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BACKGROUND AND AIMS Acute kidney injury (AKI) accounts for 13.3 million cases per year worldwide and further responsible for 1.7 million deaths per year [1]. Increasing incidence of AKI, progression toward CKD or end-stage renal disease (ESRD), affect on long term health, high mortality, morbidity and high costs are all accountable for the need to analyze AKI at a closer level [2]. Rhabdomyolysis (RM) accounting for 10% of AKI cases is due to damage of the skeletal muscle causing a release of muscle cell contents like myoglobin and toxins into the bloodstream leading to AKI, renal failure and possibly death. RM-induced AKI (RIAKI) occurs due to a variety of causes like fall risk, exposure to drugs, infections, muscle hypoxia, infections, body-temperature changes and metabolic and electrolyte disorders [3]. The immune system has been reported to play a role in the pathogenesis of AKI through inflammatory pathways. Macrophages are known to be involved in AKI to CKD transition [4]. Macrophages play an important role in the pathogenesis of RIAKI. While macrophage subtypes (inflammatory type) have been reported to drive RIAKI lesions and prognosis [5], the role of the other immune cells needs to be explored. The aim of this study was to analyze the recruitment of the whole immune system during RIAKI through single-cell RNA sequencing. METHOD Two month old C57BL/6J male mice were used for this study. Mice were administered saline (control) or glycerol i.m (7.5 mL/kg 50% glycerol diluted in saline) for induction of RM. Mouse tail vein blood was collected at 6, 24 and 48 h and serum was obtained after centrifugation (5 min at 2000 rpm). Biochemical analysis tests were used to evaluate RM intensity (creatinine phosphokinase, CPK) and renal function (blood urea nitrogen, BUN) using a Pentra 400 analyzer. To analyze the immune cell recruitment in kidney, kidneys from control and glycerol treated mice were decapsulated, minced and incubated with collagenase and DNase. The dissociated cells were then incubated in red cell lysis buffer and passed through a 40 µm filter. The dissociated cells were further incubated with CD45 antibodies and a viability marker. Kidney CD45 live cells were sorted using BD Influx cell sorter. Preparation of single cell libraries was done and single-cell RNA sequencing was performed according to 10× Genomics protocol. RESULTS Eight clusters (macrophages, monocytes, endothelial cells, neutrophils, natural killer T cells, B cells, T cells and dendritic cells) were revealed after single-cell RNA sequencing of CD45+ renal cells. Recruitment of macrophages, monocytes and neutrophils were revealed to be modified by RIAKI. As expected, NF-kB signaling pathway was the major pathway upregulated in multiple clusters. Pathway analysis in T cell cluster showcased T cell selection and differentiation and T cell receptor signaling pathway. AA467197 (a long non-coding RNA which has been reported in renal ischemia–reperfusion) was upregulated through differential expression analysis in multiple clusters. Macrophage subclustering revealed a high macrophage diversity in the seven macrophage subclusters that shared markers for macrophage subtypes (M1, M2) and pro-inflammatory markers. KLf9 was upregulated in cluster 7, which has been reported to be a stress-responsive gene associated having pro-inflammatory effect. NF-kB and IL-2 STAT5 signaling pathways were the major pathways upregulated in multiple clusters. Drug repositioning studies with connectivity map showcased SRC inhibitions as potent pharmacological intervention strategy. CONCLUSION This study revealed the immune landscaping and pathways associated in RIAKI. Single-cell approach illustrated macrophages diversity in pathological context and suggested candidates for further therapeutic approaches.

International audience; Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1β (HNF-1β) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1β deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined 1 H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b-/- ), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1β in metabolism. Hnf1b-/- cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b-/- cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b-/- cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1β. HNF-1β shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI.

International audience; 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.

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-β4. 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-β4 abundance was confirmed with ELISA. Knockout of thymosin-β4 in zebrafish altered proximal and distal tubule pronephros growth suggesting a possible role of thymosin β4 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. ispartof: KIDNEY INTERNATIONAL vol:99 issue:3 pages:737-749 ispartof: location:United States status: published

