome-associated protein(Cwc22) 3557,8 8 1,18 0, 7,2 6 3,8E -13 2,4E-09 ENSMUSG0000 0015340 Cybb Cytochrome b-245, beta polypeptide 747,51 0,62 0, 3,6 7 2,4E -04 6,9E-02 ENSMUSG0000 0074254 Cyp2a4 cytochrome P450, family 2, subfamily a, polypeptide 4(Cyp2a4) 9870,2 5 -0,53 0, -3,8 5 1,2E -04 4,2E-02 ENSMUSG0000 0108332 D530033 B14Rik RIKEN cDNA D530033B14 gen 751,19 -0,83 0, -4,6 6 3,1E -06 2,4E-03 ENSMUSG0000 D630029K 7305,0 0, -3,7 1,8E 0025279 Dnase1l3 Deoxyribonuclease 1-like 3 124,40 0,77 0, 3,5 1 4,4E -04 9,6E-02 ENSMUSG0000 0040658 Dnph1 2,-deoxynucleoside 5,-phosphate N-hydrolase 1 926,16 -0,72 0, -3,7 1 2,1E -04 6,4E-02 ENSMUSG0000 0020143 Dock2 Dedicator of cyto-kinesis 2 420,46 0,84 0, 4,2 5 2,1E -05 1,1E-02 ENSMUSG0000 0025153 Fasn fatty acid synthase(Fasn) 17210, 76 0,94 0, 5,8 3 5,6E -09 1,5E-05 ENSMUSG0000 14127, 5,8E-ENSMUSG0000 0003420 Fcgrt Fc receptor, IgG, alpha chain transporter(Fcgrt) 59 -
	0031294	05Rik	RIKEN cDNA D630029K05 gene(D630029K05Rik)	9	-0,65	5	-04

