Three-dimensional architecture of nephrons in the normal and cystic kidney.

Kidney function is crucially dependent on the complex three-dimensional structure of nephrons. Any distortion of their shape may lead to kidney dysfunction. Traditional histological methods present major limitations for three-dimensional tissue reconstruction. Here, we combined tissue clearing, multi-photon microscopy and digital tracing for the reconstruction of single nephrons under physiological and pathological conditions. Sets of nephrons differing in location, shape and size according to their function were identified. Interestingly, nephrons tend to lie in planes. When this technique was applied to a model of cystic kidney disease, cysts were found to develop only in specific nephron segments. Along the same segment, cysts are contiguous within normal non-dilated tubules. Moreover, the shapes of cysts varied according to the nephron segment. Thus, our findings provide a valuable strategy for visualizing the complex structure of kidneys at the single nephron level and, more importantly, provide a basis for understanding pathological processes such as cystogenesis.

MITF - A controls branching morphogenesis and nephron endowment.

Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment.

Human mutations affect the epigenetic/bookmarking function of HNF1B.

Bookmarking factors are transcriptional regulators involved in the mitotic transmission of epigenetic information via their ability to remain associated with mitotic chromatin. The mechanisms through which bookmarking factors bind to mitotic chromatin remain poorly understood. HNF1ß is a bookmarking transcription factor that is frequently mutated in patients suffering from renal multicystic dysplasia and diabetes. Here, we show that HNF1ß bookmarking activity is impaired by naturally occurring mutations found in patients. Interestingly, this defect in HNF1ß mitotic chromatin association is rescued by an abrupt decrease in temperature. The rapid relocalization to mitotic chromatin is reversible and driven by a specific switch in DNA-binding ability of HNF1ß mutants. Furthermore, we demonstrate that importin-ß is involved in the maintenance of the mitotic retention of HNF1ß, suggesting a functional link between the nuclear import system and the mitotic localization/translocation of bookmarking factors. Altogether, our studies have disclosed novel aspects on the mechanisms and the genetic programs that account for the mitotic association of HNF1ß, a bookmarking factor that plays crucial roles in the epigenetic transmission of information through the cell cycle.

Stat3 Controls Tubulointerstitial Communication during CKD.

In CKD, tubular cells may be involved in the induction of interstitial fibrosis, which in turn, leads to loss of renal function. However, the molecular mechanisms that link tubular cells to the interstitial compartment are not clear. Activation of the Stat3 transcription factor has been reported in tubular cells after renal damage, and Stat3 has been implicated in CKD progression. Here, we combined an experimental model of nephron reduction in mice from different genetic backgrounds and genetically modified animals with in silico and in vitro experiments to determine whether the selective activation of Stat3 in tubular cells is involved in the development of interstitial fibrosis. Nephron reduction caused Stat3 phosphorylation in tubular cells of lesion-prone mice but not in resistant mice. Furthermore, specific deletion of Stat3 in tubular cells significantly reduced the extent of interstitial fibrosis, which correlated with reduced fibroblast proliferation and matrix synthesis, after nephron reduction. Mechanistically, in vitro tubular Stat3 activation triggered the expression of a specific subset of paracrine profibrotic factors, including Lcn2, Pdgfb, and Timp1. Together, our results provide a molecular link between tubular and interstitial cells during CKD progression and identify Stat3 as a central regulator of this link and a promising therapeutic target.

Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of ß2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

Hepatocyte nuclear factor 1ß controls nephron tubular development.

Nephron morphogenesis is a complex process that generates blood-filtration units (glomeruli) connected to extremely long and patterned tubular structures. Hepatocyte nuclear factor 1ß (HNF1ß) is a divergent homeobox transcription factor that is expressed in kidney from the first steps of nephrogenesis. Mutations in HNF1B (OMIM #137920) are frequently found in patients with developmental renal pathologies, the mechanisms of which have not been completely elucidated. Here we show that inactivation of Hnf1b in the murine metanephric mesenchyme leads to a drastic tubular defect characterized by the absence of proximal, distal and Henle's loop segments. Nephrons were eventually characterized by glomeruli, with a dilated urinary space, directly connected to collecting ducts via a primitive and short tubule. In the absence of HNF1ß early nephron precursors gave rise to deformed S-shaped bodies characterized by the absence of the typical bulge of epithelial cells at the bend between the mid and lower segments. The lack of this bulge eventually led to the absence of proximal tubules and Henle's loops. The expression of several genes, including Irx1, Osr2 and Pou3f3, was downregulated in the S-shaped bodies. We also observed decreased expression of Dll1 and the consequent defective activation of Notch in the prospective tubular compartment of comma- and S-shaped bodies. Our results reveal a novel hierarchical relationship between HNF1ß and key genes involved in renal development. In addition, these studies define a novel structural and functional component of S-shaped bodies at the origin of tubule formation.

