Chrystelle Lacroix

Cécile Caubet

Anne Gonzalez-De-Peredo

Benjamin Breuil

David Bouyssié

Alexandre Stella

Luc Garrigues

Caroline Le Gall

Anthony Raevel

Angelique Massoubre

Julie Klein

Stéphane Decramer

Frédérique Sabourdy

Flavio Bandin

Odile Burlet-Schiltz

Bernard Monsarrat
email: bernard.monsarrat@ipbs.fr.

Joost-Peter Schanstra
email: joost-peter.schanstra@inserm.fr

Jean-Loup Bascands
email: jean-loup.bascands@inserm.fr

Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy

Keywords: ARG, Arginase, ASL, Arginine succinate lyase, ASS, Arginine succinate synthase, CAT Epidermal growth factor, FASP, Filter-Aided Sample Preparation, UreteroPelvic Junction, UUO, Unilateral ureteral obstruction, XIC, Extracted Ion Chromatogram

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 obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children [START_REF] Harambat | Epidemiology of chronic kidney disease in children[END_REF]. The most frequently found cause of congenital obstructive nephropathy is ureteropelvic junction (UPJ) obstruction with an estimated incidence of 1 in 1000-1500 births.

Milder forms of UPJ obstruction often progress to the spontaneous resolution of the pathology over time. This has led to a watchful waiting approach with surgical intervention only if renal deterioration is detected [START_REF] Heinlen | Operative versus nonoperative management of ureteropelvic junction obstruction in children[END_REF]. Although this medical surveillance prevents unnecessary surgery, it mostly relies on invasive follow-up. Consequently with the aim to reduce this invasive follow-up, several groups have initiated research to identify non-invasive urinary biomarkers of UPJ obstruction using both targeted and non-targeted (e.g. proteome analysis based) strategies. Targeted strategies including urinary cytokine expression analyses failed to clearly determine the need for surgery in UPJ obstruction [START_REF] Decramer | Non-invasive markers of ureteropelvic junction obstruction[END_REF][START_REF] Klein | Congenital ureteropelvic junction obstruction: human disease and animal models[END_REF]. On the other hand untargeted strategies have been more successful and by using urinary proteomics, biomarkers for renal and non-renal diseases have been identified [START_REF] Decramer | Urine in clinical proteomics[END_REF][START_REF] Hanash | Disease proteomics[END_REF][START_REF] Drube | Urinary proteome analysis identifies infants but not older children requiring pyeloplasty[END_REF][START_REF] Patel | Intraand interobserver variability in interpretation of DMSA scans using a set of standardized criteria[END_REF][START_REF] Mackenzie | A review of renal scarring in children[END_REF]. Using urinary peptidome analysis, we identified and validated a urinary peptide panel that predicted the clinical outcome of newborns with UPJ obstruction with 97% accuracy several months in advance [START_REF] Decramer | Non-invasive markers of ureteropelvic junction obstruction[END_REF][START_REF] Decramer | Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis[END_REF]. An independent small-scale study confirmed the efficiency of this biomarker panel [START_REF] Drube | Urinary proteome analysis identifies infants but not older children requiring pyeloplasty[END_REF]. These studies indicate the potential of urinary proteomics to predict the clinical fate of patients with UPJ obstruction. Although these endogenous urinary peptide biomarkers are of great potential clinical value, sequencing of these biomarkers mainly identified collagen fragments that are less informative on the pathophysiology of the disease. In contrast studies of the high molecular weight urinary proteome (i.e. proteins) might be more informative on the pathophysiology of disease. Different approaches have been used in the past to characterize the urinary proteome, either by 2D-gel electrophoresis coupled to mass spectrometry [START_REF] Smith | Development of a high-throughput method for preparing human urine for two-dimensional electrophoresis[END_REF][START_REF] Zerefos | Characterization of the human urine proteome by preparative electrophoresis in combination with 2-DE[END_REF] or reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis [START_REF] Adachi | The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins[END_REF][START_REF] Castagna | Exploring the hidden human urinary proteome via ligand library beads[END_REF][START_REF] Kentsis | Urine proteomics for profiling of human disease using high accuracy mass spectrometry[END_REF][START_REF] Marimuthu | A comprehensive map of the human urinary proteome[END_REF]. In-depth proteome analysis using extensive fractionation of the sample and high resolution, fast sequencing mass spectrometers have reported the identification of >2000 proteins in normal human urine [START_REF] Adachi | The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins[END_REF][START_REF] Kentsis | Urine proteomics for profiling of human disease using high accuracy mass spectrometry[END_REF][START_REF] Marimuthu | A comprehensive map of the human urinary proteome[END_REF]. Here, we applied quantitative high-resolution label free LC-MS/MS analysis for the identification of urinary proteins associated to UPJ obstruction in newborns. Among a number of proteins uniquely associated with severe UPJ obstruction we identified Arginase 1, not previously recognized in UPJ obstruction. Using an independent larger cohort, we further verified reduced urinary abundance of Arginase 1 using both Western blot and multiple reaction monitoring (MRM). Using the mouse model of obstructive nephropathy, we observed that the expression of arginases is modulated in situ in obstructed kidneys. Further gene expression analysis of the arginase pathway allowed us to hypothesize for a role of arginases in the development of fibrotic lesions in obstructive nephropathy.

EXPERIMENTAL PROCEDURES

Participants and urine collection

After local ethics committee approval, informed consent was obtained from all participants (parents of the newborns). All urine samples were from male newborns of less than one year old. A total of 49 UPJ obstruction patients were studied. In addition, healthy individuals were used to establish normal urinary protein patterns. Details on urine collection procedures can be found in the supplementary experimental procedures.

Clinical analysis methods

DMSA scans were performed to determine differential renal function as described [START_REF] Mackenzie | A review of renal scarring in children[END_REF] and interpreted as described by Patel et al [START_REF] Patel | Intraand interobserver variability in interpretation of DMSA scans using a set of standardized criteria[END_REF]. MAG3-scans to determine the degree of obstruction were performed as described by Wong et al [START_REF] Wong | Utility of technetium-99m-MAG3 diuretic renography in the neonatal period[END_REF].

