PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

PATJ inhibits histone deacetylase 7 to control tight junction formation and cell polarity.

The conserved multiple PDZ-domain containing protein PATJ stabilizes the Crumbs-Pals1 complex to regulate apical-basal polarity and tight junction formation in epithelial cells. However, the molecular mechanism of PATJ's function in these processes is still unclear. In this study, we demonstrate that knockout of PATJ in epithelial cells results in tight junction defects as well as in a disturbed apical-basal polarity and impaired lumen formation in three-dimensional cyst assays. Mechanistically, we found PATJ to associate with and inhibit histone deacetylase 7 (HDAC7). Inhibition or downregulation of HDAC7 restores polarity and lumen formation. Gene expression analysis of PATJ-deficient cells revealed an impaired expression of genes involved in cell junction assembly and membrane organization, which is rescued by the downregulation of HDAC7. Notably, the function of PATJ regulating HDAC7-dependent cilia formation does not depend on its canonical interaction partner, Pals1, indicating a new role of PATJ, which is distinct from its function in the Crumbs complex. By contrast, polarity and lumen phenotypes observed in Pals1- and PATJ-deficient epithelial cells can be rescued by inhibition of HDAC7, suggesting that the main function of this polarity complex in this process is to modulate the transcriptional profile of epithelial cells by inhibiting HDAC7.

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy.

SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

The transcription factor ATF4 mediates endoplasmic reticulum stress-related podocyte injury and slit diaphragm defects.

Mutations in OSGEP and four other genes that encode subunits of the KEOPS complex cause Galloway-Mowat syndrome, a severe, inherited kidney-neurological disease. The complex catalyzes an essential posttranscriptional modification of tRNA and its loss of function induces endoplasmic reticulum (ER) stress. Here, using Drosophila melanogaster garland nephrocytes and cultured human podocytes, we aimed to elucidate the molecular pathogenic mechanisms of KEOPS-related glomerular disease and to test pharmacological inhibition of ER stress-related signaling as a therapeutic principle. We found that ATF4, an ER stress-mediating transcription factor, or its fly orthologue Crc, were upregulated in both fly nephrocytes and human podocytes. Knockdown of Tcs3, a fly orthologue of OSGEP, caused slit diaphragm defects, recapitulating the human kidney phenotype. OSGEP cDNA with mutations found in patients lacked the capacity for rescue. Genetic interaction studies in Tcs3-deficient nephrocytes revealed that Crc mediates not only cell injury, but surprisingly also slit diaphragm defects, and that genetic or pharmacological inhibition of Crc activation attenuates both phenotypes. These findings are conserved in human podocytes where ATF4 inhibition improved the viability of podocytes with OSGEP knockdown, with chemically induced ER stress, and where ATF4 target genes and pro-apoptotic gene clusters are upregulated upon OSGEP knockdown. Thus, our data identify ATF4-mediated signaling as a molecular link among ER stress, slit diaphragm defects, and podocyte injury, and our data suggest that modulation of ATF4 signaling may be a potential therapeutic target for certain podocyte diseases.

Hypoxia hits APOL1 in the kidney.

Individuals of African ancestry carrying two pathogenic variants of apolipoprotein 1 (APOL1) have a substantially increased risk for developing chronic kidney disease. The course of APOL1 nephropathy is extremely heterogeneous and shaped by systemic factors such as a response to interferon. However, additional environmental factors operating in this second-hit model have been less well defined. Here, we reveal that stabilization of hypoxia-inducible transcription factors (HIF) by hypoxia or HIF prolyl hydroxylase inhibitors activates transcription of APOL1 in podocytes and tubular cells. An active regulatory DNA-element upstream of APOL1 that interacted with HIF was identified. This enhancer was accessible preferentially in kidney cells. Importantly, upregulation of APOL1 by HIF was additive to the effects of interferon. Furthermore, HIF stimulated expression of APOL1 in tubular cells derived from the urine of an individual carrying a risk variant for kidney disease. Thus, hypoxic insults may serve as important modulators of APOL1 nephropathy.

Development and Characterization of Hybrid, Temperature Sensing and Heating Yarns with Color Change.