Table 1g . Human orthologs for differentially expressed genes Human Gene symbol

 1g

		HLA-A		ENSMUSG00000067235	H2-Q10
	845 846	,8E-02 2,2E-02 2,8E-03 6,1E-03 4,8E-02 9,6E-03 6,8E-03 6,9E-02 4,1E-02 5,8E-02 9,6E-02 4,1E-02 5,5E-04 9,6E-02 2,4E-03 9,2E-ENSMUSG0000 ENSMUSG0000 0021913 Ogdhl Oxoglutarate dehydrogenase-like 276,39 0,86 0, 4,0 4 5,3E -05 ENSMUSG0000 0038910 Plcl2 Phospholipase C-like 2 782,25 -0,82 0, -4,6 3 3,7E -06 ENSMUSG0000 0052160 Pld4 Phospholipase D family, member 4 403,71 0,79 0, 4,4 1 1,0E -05 ENSMUSG0000 0042251 Pm20d1 Peptidase M20 domain containing 1 9189,1 2 -0,67 0, -3,8 1 1,4E -04 ENSMUSG0000 0030423 Pop4 Processing of precursor 4, ribonuclease P/MRP family 634,04 0,72 0, 4,2 9 1,8E -05 ENSMUSG0000 0, 4,8 1,4E 0021876 Rnase4 ribonuclease, RNase A family 4(Rnase4) 4455,7 6 -0,66 0, -4,3 8 1,2E -05 ENSMUSG0000 0031609 Sap30 sin3 associated polypeptide(Sap30) 3295,6 3 -0,59 0, -3,6 7 2,4E -04 ENSMUSG0000 0038224 Serpinf2 serine (or cysteine) peptidase inhibitor, clade F, member 2(Serpinf2) 21474, 96 -0,78 0, -3,8 6 1,1E -04 ENSMUSG0000 0035699 Slc51a solute carrier family 51, alpha subunit(Slc51a) 6523,7 0 -0,65 0, -3,7 5 1,8E -04 ENSMUSG0000 0025790 Slco3a1 Solute carrier organic anion transporter family, member 3a1 11292, 58 -0,67 0, -3,5 0 4,7E -04 ENSMUSG0000 0040693 Slco4c1 Solute carrier organic anion transporter family, member 4C1 324,30 0,76 0, 3,8 6 1,1E -04 ENSMUSG0000 0044349 Snhg11 Small nucleolar RNA host gene 11 2094,3 2 0,99 0, 5,1 1 3,2E -07 ENSMUSG0000 0096054 Syne1 Spectrin repeat containing, nuclear envelope 1 1889,0 3 0,59 0, 3,5 2 4,3E -04 ENSMUSG0000 0056296 Synpr Synaptoporin 1611,7 8 -0,90 0, -4,6 6 3,1E -06 ENSMUSG0000 5200,8 0, -4,3 1,7E 0044583 Tlr7 Toll-like receptor 7 165,79 0,86 0, 4,0 5 5,1E -05 2,2E-02 ENSMUSG0000 0040883 Tmem205 Transmembrane protein 205 4488,1 0 -0,59 0, -3,6 6 2,6E -04 7,2E-02 ENSMUSG0000 0048572 Tmem252 transmembrane protein 252(Tmem252) 6035,7 0 -0,70 0, -3,4 9 4,8E -04 9,6E-02 ENSMUSG0000 0010307 Tmem86a transmembrane protein 86A(Tmem86a) 5856,7 4 -0,74 0, -3,8 4 1,2E -04 4,3E-02 ENSMUSG0000 0016942 Tmprss6 Transmembrane serine protease 6 518,88 0,74 0, 3,6 3 2,8E -04 7,6E-02 ENSMUSG0000 0043621 Ubxn10 UBX domain protein 10 675,26 -0,80 0, -3,8 8 1,1E -04 4,1E-02 ENSMUSG0000 0031710 Ucp1 uncoupling protein 1 (mitochondrial, proton carrier)(Ucp1) 932,71 -0,94 0, -4,5 3 5,9E -06 3,9E-03 ENSMUSG0000 0027962 Vcam1 Vascular cell adhesion molecule 1 1251,8 8 0,77 0, 4,8 1 1,5E -06 1,5E-03 ENSMUSG0000 0056592 Zfp658 Zinc finger protein 658 228,62 0,98 0, 4,9 2 8,8E -07 1,0E-03 Ensembl mouse ID Mouse Gene symbol C19orf12 ENSMUSG00000054676 1600014C10Rik