Patients

All patients with unilateral antenatally detected hydronephrosis, defined by a renal pelvic diameter >5 mm, were treated at the Children's Hospital Purpan, Toulouse, France. All patients underwent an ultrasound examination within the first month of life. The degree of hydronephrosis was classified between grades I and IV according to the system developed by the Society for Fetal Urology and the ultrasound appearance of the renal parenchyma and pelvicalyceal system on longitudinal ultrasonic section [START_REF] Grignon | Urinary tract dilatation in utero: classification and clinical applications[END_REF][START_REF] Fernbach | Ultrasound grading of hydronephrosis: introduction to the system used by the Society for Fetal Urology[END_REF][START_REF] Maizels | Outcome of nonspecific hydronephrosis in the infant: a report from the Registry of the Society for Fetal Urology[END_REF]. A voiding cystourethrogram (VCUG) was systematically performed at the first visit. Patients were excluded if they had a vesicoureteric reflux (VUR), solitary kidney, bilateral hydronephrosis, ureteral dilatation or lower urinary tract abnormalities. After the first visit patients were classified into "Mild" or "Severe". A detailed clinical description of the "Mild" or "Severe" UPJ obstruction patients can be found in the supplementary experimental procedures and in [START_REF] Decramer | Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis[END_REF]. For the patients classified as "Severe", urine samples were collected as described above with a collection pouch, or directly obtained from the pelvis during pyeloplasty. These samples were studied as two different subgroups according to the origin of urine, designated "Severe-Bladder" or "Severe-Pelvis". The median age (±SD) of newborns of each group when urinary samples were collected was 22±11.5 weeks in the "Healthy" group (n=22), 15±13.7 weeks in the "Mild" group (n=27), 11±6.73 weeks in the "Severe-Bladder" group (n=22), and 13±8 weeks in the "Severe-Pelvis" group (n=6). Details regarding the age of the newborns included in the study can be found in the supplementary experimental procedures.

Label-free quantification workflow

Urinary sample preparation for NanoLC-MS/MS analysis by guest on December 17, 2020 https://www.mcponline.org

Downloaded from

For the discovery phase, 20 urine samples (n=5/group) were used. Urine samples (5-15 ml) were centrifuged at 3000g for 10 min at 4°C to remove cell debris. The supernatants were concentrated and digested using a protocol adapted from the Filter-Aided based Sample Preparation (FASP) method [START_REF] Wisniewski | Universal sample preparation method for proteome analysis[END_REF][START_REF] Wisniewski | High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers[END_REF]. For details of the FASP method and deviation from the published FASP protocol see the supplementary experimental procedures.

NanoLC-MS/MS analysis

The resulting peptides were analyzed by nanoLC-MS/MS using an Ultimate3000 system (Dionex, Amsterdam, Netherlands) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Each sample was analyzed in triplicate, resulting in 60 analytical runs (3 replicates x 4 groups x 5 samples). The order of the injections was randomized over all the samples. Further details on NanoLC-MS/MS analysis can be found in the supplementary experimental procedures.

Database search and protein validation

Database searches were performed using the Mascot Daemon software (version 2.3.2 Matrix Science, London, UK) using procedures described in the supplementary experimental procedures.

Quantification from raw MS data

Quantification was performed based on all peptide identifications derived from the 60 analytical runs, using the label-free quantitative module implemented in the MFPaQ v4.0.0 software (Mascot File Parsing and Quantification) (http://mfpaq.sourceforge.net/) as described in the supplementary experimental procedures. Quantification of peptide ions was performed based on calculated XIC areas values.

Data processing and statistical analysis

Protein relative quantification was performed by pairwise comparisons of different groups, containing each 5 samples analyzed in triplicate. The method used for missing intensity values imputation for each peptide ion is described in the supplementary experimental procedures. Based on the peptide quantitative data, a Protein Abundance Index (PAI) was calculated, defined as the total sum of all the XIC areas for all the tryptic peptide ions identified for this protein. To perform normalization of a group of analytical runs, we computed abundances ratios for all the PAI values between a reference run and all the other runs, and used the median of the PAI ratios as a normalization factor for each run. Statistical analysis was then performed for each pairwise comparison of sample groups. For each sample, the mean of normalized PAI values retrieved from triplicate nanoLC-MS by guest on December 17, 2020 measurements were calculated, and a Student's t test on log transformed mean normalized PAI values was used for statistical evaluation of the significance of expression level variations (assuming non-equal variances). P-values were adjusted for multiple testing using the Benjamini-Hochberg procedure [START_REF] Benjamini | Controlling the false discovery rate in behavior genetics research[END_REF]. All procedures were performed using R packages which can be provided upon request.

Targeted mass spectrometry analysis-Multiple Reaction Monitoring (MRM) for biomarker verification

The design of MRM assays and urinary sample preparation for MRM analysis was performed as described in the supplementary experimental procedures. Each dried digested urinary sample spiked with yeast exogenous proteins was suspended with 14 µl of a mixture of synthetic stable isotope-labeled peptides (partially purified PEPotec synthetic peptides or purified AQUA Ultimate peptides, Thermo Scientific, see the supplementary experimental procedures) in 2% acetonitrile, 0.05% trifluoroacetic acid. Half of the preparation was then loaded on the system and analyzed on the 5500 QTrap as described for the design of MRM assays (supplementary experimental procedures), using the optimized transitions list (Sup. Data 1). Quantitative measurements were performed via time-scheduled MRM acquisition, using a 4 min retention time window and a cycle time of 3s. Details of the MRM acquisition method are given in Sup. Table 1. For assessment of the repeatability of the entire analytical workflow (sample concentration on SDS-PAGE + in-gel digestion + LC-MRM analysis), replicate sample preparations and LC-MRM analysis were performed on aliquots of either a unique healthy urine sample from the cohort of the verification study or of a pooled urine sample from healthy donors (supplementary experimental procedures, Sup. Table 2 and Sup. Data 2-3 for details on these preliminary experiments). For the candidate proteins monitoring with purified AQUA peptides, response curves were generated by spiking in increasing amounts the heavy standards in a pooled urine sample from healthy donors, and the LOD/LLOQ of the assays was calculated using the QuaSAR software [START_REF] Mani | Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics[END_REF] (supplementary experimental procedures, Sup. Table 3 and Sup. Data 4-5).