AKI is a frequent condition that involves renal microcirculation impairment, infiltration of inflammatory cells with local production of proinflammatory cytokines, and subsequent epithelial disorders and mitochondrial dysfunction. Peroxisome proliferator-activated receptor γ coactivator 1-α (PPARGC1A), a coactivator of the transcription factor PPAR-γ that controls mitochondrial biogenesis and function, has a pivotal role in the early dysfunction of the proximal tubule and the subsequent renal repair. Here, we evaluated the potential role of hepatocyte nuclear factor-1β (HNF-1β) in regulating PPARGC1A expression in AKI. In mice, endotoxin injection to induce AKI also induced early and transient inflammation and PPARGC1A inhibition, which overlapped with downregulation of the HNF-1β transcriptional network. In vitro, exposure of proximal tubule cells to the inflammatory cytokines IFN-γ and TNF-α led to inhibition of HNF-1β transcriptional activity. Moreover, inhibition of HNF-1β significantly reduced PPARGC1A expression and altered mitochondrial morphology and respiration in proximal tubule cells. Chromatin immunoprecipitation assays and PCR analysis confirmed HNF-1β binding to the Ppargc1a promoter in mouse kidneys. We also demonstrated downregulation of renal PPARGC1A expression in a patient with an HNF1B germinal mutation. Thus, we propose that HNF-1β links extracellular inflammatory signals to mitochondrial dysfunction during AKI partly via PPARGC1A signaling. Our findings further strengthen the view of HNF1B-related nephropathy as a mitochondrial disorder in adulthood.

Introduction Le metabolisme energetique joue un role essentiel dans le developpement renal, la reparation tubulaire apres agression et la progression de la fibrose tubulo-interstitielle. HNF-1β est un facteur de transcription epithelial controlant la fonction mitochondriale via la regulation de PGC-1a dans les cellules tubulaires proximales. Ses variants pathogenes sont a l’origine d’anomalies developpementales renales variees et d’une fibrose renale progressive, pouvant etre expliques par des anomalies metaboliques secondaires a une dysfonction mitochondriale. Certains phenotypes renaux (progresseurs rapides) pourraient etre lies a une reponse inadaptee a une agression moderee. Description/Methodes Nous avons etudie l’effet d’une invalidation du gene Hnf1b (CrispR-Cas9) dans une lignee murine tubulaire proximale sur le metabolisme cellulaire par approche non ciblee (H1-Spectro-RMN) et des approches ciblees (dosage ATP et lactates, Red Oil) en conditions basales et d’hypoxie (1 %, 24 heures). Une re-analyse des donnees de RNASeq et ChiPSeq publiees a ete realisee et des confirmations en qPCR obtenues. Resultats En condition basale, les cellules Hnf1b-/- presentent une reduction de leur contenu intracellulaire en ATP secondaire a une utilisation reduite du pyruvate, une accumulation intracellulaire de lactate, d’intermediaires du cycle de Krebs et de gouttelettes lipidiques. Ces anomalies signent une dysfonction du metabolisme energetique mitochondrial avec reorientation vers la glycolyse. L’exposition a l’hypoxie des cellules sauvages reproduit partiellement les changements secondaires a l’invalidation d’Hnf1b tandis que les cellules Hnf1b-/- sont incapables d’accroitre plus leurs capacites glycolytiques en reponse a ce stress hypoxique. Il existe egalement des anomalies des intermediaires de biosynthese des phospholipides pouvant etre reliees a une reduction d’expression de la choline kinase α qui serait directement regulee par HNF1B. Conclusion HNF-1β controle le metabolisme energetique au sein des cellules tubulaires proximales et son invalidation conduit a une reorientation metabolique basale empechant une adaptation supplementaire en cas d’agression, confirmant notre hypothese clinique de depart.

Background Patients with HNF1B mutations develop progressive chronic renal failure, diabetes mellitus (40-50%), and liver tests abnormalities (40-70%). In HNF1B patients who reach end-stage renal disease, single kidney transplantation (SKT) or combined kidney-pancreas transplantation can be considered. Methods A retrospective multicenter study including 18 HNF1B patients receiving SKT or kidney-pancreas transplantation, and in vitro experiments including the characterization of the HNF1B expression after calcineurin inhibitor (CNI) exposure. Results After SKT, 50% of the HNF1B patients develop early posttransplantation diabetes mellitus, whereas 40% experience new-onset or severe worsening of preexisting abnormalities of liver tests, including severe cholestasis. In liver biopsies, disorders of the cholangiocytes primary cilium and various degrees of bile duct paucity and dysplasia were identified. In vitro studies combining CNI exposure and siRNA-mediated inhibition of NFATc revealed that calcineurin inhibition decreases HNF1B expression in epithelial cells but independent of NFATc. Conclusions Because HNF1B-related disease is a heterozygous condition, CNIs used to prevent rejection may induce reduced expression of the nonmutated allele of HNF1B leading to a superimposed defect of HNF-1β transcriptional activity. Taking into account the specific risk of posttransplantation diabetes mellitus and liver disorders in HNF1B patients, these findings advocate for in-depth characterization of pathways that regulate HNF1B and plead for considering individually tailored graft management that may include a CNI-free immunosuppressive regimen. Interventional studies will have to confirm this individualized approach.