ABCA13 ENSMUSG00000004668 Abca13 ADCY7 ENSMUSG00000031659 Adcy7 ADGRE1 ENSMUSG00000004730 Adgre1 ADGRG3 ENSMUSG00000060470 Adgrg3 AMBP ENSMUSG00000028356 Ambp APOB ENSMUSG00000020609 Apob APOBEC2 ENSMUSG00000040694 Apobec2 ARSG ENSMUSG00000020604 Arsg ASPDH ENSMUSG00000038704 Aspdh B4GALT5 ENSMUSG00000017929 B4galt5 ERVW-1 ENSMUSG00000090486 BC035947 BCAT1 ENSMUSG00000030268 Bcat1 C1QTNF3 ENSMUSG00000058914 C1qtnf3 CACNA1I ENSMUSG00000022416 Cacna1i CAMK4 ENSMUSG00000038128 Camk4 CD74 ENSMUSG00000024610 Cd74 CIITA ENSMUSG00000022504 Ciita CLCF1 ENSMUSG00000040663 Clcf1 COL11A2 ENSMUSG00000024330 Col11a2 CRYM ENSMUSG00000030905 Crym HDX ENSMUSG00000034551 Hdx ITGB2 ENSMUSG00000000157 Itgb2l KCNT1 ENSMUSG00000058740 Kcnt1 KLK3 ENSMUSG00000063177 Klk1b27 LAPTM5 ENSMUSG00000028581 Laptm5 LY6E ENSMUSG00000022587 Ly6e LYZ ENSMUSG00000069516 Lyz2 MOGAT1 ENSMUSG00000012187 Mogat1 MPEG1 ENSMUSG00000046805 Mpeg1 MPV17L ENSMUSG00000022679 Mpv17l MTFR2 ENSMUSG00000019992 Mtfr2 MYBPC2 ENSMUSG00000038670 Mybpc2 NHP2 ENSMUSG00000001056 Nhp2 NLRC5 ENSMUSG00000074151 Nlrc5 NPY ENSMUSG00000029819 Npy OAS2 ENSMUSG00000032690 Oas2 OGDHL ENSMUSG00000021913 Ogdhl PLCL2 ENSMUSG00000038910 Plcl2 PLD4 ENSMUSG00000052160 Pld4 PM20D1 ENSMUSG00000042251 Pm20d1 POP4 ENSMUSG00000030423 Pop4 RASL10B ENSMUSG00000020684 Rasl10b RNASE4 ENSMUSG00000021876 Rnase4 1,4E-ENSMUSG0000 0022218 Tgm1 transglutaminase 1, K polypeptide(Tgm1) 2 -0,67 1 -05 CTSS ENSMUSG00000038642 Ctss SAP30 ENSMUSG00000031609 Sap30
		0020684 CWC22 SERPINF2	Rasl10b	RAS-like, family 10, member B ENSMUSG00000027014 Cwc22 ENSMUSG00000038224 Serpinf2	149,28	1,06	3	-06
		CYBB SLC51A		ENSMUSG00000015340 ENSMUSG00000035699	Cybb Slc51a
		CYP2A13 SLCO3A1		ENSMUSG00000074254 ENSMUSG00000025790	Cyp2a4 Slco3a1
		DNASE1L3 SLCO4C1		ENSMUSG00000025279 ENSMUSG00000040693	Dnase1l3 Slco4c1
		DNPH1 SNHG11		ENSMUSG00000040658 ENSMUSG00000044349	Dnph1 Snhg11
		DOCK2 SYNE1		ENSMUSG00000020143 ENSMUSG00000096054	Dock2 Syne1
		FASN SYNPR		ENSMUSG00000025153 ENSMUSG00000056296	Fasn Synpr
		FCGRT TGM1		ENSMUSG00000003420 ENSMUSG00000022218	Fcgrt Tgm1
		G6PC1 TLR7		ENSMUSG00000078650 ENSMUSG00000044583	G6pc Tlr7
		GABRB3 TMEM205		ENSMUSG00000033676 ENSMUSG00000040883	Gabrb3 Tmem205
		GBP3 TMEM252		ENSMUSG00000040264 ENSMUSG00000048572	Gbp2b Tmem252
		GBP6 TMEM86A		ENSMUSG00000079363 ENSMUSG00000010307	Gbp4 Tmem86a
		GJB2 TMPRSS6		ENSMUSG00000046352 ENSMUSG00000016942	Gjb2 Tmprss6
		GUSB UBXN10		ENSMUSG00000025534 ENSMUSG00000043621	Gusb Ubxn10
		HLA-DQA1 UCP1		ENSMUSG00000036594 ENSMUSG00000031710	H2-Aa Ucp1
		HLA-DQB1 VCAM1		ENSMUSG00000073421 ENSMUSG00000027962	H2-Ab1 Vcam1
	858	HLA-DMA		ENSMUSG00000037649	H2-DMa