A person's body temperature is an important indicator of their health status. A deviation of that temperature by just 2 °C already has or can lead to serious consequences, such as fever or hypothermia. Hence, the development of a temperature-sensing and heatable yarn is an important step toward enabling and improving the monitoring and regulation of a person's body temperature. This technology offers benefits to several industries, such as health care and sports. This paper focuses on the characterization and development of a hybrid yarn, which can measure and visualize temperature changes through a thermoresistive and thermochromic effect. Moreover, the yarn is able to serve as a flexible heating element by connecting to a power source. The structure of the yarn is designed in three layers. Each layer and component ensures the functionality and flexibility of the yarn and additional compatibility with further processing steps. A flexible stainless steel core was used as the heat-sensitive and heat-conducting material. The layer of polyester wrapped around the stainless steel yarn improves the wearing comfort and serves as substrate material for the thermochromic coating. The resulting hybrid yarn has a reproducible sensory function and changes its resistance by 0.15 Ω between 20 and 60 °C for a length of 30 cm. In addition, the yarn has a uniform and reproducible heating power, so that temperature steps can be achieved at a defined length by selecting certain voltages. The thermochromic color change is clearly visible between 28 and 29 °C. Due to its textile structure, the hybrid sensory and actuating yarn can easily be incorporated into a woven fabric or into a textile by means of joining technology sewing.

Evolution of Renal-Disease Factor APOL1 Results in Cis and Trans Orientations at the Endoplasmic Reticulum That Both Show Cytotoxic Effects.

The recent and exclusively in humans and a few other higher primates expressed APOL1 (apolipoprotein L1) gene is linked to African human trypanosomiasis (also known as African sleeping sickness) as well as to different forms of kidney diseases. Whereas APOL1's role as a trypanolytic factor is well established, pathobiological mechanisms explaining its cytotoxicity in renal cells remain unclear. In this study, we compared the APOL family members using a combination of evolutionary studies and cell biological experiments to detect unique features causal for APOL1 nephrotoxic effects. We investigated available primate and mouse genome and transcriptome data to apply comparative phylogenetic and maximum likelihood selection analyses. We suggest that the APOL gene family evolved early in vertebrates and initial splitting occurred in ancestral mammals. Diversification and differentiation of functional domains continued in primates, including developing the two members APOL1 and APOL2. Their close relationship could be diagnosed by sequence similarity and a shared ancestral insertion of an AluY transposable element. Live-cell imaging analyses showed that both expressed proteins show a strong preference to localize at the endoplasmic reticulum (ER). However, glycosylation and secretion assays revealed that-unlike APOL2-APOL1 membrane insertion or association occurs in different orientations at the ER, with the disease-associated mutants facing either the luminal (cis) or cytoplasmic (trans) side of the ER. The various pools of APOL1 at the ER offer a novel perspective in explaining the broad spectrum of its observed toxic effects.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier.

BACKGROUND: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS). METHODS: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants. RESULTS: Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response. CONCLUSIONS: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling.

BACKGROUND: Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. METHODS: Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. RESULTS: Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)-associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue. CONCLUSIONS: Our results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.

LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes.

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. We identify CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.

α-Galactosidase a Deficiency in Fabry Disease Leads to Extensive Dysregulated Cellular Signaling Pathways in Human Podocytes.

Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.

Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Reactivation of Notch signaling in kidneys of animal models and patients with chronic kidney disease (CKD) has been shown to contribute to epithelial injury and fibrosis development. Here, we investigated the mechanisms of Notch-induced injury in renal epithelial cells. We performed genome-wide transcriptome analysis to identify Notch target genes using an in vitro system of cultured tubular epithelial cells expressing the intracellular domain of Notch1. One of the top downregulated genes was Disabled-2 ( Dab2). With the use of Drosophila nephrocytes as a model system, we found that Dab (the Drosophila homolog of Dab2) knockdown resulted in a significant filtration defect, indicating that loss of Dab2 plays a functional role in kidney disease development. We showed that Dab2 expression in cultured tubular epithelial cells is involved in endocytic regulation and that it also protects cells from TGF-ß-induced epithelial-to-mesenchymal transition. In vivo correlation studies indicated its additional role in renal ischemia-induced injury. Together, these data suggest that Dab2 plays a versatile role in the kidney and may impact on acute and CKDs.-Schütte-Nütgen, K., Edeling, M., Mendl, G., Krahn, M. P., Edemir, B., Weide, T., Kremerskothen, J., Michgehl, U., Pavenstädt, H. Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Nephrin Signaling Results in Integrin ß1 Activation.

