Integrative analysis of multi-omics data reveals importance of collagen and the PI3K AKT signalling pathway in CAKUT.

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) is the leading cause of childhood chronic kidney failure and a significant cause of chronic kidney disease in adults. Genetic and environmental factors are known to influence CAKUT development, but the currently known disease mechanism remains incomplete. Our goal is to identify affected pathways and networks in CAKUT, and thereby aid in getting a better understanding of its pathophysiology. With this goal, the miRNome, peptidome, and proteome of over 30 amniotic fluid samples of patients with non-severe CAKUT was compared to patients with severe CAKUT. These omics data sets were made findable, accessible, interoperable, and reusable (FAIR) to facilitate their integration with external data resources. Furthermore, we analysed and integrated the omics data sets using three different bioinformatics strategies: integrative analysis with mixOmics, joint dimensionality reduction and pathway analysis. The three bioinformatics analyses provided complementary features, but all pointed towards an important role for collagen in CAKUT development and the PI3K-AKT signalling pathway. Additionally, several key genes (CSF1, IGF2, ITGB1, and RAC1) and microRNAs were identified. We published the three analysis strategies as containerized workflows. These workflows can be applied to other FAIR data sets and help gaining knowledge on other rare diseases.

Improved prenatal assessment of kidney disease using multiple ultrasound features.

BACKGROUND AND HYPOTHESIS: Congenital anomalies of the kidney and the urinary tract (CAKUT), often discovered in utero, cover a wide spectrum of outcomes ranging from normal postnatal kidney function to fetal death. The current ultrasound workup does not allow for an accurate assessment of the outcome. The present study aimed to significantly improve the ultrasound-based prediction of postnatal kidney survival in CAKUT. METHODS: Histological analysis of kidneys of 15 CAKUT fetuses was performed to better standardize the ultrasound interpretation of dysplasia and cysts. Ultrasound images of 140 CAKUT fetuses with 2-year postnatal follow-up were annotated for amniotic fluid volume and kidney number, size, dysplasia and/or cysts using standardized ultrasound readout. Association of ultrasound features and clinical data (sex and age at diagnosis) with postnatal kidney function was studied using logistic regression. Amniotic fluid proteome associated to kidney dysplasia or cysts was characterized by mass spectrometry. RESULTS: Histologically, poor ultrasound corticomedullary differentiation was associated to dysplastic lesions and ultrasound hyperechogenicity was associated to the presence of microcysts. Of all ultrasound and clinical parameters, reduced amniotic volume, dysplasia and cysts were the best predictors of poor outcome (Odd ratio = 57 [95%CI: 11-481], 20 [3-225] and 7 [1-100], respectively). Their combination into an algorithm improved prediction of postnatal kidney function compared to amniotic volume alone (area under the ROC curve = 0.92 [0.86-0.98] in a 10-fold cross validation). Dysplasia and cysts were correlated (Cramer's V coefficient = 0.44, p<0.0001), but amniotic fluid proteome analysis revealed that they had distinct molecular origin (extracellular matrix and cell contacts versus cellular death, respectively), probably explaining the additivity of their predictive performances. CONCLUSION: Antenatal clinical advice for CAKUT pregnancies can be improved by a more standardized and combined interpretation of ultrasound data.

Performances of acute kidney injury biomarkers vary according to sex.

Background: Before implementing individualized strategies to treat acute kidney injury (AKI), identifying clusters of patients with divergent pathophysiological mechanisms, diagnosis criteria or outcomes is of the utmost importance. Here we studied sex-related molecular mechanisms in cardiac bypass (CBP) surgery patients developing AKI. Methods: We compared the characteristics of 1170 patients referred for CBP surgery using multivariate logistic regression and propensity score-based analysis. Performances of the candidate urinary biomarkers at <4 h post-surgery, urinary neutrophil gelatinase-associated lipocalin (uNGAL), [IGFBP7]·[TIMP-2] product (NephroCheck) and a recently developed AKI signature of 204 urinary peptides (AKI204) to predict AKI were compared in both sexes. Results: Incidence (∼25%) and severity of AKI were similar in men and women, even after adjustment for the usual risk factors of AKI, including baseline estimated glomerular filtration rate, age, diabetes mellitus, length of CBP and red blood cell transfusion. However, at the molecular level, performances of uNGAL, NephroCheck and AKI204 to predict AKI strongly diverged between men and women. In the full cohort, as well as in subgroups of men and women, the multimarker AKI204 signature outperformed uNGAL and NephroCheck and predicted the development of AKI significantly better in women than in men. Analysis of AKI204 at the single-peptide level suggested divergences of AKI mechanisms between sexes due to increased kidney inflammation in women (increased abundance of urinary fragments of osteopontin and uromodulin). Conclusions: In patients referred for CBP surgery, significant clinical and biological differences between men and women as well as sexual dimorphism of AKI biomarker performances were identified. The urinary peptide signature points to sex-related molecular mechanisms underlying AKI.