Assessment of the LC-MRM system performances during the clinical study itself was done through systematic injection of a quality control beta-galactosidase tryptic digest, interspersed between acquisition of the clinical samples from the verification cohort (Sup. Data 6), and through monitoring of the spiked-in yeast protein internal standards (Sup. Data 7). Injections corresponding to the 3 groups of samples from the verification cohort were randomized during LC-MRM analysis. The entire dataset of the MRM analysis was loaded into PeptideAtlas/PASSEL (http://www.peptideatlas.org/PASS/PASS00454). All ensure correct peak detection and integration. In addition, the AuDIT module [START_REF] Abbatiello | Automated detection of inaccurate and imprecise transitions in peptide quantification by multiple reaction monitoring mass spectrometry[END_REF] was used to assess the quality of the monitored transitions, by controlling the relative ratios between transitions in the light and heavy peptides in the preliminary triplicate test dataset (Sup. Table 2), and in the clinical dataset itself (Sup. UUO is a model of obstructive nephropathy mimicking in an accelerated manner the development of renal fibrosis [START_REF] Bascands | Obstructive nephropathy: insights from genetically engineered animals[END_REF][START_REF] Chevalier | Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy[END_REF].

Male mice 8 weeks of age were used in all experiments. Unilateral ureteral ligation was performed as previously described [START_REF] Schanstra | In vivo bradykinin B2 receptor activation reduces renal fibrosis[END_REF]. Mice were subjected to UUO during 3 or 8 days.

Details can be found in the supplementary experimental procedures.

Histological analysis and immunohistochemistry

For histology, kidney sections were fixed in Carnoy's solution for 24 h, dehydrated and embedded in paraffin. Four-micrometer paraffin-embedded sections were used for immunohistochemistry. Sections were first de-waxed in Ottix Plus (Diapath, Martinengo, Italy) and rehydrated through a series of graded ethanol washes before endogenous peroxidase blockage (S2001, DakoCytomation, Trappes, France). For detection of collagen fibril deposits, tissues sections were incubated 1 hour at room temperature with primary for morphometric analyses.

Western blot analyses

Complete details of the procedure can be found in the supplementary experimental procedures. Briefly urine samples were desalted on PD10 columns (GE Healthcare Europe GmbH, Freiburg, Germany), concentrated by lyophilisation and stored at -80°C. Renal mouse tissues were homogenized in ice-cold RIPA cell lysis buffer and then centrifuged at 13,000 g during 15 min at 4°C. The resulting supernatants were stored at -80°C and used as tissue extracts for experiments. Protein concentrations were determined by Bradford assay (Bio-Rad). For western blot analysis, ARG1 and 2 immunoblotting were performed by using specific rabbit polyclonal anti-Arginase1 (Sigma) and anti-Arginase2 (Santa-Cruz Biotechnology) antibodies. GAPDH was detected with a goat monoclonal antibody (Santa-Cruz Biotechnology). The antigen-antibody complex was detected with peroxydaseconjugated anti-rabbit or anti-goat antibodies.

Measurement of arginase activity

Arginase activity was measured using a colorimetric analysis of urea production from Larginine, as previously described [START_REF] Del Ara | Diagnostic performance of arginase activity in colorectal cancer[END_REF]. Details of the arginase activity measurement can be found in the supplementary experimental procedures.

Quantification of gene expression by real-time quantitative PCR (RT-PCR)

Total RNA from mouse kidney samples was isolated and analyzed by RT-PCR as described in the supplementary experimental procedures. Primers used are listed in Sup. Table 5.

Measurement of plasma amino acid concentration

In each group (n=8), plasma samples from two mice were pooled in order to obtain a sufficient volume for analysis. Arginine concentrations were determined in the resulting 4 samples/group by ion exchange chromatography in an Aminotac amino acid analyzer (Aminotac JLC-500/V, JEOL, Tokyo, Japan). After reaction with ninhydrin, arginine was

RESULTS

Label-free LC-MS/MS analysis of the urinary proteome of UPJ obstruction patients

We adapted the previously described filter-aided sample preparation (FASP) method ( 21) to process urinary samples on an ultrafiltration device with a 10 kDa cut-off membrane (Fig. 1).

After proteolytic digestion, tryptic peptides were recovered in the filtrate from the last centrifugation step, free of undigested protein or other high-molecular weight material.. Following this protocol, around 250 urinary proteins could be identified per nanoLC-MS/MS run on an LTQ-Orbitrap-Velos instrument. Using the same urine sample from an healthy individual, we obtained a median coefficient of variation (CV) for the PAI values of 5% for a triplicate consecutive repeat LC-MS measurement, while the variability related to a triplicate sample preparation was 14% (Sup. Data 9A). These CVs were at the lower end compared to other urinary proteome studies [START_REF] Nagaraj | Quantitative analysis of the intra-and interindividual variability of the normal urinary proteome[END_REF][START_REF] Court | Toward a standardized urine proteome analysis methodology[END_REF], and indicated that the variability of the analytical method is much lower than the inter-individual variability expected in a clinical study (see below).

For identification of urinary proteins associated with UPJ obstruction, 4 different groups of 5 newborns were studied (Fig. 1 and Materials and Methods for detailed group descriptions):

healthy individuals (Healthy), newborns with mild UPJ obstruction evolving to spontaneous resolution (Mild) and severe UPJ obstruction that needed to be surgically corrected (Severe).

In the latter, we analyzed both bladder urine samples and urine samples directly taken from the pelvis during surgery (marked Severe-Bladder and Severe-Pelvis, respectively). All samples were prepared and analyzed randomly. Each of the 20 samples were measured in triplicate which improved MS/MS sampling and increased the number of identified proteins with 30% (Sup. Data 9B). In the time course of the present clinical study (about 10 days), good chromatographic reproducibility was observed across random triplicate injections (Sup.