Hepatocyte nuclear factor 1alpha and beta control terminal differentiation and cell fate commitment in the gut epithelium.

The intestinal epithelium is a complex system characterized by massive and continuous cell renewal and differentiation. In this context, cell-type-specific transcription factors are thought to play a crucial role by modulating specific transcription networks and signalling pathways. Hnf1alpha and beta are closely related atypical homeoprotein transcription factors expressed in several epithelia, including the gut. With the use of a conditional inactivation system, we generated mice in which Hnf1b is specifically inactivated in the intestinal epithelium on a wild-type or Hnf1a(-/-) genetic background. Whereas the inactivation of Hnf1a or Hnf1b alone did not lead to any major intestinal dysfunction, the concomitant inactivation of both genes resulted in a lethal phenotype. Double-mutant animals had defective differentiation and cell fate commitment. The expression levels of markers of all the differentiated cell types, both enterocytes and secretory cells, were affected. In addition, the number of goblet cells was increased, whereas mature Paneth cells were missing. At the molecular level, we show that Hnf1alpha and beta act upstream of the Notch pathway controlling directly the expression of two crucial components: Jag1 and Atoh1. We demonstrate that the double-mutant mice present with a defect in intestinal water absorption and that Hnf1alpha and beta directly control the expression of Slc26a3, a gene whose mutations are associated with chloride diarrhoea in human patients. Our study identifies new direct target genes of the Hnf1 transcription factors and shows that they play crucial roles in both defining cell fate and controlling terminal functions in the gut epithelium.

A specific molecular signature in SARS-CoV-2-infected kidney biopsies.

Acute kidney injury is one of the most important complications in patients with COVID-19 and is considered a negative prognostic factor with respect to patient survival. The occurrence of direct infection of the kidney by SARS-CoV-2, and its contribution to the renal deterioration process, remain controversial issues. By studying 32 renal biopsies from patients with COVID-19, we verified that the major pathological feature of COVID-19 is acute tubular injury (ATI). Using single-molecule fluorescence in situ hybridization, we showed that SARS-CoV-2 infected living renal cells and that infection, which paralleled renal angiotensin-converting enzyme 2 expression levels, was associated with increased death. Mechanistically, a transcriptomic analysis uncovered specific molecular signatures in SARS-CoV-2-infected kidneys as compared with healthy kidneys and non-COVID-19 ATI kidneys. On the other hand, we demonstrated that SARS-CoV-2 and hantavirus, 2 RNA viruses, activated different genetic networks despite triggering the same pathological lesions. Finally, we identified X-linked inhibitor of apoptosis-associated factor 1 as a critical target of SARS-CoV-2 infection. In conclusion, this study demonstrated that SARS-CoV-2 can directly infect living renal cells and identified specific druggable molecular targets that can potentially aid in the design of novel therapeutic strategies to preserve renal function in patients with COVID-19.

A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes.

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

Genome-wide discovery of functional transcription factor binding sites by comparative genomics: the case of Stat3.

The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. However, the use of high throughput experimental methods, such as ChIP-chip and ChIP-sequencing, is limited by their high cost and strong dependence on cellular type and context. We developed a computational method for the genome-wide identification of functional transcription factor binding sites based on positional weight matrices, comparative genomics, and gene expression profiling. The method was applied to Stat3, a transcription factor playing crucial roles in inflammation, immunity and oncogenesis, and able to induce distinct subsets of target genes in different cell types or conditions. A newly generated positional weight matrix enabled us to assign affinity scores of high specificity, as measured by EMSA competition assays. Phylogenetic conservation with 7 vertebrate species was used to select the binding sites most likely to be functional. Validation was carried out on predicted sites within genes identified as differentially expressed in the presence or absence of Stat3 by microarray analysis. Twelve of the fourteen sites tested were bound by Stat3 in vivo, as assessed by Chromatin Immunoprecipitation, allowing us to identify 9 Stat3 transcriptional targets. Given its high validation rate, and the availability of large transcription factor-dependent gene expression datasets obtained under diverse experimental conditions, our approach appears to be a valid alternative to high-throughput experimental assays for the discovery of novel direct targets of transcription factors.

