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BACKGROUND: The cell-matrix adhesion between podocytes and the glomerular basement membrane is essential for the integrity of the kidney’s filtration barrier. Despite increasing knowledge about the complexity of integrin adhesion complexes, an understanding of the regulation of these protein complexes in glomerular disease remains elusive. METHODS: We mapped the in vivo composition of the podocyte integrin adhesome. In addition, we analyzed conditional knockout mice targeting a gene (Parva) that encodes an actin-binding protein (α-parvin), and murine disease models. To evaluate podocytes in vivo, we used super-resolution microscopy, electron microscopy, multiplex immunofluorescence microscopy, and RNA sequencing. We performed functional analysis of CRISPR/Cas9-generated PARVA single knockout podocytes and PARVA and PARVB double knockout podocytes in three- and two-dimensional cultures using specific extracellular matrix ligands and micropatterns. RESULTS: We found that PARVA is essential to prevent podocyte foot process effacement, detachment from the glomerular basement membrane, and the development of FSGS. Through the use of in vitro and in vivo models, we identified an inherent PARVB-dependent compensatory module at podocyte integrin adhesion complexes, sustaining efficient mechanical linkage at the filtration barrier. Sequential genetic deletion of PARVA and PARVB induces a switch in structure and composition of integrin adhesion complexes. This redistribution of these complexes translates into a loss of the ventral actin cytoskeleton, decreased adhesion capacity, impaired mechanical resistance, and dysfunctional extracellular matrix assembly. CONCLUSIONS: The findings reveal adaptive mechanisms of podocyte integrin adhesion complexes, providing a conceptual framework for therapeutic strategies to prevent podocyte detachment in glomerular disease.

Background Diseases of the kidney's glomerular filtration barrier are a leading cause of end-stage renal failure. Despite of a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in focal segmental glomerulosclerosis (FSGS) patient cohorts. However, p.R229Q has been proposed to cause disease only when trans-associated to specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results. Methods To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q). We assessed the phenotype using super-resolution microscopy and albuminuria measurements, and evaluated the stability of the mutant protein in cell culture experiments. Results Heterozygous PodR231Q/wildtype mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous Pod R231Q/wildtype mice showed a more severe course of disease compared with Podwildtype/wildtype mice. Podocin protein levels were decreased in PodR231Q/wildtype and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation Conclusions Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients.

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.

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR–dependent regulatory network for nutrient transport in renal proximal tubular cells.

Background Acute kidney injury is a frequent complication after cardiac surgery and is associated with adverse outcomes. Although short‐term calorie restriction (CR) has proven protective in rodent models of acute kidney injury, similar effects have not yet been demonstrated in humans. Methods and Results CR _KCH (Effect of a Preoperative Calorie Restriction on Renal Function After Cardiac Surgery) is a randomized controlled trial in patients scheduled for cardiac surgery. Patients were randomly assigned to receive either a formula diet containing 60% of the daily energy requirement (CR group) or ad libitum food (control group) for 7 days before surgery. In total, 82 patients were enrolled between April 16, 2012, and February 5, 2015. There was no between‐group difference in the primary end point of median serum creatinine increment after 24 hours (control group: 0.0 mg/dL [−0.1 – (+0.2) mg/dL]; CR group: 0.0 mg/dL [−0.2 – (+0.2) mg/dL]; P =0.39). CR prevented a rise in median creatinine at 48 hours (control group: +0.1 mg/dL [0.0 – 0.3 mg/dL]; CR group: −0.1 mg/dL [−0.2 – (+0.1) mg/dL]; P =0.03), with most pronounced effects observed in male patients and patients with a body mass index >25. This benefit persisted until discharge: Median creatinine decreased by 0.1 mg/dL (−0.2 – 0.0 mg/dL) in the CR group, whereas it increased by 0.1 mg/dL (0.0 – 0.3 mg/dL; P =0.0006) in the control group. Incidence of acute kidney injury was reduced by 5.8% (41.7% in the CR group compared with 47.5% in the control group). Safety‐related events did not differ between groups. Conclusions Despite disappointing results with respect to creatinine rise within the first 24 hours, the benefits observed at later time points and the subgroup analyses suggest the protective potential of short‐term CR in patients at risk for acute kidney injury, warranting further investigation. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 01534364.