Septic shock is the most common cause of acute kidney injury (AKI), but the underlying mechanisms remain unclear and no targeted therapies exist. Lysophosphatidic acid (LPA) is a bioactive lipid which in vivo administration was reported to mitigate inflammation and injuries caused by bacterial endotoxemia in the liver and lung. The objective of the present study was to determine whether LPA can protect against sepsis-associated AKI. C57BL/6 mice were treated with LPA 18:1 (5 mg/kg, i.p.) 1 h before being injected with the endotoxin lipopolysaccharide (LPS), and AKI was evaluated after 24 h. LPA significantly decreased the elevation of plasma urea and creatinine caused by LPS. In the kidney, LPA pretreatment significantly reduced the upregulation of inflammatory cytokines (IL-6, TNFα, monocyte chemoattractant protein-1 (MCP-1)), and completely prevented downregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and upregulation of heme oxygenase-1 caused by LPS. LPA also prevented LPS-mediated alterations of the renal mitochondrial ultrastructure. In vitro pretreatment with LPA 18:1 significantly attenuated LPS-induced upregulation of the inflammatory cytokines (TNFα and MCP-1) in RAW264 macrophages. Moreover, in vivo LPS treatment lowered urinary LPA concentration and reduced LPA anabolic enzymes (autotaxin and acylglycerol kinase), and increased the LPA catalytic enzyme (lipid phosphate phosphatase 2) expression in the kidney cortex. In conclusion, exogenous LPA exerted a protective action against renal inflammation and injuries caused by bacterial endotoxemia. Moreover, LPS reduces the renal production of LPA suggesting that sepsis-associated AKI could be mediated, at least in part, by alleviation of the protective action of endogenous LPA.

International audience; HNF1B-related disease is an emerging condition characterized by an autosomal-dominant inheritance, a 50% rate of de novo mutations, and a highly variable phenotype (renal involvement, maturity-onset diabetes of the young type 5, pancreatic hypoplasia, and urogenital tract and liver test abnormalities). Given the current lack of pathognomonic characteristics and the wide overlap with other conditions, a genetic test is the diagnostic gold standard. However, pre-genetic screening is mandatory because genetic testing has substantial costs. Our aim was to develop a HNF1B score, based on clinical, imaging, and biological variables, as a pivotal tool for rational genetic testing. A score was created using a weighted combination of the most discriminative characteristics based on the frequency and specificity in published series. The HNF1B score is calculated upon 17 items including antenatal discovery, family history, and organ involvement (kidney, pancreas, liver, and genital tract). The performance of the score was assessed by a ROC curve analysis in a 433-individual cohort containing 56 HNF1B cases. The HNF1B score efficiently and significantly discriminated between mutated and nonmutated cases (AUC 0.78). The optimal cutoff threshold for the negative predictive value to rule out HNF1B mutations in a suspected individual was 8 (sensitivity 98.2%, specificity 41.1%, and negative predictive value over 99%). Thus, the HNF1B score is a simple and accurate tool to provide a more rational approach to select patients for HNF1B screening.

Multiple sclerosis, the most common cause of progressive neurological disability in young adults, is a chronic inflammatory disease. There is solid evidence for a genetic influence in multiple sclerosis, and deciphering the causative genes could reveal key pathways influencing the disease. A genome region on rat chromosome 9 regulates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Using interval-specific congenic rat lines and association of single-nucleotide polymorphisms with inflammatory phenotypes, we localized the gene of influence to Vav1 , which codes for a signal-transducing protein in leukocytes. Analysis of seven human cohorts (12,735 individuals) demonstrated an association of rs2546133-rs2617822 haplotypes in the first VAV1 intron with multiple sclerosis (CA: odds ratio, 1.18; CG: odds ratio, 0.86; TG: odds ratio, 0.90). The risk CA haplotype also predisposed for higher VAV1 messenger RNA expression. VAV1 expression was increased in individuals with multiple sclerosis and correlated with tumor necrosis factor and interferon-γ expression in peripheral blood and cerebrospinal fluid cells. We conclude that VAV1 plays a central role in controlling central nervous system immune-mediated disease and proinflammatory cytokine production critical for disease pathogenesis.

