Podocytes Research Articles

Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome.

Shiga toxin (Stx)-producing Escherichia coli is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS. Mice coinjected with Stx2/LPS exhibited glomerular podocyte and tubular C3 deposits and C3aR overexpression associated with cell damage, which were limited by C3aR antagonist treatment. C3a promoted renal injury by affecting mitochondrial wellness as demonstrated by data showing that C3aR blockade reduced mitochondrial ultrastructural abnormalities and preserved mitochondrial mass and energy production. In cultured podocytes and tubular cells, C3a caused altered mitochondrial fragmentation and distribution, and reduced anti-oxidant SOD2 activity. Stx2 potentiated the responsiveness of renal cells to the detrimental effects of C3a through increased C3aR protein expression. These results indicate that C3aR may represent a novel target in Stx-associated HUS for the preservation of renal cell integrity through the maintenance of mitochondrial function.

Myeloid-derived growth factor deficiency exacerbates mitotic catastrophe of podocytes in glomerular disease

Podocytes are unique, highly specialized, terminally differentiated cells, which are restricted in a post-mitotic state with limited ability to repair or regenerate. Re-entering the mitotic phase causes podocyte mitotic catastrophe, thereby leading to podocyte death and glomerular injury. Myeloid-derived growth factor (MYDGF) is a novel secreted protein and plays an important role in the regulation of cardiovascular function. However, whether MYDGF is expressed in kidney parenchymal cells and whether it has biological functions in the kidney remain unknown. Here, we found that MYDGF was expressed in kidney parenchymal cells and was significantly reduced in podocytes from mice with models of focal segmental glomerulosclerosis and diabetic kidney disease. Podocyte-specific deletion of Mydgf in mice exacerbated podocyte injury and proteinuria in both disease models. Functionally, MYDGF protected podocytes against mitotic catastrophe by reducing accumulation of podocytes in the S phase, a portion of the cell cycle in which DNA is replicated. Mechanistically, MYDGF regulates the expression of the transcription factor RUNX2 which mediates some MYDGF effects. Importantly, a significant reduction of MYDGF was found in glomeruli from patients with glomerular disease due to focal segmental glomerulosclerosis and diabetic kidney disease and the level of MYDGF was correlated with glomerular filtration rate, serum creatinine and podocyte loss. Thus, our studies indicate that MYDGF may be an attractive therapeutic target for glomerular disease.

USP40 deubiquitinates HINT1 and stabilizes p53 in podocyte damage

Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.

Clinical Evaluation of Antiphospholipase A2 Receptor IgG4 level and Its IgG4-to-IgG Ratio Based on Quantitative Immunoassays in Idiopathic Membranous Nephropathy.

Background Phospholipase A2 receptor (PLA2R), located at the membrane of glomerular podocyte, is the major autoantigen of idiopathic membranous nephropathy (IMN), and its antibodies with a predominant IgG4 subclass lead to pathological lesions. Further studies could be performed to validate the clinical values of PLA2R-IgG, PLA2R-IgG4, and PLA2R-IgG4-to-IgG ratios, as ultrasensitive and quantitative immunoassays for PLA2R antibodies have been well established in our previous work. Methods A cohort of 58 IMN patients, 30 of whom were followed from 3 to 42 months, was assessed for serum PLA2R-IgG and -IgG4 levels, and the ratio of PLA2R-IgG4/-IgG combined with relative clinicopathological indicators. Results Serum PLA2R-IgG4 level was significantly correlated with glomerular PLA2R staining. In addition, it was strongly correlated with PLA2R-IgG and its ratio. PLA2R-IgG and -IgG4 levels were both correlated with high-density lipoprotein and erythrocyte sedimentation rates. The ratio at the first diagnosis can predict the remission, and its efficacy overmatched PLA2R-IgG4. In the survival curves, negative results for the ratio or PLA2R-IgG4 at the first diagnosis demonstrated higher remission rates. Conclusion Serum PLA2R-IgG4 concentration may replace renal PLA2R immunohistochemistry in IMN diagnosis. We propose that the PLA2R-IgG4-to-IgG ratio and PLA2R-IgG4 could be novel indicators for remission prediction in clinical practice.

The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes.

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

FC016: Nephrotoxicity of Antiangiogenic Therapies: Direct Impact of Sorafenib on the Podocyte via GSK3Β

