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BACKGROUND: The von Willebrand factor-directed nanobody caplacizumab has greatly changed the treatment of immune thrombotic thrombocytopenic purpura (iTTP) in recent years. Data from randomized controlled trials established efficacy and safety. OBJECTIVES: This study aims to address open questions regarding patient selection, tailoring of therapy duration, obstacles in prescribing caplacizumab in iTTP, effect on adjunct treatment, and outcomes in the real-world setting. METHODS: We report retrospective, observational cohorts of 113 iTTP episodes treated with caplacizumab and 119 historical control episodes treated without caplacizumab. We aggregated data from the caplacizumab phase II/III trials and real-world data from France, the United Kingdom, Germany, and Austria (846 episodes, 396 treated with caplacizumab, and 450 historical controls). RESULTS: Caplacizumab was efficacious in iTTP, independent of the timing of therapy initiation, but curtailed the time of active iTTP only when used in the first-line therapy within 72 hours after diagnosis and until at least partial ADAMTS13-activity remission. Aggregated data from multiple study populations showed that caplacizumab use resulted in significant absolute risk reduction of 2.87% for iTTP-related mortality (number needed to treat 35) and a relative risk reduction of 59%. CONCLUSION: Caplacizumab should be used in first line and until ADAMTS13-remission, lowers iTTP-related mortality and refractoriness, and decreases the number of daily plasma exchange and hospital stay. This trial is registered at www. CLINICALTRIALS: gov as #NCT04985318.

BACKGROUND Health-related quality of life (HRQL), fatigue, anxiety, and depression are crucial for the living kidney donor (LKD). Follow-up data for HRQL of LKDs comparing surgical techniques, especially regarding hand-assisted retroperitoneoscopic donor nephrectomy (HARP), are sparse. The aim of this study was to evaluate the influence of abdominal wall trauma minimized by HARP in comparison to open anterior approach donor nephrectomy (AA) on HRQL and additional psychosocial aspects of LKDs during the long-term follow-up. MATERIAL AND METHODS This is a cross-sectional study comparing psychosocial aspects of LKD between HARP and AA. RESULTS This study included 100 LKDs (68 HARP, 28 AA, and 4 were excluded secondary to incomplete data). The time to follow-up was 22.6 ± 11.7 (HARP) vs 58.7 ± 13.9 (AA) months (P < .005). Complications ≥3a° due to Clavien-Dindo classification was 0% in both groups. There were higher scores in all physical aspects for HARP donors vs AA donors at that time (physical function: 89.8 ± 14.6 vs 80.0 ± 19.9, P = .008, and the physical component score: 53.9 ± 7.6 vs 48.6 ± 8.5, P = .006). One year later (follow-up time + 12 months), HRQL for HARP donors was still higher. Mental items showed no significant differences. HARP donors showed better physical scores compared to the age-matched nondonor population (AA donors had lower scores). Neither the Multidimensional Fatigue Inventory-20 (MFI-20) or the Hospital Anxiety and Depression Scale (HADS) showed any differences between the 2 groups. Fatigue scores were higher for HARP and for AA compared to the age-matched population. CONCLUSIONS LKDs undergoing HARP showed better physical performance as part of HRQL in the long-term follow-up.

Increasing insight into the clinical phenotype and mechanisms of SARS-CoV-2 infections and COVID-19 has identified damage of the kidneys as a key player in the course of the disease. This manuscript updates our previous summary with current knowledge on kidney involvement in COVID-19 and chronic kidney disease as a risk factor for severe COVID-19, as well as recommendations regarding SARS-CoV-2 vaccination for patients suffering from chronic kidney disease and after organ transplantation, respectively. populations, SARS-CoV-2 vaccination is strongly recommended for all patients suffering from chronic kidney disease and after kidney transplantation.

Increasing insight into the clinical phenotype and mechanisms of SARS-CoV-2 infections and COVID-19 has identified damage of the kidneys as a key player in the course of the disease. This manuscript updates our previous summary with current knowledge on kidney involvement in COVID-19 and chronic kidney disease as a risk factor for severe COVID-19, as well as recommendations regarding SARS-CoV-2 vaccination for patients suffering from chronic kidney disease and after organ transplantation, respectively. populations, SARS-CoV-2 vaccination is strongly recommended for all patients suffering from chronic kidney disease and after kidney transplantation.