Renal complications of non-Hodgkin lymphoma encompass a wide spectrum of monoclonal Ig-related pathologies. Clonal circulating T cells can also be associated with non-renal autoimmune disorders induced by overproduction of specific patterns of cytokines or unbalanced lymphocytes sub-populations. Immunophenotyping of circulating T cells and TCR gene restriction analysis using Biomed-2 protocol. NF-κB staining and mRNA quantification of inflammatory genes in HK-2 epithelial renal cells exposed to supernatants of peripheral blood mononuclear cells with clonal T-cell population. Here, we could identify a persistent clonal T-cell population, only characterized by in-depth immunophenotyping of circulating lymphocytes and using multiplex PCR analysis of TCR gene rearrangements, in two patients with polymorphic inflammatory renal fibrosis of unknown origin. Using an in vitro approach, we could demonstrate that peripheral blood mononuclear cells including the clonal population can trigger a phenotype switch of epithelial renal cells from a quiescent state to a pro-inflammatory state characterized by NF-κB nuclear translocation and overexpression of inflammatory cytokine or chemokine. These preliminary data suggest that circulating T-cell clones may directly activate epithelial renal cells or promote a T-/B-cell population with autoimmune reactive properties against kidney cells, which, in the absence of overt renal lymphoma infiltration, lead to the subsequent inflammatory renal fibrotic phenotype.

AKI is a frequent condition that involves renal microcirculation impairment, infiltration of inflammatory cells with local production of proinflammatory cytokines, and subsequent epithelial disorders and mitochondrial dysfunction. Peroxisome proliferator-activated receptor γ coactivator 1-α (PPARGC1A), a coactivator of the transcription factor PPAR-γ that controls mitochondrial biogenesis and function, has a pivotal role in the early dysfunction of the proximal tubule and the subsequent renal repair. Here, we evaluated the potential role of hepatocyte nuclear factor–1β (HNF-1β) in regulating PPARGC1A expression in AKI. In mice, endotoxin injection to induce AKI also induced early and transient inflammation and PPARGC1A inhibition, which overlapped with downregulation of the HNF-1β transcriptional network. In vitro, exposure of proximal tubule cells to the inflammatory cytokines IFN-γ and TNF-α led to inhibition of HNF-1β transcriptional activity. Moreover, inhibition of HNF-1β significantly reduced PPARGC1A expression and altered mitochondrial morphology and respiration in proximal tubule cells. Chromatin immunoprecipitation assays and PCR analysis confirmed HNF-1β binding to the Ppargc1a promoter in mouse kidneys. We also demonstrated downregulation of renal PPARGC1A expression in a patient with an HNF1B germinal mutation. Thus, we propose that HNF-1β links extracellular inflammatory signals to mitochondrial dysfunction during AKI partly via PPARGC1A signaling. Our findings further strengthen the view of HNF1B-related nephropathy as a mitochondrial disorder in adulthood.

A single nucleotide polymorphism causing constitutive activation of Vav1 results in increased natural Treg generation and is responsible for the imbalance between Vav1 GEF and adaptor functions., CD4+ regulatory T cells (Treg cells) expressing the transcription factor Foxp3 play a pivotal role in maintaining peripheral tolerance by inhibiting the expansion and function of pathogenic conventional T cells (Tconv cells). In this study, we show that a locus on rat chromosome 9 controls the size of the natural Treg cell compartment. Fine mapping of this locus with interval-specific congenic lines and association experiments using single nucleotide polymorphisms (SNPs) identified a nonsynonymous SNP in the Vav1 gene that leads to the substitution of an arginine by a tryptophan (p.Arg63Trp). This p.Arg63Trp polymorphism is associated with increased proportion and absolute numbers of Treg cells in the thymus and peripheral lymphoid organs, without impacting the size of the Tconv cell compartment. This polymorphism is also responsible for Vav1 constitutive activation, revealed by its tyrosine 174 hyperphosphorylation and increased guanine nucleotide exchange factor activity. Moreover, it induces a marked reduction in Vav1 cellular contents and a reduction of Ca2+ flux after TCR engagement. Together, our data reveal a key role for Vav1-dependent T cell antigen receptor signaling in natural Treg cell development.

International audience; Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80(low)CD11b(high)Ly6b(high)CD206(low) kidney macrophages were dominant, whereas by day 8, F4/80(high)CD11b(+)Ly6b(low)CD206(high) cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80(low)CD11b(high)Ly6b(high)CD206(low) macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease.