Table 1h . Human orthologues of DEGs that are associated with renal disease and/or autism spectrum disorder

 1h

		GUSB RNASE4
		BCAT1 LAPTM5 UCP1	Crescentic Glomerulonephritis Renal Hypertension, Kidney Disease, Systemic Lupus Erythematosus Renal Cell Carcinoma (Nonpapillary),	MalaCards MalaCards, DisGenet, MalaCards
	860 861	Human Gene symbol C1QTNF3 CACNA1I CYBB CYP2A13 LY6E LYZ SAP30 HLA-DQA1 MOGAT1 SERPINF2 MPEG1 SLC51A VCAM1 C19orf12 ABCA13 ADCY7 ADGRE1 CAMK4 DNASE1L3 DNPH1 DOCK2 FASN FCGRT HLA-DQB1 SLCO3A1 HLA-DMA HLA-A MPV17L MTFR2 SLCO4C1 Abbreviations: ASD, Autism Spectrum Disorder; CKD, Chronic Kidney Disease; ESRD, End Stage Renal Disease; PKD, Polycystic Kidney disease reference AS D referenc e Genetic syndromes YE S SFARI, ESRD, Renal fibrosis AGR Renal Cell Carcinoma (Nonpapillary), Lupus Erythematosus, Nephrotic Clear Cell Renal Celle Carcinoma MalaCards Syndrome, Tubulointerstitial Nephritis AutDB with Uveitis, MalaCards, AGR Systemic Lupus Erythematosus, Kidney that associate Systemic Lupus Erythematosus, Systemic Lupus Erythematosus, CKD, Tafro Syndrome MalaCards Disease, Glomerulonephrititis, MalaCards, AGR kidney conditions and ASD YE S MalaCar ds, SFARI, AutDB Systemic Lupus Erythematosus, Kidney disease, Glomerulonephrititis, Focal Segmental Glomerulosclerosis MalaCards YE S SFARI, AutDB Renal Cell Carcinompa (nonpapillary), Kidney disease, Systemic Lupus Glomerulonephritis MalaCards Systemic Lupus Erythematosus, MalaCards Kidney Cancer, ESRD, Renal Fibrosis MalaCards, DisGenet Systemic Lupus Erythematosus MalaCards Behcet Syndrome, Chronic Chrmosome 16p13.3 Deletion YE AutDB, Rubinstein-Taybi Systemic Lupus Erythematosus, Nephrotic Syndrome, PKD, Tubulointerstitial Nephritis with Uveitis MalaCards Systemic Lupus Erythematosus, Membranous Nephropathy MalaCards Pyelonephritis, Wilms Tumor 5, Wilms Tumor 1, Renal Cell Carcinoma (nonpapillary), Membranous Nephropathy, Down Syndrome, Turner Syndrome, Nephrotic Syndrome, Kidney Cancer, ESRD, Uremia, Iga Glomerulonephritis, MalaCards YE S Syndrome Proximal MalaCards S AGR Syndrome CKD, Kidney Disease, PKD, Renal Oncocytoma Kidney Disease. MalaCards, AGR MalaCar MYBPC2 SNHG11 ds, NHP2 YE SFARI, SFARI, SYNE1 S AutDB DisGene t, AutDB Down Syndrome HDX CKD, Kidney Disease, ESRD, MalaCards SYNPR Systemic Lupus Erythematosus, Renal Cell Carcinoma (nonpapillary), Renal TGM1 ADGRG3 AMBP Hydronephosis, VUR 1, Hypertensive Nephropathy MalaCards, DisGenet, AGR APOB CKD, Nephrotic syndrome, Uremia, MalaCards, AGR YE S SFARI APOBEC2 ARSG ASPDH B4GALT5 ERVW-1 CD74 Disease MalaCards CIITA Systemic Lupus Erythematosus, Tuberous Sclerosis 1 MalaCards, AGR YE S MalaCar ds Tuberous Sclerosis 1 Malacards reference MalaCards, ClinVar MalaCards CLCF1 COL11A2 CRYM CTSS Renal Cell Carcinoma MalaCards CWC22 GABRB3 RETT Syndrome MalaCards YE S MalaCar t, AGR, AutDB RETT Syndrome MalaCards GBP3 GBP6 GJB2 Horseshoe Kidney, Branchiootorenal Syndrome MalaCards KLK3 Urinary Tract Obstruction, Nephrogenic Adenoma, Hydronephrosis, Lower Urinary Tract Calculus, Chromophobe Renal Cell Carcinoma MalaCards OGDHL PKD PLD4 Disease, CKD, RASL10B UBXN10 POP4 TMPRSS6 Kidney Disease, CKD, ESRD MalaCards PM20D1 TMEM86A MalaCards TMEM252 Nephrotic Syndrome (Type 7), Kidney TMEM205 PLCL2 TLR7 Glomerulonephritis MalaCards MalaCards Syndrome, Immune-Complex DisGene KCNT1 Fragile X Syndrome MalaCards S AutDB Fragile X Syndrome MalaCards OAS2 Glomerulonephrititis, Turner SFARI, YE NPY Turner Syndrome, Kidney Disease MalaCards S ds Behcet Syndrome, ds, ITGB2 Autoimmune Glomerulonephritis, MalaCards YE MalaCar Systemic Lupus Erythematosus, Pyelonephritis, Urolithiasis, Erythematosus, Polycystic Kidney G6PC1 NLRC5 fibrosis MalaCards