Data 10) as well as a good analytical repeatability (median PAI CV of 13 % for randomly triplicate measurements) while the inter-individual variability in a same study group was 50 to 60% (Sup. Data 11). Using the MFPaQ software (32), we could identify and quantify 970 unique urinary proteins from these 60 raw files (Sup.Table 6).

Candidate marker proteins selection

For the identification of proteins differentially secreted in obstructed kidneys, we focused on the 3 pairwise combinations Severe-Pelvis/Healthy, Severe-Bladder/Healthy and Mild/Healthy (Fig. 2A). Depending on the comparisons, 16 to 174 differentially secreted proteins were observed (p<0.05) (Fig. 2A and B and Sup. Table 7). Only the comparison Severe-Pelvis/Healthy yielded proteins that resisted to the multiple-testing correction. A major aim in obstructive nephropathy is to find biomarkers of severe obstruction versus mild self-resolving obstruction. Therefore, we focused mainly on proteins specifically involved in by guest on December 17, 2020

severe obstruction and retained proteins that were found with differential abundance in the Severe/Healthy group comparison (p-value <0.05), but that were not significantly modulated in the Mild/Healthy comparison. Among such markers, some had both a modified abundance in bladder urine (Severe-Bladder/Healthy comparison) and in urine collected directly from the pelvis of the pathological kidney (Severe-Pelvis/Healthy comparison), suggesting that the modification was originating specifically from the obstructed kidney (Table 1, Sup. Data 12).

On the contrary, markers which were found with different abundance in Severe-Bladder/Healthy comparison but were not significant in Severe-Pelvis/Healthy comparison were classified as proteins potentially modulated in the contralateral kidney (Sup. .

As a first verification of the urinary proteome-based analysis we searched for the presence of proteins known to be modified in UPJ obstruction. Epidermal growth factor (EGF) has been shown to be reduced in both kidney tissue and urine of patients with UPJ obstruction [START_REF] Grandaliano | MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy[END_REF][START_REF] Yang | Renal expression of epidermal growth factor and transforming growth factor-beta1 in children with congenital hydronephrosis[END_REF]. EGF was among the top differentially excreted proteins in Table 1. This reduced urinary EGF abundance in UPJ obstruction was verified by estimation of the EGF concentration by multiple reaction monitoring (MRM) using independent samples (~10/group, Sup. Data 14-15 and Sup. Data 8). These data on EGF clearly validate the global proteomics-based approach for the detection of differentially secreted urinary proteins in UPJ obstruction.

In the next step we selected proteins differentially expressed in the obstructed kidney for further verification. Selection for verification was based i) on the lowest p-values and highest fold increase or decrease, ii) on whether proteins were not yet shown to be involved in the obstructive nephropathy or renal disease and finally iii) whether these proteins could be potentially involved in the major events of obstructive nephropathy, including inflammation, myofibroblast accumulation and fibrosis [START_REF] Bascands | Obstructive nephropathy: insights from genetically engineered animals[END_REF]. The protein that corresponded best to those criteria was Arginase 1 (ARG1). Urinary proteome analysis detected ~12 times less ARG1 in urine from severe UPJ individuals compared to controls in both comparisons Severe-Pelvis/Healthy and Severe-Bladder/Healthy (p-value <0.05) (Table 1). ARG1 may potentially be involved in the development of fibrosis in UPJ obstruction by down regulating the production of nitric oxide (NO) and by directing the use of L-arginine to cellular proliferation and collagen synthesis (Fig. 3A). We therefore subsequently focused on studying the regulation of arginases in obstructive nephropathy.

Verification of modification of arginase expression in human obstructive nephropathy

According to the guidelines of clinical proteomics [START_REF] Mischak | Recommendations for biomarker identification and qualification in clinical proteomics[END_REF][START_REF] Mischak | Clinical proteomics: A need to define the field and to begin to set adequate standards[END_REF], we verified the reduced abundance of urinary ARG1 in UPJ obstruction using Western blot and MRM on a larger number of independent controls and UPJ obstruction patients (Fig. 1 and4). Western blot analyses showed a significantly lower urinary ARG1 abundance in newborns with UPJ obstruction compared to controls (Fig. 4A andB). This modified urinary ARG1 abundance was confirmed by MRM (Fig. 4C, Sup. Although we observed a significant difference in age between the "Healthy" and "Severe-Bladder" groups in both the discovery and verification cohorts, we did not observe a correlation between the age of the children and the level of ARG1 excretion, irrespective of the approach (i.e. LC-MS/MS, western blot or MRM) we used (Sup. Data 18). However in contrast to the initial LC-MS/MS analysis where ARG1 was decreased only during severe obstruction, here we observed reduced urinary ARG1 abundance compared to controls irrespective of the severity of UPJ obstruction and verification method used.

Analysis of arginase expression and activity in a mouse model of obstructive nephropathy

There are two distinct isoenzymes of mammalian arginases that have 58% sequence identity and that are encoded by separate genes [START_REF] Cederbaum | Arginases I and II: do their functions overlap?[END_REF]. Under physiological conditions, using Western blot analyses, we observed significant expression of ARG1 and ARG2 isoforms in the mouse kidney. Both arginase isoforms gave weak signals in the bladder and ARG2 also showed low expression in the ureter (Fig. 5A).

To further study the link between ARG1 and UPJ obstruction we studied the expression and activity of arginase in the obstructive nephropathy mouse model (n=8/group, Fig. 1).

Immunohistochemical studies on kidney sections validated the development of tubulointerstitial fibrosis as exemplified by increased collagen I staining as early as day 3 after UUO (Fig. 5B). Western blot analysis showed that UUO induced a significant reduction (~60%) in renal ARG1 expression as early as 3 days after UUO. In contrast, ARG2 expression increased 4-5 fold both at 3 and 8 days after UUO (Fig. 5C). In addition, total arginase activity was found significantly increased (6 fold) in both 3 or 8 days obstructed kidneys (Fig. 5D). These data suggest that the reduced urinary ARG1 abundance in newborns with UPJ obstruction can originate from its reduced expression in the obstructed kidney. However the significant increase in arginase activity in the obstructed mouse kidney most likely originates from ARG2 induction during obstructive nephropathy.