Introduction Des donnees in vitro suggerent que l’inhibition pharmacologique des proteines EPAC, des regulateurs du signal cytoplasmique de l’AMPc, s’accompagne d’anomalies quantitatives et qualitatives de l’expression de l’aquaporine-2 (AQP2) dans les cellules principales du canal collecteur, en particulier lors d’une stimulation prolongee des recepteurs AVPR2 par l’ADH. EPAC1 ou EPAC2 pourraient etre de nouvelles cibles therapeutiques dans les pathologies impliquant la voie de signalisation ADH/AVPR2/AMPc/AQP2 (SIADH, diabete insipide, polykystose renale). Patients et methodes Dans des souris invalidees pour Epac1 (Epac1-/-) et des souris sauvages (wt), les parametres suivants ont ete mesures a l’etat basal, apres injection de ddAVP ou de furosemide, et apres restriction hydrique prolongee (RH30) : osmolarite, sodium, potassium, magnesium, calcium, uree, creatinine (sang et urine) ; histologie renale ; expression d’AQP2 (qPCR, WB et microscopie confocale) et d’Avpr2, Nkcc, Nkcc2, UT-1, Aqp1, Aqp8 (qPCR). Resultats A l’etat basal, l’osmolarite urinaire et serique des souris Epac1-/- et wt ne different pas, tout comme les concentrations d’electrolytes. L’expression des autres acteurs de l’homeostasie hydrique n’est pas modifiee. De meme, apres stimulation aigue (ddAVP) ou prolongee (RH30) du recepteur AVPR2, les capacites de reabsorption hydrique des souris Epac1-/- et wt sont identiques. La reponse au furosemide est egalement conservee (diurese, osmolarite). De fait, l’expression (ARNm et proteine) d’AQP2, tout comme son adressage a la membrane apicale chez les souris Epac1-/- sont identiques aux controles. En revanche, on note chez les souris wt une augmentation significative de l’expression d’EPAC1 sans modification d’EPAC2 apres RH30 (WB). Chez les souris Epac1-/-, l’expression d’EPAC2 est significativement diminuee apres RH30 (WB). Discussion Contrairement aux donnees in vitro utilisant un blocage pharmacologique visant EPAC1 et EPAC2, les souris Epac1-/- ne presentent pas de trouble franc de concentration des urines, suggerant une redondance des voies de signalisation et/ou une action combinee d’EPAC1 et EPAC2. Conclusion Chez la souris, l’invalidation isolee d’EPAC1 ne s’accompagne pas d’anomalie de l’expression d’AQP2 et de la reabsorption d’eau.

Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

International audience; Gold or mercury salts trigger a dramatic IgE response and a CD4 T-cell-dependent nephropathy in Brown-Norway (BN), but not in Lewis (LEW) rats. We previously identified the 1.1-Mb Iresp3 (immunoglobin response QTL3) locus on chromosome 9 that controls these gold salt-triggered immune disorders. In the present work, we investigated the genetic control of HgCl2-induced immunological disorders and assessed the relative contribution of the CD45RC(high) and CD45RC(low) CD4 T-cell subpopulations in this control. By using interval-specific congenic lines, we narrowed down Iresp3 locus to 117-kb and showed that BN rats congenic for the LEW 117-kb were protected from HgCl2-triggered IgE response and nephropathy. This 117-kb interval also controls CD45RC expression by CD4 T cells and the ability of CD45RC(high) CD4 T cells to trigger the autoimmune disorders resulting from HgCl2 administration. This 117-kb region contains four genes, including Vav1, a strong candidate gene according to its cellular function and exclusive expression in hematopoietic cells. Thus, this study highlights the role of the CD45RC(high) CD4 T-cell subpopulation in the opposite susceptibility of BN and LEW rats to HgCl2-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

International audience; Epithelial repair following acute kidney injury (AKI) requires epithelial-mesenchyme-epithelial cycling associated with transient re-expression of genes normally expressed during kidney development as well as activation of growth factors and cytokine-induced signaling. In normal kidney, the Hnf-1β transcription factor drives nephrogenesis, tubulogenesis and epithelial homeostasis through the regulation of epithelial planar cell polarity and expression of developmental or tubular segment-specific genes. In a mouse model of ischemic AKI induced by a 2-hours hemorrhagic shock, we show that expression of this factor is tightly regulated in the early phase of renal repair with a biphasic expression profile (early down-regulation followed by transient over-expression). These changes are associated to tubular epithelial differentiation as assessed by KSP-cadherin and megalin-cubilin endocytic complex expression analysis. In addition, early decrease in Hnf1b expression is associated with the transient over-expression of one of its main target genes, the suppressor of cytokine signaling Socs3, which has been shown essential for renal repair. In vitro, hypoxia induced early up-regulation of Hnf-1β from 1 to 24 hours, independently of the hypoxia-inducible factor Hif-1α. When prolonged, hypoxia induced Hnf-1β down-regulation while normoxia led to Hnf-1β normalization. Last, Hnf-1β down-regulation using RNA interference in HK-2 cells led to phenotype switch from an epithelial to a mesenchyme state. Taken together, we showed that Hnf-1β may drive recovery from ischemic AKI by regulating both the expression of genes important for homeostasis control during organ repair and the state of epithelial cell differentiation.