BACKGROUND: Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. METHODS: To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. RESULTS: Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin ß1 in cultured podocytes. CONCLUSIONS: Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin ß1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.

WW and C2 domain-containing proteins regulate hepatic cell differentiation and tumorigenesis through the hippo signaling pathway.

The Hippo pathway regulates cell differentiation, proliferation, and apoptosis. Upon activation, it inhibits the import of the transcriptional coactivator yes-associated protein (YAP) into the nucleus, thus suppressing transcription of pro-proliferative genes. Hence, dynamic and precise control of the Hippo pathway is crucial for organ size control and the prevention of tumor formation. Hippo signaling is controlled by a growing number of upstream regulators, including WW and C2 domain-containing (WWC) proteins, which trigger a serine/threonine kinase pathway. One component of this is the large tumor suppressor (LATS) kinase, which phosphorylates YAP, trapping it in the cytoplasm. WWC proteins have been shown to interact with LATS in vitro and stimulate its kinase activity, thus directly promoting cytoplasmic accumulation of phosphorylated YAP. However, the function of the WWC proteins in the regulation of cell proliferation, organ size control, and tumor prevention in vivo has not yet been determined. Here, we show that loss of hepatic WWC expression in mice leads to tissue overgrowth, inflammation, fibrosis, and formation of liver carcinoma. WWC-deficient mouse livers display reduced LATS activity, increased YAP-mediated gene transcription, and enhanced proliferation of hepatic progenitor cells. In addition, loss of WWC expression in the liver accelerates the turnover of angiomotin proteins, which act as negative regulators of YAP activity. CONCLUSION: Our data define an essential in vivo function for WWC proteins as regulators of canonical and noncanonical Hippo signaling in hepatic cell growth and liver tumorigenesis. Thus, expression of WWC proteins may serve as novel prognostic factors in human liver carcinoma. (Hepatology 2018;67:1546-1559).

Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

Podocyte malfunction is central to glomerular diseases and is marked by defective podocyte intercellular junctions and actin cytoskeletal dynamics. Podocytes share many morphologic features with neurons, so that similar sets of proteins appear to regulate cell process formation. One such protein is the tropomyosin-related kinase C (TrkC). TrkC deficiency in mice leads to proteinuria as a surrogate of defective kidney filter function. Activation of endogenous TrkC by its ligand neurotrophin-3 resulted in increased podocyte migration-a surrogate of podocyte actin dynamics in vivo. Employing a mutagenesis approach, we found that the Src homologous and collagen-like (Shc) binding site Tyr516 within the TrkC cytoplasmic domain was necessary for TrkC-induced migration of podocytes. TrkC activation led to a mobility shift of Wiskott-Aldrich syndrome family verprolin-homologous protein (WAVE)-2 which is known to orchestrate Arp2/3 activation and actin polymerization. Chemical inactivation of Erk or mutagenesis of 2 of 4 known Erk target sites within WAVE2, Thr346 and Ser351, abolished the TrkC-induced WAVE2 mobility shift. Knockdown of WAVE2 by shRNA abolished TrkC-induced podocyte migration. In summary, TrkC signals to the podocyte actin cytoskeleton to induce migration by phosphorylating WAVE2 Erk dependently. This signaling mechanism may be important for TrkC-mediated cytoskeletal dynamics in podocyte disease.-Gromnitza, S., Lepa, C., Weide, T., Schwab, A., Pavenstädt, H., George, B. Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

Cellular vacuolization caused by overexpression of the PIKfyve-binding deficient Vac14L156R is rescued by starvation and inhibition of vacuolar-ATPase.

