Unraveling the role of natriuretic peptide clearance receptor (NPR3) in glomerular diseases.

Natriuretic peptides (NPs) are cardio-derived hormones that have a crucial role in maintaining cardiovascular homeostasis. Physiological effects of NPs are mediated by binding to natriuretic peptide receptors 1 and 2 (NPR1/2), whereas natriuretic peptide receptor 3 (NPR3) acts as a clearance receptor that removes NPs from the circulation. Mouse studies have shown that local NP-signaling in the kidney glomerulus is important for the maintenance of renal homeostasis. In this study we examined the expression of NPR3 in kidney tissue and explored its involvement in renal physiology and disease by generating podocyte-specific knockout mice (NPR3podKO) as well as by using an NPR3 inhibitor (NPR3i) in rodent models of kidney disease. NPR3 was highly expressed by podocytes. NPR3podKO animals showed no renal abnormalities under healthy conditions and responded similarly to nephrotoxic serum (NTS) induced glomerular injury. However, NPR3i showed reno-protective effects in the NTS-induced model evidenced by decreased glomerulosclerosis and reduced podocyte loss. In a ZSF1 rat model of diabetic kidney injury, therapy alone with NPR3i did not have beneficial effects on renal function/histology, but when combined with losartan (angiotensin receptor blocker), NPR3i potentiated its ameliorative effects on albuminuria. In conclusion, these results suggest that NPR3 may contribute to kidney disease progression.

Surveillance for Nonmuscle Invasive Bladder Cancer: Identifying the Point of Diminishing Returns.

OBJECTIVE: To characterize first and second recurrence patterns using 26years of cohort-level follow-up and microsimulation modeling. METHODS: Patients diagnosed with nonmuscle-invasive bladder cancer in Stockholm County between 1995 and 1996 were included. Clinical, pathological, and longitudinal follow-up data were gathered. Logistic regressions, Kaplan Meier curves, and Cox proportional hazards models were run to generate assumptions for a microsimulation model, simulating first and second recurrence and progression for 10,000 patients. RESULTS: Three hundred eighty-six patients were included: 67.4% were male; >50% were TaLG; and 37.5% were American Urological Association high-risk. Median time to recurrence was 300days. Three patients had missing data. Cohort follow-up has been carried out for 26years. For simulated first-recurrences, low-risk patients recurred at 56.6% over 15years of follow-up, with 2.2% muscle-invasive (MI) progression; intermediate-risk patients recurred at 62.8%, with 4.3% MI progression; high-risk patients recurred at 48.7% over 15years, with MI progression at 14.3%. For second recurrences, 70.7%, 75.7%, and 84.7% of low, medium, and high-risk patients recurred. No patients were seen to have first recurrences after 9years, with low, but notable, rates beyond 5years. CONCLUSION: These data suggest that low-, intermediate-, and high-risk patients without recurrence at 5years may be potentially transitioned to less invasive monitoring.

A sulfonamide chalcone AMPK activator ameliorates hyperglycemia and diabetic nephropathy in db/db mice.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM), which currently lacks effective treatments. AMP-activated protein kinase (AMPK) stimulation by chalcones, a class of polyphenols abundantly found in plants, is proposed as a promising therapeutic approach for DM. This study aimed to identify novel chalcone derivatives with improved AMPK-stimulating activity in human podocytes and evaluate their mechanisms of action as well as in vivo efficacy in a mouse model of DN. Among 133 chalcone derivatives tested, the sulfonamide chalcone derivative IP-004 was identified as the most potent AMPK activator in human podocytes. Western blot analyses, intracellular calcium measurements and molecular docking simulation indicated that IP-004 activated AMPK by mechanisms involving direct binding at allosteric site of calcium-dependent protein kinase kinase ß (CaMKKß) without affecting intracellular calcium levels. Interestingly, eight weeks of intraperitoneal administration of IP-004 (20 mg/kg/day) significantly decreased fasting blood glucose level, activated AMPK in the livers, muscles and glomeruli, and ameliorated albuminuria in db/db type II diabetic mice. Collectively, this study identifies a novel chalcone derivative capable of activating AMPK in vitro and in vivo and exhibiting efficacy against hyperglycemia and DN in mice. Further development of AMPK activators based on chalcone derivatives may provide an effective treatment of DN.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

Deep learning-based segmentation and quantification of podocyte foot process morphology suggests differential patterns of foot process effacement across kidney pathologies.