CCL7 Chemokine Is a Marker but Not a Therapeutic Target of Acute Kidney Injury.

BACKGROUND: Chemokines orchestrate immune cells activation and infiltration during acute kidney injury (AKI). OBJECTIVES: We aim to test whether deletion of C-C chemokine ligand 7 (CCL7), a small chemokine related to CCL2 (MCP-1), may modulate AKI development and progression toward kidney fibrosis. METHOD: Expression of CCL7 was quantified in murine cortical tubular (MCT) cells exposed to myoglobin or lipopolysaccharide or submitted to metabolic reprogramming. Kidney function (BUN, glomerular filtration rate), expression of CCL7 receptors, and kidney infiltration by inflammatory cells (F4/80+ macrophages, MPO+ neutrophils, and B220+ B-cells) were assessed in wt and Ccl7-/- mice submitted to 3 different models of AKI or kidney fibrosis (uni/bilateral ischemia/reperfusion injury (u/bIRI) and rhabdomyolysis). RESULTS: Toxin exposure of MCT cells, as well as metabolic reprogramming recapitulating AKI changes, led to a dramatic up-regulation of CCL7. In vivo, kidney expression of Ccl7 and Ccl2 significantly increased after AKI and remained increased beyond the acute phase (30 days after uIRI). The expression of the CCL7 receptors was heterogeneous and varied with time. Kidney function, expression of CCL7 receptors and Ccl2, and the number of inflammatory cells within kidneys were similar in wt and Ccl7-/- mice at baseline and at day 2 after AKI. Thirty days after uIRI, kidney fibrosis was similar in both mouse strains. CONCLUSIONS: Despite strong induction of CCL7 after AKI, CCL7 deficiency does not prevent AKI and the transition toward kidney fibrosis and should probably not be further explored as a potential target to prevent or treat AKI.

Fetal biomarkers for lower urinary tract obstruction secondary to posterior urethral valves.

Today, prenatal diagnosis of congenital urogenital malformations is mostly dependent on anatomical variations found on imaging. However, these findings can mislead us in telling us when to intervene, and about post-natal prognosis. Since many findings are dependent on multiple assessments, delayed diagnosis can occur, leading to less optimal outcomes compared to early intervention. Analyses of fetal urinary biomarkers have been proposed as a method of finding biological changes that are predictive for diagnosis and prognosis in fetuses at risk of kidney disease. We interviewed a group of researchers that have demonstrated that by combining multiple omics traits extracted from fetal urine, the biological variability found in single omics data can be circumvented. By analyzing multiple fetal urine peptides and metabolites at single time point, the prognostic power of postnatal renal outcome in fetuses with lower urinary tract obstruction is significantly increased. In this interview, we inquired about the technical aspects of the tests, challenges, and limitations the research group have come across, and how they envision the future for multi-omics fetal analysis in the clinic.

Leucine-Rich Alpha-2 Glycoprotein 1 Accumulates in Complicated Atherosclerosis and Promotes Calcification.

Atherosclerosis is the primary cause of cardiovascular disease. The development of plaque complications, such as calcification and neo-angiogenesis, strongly impacts plaque stability and is a good predictor of mortality in patients with atherosclerosis. Despite well-known risk factors of plaque complications, such as diabetes mellitus and chronic kidney disease, the mechanisms involved are not fully understood. We and others have identified that the concentration of circulating leucine-rich α-2 glycoprotein 1 (LRG1) was increased in diabetic and chronic kidney disease patients. Using apolipoprotein E knockout mice (ApoE-/-) (fed with Western diet) that developed advanced atherosclerosis and using human carotid endarterectomy, we showed that LRG1 accumulated into an atherosclerotic plaque, preferentially in calcified areas. We then investigated the possible origin of LRG1 and its functions on vascular cells and found that LRG1 expression was specifically enhanced in endothelial cells via inflammatory mediators and not in vascular smooth muscle cells (VSMC). Moreover, we identified that LRG1 was able to induce calcification and SMAD1/5-signaling pathways in VSMC. In conclusion, our results identified for the first time that LRG1 is a direct contributor to vascular calcification and suggest a role of this molecule in the development of plaque complications in patients with atherosclerosis.

