Abstract
Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.
Highlights
Congenital obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children [1]
Around 250 urinary proteins could be identified per nanoLC-Mass Spectrometry/Mass Spectrometry (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 Protein Abundance Index (PAI) values of 5% for a triplicate consecutive repeat Liquid Chromatography/Mass Spectrometry (LC-MS) measurement, while the variability related to a triplicate sample preparation was 14%
Summary
Congenital obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children [1]. 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 [2]. An independent small-scale study confirmed the efficiency of this biomarker panel [7] These studies indicate the potential of urinary proteomics to predict the clinical fate of patients with UPJ obstruction. 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. We applied quantitative high-resolution label free LC-MS/MS analysis for the identification of urinary proteins associated to UPJ obstruction in newborns. 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
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