Phosphoinositides (PI) and converting enzymes are crucial determinants of organelle identity and morphology. One important endolysosomal specific PI is PI(3,5)P2, generated by the PIKfyve kinase, which orchestrates in combination with Vac14 and Fig4. Dysfunction of this complex leads to large intracellular vacuoles in various cell types and is linked to neurological diseases. Here, we characterize the vacuolization phenotype caused by overexpression of the PIKfyve binding deficient mutant Vac14L156R in podocytes, which represent specialized cells of the kidney. Vacuolization of podocytes, which was associated with strong maturation defects in the endolysosomal system, could be completely rescued by starvation or treatment of cells with the v-ATPase inhibitor Bafilomycin A1. Moreover, we elucidated a strong and reversible de-vacuolization effect of the cholesterol export inhibitor U18666A, which was accompanied by increased basification of the lysosomal pH values. Taken together, our data give new hints to potential therapeutic targets in the treatment of disease linked to intracellular vacuolization.

Nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant results in albuminuria and mesangial expansion.

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles-observed in many histologic analyses of renal diseases-is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant led to albuminuria, accompanied by mesangial expansion, increased glomerular size, and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14L156R in Madin-Darby canine kidney cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca2+ depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14L156R-overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.

Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, l-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In addition, treatment with AngII led to LCP1 redistribution to the cell margins, membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in podocytes.-Schenk, L. K., Möller-Kerutt, A., Klosowski, R., Wolters, D., Schaffner-Reckinger, E., Weide, T., Pavenstädt, H., Vollenbröker, B. Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Distinct functions of Crumbs regulating slit diaphragms and endocytosis in Drosophila nephrocytes.

Mammalian podocytes, the key determinants of the kidney's filtration barrier, differentiate from columnar epithelial cells and several key determinants of apical-basal polarity in the conventional epithelia have been shown to regulate podocyte morphogenesis and function. However, little is known about the role of Crumbs, a conserved polarity regulator in many epithelia, for slit-diaphragm formation and podocyte function. In this study, we used Drosophila nephrocytes as model system for mammalian podocytes and identified a conserved function of Crumbs proteins for cellular morphogenesis, nephrocyte diaphragm assembly/maintenance, and endocytosis. Nephrocyte-specific knock-down of Crumbs results in disturbed nephrocyte diaphragm assembly/maintenance and decreased endocytosis, which can be rescued by Drosophila Crumbs as well as human Crumbs2 and Crumbs3, which were both expressed in human podocytes. In contrast to the extracellular domain, which facilitates nephrocyte diaphragm assembly/maintenance, the intracellular FERM-interaction motif of Crumbs is essential for regulating endocytosis. Moreover, Moesin, which binds to the FERM-binding domain of Crumbs, is essential for efficient endocytosis. Thus, we describe here a new mechanism of nephrocyte development and function, which is likely to be conserved in mammalian podocytes.

Intracellular APOL1 Risk Variants Cause Cytotoxicity Accompanied by Energy Depletion.

Population genetic approaches have uncovered a strong association between kidney diseases and two sequence variants of the APOL1 gene, called APOL1 risk variant G1 and variant G2, compared with the nonrisk G0 allele. However, the mechanism whereby these variants lead to disease manifestation and, in particular, whether this involves an intracellular or extracellular pool of APOL1 remains unclear. Herein, we show a predominantly intracellular localization of APOL1 G0 and the renal risk variants, which localized to membranes of the endoplasmic reticulum in podocyte cell lines. This localization did not depend on the N-terminal signal peptide that mediates APOL1 secretion into the circulation. Additionally, a fraction of these proteins localized to structures surrounding mitochondria. In vitro overexpression of G1 or G2 lacking the signal peptide inhibited cell viability, triggered phosphorylation of stress-induced kinases, increased the phosphorylation of AMP-activated protein kinase, reduced intracellular potassium levels, and reduced mitochondrial respiration rates. These findings indicate that functions at intracellular membranes, specifically those of the endoplasmic reticulum and mitochondria, are crucial factors in APOL1 renal risk variant-mediated cell injury.