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

Increasing insight into the clinical phenotype and mechanisms of SARS-CoV-2 infections and COVID-19 has identified damage of the kidneys as a key player in the course of the disease. This manuscript summarizes the current knowledge on direct viral infection of kidney tissue, proteinuria and acute kidney injury in COVID-19, and management of patients on chronic dialysis as well as after kidney transplantation. Direct infection of podocytes and proximal tubular cells by SARS-CoV-2 has been confirmed and results in proteinuria and hematuria at an early stage of COVID-19. In this context, any kidney affection is a predictor of worse outcomes among COVID-19 patients irrespective of the initial presentation and increases the risk of acute kidney injury. Specific therapies for kidney damage and acute kidney injury within COVID-19 that could be generally recommended are currently lacking. Patients on chronic hemodialysis in particular are at risk for contracting SARS-CoV-2 infections as indicated by outbreaks and super-spreading events in hemodialysis facilities. Immunosuppressive therapy after kidney transplantation needs to be adapted upon diagnosis of COVID-19 depending on the severity of the initial presentation.

Increasing insight into the clinical phenotype and mechanisms of SARS-CoV-2 infections and COVID-19 has identified damage of the kidneys as a key player in the course of the disease. This manuscript summarizes the current knowledge on direct viral infection of kidney tissue, proteinuria and acute kidney injury in COVID-19, and management of patients on chronic dialysis as well as after kidney transplantation. Direct infection of podocytes and proximal tubular cells by SARS-CoV-2 has been confirmed and results in proteinuria and hematuria at an early stage of COVID-19. In this context, any kidney affection is a predictor of worse outcomes among COVID-19 patients irrespective of the initial presentation and increases the risk of acute kidney injury. Specific therapies for kidney damage and acute kidney injury within COVID-19 that could be generally recommended are currently lacking. Patients on chronic hemodialysis in particular are at risk for contracting SARS-CoV-2 infections as indicated by outbreaks and super-spreading events in hemodialysis facilities. Immunosuppressive therapy after kidney transplantation needs to be adapted upon diagnosis of COVID-19 depending on the severity of the initial presentation.

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification,mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo. We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, a-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of a-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

Background Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown. Methods We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli. Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations. Conclusions Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

The transcription factor Wilms’ tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay.

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor(1). Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB)(2). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated(3,4). In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.

In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This “one-segment-one-proteome-approach” defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins. In an attempt to target this protein co-expression module, genetic ablation of LAMP1-correlated lysosomal proteases in mice could ameliorate glomerular damage. Furthermore, individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases demonstrated increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene products. Therefore, increased glomerular lysosomal load is a conserved key mechanism in proteinuric kidney diseases, and the technology applied here can be implemented to address heterogeneous pathophysiology in a variety of diseases at a sub-biopsy scale

Background: The DNA-binding transcription factor Wilms' Tumor Suppressor-1 (WT1) plays an essential role in nephron progenitor differentiation during renal development. We previously used Wt1 chromatin-immunoprecipitation coupled to microarray (ChIP-chip) to identify novel Wt1 target genes that may regulate nephrogenesis in vivo. We discovered that all three members of the SoxC subfamily, namely, Sox4, Sox11, and Sox12, are bound by Wt1 in mouse embryonic kidneys in vivo. SoxC genes play master roles in determining neuronal and mesenchymal progenitor cell fate in a multitude of developmental processes, but their function in the developing kidney is largely unknown. Results: Here we show that all three SoxC genes are expressed in the nephrogenic lineages during renal development. Conditional ablation of Sox4 in nephron progenitors and their cellular descendants (Sox4nephron- mice) results in a significant reduction in nephron endowment. By postnatal day (P)7, Sox4nephron- renal corpuscles exhibit reduced numbers of Wt1+ podocytes together with loss of expression of the slit diaphragm protein nephrin. Sox4nephron- mice develop early-onset proteinacious glomerular injury within 2 weeks of birth progressing to end-stage renal failure within 5–9 months. Conclusions: Collectively, our results demonstrate an essential requirement of Sox4 for normal renal development in vivo. Developmental Dynamics 242:790–799, 2013. © 2013 Wiley Periodicals, Inc.

Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target CTGF was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases. 2017

Autosomal-dominant hypertension and brachydactyly (Online Mendelian Inheritance in Man 112410) is a prototype-translational research project. We used interphase fluorescent in situ hybridization and discovered complex rearrangements on chromosome 12p in 5 families but elucidated a common inverted region in the linkage interval. The inversion contains no known gene. However, we found 5 expressed sequence tags in databases. We used 5′- and 3′-Rapid Amplification of cDNA Ends PCR for elongation of the transcripts in phenotype-relevant tissue (fetal aorta, fetal brain, and fetal cartilage). We detected tissue-specific multiple splicing with different exon usage of 32 exons in the gene-related structure. These different transcripts lack both open reading frames and Kozak sequences. In vitro transcription/translation experiments did not identify any peptide-related molecules. We then performed quantitative RT-PCR to test for differential expression of the various spliced transcripts in the total fibroblast RNA of affected and nonaffected Turkish family members. Skin fibroblasts of affected individuals have a significantly increased proliferation rate compared with nonaffected individuals. Ten of 12 spliced exon combinations representing all of the spliced variants do not show a significantly different RNA expression rate. However, 2 RT-PCR products are exclusively expressed in nonaffected individuals. Both reverse transcription amplicons share 1 exon. This result is surprising because of the autosomal-dominant mode of inheritance of the trait. RNA secondary prediction of this single exon results in a stable stem-loop structure known to be essential for microRNA processing. We are pursuing the possibility of microRNA expression in affected patients that leads to complete down regulation of a spliced transcript.

Development of the metanephric kidney depends on tightly regulated interplay between self-renewal and differentiation of a nephron progenitor cell (NPC) pool. Several key factors required for the survival of NPCs have been identified, including fibroblast growth factor (FGF) signaling and the transcription factor Wilms' tumor suppressor 1 (WT1). Here, we present evidence that WT1 modulates FGF signaling by activating the expression of growth arrest-specific 1 (Gas1), a novel WT1 target gene and novel modulator of FGF signaling. We show that WT1 directly binds to a conserved DNA binding motif within the Gas1 promoter and activates Gas1 mRNA transcription in NPCs. We confirm that WT1 is required for Gas1 expression in kidneys in vivo. Loss of function of GAS1 in vivo results in hypoplastic kidneys with reduced nephron mass due to premature depletion of NPCs. Although kidney development in Gas1 knockout mice progresses normally until E15.5, NPCs show decreased rates of proliferation at this stage and are depleted as of E17.5. Lastly, we show that Gas1 is selectively required for FGF-stimulated AKT signaling in vitro. In summary, our data suggest a model in which WT1 modulates receptor tyrosine kinase signaling in NPCs by directing the expression of Gas1.

Early-onset parkinsonism is frequently reported in connection with mutations in the parkin gene. In this study, we present the results of extensive genetic screening for parkin mutations in 111 community-derived early-onset parkinsonism patients (age of onset

Early-onset parkinsonism (EOP) may be associated with different mutations in the parkin gene, including exon deletions and duplications. To test for gene dosage alterations, we developed a new method of quantitative duplex PCR using the fluorescence resonance energy transfer technique on the LightCycler (Roche Diagnostics). In 21 patients with EOP, three mutations (a single base pair substitution in exon 3 and small deletions in exon 9) were detected by conventional mutational screening (single-strand conformation polymorphism and sequence analysis), while alterations of gene dosage were found in seven patients. We identified heterozygous and compound heterozygous deletions of exons 2, 3, 5 and 7. The latter was also found in the homozygous state. In addition, two heterozygous duplications of exon 4 were observed. Remarkably, two patients carried more than two parkin mutations. This is the first study systematically screening all 12 exons of parkin by real-time, kinetic quantification and clearly shows that mutational analysis of the parkin gene should include gene dosage studies. Furthermore, our method of quantitative PCR is easily applicable to any other gene to be screened for deletions or duplications of whole exons.