Analysis of the expression of genes involved in the pro-fibrotic arginase pathway in the mouse model of obstructive nephropathy

We studied mRNA expression of different enzymes and proteins leading to proline and polyamine synthesis in the pro-fibrotic arginase pathway (Fig. 3A) in the mouse model. expression). We next studied the expression of genes involved in L-Arginine availability (Fig. 3A). Two genes (Ass and Asl) involved in the transformation of citruline in L-arginine were down-regulated and genes coding for L-arginine transporters (Cat1 and 2) were up-regulated (Fig. 6B). In parallel to these measurements we also observed that plasma arginine concentrations were significantly increased in obstructed mice (Fig. 6C). Taken together, this animal model data suggests that obstructive nephropathy is associated with increased intrarenal L-Arginine availability and potential activation of the pro-fibrotic arginase pathway (Fig. 3B).

by guest on December 17, 2020 https://www.mcponline.org Downloaded from

DISCUSSION

Using label-free LC-MS/MS-based urinary proteome analysis and additional studies in the mouse obstructive nephropathy model, we identified the arginase pathway as a potential player in obstructive nephropathy.

One of the main issues in urinary proteome analysis is to maximize the number of detected proteins, while using an analytical workflow compatible with relative quantitative comparison of the samples, in order to define potential biomarkers. Many in-depth proteomic studies on urine are based on extensive fractionation of the samples, final quantification of the proteins being usually performed by spectral counting [START_REF] Adachi | The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins[END_REF][START_REF] Kentsis | Urine proteomics for profiling of human disease using high accuracy mass spectrometry[END_REF][START_REF] Goo | Urinary proteome analysis of irritable bowel syndrome (IBS) symptom subgroups[END_REF][START_REF] Kentsis | Urine proteomics for discovery of improved diagnostic markers of Kawasaki disease[END_REF][START_REF] Wright | Label-free quantitative proteomics reveals differentially regulated proteins influencing urolithiasis[END_REF][START_REF] Zoidakis | Profilin 1 is a potential biomarker for bladder cancer aggressiveness[END_REF]. However, such approaches are in general time consuming which cannot be overcome with pooling of samples since this results in loss of statistical power. Other strategies to increase the depth of the proteome analysis are based on either immunodepletion of abundant urinary proteins or sample treatment with combinatorial peptide ligand libraries but require in general large quantities of starting material [START_REF] Castagna | Exploring the hidden human urinary proteome via ligand library beads[END_REF]. Single-run LC-MS/MS label-free quantification of proteins based on peptide ion intensity (30) allowed the identification of ~400 urinary proteins per run. We used a similar method based on single-run analysis of individual samples, and label-free quantitative analysis using the MFPaQ software.

An additional difficulty in our study was the very low amount of protein obtained from urine samples of newborn (protein concentration typically around 5-30 µg/mL versus 100 µg/mL in adults). In addition, typically, only a very limited volume (5-10 ml) of urine was obtained in newborns. A large number of protocols have been described and compared to prepare urinary proteins for proteomic analysis, consisting generally in a concentration step based on either lyophilisation, precipitation, solid-phase extraction or ultrafiltration of urine, followed by desalting and protein digestion with trypsin [START_REF] Court | Toward a standardized urine proteome analysis methodology[END_REF][START_REF] Lee | Optimizing sample handling for urinary proteomics[END_REF][START_REF] Thongboonkerd | Practical points in urinary proteomics[END_REF]. In our hands, best results were obtained using an ultrafiltration protocol adapted from the (FASP) method.

Finally, our analytical workflow resulted in a fairly robust and repeatable measurement of protein abundance by nanoLC-MS/MS, with typical CVs around 15% for triplicate preparation and MS analysis. In the discovery step of the study, four different groups (n=5/group) were defined and 970 unique urinary proteins were identified using this label-free LC-MS/MS protocol, that could be quantified using the cross-assignment procedure of MFPaQ.

Using this approach, a number of proteins with significant differences in urinary abundance originating from the obstructed kidney were identified including ARG1, EGF, LRCC15, TGM3

and PTGDS (Table 1) and also proteins modulated in contralateral kidney including CDH13 and HSPA5 (Sup. Data 13). While the urinary abundance of TGM3 was clearly decreased in the obstructed kidney, TGM3 was not selected for our first verification set because we suspected that this change in abundance was due to epidermal contamination of the by guest on December 17, 2020

samples. Indeed, expression of TGM3 is normally restricted to superficial epidermis and to hair follicles.

Given the low number of samples per group in the discovery study, we verified these differences in new samples using targeted proteomics via Western-blot and MRM analysis.

Until now only a few small-scale studies have described the successful use of MRM for the multiplexed quantification of potential clinical biomarkers in urine [START_REF] Chen | Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients[END_REF][START_REF] Chen | Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers[END_REF][START_REF] Huttenhain | Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics[END_REF]. In addition, in these studies, at least 10 ml of urine were used for reproducible MRM analysis. Here, we wanted to take advantage of the high sensitivity of the MRM technique to perform the verification on a number of candidate biomarkers of a larger cohort of children from which often only low volumes of urine were available (3-5 mL). We therefore developed an MRM protocol for the detection of proteins in 3 ml of urine. While the ultrafiltration method (e.g. FASP-based) was reproducible in our hands in the discovery phase using larger volumes of urine (≥10 mL), it gave less reproducible results with small sample volumes (3 mL). This is most probably due to loss of material on the membrane that appears to be critical when dealing with small amounts of protein. We thus switched to a processing method using direct desalting, lyophilisation of samples, and concentration of proteins in one gel band by 1D SDS-PAGE followed by in-gel digestion, which allowed to minimize sample loss and to optimize the final yield of tryptic peptides available for MRM analysis. Using this procedure, the targeted proteins could be detected with improved sensitivity and repeatability, and particularly ARG1 was quantified with good precision in 31 individuals starting with 3 ml of urine.