Calprotectin is a contributor to and potential therapeutic target for vascular calcification in chronic kidney disease.

Vascular calcification is an important risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). It is also a complex process involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. In an observational, multicenter European study, including 112 patients with CKD from Spain and 171 patients on dialysis from France, we used serum proteome analysis and further validation by ELISA to identify calprotectin, a circulating damage-associated molecular pattern protein, as being independently associated with CV outcome and mortality. This was confirmed in an additional cohort of 170 patients with CKD from Sweden, where increased serum calprotectin concentrations correlated with increased vascular calcification. In primary human VSMCs and mouse aortic rings, calprotectin exacerbated calcification. Treatment with paquinimod, a calprotectin inhibitor, as well as pharmacological inhibition of the receptor for advanced glycation end products and Toll-like receptor 4 inhibited the procalcifying effect of calprotectin. Paquinimod also ameliorated calcification induced by the sera of uremic patients in primary human VSMCs. Treatment with paquinimod prevented vascular calcification in mice with chronic renal failure induced by subtotal nephrectomy and in aged apolipoprotein E-deficient mice as well. These observations identified calprotectin as a key contributor of vascular calcification, and increased circulating calprotectin was strongly and independently associated with calcification, CV outcome, and mortality in patients with CKD. Inhibition of calprotectin might therefore be a promising strategy to prevent vascular calcification in patients with CKD.

Machine Learning-Based Urine Peptidome Analysis to Predict and Understand Mechanisms of Progression to Kidney Failure.

Introduction: The identification of patients with chronic kidney disease (CKD) at risk of progressing to kidney failure (KF) is important for clinical decision-making. In this study we assesed whether urinary peptidome (UP) analysis may help classify patients with CKD and improve KF risk prediction. Methods: The UP was analyzed using capillary electrophoresis coupled to mass spectrometry in a case-cohort sample of 1000 patients with CKD stage G3 to G5 from the French CKD-Renal Epidemiology and Information Network (REIN) cohort. We used unsupervised and supervised machine learning to classify patients into homogenous UP clusters and to predict 3-year KF risk with UP, respectively. The predictive performance of UP was compared with the KF risk equation (KFRE), and evaluated in an external cohort of 326 patients. Results: More than 1000 peptides classified patients into 3 clusters with different CKD severities and etiologies at baseline. Peptides with the highest discriminative power for clustering were fragments of proteins involved in inflammation and fibrosis, highlighting those derived from α-1-antitrypsin, a major acute phase protein with anti-inflammatory and antiapoptotic properties, as the most significant. We then identified a set of 90 urinary peptides that predicted KF with a c-index of 0.83 (95% confidence interval [CI]: 0.81-0.85) in the case-cohort and 0.89 (0.83-0.94) in the external cohort, which were close to that estimated with the KFRE (0.85 [0.83-0.87]). Combination of UP with KFRE variables did not further improve prediction. Conclusion: This study shows the potential of UP analysis to uncover new pathophysiological CKD progression pathways and to predict KF risk with a performance equal to that of the KFRE.

Plantar osteochondral fragments in young Standardbreds are associated with minimal joint inflammation at the time of surgical removal.