A gene for autosomal recessive parkinsonism, PARK2 (parkin), has recently been identified on chromosome 6q and shown to be mutated in Japanese and European families, mostly with early-onset parkinsonism. Here we present a large pedigree from South Tyrol (a region of northern Italy) with adult-onset, clinically typical tremor-dominant parkinsonism of apparently autosomal dominant inheritance. Haplotype analysis excluded linkage to the chromosome 2p, 4p, and 4q regions that harbor genes associated with autosomal dominant parkinsonism, but implicated the parkin locus on chromosome 6q. Compound heterozygous deletions in the parkin gene (one large and one truncating) were identified in 4 affected male siblings. The patients were clinically indistinguishable from most patients with idiopathic Parkinson's disease. None of them displayed any of the clinical hallmarks described in patients with previously reported parkin mutations, including diurnal fluctuations, benefit from sleep, foot dystonia, hyperreflexia, and early susceptibility to levodopa-induced dyskinesias. Two affected female individuals carried one (truncating) of the two deletions in a heterozygous state with an apparently normal allele. We conclude that the phenotypic spectrum associated with mutations in the parkin gene is broader than previously reported, suggesting that this gene may be important in the etiology of the more frequent late-onset typical Parkinson's disease.

Agents that target the activity of the mammalian target of rapamycin (mTOR) kinase in humans are associated with proteinuria. However, the mechanisms underlying mTOR activity and signaling within the kidney are poorly understood. In this study, we developed a sensitive immunofluorescence technique for the evaluation of activated pmTOR and its associated signals in situ. While we find that pmTOR is rarely expressed in normal non-renal tissues, we consistently find intense expression in glomeruli within normal mouse and human kidneys. Using double staining, we find that the expression of pmTOR co-localizes with nephrin in podocytes and expression appears minimal within other cell types in the glomerulus. In addition, we found that pmTOR was expressed on occasional renal tubular cells within mouse and human kidney specimens. We also evaluated mTOR signaling in magnetic bead-isolated glomeruli from normal mice and, by Western blot analysis, we confirmed function of the pathway in glomerular cells vs. interstitial cells. Furthermore, we found that the activity of the pathway as well as the expression of VEGF, a target of mTOR-induced signaling, were reduced within glomeruli of mice following treatment with rapamycin. Collectively, these findings demonstrate that the mTOR signaling pathway is constitutively hyperactive within podocytes. We suggest that pmTOR signaling functions to regulate glomerular homeostasis in part via the inducible expression of VEGF.

The DNA-binding transcription factor Wilms' Tumor Suppressor-1 (WT1) plays an essential role in nephron progenitor differentiation during renal development. We previously used Wt1 chromatin-immunoprecipitation coupled to microarray (ChIP-chip) to identify novel Wt1 target genes that may regulate nephrogenesis in vivo. We discovered that all three members of the SoxC subfamily, namely, Sox4, Sox11, and Sox12, are bound by Wt1 in mouse embryonic kidneys in vivo. SoxC genes play master roles in determining neuronal and mesenchymal progenitor cell fate in a multitude of developmental processes, but their function in the developing kidney is largely unknown.Here we show that all three SoxC genes are expressed in the nephrogenic lineages during renal development. Conditional ablation of Sox4 in nephron progenitors and their cellular descendants (Sox4(nephron-) mice) results in a significant reduction in nephron endowment. By postnatal day (P)7, Sox4(nephron-) renal corpuscles exhibit reduced numbers of Wt1+ podocytes together with loss of expression of the slit diaphragm protein nephrin. Sox4(nephron-) mice develop early-onset proteinacious glomerular injury within 2 weeks of birth progressing to end-stage renal failure within 5-9 months.Collectively, our results demonstrate an essential requirement of Sox4 for normal renal development in vivo.