Morphological alterations at the kidney filtration barrier increase intrinsic capillary wall permeability resulting in albuminuria. However, automated, quantitative assessment of these morphological changes has not been possible with electron or light microscopy. Here we present a deep learning-based approach for segmentation and quantitative analysis of foot processes in images acquired with confocal and super-resolution fluorescence microscopy. Our method, Automatic Morphological Analysis of Podocytes (AMAP), accurately segments podocyte foot processes and quantifies their morphology. AMAP applied to a set of kidney diseases in patient biopsies and a mouse model of focal segmental glomerulosclerosis allowed for accurate and comprehensive quantification of various morphometric features. With the use of AMAP, detailed morphology of podocyte foot process effacement was found to differ between categories of kidney pathologies, showed detailed variability between diverse patients with the same clinical diagnosis, and correlated with levels of proteinuria. AMAP could potentially complement other readouts such as various omics, standard histologic/electron microscopy and blood/urine assays for future personalized diagnosis and treatment of kidney disease. Thus, our novel finding could have implications to afford an understanding of early phases of kidney disease progression and may provide supplemental information in precision diagnostics.

Soluble Klotho protects against glomerular injury through regulation of ER stress response.

αKlotho (Klotho) has well established renoprotective effects; however, the molecular pathways mediating its glomerular protection remain incompletely understood. Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases.

The role of dendrin in IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) and its systemic variant IgA vasculitis (IgAV) damage the glomeruli, resulting in proteinuria, hematuria and kidney impairment. Dendrin is a podocyte-specific protein suggested to be involved in the pathogenesis of IgAN. Upon cell injury, dendrin translocates from the slit diaphragm to the nucleus, where it is suggested to induce apoptosis and cytoskeletal changes, resulting in proteinuria and accelerated disease progression in mice. Here we investigated gene and protein expression of dendrin in relation to clinical and histopathological findings to further elucidate its role in IgAN/IgAV. METHODS: Glomerular gene expression was measured using microarray on 30 IgAN/IgAV patients, 5 patients with membranous nephropathy (MN) and 20 deceased kidney donors. Dendrin was spatially evaluated on kidney tissue sections by immunofluorescence (IF) staining (IgAN patients, n = 4; nephrectomized kidneys, n = 3) and semi-quantified by immunogold electron microscopy (IgAN/IgAV patients, n = 21; MN, n = 5; living kidney donors, n = 6). Histopathological grading was performed according to the Oxford and Banff classifications. Clinical data were collected at the time of biopsy and follow-up. RESULTS: Dendrin mRNA levels were higher (P = .01) in IgAN patients compared with MN patients and controls and most prominently in patients with preserved kidney function and fewer chronic histopathological changes. Whereas IF staining did not differ between groups, immunoelectron microscopy revealed that a higher relative nuclear dendrin concentration in IgAN patients was associated with a slower annual progression rate and milder histopathological changes. CONCLUSION: Dendrin messenger RNA levels and relative nuclear protein concentrations are increased and associated with a more benign phenotype and progression in IgAN/IgAV patients.

A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

Molecular insights into the early stage of glomerular injury in IgA nephropathy using single-cell RNA sequencing.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and defined by the presence of IgA-containing immune complexes in the mesangium that induce an inflammation leading to glomerulonephritis. Since we poorly understand early mechanisms of glomerular injury in IgAN we performed single-cell RNA sequencing (scRNA-seq) analysis of glomerulus-associated cells using SMARTseq2-technology at the early stage of IgAN in grouped ddY-mice. Cell-specific molecular signatures unraveled a key role of endothelial cells in the early pathogenesis of IgAN, especially in the recruitment and infiltration of immune cells. Mesangial and podocyte cells demonstrated less molecular changes. Several intra-glomerular paracrine pathways were detected, such as mesangial cell-derived Slit3 potentially activating Robo-receptors in podocyte/endothelial cells. Surprisingly, proximal tubular cells were strongly affected at the early stage and potential glomerulo-tubular cell-cell crosstalk pathways were identified. Importantly, many of the cellular transcriptomic signatures identified in this well-established mouse model were also detected in published bulk transcriptomic data in human IgAN. Moreover, we validated the functionality of key cell-cell crosstalk pathways using cell culture models, such as the effect of the Slit-Robo signalling axis. Thus, our study provides important novel molecular insights into the pathogenesis of early IgAN-associated glomerulopathy.

Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway.

Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.

Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes.

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.

A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales.

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines.

Inactivation of mediator complex protein 22 in podocytes results in intracellular vacuole formation, podocyte loss and premature death.

Podocytes are critical for the maintenance of kidney ultrafiltration barrier and play a key role in the progression of glomerular diseases. Although mediator complex proteins have been shown to be important for many physiological and pathological processes, their role in kidney tissue has not been studied. In this study, we identified a mediator complex protein 22 (Med22) as a renal podocyte cell-enriched molecule. Podocyte-specific Med22 knockout mouse showed that Med22 was not needed for normal podocyte maturation. However, it was critical for the maintenance of podocyte health as the mice developed progressive glomerular disease and died due to renal failure. Detailed morphological analyses showed that Med22-deficiency in podocytes resulted in intracellular vacuole formation followed by podocyte loss. Moreover, Med22-deficiency in younger mice promoted the progression of glomerular disease, suggesting Med22-mediated processes may have a role in the development of glomerulopathies. This study shows for the first time that mediator complex has a critical role in kidney physiology.