BACKGROUND: Plantar osteochondral fragments (POF) are common but their effect on joint health of young Standardbreds in race training is largely unknown. OBJECTIVES: Evaluate the inflammatory effects of POF in metatarsophalangeal joints of young Standardbreds as a step towards developing evidence-based recommendations for surgical removal. STUDY DESIGN: Cohort study. METHODS: Forty-nine Standardbred horses (age 11-33 months) presented for surgical removal of POF from 56 metatarsophalangeal joints. Synovial tissue collected at arthroscopy was subjected to histopathology. IL-1ß, TNF-α, and PGE-2 were measured in synovial fluid using ELISA. Digital arthroscopy images were scored for inflammation. Racing performance data were retrieved from a public database. RESULTS: Median time in race training prior to surgery was 8 weeks (IQR 4-12; range 0-40). There was minimal evidence of synovial inflammation as assessed by histopathology (median total score 2/20, IQR 0-2, range 0-5) or arthroscopy (median average total score 2.67/15, IQR 1.79-4, range 0-8.83). IL-1ß was not detected in any sample. TNF-α (median 0 pg/mL, IQR 0-0) and PGE-2 (median 56.6 pg/mL, IRQ 40.5-99.8) were measured at low levels. Weeks in training prior to surgery was associated with the number of starts in the season after surgery (incidence rate ratio 1.02, 95% CI 1.00, 1.04, P = .03). MAIN LIMITATIONS: Small sample size from a single breed with a relatively short training time prior to surgery. CONCLUSIONS: There was minimal evidence of synovial inflammation in the metatarsophalangeal joints in this population of young Standardbred horses with POF. It is possible that POF may result in a different inflammatory response than other fragments because they are generally well-embedded in situ. These findings suggest that, in Standardbreds, race training can commence several weeks prior to surgical removal of POF with minimal detrimental effects on joint health, although further investigation of long-term effects of POF on joint health is warranted.

INTRODUCTION/CONTEXTE: Les fragments plantaires ostéochondraux (POF) sont communs mais leur effet au niveau sur la santé articulaire chez les jeunes Standardbreds en entraînement de course demeure inconnu. OBJECTIFS: Évaluer les effets inflammatoires des POF des articulations métatarsophalangiennes chez les jeunes Standardbreds dans le but d'ajouter à l'évidence disponible concernant les recommandations pour leur retrait chirurgical. TYPE D'ÉTUDE: Étude de cohorte descriptive clinique. MÉTHODES: Quarante-neuf chevaux Standardbreds (âgés 11-33 mois) ont été présentés pour retrait chirurgical de POF en provenance de 56 articulations métatarsophalangiennes. Un échantillon de membrane synoviale recueilli au moment de l'arthroscopie a été soumis en histopathologie. IL-1ß, TNF-α, and PGE-2 ont été mesurés dans le liquide synovial par ELISA. Les images digitales d'arthroscopie ont été évaluées pour la présence d'inflammation. Les données de performance en course ont été retrouvées via une base de données publique. RÉSULTATS: Le temps médian de retour à l'entraînement suivant la procédure chirurgicale était de 8 semaines (IQR 4-12; étendu 0-40). Peu d'inflammation synoviale a été détectée en histopathologie (score médian total 2/20, IQR 0-2, étendu 0-5) ou arthroscopie (score médian total 2.67/15, IQR 1.79-4, étendu 0-8.83). IL-1ß a été détectée dans aucun échantillon. TNF-α (médiane 0 pg/mL, IQR 0-0) et PGE-2 (médiane 56.6 pg/mL, IQR 40.5-99.8) ont été détectés en faible quantité. Le nombre de semaines à l'entraînement avant la procédure chirurgicale était associé au nombre de départs pour la saison suivant la chirurgie (IRR 1.02, P = 0.03). LIMITES PRINCIPALES: Petite taille d'échantillon provenant d'une seule race de chevaux ayant une période d'entraînement relativement courte avant la procédure chirurgicale. CONCLUSIONS: Il y a peu d'évidence d'inflammation synoviale dans les articulations métatarsophalangiennes chez cette population de jeunes chevaux Standardbreds ayant des POF. Il est possible que les POF entraînent une réponse inflammatoire différente des autres fragments puisqu'ils sont généralement bien attachés dans l'articulation. Ces résultats suggèrent que chez les Standardbreds, l'entraînement de course puisse commencer plusieurs semaines avant le retrait chirurgical des POF en ayant des effets délétères minimaux pour la santé articulaire. Ceci dit, davantage de recherche est nécessaire pour établir les effets à long-terme de ces POF sur la santé articulaire.

A universal predictive and mechanistic urinary peptide signature in acute kidney injury.