ARG1 was verified as a protein with modified abundance among the proteins specifically originating from the obstructed kidney. Modified EGF excretion was also verified by MRM, confirming the previous observation of modified urinary EGF abundance in obstructive nephropathy [START_REF] Grandaliano | MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy[END_REF][START_REF] Yang | Renal expression of epidermal growth factor and transforming growth factor-beta1 in children with congenital hydronephrosis[END_REF][START_REF] Li | Prediction of the outcome of antenatal hydronephrosis: significance of urinary EGF[END_REF]. According to the study of Li et al., urinary EGF could allow discrimination between mild or severe obstruction in infants [START_REF] Li | Prediction of the outcome of antenatal hydronephrosis: significance of urinary EGF[END_REF], however, in our study, this difference, using MRM, between the "Severe-Bladder" and "Mild" groups lost significance after correction for multiple testing (Sup. Data 8). Thus, the relation between the urinary EGF abundance and the severity of the pathology should be investigated on a larger cohort of patients. The decrease in urinary abundance of LRCC15 was also confirmed, but this difference lost significance in the comparison "Severe-Bladder/Mild" after BH correction.

Although the abundance of urinary ARG1 is decreased in UPJ obstruction patients compared to healthy newborns, there is no significant difference of its abundance between mild and severe UPJ obstruction both measured by Western blot and MRM (Fig. 4B and 4C and Sup. Data 8). So, while urinary ARG1 is not a good clinical biomarker for differentiating between the severity of UPJ obstruction, the protein is potentially involved in the pathophysiology of the disease. Since biopsies from newborns with UPJ obstruction are not available, we used the mouse obstructive nephropathy model to further study the role of arginase in obstructive by guest on December 17, 2020 nephropathy. We observed that ARG1 expression was also reduced in situ, in the obstructed kidney of mice. Based on this result and because it is known that most of the excreted urinary proteins originate from the kidney and urinary tract [START_REF] Thongboonkerd | Renal and urinary proteomics: current applications and challenges[END_REF], it is most likely that the modified urinary ARG1 abundance in UPJ obstruction patients reflects reduced ARG1 expression in the kidney.

Two isoforms of arginases exist: ARG1 and 2. To study the overall effects of obstructive nephropathy on arginases, we also studied the ARG2 expression and total arginase activity.

Interestingly, in contrast to ARG1, UUO strongly increased renal ARG2 expression and total renal arginase activity. While ARG2 is the predominant arginase isoform expressed in the kidney, ARG2 was not detected in the initial LC-MS/MS discovery study. This could be explained either by an absence of excretion due to differences in the protein distribution (ARG1 is expressed in cytoplasm whereas ARG2 is mitochondrial), or, degradation of the ARG2 protein in urine or by absence of identification of the protein due to the physicochemical characteristics of ARG2 tryptic-peptides. Consistent with our observation, ARG1, but not ARG2, was already identified in urine from healthy individuals by other laboratories using urinary proteome analysis [START_REF] Marimuthu | A comprehensive map of the human urinary proteome[END_REF]. In contrast with decreased ARG1 expression after obstruction observed in our study, Schwartz et al [START_REF] Schwartz | Arginine transport is augmented, through modulation of cationic amino acid transporter-1, in obstructive uropathy in rats[END_REF] reported that both isoforms of arginases were up-regulated in a rat model of bilateral obstructive nephropathy.

This difference observed for ARG1 expression in their study can have different origins. They: i) used rats and not mice, ii) used a bilateral, instead of a unilateral, model of ureteral obstruction, iii) studied arginase expression in glomeruli while we used the total renal cortex and iv) studied arginase expression 1 day after obstruction while we studied arginase expression 3 and 8 days post-obstruction. We ruled out the latter since we observed initiation of reduction of renal ARG1 expression one day after unilateral obstruction in our mouse model (results not shown).

In adult mammals, the majority of endogenous L-arginine synthesis takes place in the proximal tubules of the kidney where citrulline, originally released from small intestine, is metabolized into arginine by the enzymes ASS and ASL and can be subsequently exported to the systemic circulation [START_REF] Wu | Arginine metabolism: nitric oxide and beyond[END_REF]. Arginase activity represents a possible route of the L-arginine metabolism [START_REF] Morris | Arginine metabolism: boundaries of our knowledge[END_REF] and has already been associated to pathogenesis of various disorders such as allergic asthma, airway inflammation, diabetes, vascular dysfunction and atherosclerosis [START_REF] Yang | Renal expression of epidermal growth factor and transforming growth factor-beta1 in children with congenital hydronephrosis[END_REF][START_REF] Ming | Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis[END_REF](53)[START_REF] Gronros | Arginase inhibition restores in vivo coronary microvascular function in type 2 diabetic rats[END_REF][START_REF] Olivon | Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/-mice[END_REF][START_REF] Maarsingh | Arginase and pulmonary diseases[END_REF][START_REF] Bratt | Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin[END_REF], among others. Data from our studies on the animal model of obstructive nephropathy show the potential ability to up-regulate L-arginine transport (via Cat transporters) and to direct its metabolism to L-proline via "the pro-fibrotic arginase pathway" (Fig. 3B).