The transcription factor HNF1α regulates expression of chloride-proton exchanger ClC-5 in the renal proximal tubule.

The Cl(-)/H(+) exchanger ClC-5 is essential for the endocytic activity of the proximal tubule cells and the tubular clearance of proteins filtered in the glomeruli. The mechanisms that regulate the expression of ClC-5 in general and its specific expression in the proximal tubule are unknown. In this study, we investigated the hypothesis that the hepatocyte nuclear transcription factor HNF1α, which is predominantly expressed in proximal tubule segments, may directly regulate the expression of ClC-5. In situ hybridization demonstrated that the expression of Clcn5 overlaps with that of Hnf1α in the developing kidney as well as in absorptive epithelia, including the digestive tract and yolk sac. Multiple binding sites for HNF1 were mapped in the 5'-regulatory sequences of the mouse and human Clcn5/CLCN5 genes. The transactivation of the Clcn5/CLCN5 promoter by HNF1α was verified in vitro, and the binding of HNF1α to the Clcn5 promoter in vivo was confirmed by chromatin immunoprecipitation in mouse kidney. The expression of Clcn5 was reduced in the proximal tubule segments of HNF1α-null kidneys, and it was rescued upon transfection of HNF1α-null cells with wild-type but not with mutant HNF1α. These data demonstrate that HNF1α directly regulates the expression of ClC-5 in the renal proximal tubule and yield insights into the mechanisms governing epithelial differentiation and specialized transport activities in the kidney.

Developmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease.

The architecture of renal glomeruli is acquired through intricate and still poorly understood developmental steps. In our study we identify a crucial glomerular morphogenetic event in nephrogenesis that drives the remodeling/separation of the prospective vascular pole (the future entrance of the glomerular arterioles) and the urinary pole (the tubular outflow). We demonstrate that this remodeling is genetically programmed. In fact, in mouse and human, the absence of HNF1B impairs the remodeling/separation of the two poles, leading to trapping and constriction of the tubular outflow inside the glomerulus. This aberration gives rise to obstructive glomerular dilations upon the initiation of primary urine production. In this context, we show that pharmacological decrease of glomerular filtration significantly contains cystic expansion. From a developmental point of view, our study discloses a crucial event on glomerular patterning affecting the "inside-outside" fate of the epithelia in the renal glomerulus.

Signaling pathways predisposing to chronic kidney disease progression.

The loss of functional nephrons after kidney injury triggers the compensatory growth of the remaining ones to allow functional adaptation. However, in some cases, these compensatory events activate signaling pathways that lead to pathological alterations and chronic kidney disease. Little is known about the identity of these pathways and how they lead to the development of renal lesions. Here, we combined mouse strains that differently react to nephron reduction with molecular and temporal genome-wide transcriptome studies to elucidate the molecular mechanisms involved in these events. We demonstrated that nephron reduction led to 2 waves of cell proliferation: the first one occurred during the compensatory growth regardless of the genetic background, whereas the second one occurred, after a quiescent phase, exclusively in the sensitive strain and accompanied the development of renal lesions. Similarly, clustering by coinertia analysis revealed the existence of 2 waves of gene expression. Interestingly, we identified type I interferon (IFN) response as an early (first-wave) and specific signature of the sensitive (FVB/N) mice. Activation of type I IFN response was associated with G1/S cell cycle arrest, which correlated with p21 nuclear translocation. Remarkably, the transient induction of type I IFN response by poly(I:C) injections during the compensatory growth resulted in renal lesions in otherwise-resistant C57BL6 mice. Collectively, these results suggest that the early molecular and cellular events occurring after nephron reduction determine the risk of developing late renal lesions and point to type I IFN response as a crucial event of the deterioration process.

A genomic map of p53 binding sites identifies novel p53 targets involved in an apoptotic network.

