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. 30 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.

Single-cell RNA sequencing identifies senescence as therapeutic target in rhabdomyolysis-induced acute kidney injury.

BACKGROUND: The role of macrophages in the development of rhabdomyolysis-induced acute kidney injury (RM-AKI) has been established, but an in-depth understanding of the changes in the immune landscape could help to improve targeted strategies. Whereas senescence is usually associated with chronic kidney processes, we also wished to explore whether senescence could also occur in AKI and whether senolytics could act on immune cells. METHODS: Single-cell RNA sequencing was used in the murine glycerol-induced RM-AKI model to dissect the transcriptomic characteristics of CD45+ live cells sorted from kidneys 2 days after injury. Public datasets from murine AKI models were reanalysed to explore cellular senescence signature in tubular epithelial cells (TECs). A combination of senolytics (dasatinib and quercetin, DQ) was administered to mice exposed or not to RM-AKI. RESULTS: Unsupervised clustering of nearly 17 000 single-cell transcriptomes identified seven known immune cell clusters. Sub-clustering of the mononuclear phagocyte cells revealed nine distinct cell sub-populations differently modified with RM. One macrophage cluster was particularly interesting since it behaved as a critical node in a trajectory connecting one major histocompatibility complex class IIhigh (MHCIIhigh) cluster only present in Control to two MHCIIlow clusters only present in RM-AKI. This critical cluster expressed a senescence gene signature, that was very different from that of the TECs. Senolytic DQ treatment blocked the switch from a F4/80highCD11blow to F4/80lowCD11bhigh phenotype, which correlated with prolonged nephroprotection in RM-AKI. CONCLUSIONS: Single-cell RNA sequencing unmasked novel transitional macrophage subpopulation associated with RM-AKI characterized by the activation of cellular senescence processes. This work provides a proof-of-concept that senolytics nephroprotective effects may rely, at least in part, on subtle immune modulation.

Abcc6 deficiency prevents rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis is a risk factor for acute kidney injury, transition towards chronic kidney disease, and death. The role of calcium phosphate deposits in the mechanisms of rhabdomyolysis-induced acute kidney injury (RAKI) is still unclear. Better insight of the role calcium in RAKI could lead to new therapeutic avenues. Here, we show in a mice model of RAKI that calcium phosphate deposits were frequent in the kidney (hydroxyapatite) and partly correlated with the severity of the kidney injury. However, the intensity of deposits was highly heterogeneous between mice. Treatment with sodium chloride, sodium bicarbonate or inorganic pyrophosphate (PPi; an inhibitor of the calcium phosphate crystallization), or combinations thereof, did not improve kidney outcomes and hydroxyapatite deposition during RAKI. Unexpectedly, Abcc6 knockout mice (ko), characterized by PPi deficiency, developed less severe RAKI despite similar rhabdomyolysis severity, and had similar hydroxyapatite deposition suggesting alternative mechanisms. This improved kidney outcome at day 2 translated to a trend in improved glomerular filtration rate at month 2 in Abcc6-/-mice and to significantly less interstitial fibrosis. In addition, whereas the pattern of infiltrating cells at day 2 was similar between wt and ko mice, kidneys of Abcc6-/- mice were characterized by more CD19+ B-cells, less CD3+ T-cells and a lower R1/R2 macrophage ratio at month 2. In summary, kidney calcium phosphate deposits are frequent in RAKI but hydration with sodium bicarbonate or sodium chloride does not modify the kidney outcome. Blocking ABCC6 emerges as a new option to prevent RAKI and subsequent transition toward kidney fibrosis.

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.

Hepatocyte nuclear factor-1ß shapes the energetic homeostasis of kidney tubule cells.

Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1ß (HNF-1ß) 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-1ß 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-1ß 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-1ß. HNF-1ß 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.

Ibrutinib does not prevent kidney fibrosis following acute and chronic injury.

Recent studies suggested that ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, developed for the treatment of chronic lymphocytic leukemia, may prevent NLRP3 inflammasome activation in macrophages, IL-1ß secretion and subsequent development of inflammation and organ fibrosis. The role of NLRP3 has been underlined in the various causes of acute kidney injury (AKI), a pathology characterized by high morbimortality and risk of transition toward chronic kidney disease (CKD). We therefore hypothesized that the BTK-inhibitor ibrutinib could be a candidate drug for AKI treatment. Here, we observed in both an AKI model (glycerol-induced rhabdomyolysis) and a model of rapidly progressive kidney fibrosis (unilateral ureteral obstruction), that ibrutinib did not prevent inflammatory cell recruitment in the kidney and fibrosis. Moreover, ibrutinib pre-exposure led to high mortality rate owing to severer rhabdomyolysis and AKI. In vitro, ibrutinib potentiated or had no effect on the secretion of IL-1ß by monocytes exposed to uromodulin or myoglobin, two danger-associated molecule patterns proteins involved in the AKI to CKD transition. According to these results, ibrutinib should not be considered a candidate drug for patients developing AKI.

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.

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.

A single dose of estrogen during hemorrhagic shock protects against Kidney Injury whereas estrogen restoration in ovariectomized mice is ineffective.

The protective effect of estrogens against chronic glomerular diseases is admitted but remains debated during acute kidney injury (AKI). Using a model of resuscitated hemorrhagic shock in C57/Bl6 female mice, this study evaluated at 1 and 21 days the renal effect of (1) endogenous estrogen, using ovariectomized mice with or without chronic estrogen restoration, or (2) exogenous estrogen, using a single administration of a pharmacological dose during shock resuscitation. In both ovariectomized and intact mice, hemorrhagic shock induced epithelial cell damages (assessed by KIM-1 renal expression) with secondary renal fibrosis but without significant decrease in GFR at day 21. Ovariectomy with or without estrogen restoration have no significant effect on renal damages and dysfunction. This lack of effect was associated with a marked (> 80%) reduction of total kidney GPR30 expression. By contrast, a single high dose of estradiol in intact mice reduced renal KIM-1 expression by 2/3, attenuated the severity of cell death related to pyroptosis, and prevented the increase of fibrosis by 1/3. This provides a rationale to investigate the benefits of a single administration of estrogen or estrogen modulators during acute kidney injuries in males. Furthermore, the cost/benefit ratio of such administration should be investigated in Human.

Lysophosphatidic Acid Protects Against Endotoxin-Induced Acute Kidney Injury.

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.

Hepatocyte Nuclear Factor-1ß Controls Mitochondrial Respiration in Renal Tubular Cells.

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-1ß (HNF-1ß) 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-1ß transcriptional network. In vitro, exposure of proximal tubule cells to the inflammatory cytokines IFN-γ and TNF-α led to inhibition of HNF-1ß transcriptional activity. Moreover, inhibition of HNF-1ß significantly reduced PPARGC1A expression and altered mitochondrial morphology and respiration in proximal tubule cells. Chromatin immunoprecipitation assays and PCR analysis confirmed HNF-1ß 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-1ß 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.

Systems biology combining human- and animal-data miRNA and mRNA data identifies new targets in ureteropelvic junction obstruction.

BACKGROUND: Although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. RESULTS: We have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. CONCLUSIONS: Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.

Asymptomatic circulating T-cell clone cause renal polymorphic inflammatory fibrosis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Calcineurin Inhibitors Downregulate HNF-1ß and May Affect the Outcome of HNF1B Patients After Renal Transplantation.

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-1ß 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.

Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

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, ß-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 ß-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 ß-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.

Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury.

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.

The HNF1B score is a simple tool to select patients for HNF1B gene analysis.

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.