Introduction L’uromoduline (UMOD), une proteine normalement excretee dans la lumiere de la branche ascendante large de Henle, peut induire la synthese d’IL-1β par les macrophages (MΦ) via la voie de l’inflammasome NLRP3. Alors qu’un mauvais adressage d’UMOD vers l’interstitium (mutation ponctuelle, agression renale aigue ou chronique) s’accompagne d’une fibrose inflammatoire, les souris Umod-/- soumises a une agression renale aigue ont une inflammation interstitielle plus severe, suggerant donc un role a la fois pro- et anti-inflammatoire d’UMOD. L’effet d’UMOD sur la polarisation des MΦ (phenotype M1, pro-inflammatoire, ou M2, anti-inflammatoire) est encore inconnu, et pourrait etre implique dans la physiopathologie de la tubulopathie myelomateuse (TM). Materiels et methodes Dans des MΦ obtenus a partir de PBMC humains (MΦ non polarises M0, polarises M1 ou M2), nous avons quantifie l’expression genique des marqueurs M1 (CCR7, CD86) et M2 (CD163, TGM2) et des cytokines TNFα et IL-10 ; dose la production d’IL-1β. Sur des biopsies renales de patients avec une TM, nous avons quantifie la fibrose inflammatoire satellite de cylindres d’UMOD et de chaines legeres (CL), et realise des marquages anti-UMOD, CCR7 et CD163. Resultats In vitro, UMOD induit une secretion precoce (H14) et dose-dependante d’IL-1β par les MΦ M0, suggerant l’acquisition d’un phenotype M1 initial. Cependant, de H14 a H48, les MΦ M0 semblent s’orienter vers un phenotype M2b (CD68+ CD163+ TGM2+ IL10+), anti-inflammatoire et profibrosant. L’effet est identique sur des MΦ pre-polarises en M1. Dans les biopsies de 15 patients avec TM, on observe des MΦ M2 CD68+ CD163+ en exces dans l’interstitium, en particulier au contact des cylindres UMOD-CL. Le nombre de MΦ M1 CCR7+ est faible. In vitro, l’ajout de CL nephrotoxiques a UMOD n’augmente pas la synthese d’IL-1β et n’a pas d’effet sur l’expression des marqueurs de polarisation des MΦ etudies. Discussion L’obstruction tubulaire par les complexes CL–UMOD reste le facteur declenchant de la TM mais UMOD seule semble un acteur puissant de l’activation et de la polarisation macrophagique. Conclusion UMOD active rapidement les MΦ (secretion d’IL-1β, pro-inflammatoire) mais les polarise secondairement vers un phenotype anti-inflammatoire profibrosant. Ces resultats sont en defaveur d’une approche pharmacologique ciblant uniquement IL-1β au cours de la TM.

International audience; The information gathered from the large number of omics experiments in renal biology is underexplored, as it is scattered over many publications or held in supplemental data. To address this, we have developed an open-source Kidney and Urinary Pathway Knowledge Base (KUPKB) that facilitates simple exploration of these omics data. The KUPKB currently comprises 220 data sets (miRNA, mRNA, proteins, and metabolites) extracted from existing publications or databases. Researchers can explore the integrated data using the iKUP browser, and a simple template is provided to submit new omics data sets to the knowledge base. As an example of iKUP's use, we show how we identified, in silico, calreticulin as a protein induced in human interstitial fibrosis and tubular atrophy (IFTA) in chronic kidney transplant rejection; a link that would have been difficult to establish using existing Web-based tools. Using immunohistochemistry, we validated in vivo this in silico result in human and rat biopsies of IFTA, thus identifying calreticulin as a potential new player in chronic kidney transplant rejection. The KUPKB provides a simple tool that enables users to quickly survey a wide range of omics data sets and has been shown to facilitate rapid hypothesis generation in the context of renal pathophysiology.