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.

Many genes are responsible for the modulation of lifespan in model organisms. In addition to regulating adaptive biologic responses that control stress signaling and longevity, some of these genes participate in tumor formation. The mechanisms that determine longevity and link regulation of lifespan with tumorigenesis are poorly understood. Here, we show that the tumor suppressor von Hippel-Lindau (VHL), which has widely known roles in renal carcinogenesis and the formation of kidney cysts, controls longevity in Caenorhabditis elegans. Loss of vhl-1 significantly increased lifespan and resulted in accelerated basal signaling of the p38 mitogen-activated protein kinase PMK-3. Furthermore, the VHL-1 effect on the regulation of lifespan was independent of the insulin/IGF-1-like signaling pathway, suggesting a mechanism for stress resistance that controls both lifespan and tumorigenesis. These findings define VHL-1 as a player in longevity signaling and connect aging, regulation of lifespan, and stress responses with formation of renal cell carcinomas.

Podocytes are visceral epithelial cells supporting the function of the glomerular filter. Interdigitating foot processes of podocytes enwrap the glomerular capillaries and are connected by a highly specialized cell junction, the slit diaphragm. Signal transduction at the slit diaphragm is essential for the proper function of the kidney filtration barrier. The slit diaphragm constitutes a dynamic multiprotein signaling complex that contains structural proteins, receptors, signaling adaptors, ion channels, and scaffolding proteins. Function of some of these proteins requires cholesterol attached to the multiprotein complex. Recruitment of cholesterol is achieved through the PHB domain protein podocin, a member of a novel family of lipid-binding proteins that are conserved through evolution. The finding that cholesterol interaction regulates the activity of ion channels at the glomerular filtration barrier has important implications for renal physiology and pathophysiology.

Wnt signaling cascades activate morphogenetic programs that range from cell migration and proliferation to cell fate determination and stem cell renewal. These pathways enable cells to translate environmental cues into the complex cellular programs that are needed to organize tissues and build organs. Wnt signaling is essential for renal development; however, the specific molecular underpinnings involved are poorly understood. Recent research has revealed an unexpected intersection between Wnt signaling and polycystic kidney disease. Some polycystic kidney disease proteins, such as Inversin and Bardet-Biedl syndrome family members, were found to use components of the Wnt signaling cascade to orient cells along a secondary polarity axis within the plane of the epithelium. These spatial cues may be needed to position nascent tubules with a defined geometry.

The NEPH family comprises three transmembrane proteins of the Ig superfamily interacting with the glomerular slit diaphragm proteins podocin and ZO-1. NEPH1 binds to nephrin, another component of the slit diaphragm, and loss of either partner causes heavy proteinuria. NEPH2, which is strongly conserved among a large number of species, is also expressed in the kidney; however, its function is unknown. The authors raised NEPH2 antisera to demonstrate NEPH2 expression in a variety of mouse tissues, including the kidney and a podocyte cell line. The authors localized the expression of NEPH2 to the glomerular slit diaphragm by electron microscopy and show NEPH2 homodimerization and specific interactions with the extracellular domain of nephrin in vitro and in vivo. NEPH1, however, failed to interact with NEPH2. The authors detected immunoreactive NEPH2 in urine of healthy subjects, suggesting that the extracellular domain is cleaved under physiologic conditions. These findings were confirmed in vitro in podocyte cell culture. Shedding is increased by tyrosine phosphatase inhibitors and diminished by GM6001, an inhibitor of metalloproteinases. Overexpression experiments indicate an involvement of the MT1-matrix metalloproteinase. The results suggest a role for NEPH2 in the organization and/or maintenance of the glomerular slit diaphragm that may differ from the functions of NEPH1 and nephrin.