The transcriptional activity of the p53 protein is central to its role in tumor suppression. Identification of the complete repertoire of p53-regulated genes is critical for dissecting the complexity of the p53 network. Although several different approaches have been used to characterize the p53 genetic program, we still lack a comprehensive molecular understanding of how p53 prevents cancer. Using a computational approach, we generated a genome-wide map of p53 binding sites (p53BS) to identify novel p53 target genes. We show that the presence of nearby p53BS can identify new proapoptotic members of the Bcl2 family. We show that p53 binds to p53BS identified in the BCL-G/BCL2L14 gene and that induction of this gene contributes to p53-mediated apoptosis. We found that p53 activates the COL18A1 gene encoding the precursor for the antiangiogenic factor endostatin. We also show that p53 up-regulates the MAP4K4 gene and activates the c-Jun NH2-terminal kinase (JNK) pathway to drive apoptosis. Thus, unbiased mapping of the genomic landscape of p53BS provides a systematic and complementary approach to identify novel factors and connections in the p53 genetic network. Our study illustrates how systematic genomic approaches can identify binding sites that are functionally relevant for a p53 transcriptional program. The genetic link among p53, antiangiogenic factors, and the JNK signaling pathway adds new dimensions to understanding p53 function in highly connected genetic networks.

A suppressor locus for MODY3-diabetes.

Maturity Onset Diabetes of the Young type 3 (MODY3), linked to mutations in the transcription factor HNF1A, is the most prevalent form of monogenic diabetes mellitus. HNF1alpha-deficiency leads to defective insulin secretion via a molecular mechanism that is still not completely understood. Moreover, in MODY3 patients the severity of insulin secretion can be extremely variable even in the same kindred, indicating that modifier genes may control the onset of the disease. With the use of a mouse model for HNF1alpha-deficiency, we show here that specific genetic backgrounds (C3H and CBA) carry a powerful genetic suppressor of diabetes. A genome scan analysis led to the identification of a major suppressor locus on chromosome 3 (Moda1). Moda1 locus contains 11 genes with non-synonymous SNPs that significantly interacts with other loci on chromosomes 4, 11 and 18. Mechanistically, the absence of HNF1alpha in diabetic-prone (sensitive) strains leads to postnatal defective islets growth that is remarkably restored in resistant strains. Our findings are relevant to human genetics since Moda1 is syntenic with a human locus identified by genome wide association studies of fasting glycemia in patients. Most importantly, our results show that a single genetic locus can completely suppress diabetes in Hnf1a-deficiency.

A mitotic transcriptional switch in polycystic kidney disease.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD). We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemia-reperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1beta. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin. We propose that transcription factors such as HNF-1beta might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1beta remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1beta is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing.

The SWI/SNF chromatin-remodeling complex subunit SNF5 is essential for hepatocyte differentiation.

Regulation of gene expression underlies cell differentiation and organogenesis. Both transcription factors and chromatin modifiers are crucial for this process. To study the role of the ATP-dependent SWI/SNF chromatin-remodeling complex in cell differentiation, we inactivated the gene encoding the core complex subunit SNF5/INI1 in the developing liver. Hepatic SNF5 deletion caused neonatal death due to severe hypoglycemia; mutant animals fail to store glycogen and have impaired energetic metabolism. The formation of a hepatic epithelium is also affected in SNF5-deficient livers. Transcriptome analyses showed that SNF5 inactivation is accompanied by defective transcriptional activation of 70% of the genes that are normally upregulated during liver development. These include genes involved in glycogen synthesis, gluconeogenesis and cell-cell adhesion. A fraction of hepatic developmentally activated genes were normally expressed, suggesting that cell differentiation was not completely blocked. Moreover, SNF5-deleted cells showed increased proliferation and we identified several misexpressed genes that may contribute to cell cycle deregulation in these cells. Our results emphasize the role of chromatin remodeling in the activation of cell-type-specific genetic programs and driving cell differentiation.

A transcriptional network in polycystic kidney disease.

Mutations in cystic kidney disease genes represent a major genetic cause of end-stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1beta (HNF1beta) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal-specific inactivation of HNF1beta develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1beta binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1beta and cystic disease genes. Interestingly, most of the identified HNF1beta target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1beta.