Introduction L’insuffisance renale aigue (IRA) secondaire a une rhabdomyolyse est frequente et represente 10 % des IRA. La tubulopathie est principalement attribuee au role deletere de la myoglobine. Nous avons fait l’hypothese que cette agression renale impliquait des macrophages, polarises vers le phenotype M1 (pro-inflammatoire) ou M2 (reparateur). Materiels et methodes Dans un modele murin, la rhabdomyolyse est induite par injection intramusculaire de glycerol. Les macrophages renaux sont caracterises a l’aide de 9 marqueurs de surface, et par PCR quantitative sur cellules uniques pour analyse transcriptionnelle. Le clodronate liposomal (CL) est utilise pour depleter les macrophages. L’effet de la myoglobine sur les cellules epitheliales et des macrophages en culture primaires est evalue in vitro. Resultats Le phenotype des macrophages du rein varie en fonction du temps. A j2 apres la rhabdomyolyse, des macrophages pro-inflammatoires M1 sont massivement recrutes, alors qu’a j8, d’autres macrophages de phenotype M2 reparateur, deviennent predominants. Le traitement par CL reduit la mortalite par insuffisance renale des souris, et revele le role-cle des macrophages pro-inflammatoires secretant des composants de la matrice extracellulaire (fibronectine, collagene III), et des chemo-attractants (CCL-2, CCL-7). La myoglobine per se entraine la production de chemo-attractants par les tubulocytes renaux et les macrophages, et polarise ces derniers vers un phenotype M1. A J30, alors que la fonction renale est globalement restauree, on note une fibrose tubulo-interstitielle majeure, qui est significativement prevenue par le traitement par CL. Discussion et conclusion Ces donnees mettent en evidence : (i) le role determinant des macrophages inflammatoires M1 dans la physiopathologie et le pronostic de l’IRA post-rhabdomyolyse murine ; (ii) l’existence de lesions de fibrose renale persistante apres recuperation fonctionnelle.

In early human pregnancy, uterine decidual NK cells (dNK) are abundant and considered as cytokine producers but poorly cytotoxic despite their cytolytic granule content, suggesting a negative control of this latter effector function. To investigate the basis of this control, we examined the relative contribution to the cytotoxic function of different activating receptors expressed by dNK. Using a multicolor flow cytometry analysis, we found that freshly isolated dNK exhibit a unique repertoire of activating and inhibitory receptors, identical among all the donors tested. We then demonstrated that in fresh dNK, mAb-specific engagement of NKp46-, and to a lesser extent NKG2C-, but not NKp30-activating receptors induced intracellular calcium mobilization, perforin polarization, granule exocytosis and efficient target cell lysis. NKp46-mediated cytotoxicity is coactivated by CD2 but dramatically blocked by NKG2A coengagement, indicating that the dNK cytotoxic potential could be tightly controlled in vivo. We finally found that in dNK, mAb-specific engagement of NKp30, but not NKp46, triggered the production of IFN-γ, TNF-α, MIP-1α, MIP-1β, and GM-CSF proinflammatory molecules. These data demonstrate a differential, controlled role of NKp46- and NKp30-activating receptors expressed by dNK that could be critical for the outcome of pregnancy and the killing of uterine cells infected by pathogens.

Introduction Le shear stress (FSS) urinaire, genere par l’ecoulement de l’urine dans le tubule renal, est modifie precocement dans la majorite des nephropathies. De recents resultats suggerent que le FSS urinaire pourrait contribuer au developpement de l’inflammation tubulo-interstitielle dans les maladies renales. En revanche, son impact sur l’integrite de l’epithelium tubulaire est largement meconnu. Le but de cette etude est donc d’evaluer par une approche in vitro les effets du FSS sur le phenotype epithelial des cellules tubulaires renales. Materiels et methodes Des cellules HK-2 cultivees a forte confluence sur du collagene IV sont exposees a un FSS laminaire et continu. Les cellules servant de controle sont maintenues en conditions statiques. La structure epitheliale des cellules est evaluee en etudiant par PCR quantitative, western blot ou immunofluorescence les composants des jonctions serrees (claudine-2, Zo-1), des jonctions d’ancrage (E-cadherine, beta-catenine), du complexe Par de polarite apicale (pard3, pard6, cdc42 et PKC-zeta) et du cil primaire (tubuline alpha-acetylee). Resultats Dans les cellules HK-2 soumises pendant 48 h a un FSS d’intensite 0,5 Pa, on observe la disparition de claudine-2. La distribution de ZO-1 est modifiee, son marquage a la membrane devenant discontinu. La quantite d’E-cadherine est diminuee et l’expression de ses represseurs Snail1 et Slug est au contraire augmentee. Le FSS entraine egalement une relocalisation de beta-catenine de la membrane vers le cytoplasme. Il reduit fortement le niveau d’ARNm codant pard6 sans changer l’expression de pard3, cdc42 et PKC-zeta. Enfin, le FSS provoque la perte du cil primaire apical. L’ensemble de ces effets est attenue par des intensites plus faibles de FSS (0,01 Pa et 0,1 Pa) et pour des temps plus courts (5 h et 24 h). Discussion et conclusion Ainsi, en reponse au FSS, les cellules tubulaires perdent un nombre important de leurs caracteristiques epitheliales. Les variations du FSS urinaire pourraient donc jouer un role dans la progression des nephropathies, via la desorganisation de l’epithelium tubulaire.