The discovery of nephrin and other slit diaphragm proteins dramatically expanded our knowledge of the molecular make-up of the glomerular filter of the kidney. This review focuses on the most recent evidence elucidating the dynamic functions of the slit diaphragm and stresses the importance of slit diaphragm proteins in mediating signal transduction in the podocyte.Nephrin and neph molecules form specialized cell junctions in various systems in different species. The organization of these specialized cell junctions is dependent on intracellular signaling networks. There is a rapidly growing number of identified adapter and signaling molecules that are recruited to this signaling network. These proteins help to maintain programs for cell survival, cell polarity and endocytosis. The latest examples of identified signaling proteins at the slit diaphragm are fyn, p85, and calcium/calmodulin-dependent serine protein kinase. At the level of the slit insertion site, podocin seems to play a pivotal role in the establishment of a lipid-enriched signaling environment. Moreover, the protocadherin FAT1 has been identified as an organizer of actin polymerization and could therefore serve as a linker of the slit diaphragm to cytoskeletal organization.From recent data, a novel concept of a highly dynamic slit protein complex is emerging. Slit diaphragm proteins are contributing to the regulation of cell polarity, cell survival and cytoskeletal organization. This concept is further supported by the fact that many clinically relevant mutations of slit diaphragm proteins interfere with signaling processes at the slit diaphragm and cause proteinuria and progressive kidney disease.

Diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. Recent studies have emphasized the critical role of the slit diaphragm of podocytes for the size-selective filtration barrier of the kidney and revealed novel aspects of the mechanisms that lead to

Nephrin and NEPH1, the gene products of NPHS1 and NEPH1, are podocyte membrane proteins of the Ig super- family. Similar to the nephrin knockout, mice lacking NEPH1 show severe proteinuria leading to perinatal death. To identify the ligand of NEPH1, the extracellular domain of NEPH1 was fused to human IgG. This NEPH1-Ig fusion protein labeled the glomerular capillary wall of mouse kidneys in a staining pat- tern identical to NEPH1 and nephrin, prompting speculation that that NEPH1 might form homodimers and/or heterodimers with nephrin. In coimmunoprecipitation and pull-down assays, the NEPH1-Ig fusion protein precipitated wild-type NEPH1 from overexpressing HEK 293T cells. Truncational analysis revealed that the adhesive properties were not confined to a single Ig domain of NEPH1. Fusion proteins containing two Ig domains of NEPH1 were sufficient to immobilize NEPH1, but they failed to interact with control protein containing the phy- logenetically related PKD repeats of polycystin-1. NEPH1 also precipitated nephrin, a protein with eight Ig domains and a fibronectin-like domain. Truncational analysis of nephrin re- vealed a very similar mode of interaction, i.e., two nephrin Ig domains fused to human IgG precipitated either nephrin or NEPH1, but not the control protein. Both NEPH1 and nephrin interactions were strictly dependent upon posttranslational gly- cosylation, and bacterially expressed protein failed to bind NEPH1. These findings demonstrate that the Ig domains of NEPH1 and nephrin form promiscuous homodimeric and het- erodimeric interactions that may facilitate cis- and trans- ho- modimerizations and heterodimerizations of these molecules at the glomerular slit diaphragm. Renal filtration of small solutes and water without loss of larger molecules is intimately linked to the glomerular base- ment membrane and the slit diaphragm between interdigitating podocytes. Alterations of these structures, either acquired or hereditary, commonly lead to proteinuria. Hereditary nephrotic syndromes are a heterogeneous group, displaying severe proteinuria and renal failure. Best-character- ized is the congenital nephrotic syndrome of the Finnish type, caused by mutations in NPHS1, the gene encoding nephrin. Affected individuals exhibit massive proteinuria in utero and nephrosis at birth (1). Nephrin is an integral membrane protein located at adjacent sites of secondary foot processes of podo- cytes, a specialized epithelial cell that ensures size and charge selective ultrafiltration (reviewed in reference 2). The precise function of nephrin is unknown; however, it appears to form a zipper-like filter structure within the approximately 40-nm- wide slits between two foot processes (3).