BACKGROUND: The delayed diagnosis of acute kidney injury (AKI) episodes and the lack of specificity of current single AKI biomarkers hamper its management. Urinary peptidome analysis may help to identify early molecular changes in AKI and grasp its complexity to identify potential targetable molecular pathways. METHODS: In derivation and validation cohorts totalizing 1170 major cardiac bypass surgery patients and in an external cohort of 1569 intensive care unit (ICU) patients, a peptide-based score predictive of AKI (7-day KDIGO classification) was developed, validated, and compared to the reference biomarker urinary NGAL and NephroCheck and clinical scores. RESULTS: A set of 204 urinary peptides derived from 48 proteins related to hemolysis, inflammation, immune cells trafficking, innate immunity, and cell growth and survival was identified and validated for the early discrimination (< 4 h) of patients according to their risk to develop AKI (OR 6.13 [3.96-9.59], p < 0.001) outperforming reference biomarkers (urinary NGAL and [IGFBP7].[TIMP2] product) and clinical scores. In an external cohort of 1569 ICU patients, performances of the signature were similar (OR 5.92 [4.73-7.45], p < 0.001), and it was also associated with the in-hospital mortality (OR 2.62 [2.05-3.38], p < 0.001). CONCLUSIONS: An overarching AKI physiopathology-driven urinary peptide signature shows significant promise for identifying, at an early stage, patients who will progress to AKI and thus to develop tailored treatments for this frequent and life-threatening condition. Performance of the urine peptide signature is as high as or higher than that of single biomarkers but adds mechanistic information that may help to discriminate sub-phenotypes of AKI offering new therapeutic avenues.

The tryptophan pathway and nicotinamide supplementation in ischaemic acute kidney injury.

BACKGROUND: Down-regulation of the enzymes involved in tryptophan-derived nicotinamide (NAM) adenine dinucleotide (NAD+) production was identified after acute kidney injury (AKI), leading to the hypothesis that supplementation with NAM may increase the kidney NAD+ content, rescuing tryptophan pathways and subsequently improving kidney outcomes. METHODS: Urinary measurement of tryptophan and kynurenin using liquid chromatography-mass spectrometry metabolomics was used in a cohort of 167 cardiac bypass surgery patients along with tests for correlation to the development of postoperative AKI. A mouse model of ischaemic AKI using ischaemia-reperfusion injury (bilateral clamping of renal arteries for 25 min) was also used. RESULTS: We identified a significant decrease in urinary tryptophan and kynurenin in patients developing AKI, irrespective of the Kidney Disease: Improving Global Outcomes (KDIGO) stage. Although a significant difference was observed, tryptophan and kynurenin moderately discriminated for the development of all AKI KDIGO stages {area under the curve [AUC] 0.82 [95% confidence interval (CI) 0.75-0.88] and 0.75 [0.68-0.83], respectively} and severe KDIGO Stages 2-3 AKI [AUC 0.71 (95% CI 0.6-0.81) and 0.66 (0.55-0.77), respectively]. Sparked by this confirmation in humans, we aimed to confirm the potential preventive effect of NAM supplementation in wild-type male and female C57BL/6 mice subjected to ischaemic AKI. NAM supplementation had no effect on renal function (blood urea nitrogen at Day 1, sinistrin-fluorescein isothiocyanate glomerular filtration rate), architecture (periodic acid-Schiff staining) and injury or inflammation (kidney injury molecule 1 and IL18 messenger RNA expression). In addition, NAM supplementation did not increase post-AKI NAD+ kidney content. CONCLUSION: Notwithstanding the potential role of NAM supplementation in the setting of basal NAD+ deficiency, our findings in mice and the reanalysis of published data do not confirm that NAM supplementation can actually improve renal outcomes after ischaemic AKI in unselected animals and probably patients.

How can artificial intelligence be used for peptidomics?

INTRODUCTION: Peptidomics is an emerging field of omics sciences using advanced isolation, analysis, and computational techniques that enable qualitative and quantitative analyses of various peptides in biological samples. Peptides can act as useful biomarkers and as therapeutic molecules for diseases. AREAS COVERED: The use of therapeutic peptides can be predicted quickly and efficiently using data-driven computational methods, particularly artificial intelligence (AI) approach. Various AI approaches are useful for peptide-based drug discovery, such as support vector machine, random forest, extremely randomized trees, and other more recently developed deep learning methods. AI methods are relatively new to the development of peptide-based therapies, but these techniques already become essential tools in protein science by dissecting novel therapeutic peptides and their functions (Figure 1). EXPERT OPINION: Researchers have shown that AI models can facilitate the development of peptidomics and selective peptide therapies in the field of peptide science. Biopeptide prediction is important for the discovery and development of successful peptide-based drugs. Due to their ability to predict therapeutic roles based on sequence details, many AI-dependent prediction tools have been developed (Figure 1).