Introduction Le pronostic des patients porteurs d’une mutation d’HNF1B apres transplantation renale (TR) ou rein/pancreas (TRP) est mal connu. Patients et methodes Etude retrospective evaluant le devenir des patients HNF1B beneficiant d’une TR/TRP. Caracterisation in vitro de l’impact des anticalcineurines (ACN) sur l’expression d’HNF1B. Resultats 15 patients HNF1B ont ete inclus (13 TR, 2 TRP ; âge moyen a la greffe 28 ans [5–53]). Avant greffe, 9 patients n’etaient pas diabetiques et 8 n’avaient pas d’anomalies des tests hepatiques. L’immunosuppression etait la suivante : induction (n = 10), ACN (tacrolimus [n = 11] ou CsA [n = 4]), MMF (n = 14) ou AZA (n = 1), et prednisone (n = 15). Apres un suivi de 73 ± 56 mois [17–180], le DFGe etait a 65 ± 29 mL/min/1,73 m2. Apres TR, 5/9 patients (55 %) ont developpe un diabete precoce, 4/8 patients (50 %) ont developpe des anomalies hepatiques et 2/7 patients ont tres nettement aggrave leurs tests hepatiques (cholestase non icterique en particulier) sans explication a la biopsie hepatique. In vitro, la culture de cellules HK2 en presence d’inhibiteurs de la calcineurine (CsA ou tacrolimus) induit une diminution dose-dependante de l’expression d’HNF1B et de ses genes cibles (TMEM27, CDH16, PKD2). L’inhibition de la calcineurine bloque normalement la translocation des facteurs de transcription NFAT dans le noyau. Pour autant, malgre la liaison du facteur de transcription NFATc1 en amont d’HNF1B, l’inhibition selective des facteurs NFATc1-4 ne s’accompagne pas dans ces cellules de la perte d’expression d’HNF1B. L’impact des ACN sur la ciliogenese dependante d’HNF1B dans les cholangiocytes, est en cours de caracterisation. Discussion et conclusion Chez les patients mutes pour HNF1B, un diabete et une cholestase precoces ou severes sont frequents apres transplantation avec prise d’ACN. Dans un contexte de mutation preexistante d’HNF1, les ACN diminuent l’expression d’HNF1B, et miment les consequences de la deletion bi-allelique d’Hnf1B chez la souris (dysfonction pancreatique et paucite des voies biliaires).

Introduction L’IRA septique est caracterisee par une dysfonction tubulaire adaptative, controlee par les mitochondries et induite par l’association d’une dysfonction microvasculaire, d’un stress oxydant et d’une inflammation intra-renale (impliquant les lymphocytes NKT IFNg + a la phase aigue). Les mecanismes moleculaires precis liant la reponse inflammatoire systemique et la dysfonction tubulaire sont encore mal connus. Materiels et methodes Caracterisation de la reponse de l’epithelium tubulaire (reseaux transcriptionnels, oxydoreduction) a une agression aigue inflammatoire in vivo (modele murin de choc endotoxinique) et in vitro (cellules HK2 avec surnageant de PBMC active (LPS) ou avec IFN g, siRNA anti-STAT3). Resultats Chez la souris, on observe a la phase aigue de l’IRA septique (H6-24) une inflammation intra-renale (hyperproduction d’IL-6, IFN g, IL-18), une dysfonction epitheliale proximale (inhibition du complexe megaline-cubline, des transporteurs OAT1 et SGLT2), un desequilibre du systeme d’oxydoreduction (SOD2, CATALASE), et une modification des reseaux transcriptionnels des facteurs de transcription HNF (HNF-1A, -1B et -4A) en faveur de l’inhibition de leur activite transcriptionnelle. In vitro, ces donnees sont reproduites par l’application de surnageant de PBMC stimulees par du LPS ou de l’IFNg seul (effet dose). L’inhibition isolee de STAT3 (normalement active par la voie du recepteur de l’IFNg) ne modifie pas l’action de l’IFNg sur le reseau transcriptionnel des HNF. L’inhibition isolee ou combinee de STAT1 est en cours d’etude. Discussion et conclusion La secretion d’IFN g dans le rein aux temps precoces de l’agression septique induit directement une dysfonction tubulaire proximale, impliquant une inhibition de l’activite transcriptionnelle des HNF (independante de l’activation de STAT3). Les mecanismes precis liant l’IFNg et l’activite transcriptionnelle des HNF sont en cours d’exploration.