Novel insights into the disease transcriptome of human diabetic glomeruli and tubulointerstitium.

BACKGROUND: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown. METHODS: RNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30-85) years, chronic kidney disease stages 1-4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30-70) years]. RESULTS: Principal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients. CONCLUSIONS: Here we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.

GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway.

BACKGROUND: Inflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood. METHODS: We performed large-scale gene expression profiling of glomerulus-associated G protein-coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes. RESULTS: We identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-κB signaling. CONCLUSIONS: GPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-κB signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.

Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement.

Podocytes have an important role in the pathogenesis of diabetic nephropathy (DN). Podocyte foot process effacement, mediated largely by the actin-based cytoskeleton of foot processes, is commonly detected in DN and is believed to be a key pathogenic event in the development of proteinuria. In this study, we identified coronin 2b (Coro2b), a member of known actin-regulating proteins, the coronins, as a highly podocyte-enriched molecule located at the cytoplasmic side of the apical plasma membrane. Studies in human renal biopsies show that glomerular Coro2b expression is significantly down-regulated in patients with DN. Studies in knockout mice indicate that Coro2b is not required for the development or maintenance of the glomerular filtration barrier. Moreover, inactivation of Coro2b specifically in podocytes does not affect the outcome of nephropathy in a streptozotocin-induced diabetes model. However, Coro2b seems to modulate the reorganization of foot processes under pathological conditions as Coro2b knockout podocytes are partially protected from protamine sulfate perfusion-induced foot process effacement. Taken together, our study suggests a role for Coro2b in the pathogenesis of glomerulopathies. Further studies regarding the involvement of Coro2b in podocyte health and diseases are warranted.

A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell-derived kidney model for drug discovery.

Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.

Understanding Podocyte Biology to Develop Novel Kidney Therapeutics.

Over the past two decades it has become increasing clear that injury and loss of podocytes is an early and common clinical observation presented in many forms of glomerulopathy and chronic kidney disease. Identification of disease-causing monogenic mutations in numerous podocyte-expressed genes as well as studies conducted using preclinical animal models have shown that the podocyte plays a central role in establishing kidney dysfunction. In this review, we summarize current knowledge regarding the potential for podocyte-targeted therapies and give our view on how a deeper understanding of the molecular makeup of the podocyte will enable future therapeutic interventions. Specifically, we recount some of the currently described podocentric strategies for therapy and summarize the status and evolution of various model systems used to facilitate our understanding of the molecular and functional underpinnings of podocyte biology.

Association of crumbs homolog-2 with mTORC1 in developing podocyte.

The evidence that gene mutations in the polarity determinant Crumbs homologs-2 (CRB2) cause congenital nephrotic syndrome suggests the functional importance of this gene product in podocyte development. Because another isoform, CRB3, was reported to repress the mechanistic/mammalian target of the rapamycin complex 1 (mTORC1) pathway, we examined the role of CRB2 function in developing podocytes in relation to mTORC1. In HEK-293 and MDCK cells constitutively expressing CRB2, we found that the protein localized to the apicolateral side of the cell plasma membrane and that this plasma membrane assembly required N-glycosylation. Confocal microscopy of the neonate mouse kidney revealed that both the tyrosine-phosphorylated form and non-phosphorylated form of CRB2 commence at the S-shaped body stage at the apicolateral side of podocyte precursor cells and move to foot processes in a capillary tuft pattern. The pattern of phosphorylated mTOR in developing podocytes was similar to that of CRB2 tyrosine phosphorylation. Additionally, the lack of a tyrosine phosphorylation site on CRB2 led to the reduced sensitivity of mTORC1 activation in response to energy starvation. CRB2 may play an important role in the mechanistic pathway of developing podocytes through tyrosine phosphorylation by associating with mTORC1 activation.

Depletion of Gprc5a Promotes Development of Diabetic Nephropathy.

Background Renal glomeruli are the primary target of injury in diabetic nephropathy (DN), and the glomerular podocyte has a key role in disease progression.Methods To identify potential novel therapeutic targets for DN, we performed high-throughput molecular profiling of G protein-coupled receptors (GPCRs) using human glomeruli.Results We identified an orphan GPCR, Gprc5a, as a highly podocyte-specific gene, the expression of which was significantly downregulated in glomeruli of patients with DN compared with those without DN. Inactivation of Gprc5a in mice resulted in thickening of the glomerular basement membrane and activation of mesangial cells, which are two hallmark features of DN in humans. Compared with wild-type mice, Gprc5a-deficient animals demonstrated increased albuminuria and more severe histologic changes after induction of diabetes with streptozotocin. Mechanistically, Gprc5a modulated TGF-ß signaling and activation of the EGF receptor in cultured podocytes.Conclusions Gprc5a has an important role in the pathogenesis of DN, and further study of the podocyte-specific signaling activity of this protein is warranted.