Mapping of the amniotic fluid proteome of fetuses with congenital anomalies of the kidney and urinary tract identifies plastin 3 as a protein involved in glomerular integrity.

Congenital anomalies of the kidney and the urinary tract (CAKUT) are the first cause of chronic kidney disease in childhood. Several genetic and environmental origins are associated with CAKUT, but most pathogenic pathways remain elusive. Considering the amniotic fluid (AF) composition as a proxy for fetal kidney development, we analyzed the AF proteome from non-severe CAKUT (n = 19), severe CAKUT (n = 14), and healthy control (n = 22) fetuses using LC-MS/MS. We identified 471 significant proteins that discriminated the three AF groups with 81% precision. Among them, eight proteins independent of gestational age (CSPG4, LMAN2, ENDOD1, ANGPTL2, PRSS8, NGFR, ROBO4, PLS3) were associated with both the presence and the severity of CAKUT. Among those, five were part of a protein-protein interaction network involving proteins previously identified as being potentially associated with CAKUT. The actin-bundling protein PLS3 (plastin 3) was the only protein displaying a gradually increased AF abundance from control, via non-severe, to severe CAKUT. Immunohistochemistry experiments showed that PLS3 was expressed in the human fetal as well as in both the fetal and the postnatal mouse kidney. In zebrafish embryos, depletion of PLS3 led to a general disruption of embryonic growth including reduced pronephros development. In postnatal Pls3-knockout mice, kidneys were macroscopically normal, but the glomerular ultrastructure showed thickening of the basement membrane and fusion of podocyte foot processes. These structural changes were associated with albuminuria and decreased expression of podocyte markers including Wilms' tumor-1 protein, nephrin, and podocalyxin. In conclusion, we provide the first map of the CAKUT AF proteome that will serve as a reference for future studies. Among the proteins strongly associated with CAKUT, PLS3 did surprisingly not specifically affect nephrogenesis but was found as a new contributor in the maintenance of normal kidney function, at least in part through the control of glomerular integrity. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PRYNT: a tool for prioritization of disease candidates from proteomics data using a combination of shortest-path and random walk algorithms.

The urinary proteome is a promising pool of biomarkers of kidney disease. However, the protein changes observed in urine only partially reflect the deregulated mechanisms within kidney tissue. In order to improve on the mechanistic insight based on the urinary protein changes, we developed a new prioritization strategy called PRYNT (PRioritization bY protein NeTwork) that employs a combination of two closeness-based algorithms, shortest-path and random walk, and a contextualized protein-protein interaction (PPI) network, mainly based on clique consolidation of STRING network. To assess the performance of our approach, we evaluated both precision and specificity of PRYNT in prioritizing kidney disease candidates. Using four urinary proteome datasets, PRYNT prioritization performed better than other prioritization methods and tools available in the literature. Moreover, PRYNT performed to a similar, but complementary, extent compared to the upstream regulator analysis from the commercial Ingenuity Pathway Analysis software. In conclusion, PRYNT appears to be a valuable freely accessible tool to predict key proteins indirectly from urinary proteome data. In the future, PRYNT approach could be applied to other biofluids, molecular traits and diseases. The source code is freely available on GitHub at: https://github.com/Boizard/PRYNT and has been integrated as an interactive web apps to improved accessibility ( https://github.com/Boizard/PRYNT/tree/master/AppPRYNT ).

Amniotic fluid peptides predict postnatal kidney survival in developmental kidney disease.

Although a rare disease, bilateral congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of end stage kidney disease in children. Ultrasound-based prenatal prediction of postnatal kidney survival in CAKUT pregnancies is far from accurate. To improve prediction, we conducted a prospective multicenter peptidome analysis of amniotic fluid spanning 140 evaluable fetuses with CAKUT. We identified a signature of 98 endogenous amniotic fluid peptides, mainly composed of fragments from extracellular matrix proteins and from the actin binding protein thymosin-ß4. The peptide signature predicted postnatal kidney outcome with an area under the curve of 0.96 in the holdout validation set of patients with CAKUT with definite endpoint data. Additionally, this peptide signature was validated in a geographically independent sub-cohort of 12 patients (area under the curve 1.00) and displayed high specificity in non-CAKUT pregnancies (82 and 94% in 22 healthy fetuses and in 47 fetuses with congenital cytomegalovirus infection respectively). Change in amniotic fluid thymosin-ß4 abundance was confirmed with ELISA. Knockout of thymosin-ß4 in zebrafish altered proximal and distal tubule pronephros growth suggesting a possible role of thymosin ß4 in fetal kidney development. Thus, recognition of the 98-peptide signature in amniotic fluid during diagnostic workup of prenatally detected fetuses with CAKUT can provide a long-sought evidence base for accurate management of the CAKUT disorder that is currently unavailable.

Apelin affects the mouse aging urinary peptidome with minimal effects on kidney.

Kidney function is altered by age together with a declined filtration capacity of 5-10% per decade after 35 years. Renal aging shares many characteristics with chronic kidney disease. Plasma levels of the bioactive peptide apelin also decline with age and apelin has been shown to be protective in chronic kidney disease. Therefore we evaluated whether apelin could also improve aging-induced renal lesions and function in mice. Since urine is for the major part composed of proteins and peptides originating from the kidney, we first studied apelin-induced changes, in the aging urinary peptidome. Despite the recently published age-associated plasma decrease of apelin, expression of the peptide and its receptor was increased in the kidneys of 24 months old mice. Twenty-eight days treatment with apelin significantly modified the urinary peptidome of 3 and 24 months old mice towards a signature suggesting more advanced age at 3 months, and a younger age at 24 months. The latter was accompanied by a decreased staining of collagen (Sirius red staining) in 24 months old apelin-treated mice, without changing aging-induced glomerular hypertrophy. In addition, apelin was without effect on aging-induced renal autophagy, apoptosis, inflammation and reduced renal function. In conclusion, treatment of aged mice with apelin had a limited effect on kidney lesions although modifying the urinary peptidome towards a younger signature. This supports evidence of apelin inducing more general beneficial effects on other aging organs, muscles in particular, as recently shown for sarcopenia, markers of which end up via the glomerular filtration in urine.

A capillary electrophoresis coupled to mass spectrometry pipeline for long term comparable assessment of the urinary metabolome.

Although capillary electrophoresis coupled to mass spectrometry (CE-MS) has potential application in the field of metabolite profiling, very few studies actually used CE-MS to identify clinically useful body fluid metabolites. Here we present an optimized CE-MS setup and analysis pipeline to reproducibly explore the metabolite content of urine. We show that the use of a beveled tip capillary improves the sensitivity of detection over a flat tip. We also present a novel normalization procedure based on the use of endogenous stable urinary metabolites identified in the combined metabolome of 75 different urine samples from healthy and diseased individuals. This method allows a highly reproducible comparison of the same sample analyzed nearly 130 times over a range of 4 years. To demonstrate the use of this pipeline in clinical research we compared the urinary metabolome of 34 newborns with ureteropelvic junction (UPJ) obstruction and 15 healthy newborns. We identified 32 features with differential urinary abundance. Combination of the 32 compounds in a SVM classifier predicted with 76% sensitivity and 86% specificity UPJ obstruction in a separate validation cohort of 24 individuals. Thus, this study demonstrates the feasibility to use CE-MS as a tool for the identification of clinically relevant urinary metabolites.