Data bases: MALACARDS (https://www.malacards.org); SFARI (https://gene.sfari.org); ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/); DisGenet (https://www.disgenet.org); AutDB (http://autism.mindspec.org/autdb/Welcome.do); AGR (Alliance of Genome Resources) (https://www.alliancegenome.org).

Table 1i . Detailed of proteins origin of the 33 differentially excreted urinary peptides obtained by the comparison between Tshz3 +/lacZ heterozygous and WT control mice

 1i

	863	Pept ideI D 2234 0 1394 2 1568 1 1662 0 4260 7480 1955 6 1232 5 1971 1 8738 1928 5 972 9385 1960 6 1193 6 1101 8 1339 9 2167 3 1934 5 1999 5 931 9525 7284 7252 8447 1502 932 7194 1522 1 8166 7629 1544 7 2133 3 1498 7	p_valu e_BH 0.0129 26897 0.0160 90788 0.0256 78503 0.0290 89955 0.0416 91443 0.0334 17318 0.0278 12250 0.0457 81039 0.0417 23205 0.0406 02421 0.0110 84765 0.0278 12250 0.0182 50194 0.0026 81798 0.0129 26897 0.0365 46003 0.0129 26897 0.0201 69148 0.0423 16711 0.0288 18934 0.0335 45397 0.0297 58618 0.0129 26897 0.0417 23205 0.0129 26897 0.0278 12250 0.0365 46003 0.0268 16915 0.0129 26897 0.0278 12250 0.0457 81039 0.0417 23205 0.0301 31027	Mas se 179 3,90 2 137 9,65 3 145 2,78 4 149 4,77 7 100 2,54 113 0,59 7 164 6,89 8 131 5,63 8 165 5,83 7 118 0,58 3 163 3,82 859, 435 6 120 8,60 3 164 9,81 5 130 2,66 127 1,61 6 135 7,64 9 175 9,90 3 163 6,82 8 167 1,65 857, 445 6 121 4,59 1 112 3,55 3 112 2,56 7 116 8,60 9 152 0,76 3 111 9,53 7 143 3,72 5 115 7,54 3 113 6,51 144 1,68 9 174 1,98 4 142 1,71 9	CE_ t 34,1 747 6 48,0 048 2 32,3 792 9 32,1 192 6 28,2 032 2 KGEpGDTGVK Sequence EEASSTGRNF NVEKIN EPGVTGLQGQ AGPpG ILGGQEAAAHA RPY SPGRHEAQEP FLQ 29,3 769 4 ERIASEASRL 28,0 235 7 GEAGKPGRpG ERGPpGP 38,8 357 5 DGTpGGpGIR GMpG 33,5 862 GQPGAKGEpG DTGVKGDA 37,8 385 4 ApGEKGEGGP pGP 41,1 177 4 EVGPpGPPGp AGEKGSpG 34,1 979 8 GTAGEpGKAG 38,3 618 3 ApGDRGEAGP pGP 41,2 899 7 EVGPpGPpGp AGEKGSpG 31,7 936 9 GAKGEpGDTG VKGD 47,9 784 2 GPpGPpGPpG PPSGG 31,5 735 8 NGDDGEAGKp GRpG 34,0 971 GRpGEVGPpG PpGPAGEKG 40,7 328 5 GSpGSpGPDG KTGPpGPAG 48,0 321 4 TGDSGPAGpP GPpGppGPP 34,8 108 6 INKEIQN 47,9 075 1 GPpGPpGpPG PSGP 36,2 466 4 GGKGEQGPA GPpG 36,7 175 8 GPpGPpGAAG KEG 37,4 637 KpGEQGVPGD LG 40,0 275 7 VGPpGPpGpA GEKGSpG 47,6 387 6 GEVGPpGPpG PAG 38,8 085 3 GEVGPpGPpG PAGEKG 47,7 683 2 GPpGPpGPpG PpA 47,7 381 9 GQpGPpGPpG TAG 48,1 270 4 GPPGPEGPpG PpGLQG 34,2 490 8 FIDQTRVLNLG PITR 39,3 592 1 RGETGpAGpQ GPpGP p = hydroxylated proline	Prot_Name Major urinary protein 17 (MUP 17) Collagen alpha-1(VII) chain (Long-chain collagen) (LC collagen) Complement factor D (EC 3.4.21.46) (28 kDa adipocyte protein) (Adipsin) (C3 convertase activator) (Properdin factor D) Tripeptidyl-peptidase 1 (TPP-1) (EC 3.4.14.9) (Lysosomal pepstatin-insensitive protease) (LPIC) (Tripeptidyl aminopeptidase) (Tripeptidyl-peptidase I) (TPP-I) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Histone H2B type 1-F/J/L (H2B 