The increase in ARG2 expression/activity that we have observed in the obstructive nephropathy model could represent a key event for the development of renal fibrosis. First, it by guest on December 17, 2020 could lead to shunting of the NOS pathway [START_REF] Durante | Arginase: a critical regulator of nitric oxide synthesis and vascular function[END_REF][START_REF] Mori | Regulation of nitric oxide synthesis and apoptosis by arginase and arginine recycling[END_REF] and it has been shown that NOS inhibition leads to increased fibrosis in the UUO model [START_REF] Morrissey | Nitric oxide generation ameliorates the tubulointerstitial fibrosis of obstructive nephropathy[END_REF][START_REF] Miyajima | Role of nitric oxide in renal tubular apoptosis of unilateral ureteral obstruction[END_REF][START_REF] Manucha | Cytoprotective role of nitric oxide associated with Hsp70 expression in neonatal obstructive nephropathy[END_REF]. Interestingly, recent studies reported that deficiency of ARG2 confers kidney protection in diabetic mouse model and that arginase 2 represents an important mechanism in inducing e-NOS uncoupling leading to oxidative stress and vascular inflammatory responses [START_REF] Ming | Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis[END_REF]53,[START_REF] You | Arginase inhibition mediates renal tissue protection in diabetic nephropathy by a nitric oxide synthase 3-dependent mechanism[END_REF][START_REF] Yang | Arginase: the emerging therapeutic target for vascular oxidative stress and inflammation[END_REF]. Second, increased arginase activity could induce the production of proline, essential for synthesis and deposition of collagen a major component of the extracellular matrix (e.g. fibrosis). There is a growing interest in the role of arginase in tissue remodeling and fibrosis, particularly in the lung [START_REF] Maarsingh | Arginase and pulmonary diseases[END_REF]. Up-regulation of both ARG1 and ARG2 isoforms has already been reported in bleomycin-induced lung fibrosis in mice and collagen synthesis from murine pulmonary fibroblasts can be blocked by arginase inhibitors [START_REF] Kitowska | Functional role and species-specific contribution of arginases in pulmonary fibrosis[END_REF][START_REF] Endo | Induction of arginase I and II in bleomycin-induced fibrosis of mouse lung[END_REF]. The role of ARG1 versus ARG2 in obstructive nephropathy remains to be clarified. Since ARG2 co-localizes with OAT in mitochondria it has already been proposed that ARG2 would preferentially be the arginase isoform involved in proline production, whereas the co-localization of ARG1 and ODC would favour polyamine synthesis [START_REF] Ozaki | Expression of arginase II and related enzymes in the rat small intestine and kidney[END_REF].

Collectively our data, starting with urinary proteome analysis in human samples of UPJ obstruction patients and combining those results with data obtained in the animal obstructive nephropathy model suggest that the arginine metabolism is disturbed in obstructive nephropathy. Induction of ARG2 could lead to activation of the proline formation pathway instead of a synthesis of polyamines that would ultimately result in synthesis/deposition of collagen. In the long term targeting the arginase pathway could have a therapeutic potential in kidney fibrosis.

by guest on December 17, 2020 Urine samples from newborns less than 1 year old were divided into 4 groups: bladder urine from healthy individuals (Healthy), bladder urine from patients with mild obstruction that evolve towards spontaneous resolution of the obstruction (Mild), bladder urine from patients with severe obstruction that need surgery (Severe-Bladder) and pelvis urine from patients with severe obstruction that was collected during surgery (Severe-Pelvis). A, Urine protein biomarker candidates discovery by label-free quantitative LC-MS/MS analysis of urinary proteome of the 4 groups (n=5/group). Urinary samples were centrifuged to remove cell debris then subjected to an adaptation of the filter-aided sample preparation (FASP) method ARG1 is a cytosolic enzyme known to be expressed in the kidney [START_REF] Spector | Immunologic studies of arginase in tissues of normal human adult and arginase-deficient patients[END_REF][START_REF] Choi | Immunohistochemical study of arginase 1 and 2 in various tissues of rats[END_REF]. It was originally identified in liver hepatocytes, where it catalyses the hydrolysis of L-arginine to L-ornithine and urea in the final step of the urea cycle that allows ammonia detoxification [START_REF] Crombez | Hyperargininemia due to liver arginase deficiency[END_REF]. Free

  by guest onDecember 17, 2020

  by guest onDecember 17, 2020 antibody anti-collagen type I (1:250, CL50151AP1 Cedarlane, Ontario, Canada) and further with the secondary anti-rabbit IgG Dako Envision HRP system (K4002, DakoCytomation) during 30 min. Immunological complexes were visualized by the addition of the DAB substrate during 10 min (TA-125-HDX, Thermo Fisher Scientific). Sections were counterstained with hematoxylin and mounted. Kidney sections were scanned using a Nanozoomer 2.0 RS (Hamamatsu Photonics SARL, Massy, France) and treated with the Morpho-expert image-analysis software (version 1.00, Explora Nova, La Rochelle, France)

  The gene expression profiles (Fig. 6A) suggest activation of the proline formation pathway (increased Oat, P5cr and P5cs and reduced proDH and P5cd mRNA expression at both 3 and 8 days post UUO) accompanied by reduced polyamine synthesis (reduced Odc mRNA by guest on December 17, 2020 https://www.mcponline.org Downloaded from

Figure 1 :

 1 Figure 1: Study layout for the identification of new proteins involved in the pathophysiology of congenital unilateral UPJ obstruction.

( 21 )

 21 to concentrate, desalt, reduce, alkylate, and digest urine proteins in an ultrafiltration device. Tryptic urine peptides were analyzed in triplicate by nanoLC-MS/MS analysis and label-free quantification of urinary protein candidates was performed as described. B, Selected candidates from the discovery phase were verified by Western blot analysis and targeted proteomics using a new cohort of patients. C, Unilateral ureteral obstruction (UUO), an experimental mouse model of obstructive nephropathy was used to assess renal expression of a candidate protein biomarker and its modulation during pathology.

Figure 2 :

 2 Figure 2: Quantitative analysis of urinary proteome following UPJ obstruction. A, Statistical analysis of pairwise analysis of Severe-Pelvis or Severe-Bladder or Mild versus Healthy. The number of peptides before and after missing value imputation and the total number of proteins statistically analyzed are indicated for each pairwise comparison. The total number of proteins detected as differentially secreted (Student's t test with p < 0.05) is indicated as well as the number of proteins under-and over-secreted. Proteins that survived multiple testing correction (Benjamini-Hochberg adjustment) are also indicated. B, Volcano plots showing t test p-values (-log10) versus protein ratios between control and patients (log2). All proteins with a Student's t test p-value below 0.05 were considered significant. Red and green dots indicate proteins with increased or decreased urinary abundance, respectively. Grey dots indicate proteins that not significantly changed in the different comparisons.