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.

Comparison of the amniotic fluid and fetal urine peptidome for biomarker discovery in renal developmental disease.

Production of amniotic fluid (AF) is view as predominately driven by excretion of fetal urine (FU). However, the origin of AF peptides, often considered as potential biomarkers of developmental diseases, has never been investigated. Here, we evaluated the FU origin of AF peptides and if the AF peptide content can be used as a surrogate of FU. The abundance of endogenous peptides was analyzed by capillary electrophoresis coupled to mass spectrometry in 216 AF and 64 FU samples. A total of 2668 and 3257 peptides was found in AF and FU respectively. The AF peptidome largely overlapped with the FU peptidome, ranging from 54% in the second pregnancy trimester to 65% in the third trimester. Examination of a subset of 16 paired AF and FU samples revealed that 67 peptides displayed a significant positively correlated abundance in AF and FU, strongly suggesting that their presence in AF was directly associated to FU excretion. As proof-of-concept we showed that measuring the AF abundance of these 67 peptides of FU origin allowed prediction of postnatal renal survival in fetuses with posterior urethral valves. These results demonstrate that the AF peptidome can be considered as a good surrogate of the FU peptidome.

The low affinity p75 neurotrophin receptor is down-regulated in congenital anomalies of the kidney and the urinary tract: Possible involvement in early nephrogenesis.

Congenital Anomalies of the Kidney and of the Urinary Tract (CAKUT) cover a broad range of disorders including abnormal kidney development caused by defective nephrogenesis. Here we explored the possible involvement of the low affinity p75 neurotrophin receptor (p75NTR) in CAKUT and nephrogenesis. In mouse, p75NTR was highly expressed in fetal kidney, located within cortical early nephrogenic bodies, and decreased rapidly after birth. In human control fetal kidney, p75NTR was also located within the early nephrogenic bodies as well as in the mature glomeruli, presumably in the mesangium. In CAKUT fetal kidneys, the kidney cortical structure and the localization of p75NTR were often disorganized, and quantification of p75NTR in amniotic fluid revealed a significant reduction in CAKUT compared to control. Finally, invalidation of p75NTR in zebrafish embryo with an antisense morpholino significantly altered pronephros development. Our results indicate that renal p75NTR is altered in CAKUT fetuses, and could participate to early nephrogenesis.

Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease.

While blocking the renin angiotensin aldosterone system (RAAS) has been the main therapeutic strategy to control diabetic kidney disease (DKD) for many years, 25-30% of diabetic patients still develop the disease. In the present work we adopted a systems biology strategy to analyze glomerular protein signatures to identify drugs with potential therapeutic properties in DKD acting through a RAAS-independent mechanism. Glomeruli were isolated from wild type and type 1 diabetic (Ins2Akita) mice treated or not with the angiotensin-converting enzyme inhibitor (ACEi) ramipril. Ramipril efficiently reduced the urinary albumin/creatine ratio (ACR) of Ins2Akita mice without modifying DKD-associated renal-injuries. Large scale quantitative proteomics was used to identify the DKD-associated glomerular proteins (DKD-GPs) that were ramipril-insensitive (RI-DKD-GPs). The raw data are publicly available via ProteomeXchange with identifier PXD018728. We then applied an in silico drug repurposing approach using a pattern-matching algorithm (Connectivity Mapping) to compare the RI-DKD-GPs's signature with a collection of thousands of transcriptional signatures of bioactive compounds. The sesquiterpene lactone parthenolide was identified as one of the top compounds predicted to reverse the RI-DKD-GPs's signature. Oral treatment of 2 months old Ins2Akita mice with dimethylaminoparthenolide (DMAPT, a water-soluble analogue of parthenolide) for two months at 10 mg/kg/d by gavage significantly reduced urinary ACR. However, in contrast to ramipril, DMAPT also significantly reduced glomerulosclerosis and tubulointerstitial fibrosis. Using a system biology approach, we identified DMAPT, as a compound with a potential add-on value to standard-of-care ACEi-treatment in DKD.