291A) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(III) chain Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(III) chain Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Clusterin (Apolipoprotein J) (Apo-J) (Clustrin) (Sulfated glycoprotein 2) (SGP-2) [Cleaved into: Clusterin beta chain; Clusterin alpha chain] Collagen alpha-1(XIII) chain Collagen alpha-2(I) chain (Alpha-2 type I collagen) Collagen alpha-2(I) chain (Alpha-2 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(I) chain (Alpha-1 type I collagen) Collagen alpha-1(XV) chain [Cleaved into: Restin (Endostatin-XV)] Collagen alpha-1(III) chain Collagen alpha-1(XIII) chain Uromodulin (Tamm-Horsfall urinary glycoprotein) (THP) [Cleaved into: Uromodulin; secreted form] Collagen alpha-1(XIV) chain	Prot_ Symb ol MUP1 7 CO7A 1 CFAD P03953 26 39 Prot_Ac cession Sta rtA A Sto pA A B5X0G 2 19 34 Q63870 125 9 127 3 TPP1 O89023 293 305 CO1A 1 P11087 815 824 H2B1 F P10853 72 81 1 P11087 222 238 CO1A CO3A 1 P08121 526 539 CO1A 1 P11087 810 827 CO3A 1 P08121 828 840 CO1A 1 P11087 910 927 CO1A 1 P11087 576 585 CO1A 1 P11087 787 799 CO1A 1 P11087 910 927 CO1A 1 P11087 813 826 CO1A 1 P11087 117 0 118 4 CO1A 1 P11087 218 231 CO1A 1 P11087 906 924 CO1A 1 P11087 531 549 CO1A 1 P11087 116 0 117 8 CLUS Q06890 41 47 CODA 1 Q9R1N 9 277 290 CO1A 2 Q01149 547 559 CO1A 2 Q01149 811 823 CO1A 1 P11087 646 657 CO1A 1 P11087 911 927 CO1A 1 P11087 909 921 CO1A 1 P11087 909 924 COFA 1 O35206 107 5 108 7 CO3A 1 P08121 329 341 CODA 1 Q9R1N 9 602 617 URO M Q91X17 593 607 COEA 1 Q80X19 149 2 150 6	Pept ideI D 223 40 139 42 156 81 166 20 426 0 748 0 56 195 25 11 8 85 5 06 36 18 99 73 45 95 5 4 2 7 2 4 21 6 9 47 33 87	Mean LacZ 196,2 79666 7 79,68 90666 7 484,8 70666 7 134,8 59866 7 118,3 42666 7 387,1 88 36 77,38 410,4 316 97,73 Mean WT 2,166 25 4,642 0833 3 30,84 65 16,18 3833 3 16,58 3833 3 55,47 6 3 16,18 9583 1149, 64313 3 3789, 51666 7 2618, 5458 3 893,8 4596, 02866 7776 7 7 322,7 4108 3 11265 ,728 1668 6,741 8 1646, 9486, 13146 7959 7 2 193,0 38266 7 56,17 025 175,4 74066 7 1161, 6598 2201, 4412 5 583,4 3496, 16666 7980 7 8 64,55 4666 7 664,5 52733 3 1316, 2705 6896, 881,2 0216 18 7 318,8 58933 3 119,3 34 917,3 95866 7 1902, 3558 3 85,35 893,7 27333 9541 3 7 477,9 65533 3 200,0 8875 2531, 80493 3 5723, 6408 3 1341, 113,7 7740 734 8 1519, 35813 3 679,1 6475 4585, 02026 7 784,5 99666 7 2096, 3034 2 392,0 4803, 53333 2158 3 3 2344, 5474 2 646,1 97533 3 658,7 40133 3 1763, 6471 7 58,21 54666 989,7 7 535 429,8 4916 7 2405, 85386 7 6710, 7721 7	Mean LacZ/Mea nWT 90,608 17,167 15,719 8,333 7,136 6,979 4,780 4,200 1,447 0,194 3,562 0,675 0,174 3,437 0,528 0,167 2,718 0,505 0,128 2,672 0,482 0,095 2,389 0,442 0,085 2,237 0,374 0,082 1,956 0,374 0,059 1,503 0,359	Fold-chang e 90,60 8 17,16 7 15,71 9 8,333 7,136 6,979 4,780 4,200 1,447 0,194 3,562 0,675 0,174 3,437 0,528 0,167 2,718 0,505 0,128 2,672 0,482 0,095 2,389 0,442 0,085 2,237 0,374 0,082 1,956 0,374 0,059 1,503 0,359