Figure 3 :

 3 Figure 3: A, Cellular arginine metabolism and putative role of arginase in the development of renal fibrosis.

  arginine is derived from diet, protein turnover or endogenous synthesis. Neo-synthesis from citrulline occurs via successive actions of Arginine succinate synthase (ASS) and Arginine succinate lyase (ASL). Cellular uptake of L-arginine is mediated by the Cationic amino acid Transporters CATs. Intracellular L-arginine can further be metabolized via two main distinct pathways: L-arginine is catabolyzed to L-citrulline and NO by NO synthases (NOS) (grey boxes) or alternatively to L-ornithine and urea by arginases (ARG) (light boxes). Evidence from literature shows that, in this context, arginase can be considered as a key regulator of pro-or anti-fibrotic pathways. First by substrate competition with nitric oxide (NO) synthases, arginases can regulate the production of NO known to have anti-fibrotic properties. Second, via ornithine production, arginase acts upstream from the mitochondrial ornithine aminotransferase (OAT) and can control proline formation essential for collagen synthesis in fibrosis. B, Proposed changes in the arginase pathway in obstructive nephropathy. The data suggest that obstructive nephropathy induces arginine transport and activation of the profibrotic arginase pathway via activation (red) of ARG2, increased expression of OAT, P5CR and P5CS and decreased expression (green) of proDH, P5CD and ODC leading to increased proline availability and collagen synthesis. Key to abbreviations: CAT, Cationic amino acid transporter; ASS, Arginine succinate synthase; ASL, Arginine succinate lyase; ARG, Arginases; NOS, Nitric oxide synthase; NOHA, N-Hydroxy-L-arginine; ODC, Ornithine decarboxylase; OAT, Ornithine aminotransferase; P5C, Pyrroline-5-carboxylate; P5CS, P5C synthase, P5CR, P5C reductase; P5CD, P5C dehydrogenase; ProDH, Proline dehydrogenase; NO, nitric oxide.

Figure 4 :

 4 Figure 4: Verification of modified urinary abundance of ARG1 by immunohistochemistry and MRM in a new patient cohort. A, Representative Western blot analysis shows a large variability in urinary ARG1 abundance in healthy subjects and a reduced urinary abundance in UPJ obstruction patients (10 µg total urinary proteins/lanes). B, Densitometric analysis of Western blot analyses. Values of anti-ARG1 immunoreactivities were normalized by the quantity of albumin present in each sample as determined by Ponceau coloration. The lines represent the mean ARG1 expression in each group. C, LC-MRM analysis shows that urinary ARG1 concentration is lower in UPJ obstruction patients. Logarithmic plots of the ARG1 protein concentration

Figure 5 :

 5 Figure 5: Arginases expression in the mouse kidney and urinary tract and in obstructive nephropathy in mice. A, Western blot analysis of arginase 1 and 2 expression in mouse kidney, bladder and ureter. B, Renal tubulo-interstitial fibrosis in the UUO mouse model. Immunohistochemical staining for collagen I (Coll I) of kidneys sections from control mice (Ctrl) or from mice obstructed for 3 or 8 days (UUO day 3 and day 8, respectively). Representative pictures are shown. Quantification was performed by morphometric analysis. C, Representatives pictures of Western blot analyses and semi-quantitative densitometric analysis of arginase 1 (left) and arginase 2 (right) expression during UUO. D, Total arginase activity as a function of the duration of obstruction was measured in kidney homogenates using a colorimetric analysis for urea production from an L-arginine. Graph values are represented as means ± SD (n=8/group). Mann-Whitney test p-value: **<0,01; ***<0,001.

Figure 6 :

 6 Figure 6: Quantification of renal mRNA expression of enzymes of the « Arginase pathway » and modification of the L-Arginine metabolism during UUO. A, Real-time PCR quantification of mRNA levels of Ornithine aminotransferase (Oat), P5C synthase, (P5cs), P5C reductase (P5cr), Ornithine decarboxylase (Odc), P5C dehydrogenase (P5cd) and Proline dehydrogenase (Prodh) genes in mouse kidneys from control mice or from mice obstructed for 3 or 8 days (n=8/group). B, Real-time PCR quantification of mRNA levels of genes involved in the L-Arginine metabolism in kidneys from control and 3 or 8 days-obstructed mice (n=8/group). Arginine succinate synthase (Ass) and Arginine succinate lyase (Asl) are enzymes responsible for cellular L-arginine production. Cationic amino acid transporters (Cat 1-2) allow L-arginine transport in cells. C, Measurement of plasma arginine (µmol/L) during UUO (n=4/group).

  Figure 3

Table 4

 4

	). Endogenous protein amounts were

Table 4 ,

 4 Sup. Data 8, MRM chromatograms views of the peptides monitored for ARG1 are provided in Sup..

Table 1 :

 1 Significant proteins that were identified as specifically modulated in the obstructed kidney.Proteins that were found to be significantly different in the comparisons Severe-Pelvis and Severe-Bladder versus Healthy (columns in grey), but that were not different in the comparison Mild versus Healthy. P-value was determined using a Student's t test and Fold changes between the patient and the control groups were calculated using the median normalized area of the 5 samples per group. The table indicates raw fold changes. For ratio values <1, we performed a 1/x transformation and indicated those as negative fold changes.

	Downloaded from
	https://www.mcponline.org
	by guest on December 17, 2020

https://www.mcponline.org Downloaded from

by guest on December 17, 2020 https://www.mcponline.org Downloaded from

by guest on December 17, 2020 https://www.mcponline.org Downloaded from

ACKNOWLEDGEMENTS

The work in this study was funded by the Agence Nationale pour la Recherche (Beyond Markers, ANR-07-PHYSIO-004-01) program. SD, FB and JLB acknowledge support by Inserm and the "Direction Régional Clinique" (CHU de Toulouse, France) under the Interface program. JPS was supported by Inserm and the "Direction de la Recherché Médicale et Innovation" (CHU de Toulouse, France) under the "Contrat Hospitalier de Recherche Translationnelle" program. The work was supported in part by grants from the Région Midi-Pyrénées, European funds (FEDER), and the French Ministery of Research (Investissements d'avenir, Proteomics French Infrastructure) to BM and OBS. We would like to acknowledge the skilled technical help of JJ Maoret from the GET TQ (www.genotoul.fr) facility for the QPCR analysis. by guest on December 17, 2020