Damage or loss of podocytes causes glomerulosclerosis in murine models, and mutations in podocyte-specific genes cause nephrotic syndrome in humans. Zebrafish provide a valuable model for kidney research, but disruption of pronephroi leads to death within a few days, thereby preventing the study of CKD. In this study, we generated an inducible model of podocyte injury in zebrafish (pod::NTR-mCherry) by expressing a bacterial nitroreductase, which converts metronidazole to a cytotoxin, specifically in podocytes under the control of the zebrafish nphs2/podocin promoter. Application of the prodrug metronidazole to the transgenic fish induces acute damage to the podocytes in pronephroi of larval zebrafish and the mesonephroi of adult zebrafish, resulting in foot-process effacement and podocyte loss. We also developed a functional assay of the glomerular filtration barrier by creating transgenic zebrafish expressing green fluorescent protein (GFP)–tagged vitamin D–binding protein (VDBP) as a tracer for proteinuria. In the VDBP-GFP and pod::NTR-mCherry double-transgenic fish, induction of podocyte damage led to whole-body edema, and the proximal tubules reabsorbed and accumulated VDBP-GFP that leaked through the glomeruli, mimicking the phenotype of human nephrotic syndrome. Moreover, expression of wt1b::GFP, a marker for the developing nephron, extended into the Bowman capsule in response to podocyte injury, suggesting that zebrafish have a podocyte-specific repair process known to occur in mammalian metanephros. These data support the use of these transgenic zebrafish as a model system for studies of glomerular pathogenesis and podocyte regeneration.

Background —ACE (kininase II) inhibitors have been shown to exert their beneficial cardiovascular effects via the inhibition of both angiotensin II formation and bradykinin breakdown. Because recent evidence suggests that ACE inhibitors may also interfere with B 2 kinin receptor signaling and thus enhance the vascular response to bradykinin, we examined whether the distribution of B 2 kinin receptors within the plasma membrane of native endothelial cells is affected by an ACE inhibitor. Methods and Results —Localization of the B 2 kinin receptor in membranes prepared from native porcine aortic endothelial cells was evaluated by means of specific [ 3 H]bradykinin binding and immunoprecipitation of the B 2 receptor from isolated membranes. Effects of bradykinin and ramiprilat on intracellular signaling were determined by monitoring the activation of the extracellularly regulated kinases Erk1 and Erk2 as well as [Ca 2+ ] i increases in fura 2–loaded endothelial cells. Stimulation of native endothelial cells with bradykinin 100 nmol/L resulted in the time-dependent sequestration of the B 2 receptor to caveolin-rich (CR) membranes, which was maximal after 5 minutes. Pretreatment with ramiprilat 100 nmol/L for 15 minutes significantly attenuated the recovery of B 2 kinin receptors in CR membranes while increasing that from membranes lacking caveolin. This effect was not due to the inhibition of bradykinin degradation, because no effect was seen in the presence of an inhibitory concentration of the synthetic ACE substrate hippuryl- l -histidyl- l -leucine. Ramiprilat also decreased [ 3 H]bradykinin binding to CR membranes when applied either before or after bradykinin stimulation. Moreover, ramiprilat resulted in reactivation of the B 2 receptor in bradykinin-stimulated cells and induced a second peak in [Ca 2+ ] i and reactivation of Erk1/2. Conclusions —The ACE inhibitor ramiprilat interferes with the targeting of the B 2 kinin receptor to CR membrane domains in native endothelial cells. Therefore, effects other than the inhibition of kininase II may account for the effects of ramiprilat and other ACE inhibitors on the vascular system.

Winkler et al raise an interesting point that perhaps lowering of soluble fms-like tyrosine kinase 1 (sFlt-1) may not have been the sole reason for the therapeutic benefit noted in our study,1 and that lowering other substances such as low-density lipoprotein cholesterol by dextran sulfate cellulose (DSC) apheresis may have contributed to the benefit we observed. In addition, Winkler et al contend that the therapeutic benefit noted in a prior study by Wang et al2 that used heparin-mediated extracorporeal low-density lipoprotein precipitation (HELP) apheresis may have been caused by lowering of low-density lipoprotein cholesterol. We agree with Winkler et al that we cannot conclude lowering sFlt-1 was the sole reason for the therapeutic benefit. Future clinical studies using specific apheretic columns (eg, sFlt-1 antibody columns) are needed to directly answer the contributing role of sFlt-1 in mediating preeclamptic signs and symptoms. We would like to point out that although lipid abnormalities frequently accompany …