Acknowledgements

We appreciate feedback on the manuscript provided by Pierre L. Roubertoux. Microscopy was performed at the imaging platform of the IBDM, supported by the ANR through the "Investments for the Future" program (France-BioImaging, ANR-10-INSB-04-01). This work was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642937 (Scientific coordinator L.F), the French National Research Agency (ANR-17-CE16-0030-01 "TSHZ3inASD" project to L.F.), the Centre National de la Recherche Scientifique (CNRS), the Institut National pour la Recherche Médicale (INSERM) and Aix-Marseille University. We acknowledge grant support from the Medical Research Council project grant (MR/T016809/1) and Kids Kidney Research/Kidney Research UK project grant (2010). I.S-M. and P.M. acknowledge financial support from the European Union's Horizon 2020 research and innovation PhD programme under the Marie Sklodowska-Curie grant agreement No 642937.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Raw data (FastQ files) from the sequencing experiment (triplicates from wild-type and Tshz3 +/lacZ adult kidney) and raw abundance measurements for genes (read counts) for each sample are available from Gene Expression Omnibus (GEO) under accession GSE182010, which should be quoted in any manuscript discussing the data.

Ethics Statement

The Experiments were ethically approved by the 1) "Comité National de Réflexion Ethique sur l'Expérimentation Animale 14" (ID numbers 57-07112012) and were in agreement with the European Communities Council Directive (2010/63/EU);

Conflict of Interest Statement

The authors declare no conflicts of interest and no competing financial interests.

Supplementary Figures Supplementary figure 1: Enrichment plots from gene enrichment analysis (GSEA).

GSEA results showing positive (A) and negative (B) enrichment in the kidneys of Tshz3 +/lacZ adult mice for gene sets related to "interferon gamma response", "epithelial to mesenchymal transition", "inflammatory response" (A) and to "Oxydative Phosphorylation", "Xenobiotic Metabolism" and Fatty Acid Metabolism (B).

Supplementary

From the interactive online database at https://shiny.mdc-berlin.de/mgsca/. Karaiskos