Abstract BACKGROUND AND AIMS Tumor angiogenesis is one of the therapeutic targets used in oncology, to limit cancer growth and spreading. The main angiogenesis signalling pathway is mediated by the vascular endothelial growth factor (VEGF) that binds to its tyrosine kinase receptor (VEGFR), mainly expressed on endothelial cells. The antiangiogenic therapy could target the VEGF ligand (anti-VEGF), or its receptors by inhibition of kinase activity (tyrosine kinase inhibitor, TKI) such as sorafenib. The toxicity of antiangiogenic therapies is a growing concern in clinical use. Indeed, nephrotoxicity is a critical side-effect that leads to discontinuation of therapy. Renal histological illustration of this toxicity is minimal change nephropathy/focal segmental glomerulosclerosis (MCNS/FSGS) and thrombotic micro-angiopathy (TMA), involving two distinct cell types, podocytes and endothelial cells respectively. Podocytes are the main source of VEGF and express VEGFR in the glomerulus. Mechanisms linking TKI therapy to podocyte dysfunction and nephrotic level proteinuria are still poorly understood. The working hypothesis is that nephrotoxicity of sorafenib is primarily through its effect on glomerular podocytes. METHOD Based on LC-MS/MS proteomic analysis, we have identified dysregulated cellular pathways on sorafenib-exposed podocytes. We validated these results by western blotting and immunofluorescence staining, on a cultured podocyte line exposed to sorafenib (2.5 µmol/L, 24-h). RESULTS Our results showed that sorafenib inhibits the downstream signalling pathways of VEGFR on podocytes, which induce injury by decreasing the expression of podocyte-specific markers (WT1, podocin). This is associated with podocyte morphology changes with podocyte cytoskeleton damages (actin and microtubules) and focal adhesions loss. We identified a novel podocyte target of sorafenib: the GSK3β. Indeed, sorafenib reduces significantly GSK3β inhibitory phosphorylation. Moreover, GSK3β regulates Tau, microtubule-associated proteins (MAP) and key regulator of microtubules remodelling. We show that tau phosphorylation increases significantly in sorafenib-treated cells. Furthermore, we found that sorafenib impairs the autophagic flux in podocytes, as indicated by an increase in LC3-II/LC3-I ratio in western blot, reflect of autophagosomes accumulation in podocytes. These results indicate that GSK3β overactivity induced by sorafenib participates in the disruption of the microtubules through tau phosphorylation, which interferes with cellular vesicle trafficking and blocks autophagic flux in podocytes. In addition, sorafenib leads to podocyte apoptosis through induction of endoplasmic reticulum (ER) stress (GADD153 protein induction and its nuclear translocation) and mitochondrial dysfunction. ER stress induced a decrease in podocyte protein synthesis with increased eIF2α phosphorylation. Sorafenib also causes mitochondrial membrane potential loss and mitochondrial depolarization. CONCLUSION Thus, in the current study, we show that sorafenib has a direct effect on podocytes, modulating specific signalling pathways to induce podocyte damage and ultimately glomerular lesions. However, we have to confirm the clinical relevance of our results on kidney biopsies from patients diagnosed with nephrotic syndrome following sorafenib treatment.

FC074: Mineralocorticoid Receptor Drives Parietal Epithelial Cell Activation and Glomerular Injury During Crescentic Glomerulonephritis

Abstract BACKGROUND AND AIMS We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury (1). Accumulating evidence has indicated the potential contributions of aldosterone and mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is strongly expressed in endothelial cells, glomerular mesangial cells, podocytes and distal tubular cells. Previous studies have shown that administration of mineralocorticoid receptor antagonists, including spironolactone and eplerenone, has beneficial effects in various renal injury animal models, such as unilateral ureteral obstruction, ischemia-reperfusion, cyclosporine-induced nephrotoxicity and hypertensive renal injury. The role of the MR in extra capillary glomerulopathies is still elusive and mechanistically unclear. METHOD To investigate the cell-specific role of the MR in PEC in the course of crescentic glomerulonephritis, we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Crescentic glomerulonephritis (GN) was induced using the antiglomerular basement membrane nephrotoxic serum (NTS) model. In vivo, biological parameters (albuminuria, blood urea nitrogen—BUN), glomerular injury, as well as PEC activation (CD44, CD9 and fibronectin staining) were assessed. RESULTS At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged using the NTS model, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function as compared to Pec Cre Nr3c2 wt/wt counterparts. Crescents were also more numerous and organized in Pec Cre Nr3c2wt/wt mice. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less activation molecule CD44 on PECs. Thus, genetic disruption of MR in PEC as well as pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC and crescents. CONCLUSION Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis along with the recently discovered CD9/EGFR/PDGFR pathway. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

FC042: Generation of Patient-Specific Hipsc-Podocytes to Study Phenotype-Specific Alterations and Test Therapeutic Targets Ex Vivo

Abstract BACKGROUND AND AIMS Genetic focal segmental glomerulosclerosis (FSGS) is related to irreversible damage of the filtration barrier and podocyte effacement. More than 40 genes with mutations, involved in slit diaphragm formation, actin cytoskeleton synthesis, podocyte adhesion or metabolic pathways, are known to induce FSGS. Up to 17% of all cases of autosomal-dominant FSGS are associated with mutations in the inverted formin 2 (INF2). To study the patient-specific alterations of the podocyte phenotype and to investigate potential treatment effects on INF2 mutant cells ex vivo we generated human induced pluripotent human stem cell (hiPSC)-derived podocytes from a patient holding an INF2 mutation and a healthy control. Wild-type and INF2 mutant hiPSCs-podocytes were compared regarding morphology, podocyte-specific marker expression and actin cytoskeleton and treated with different immunosuppressive drugs and actin modulating agents to analyse cell response. METHOD To generate hiPSCs-podocytes, fibroblasts were outgrown from a skin punch biopsy, reprogrammed into hiPSCs and subsequently stepwise differentiated into podocytes. The generated hiPSCs-podocytes were characterized by bulk sequencing, qPCR, western blot analysis and immunofluorescence staining regarding morphology (e.g. cell size and filopodia length), podocyte-specific markers and actin cytoskeleton development. Moreover, wild-type and INF2 mutant cells were treated with different stimuli [e.g. Solu-Decortin H (SDH), tacrolimus, Bis-T and ROCK inhibitor Y-27 632] and compared regarding their response. Especially filopodia length and cytoskeletal alterations were observed by quantification of total fluorescence of F-actin and cortactin and classified regarding actin filament thickness (type-A: thick bundles, type-B: thin filaments no bundles and type-C: disrupted cytoskeleton without filaments). RESULTS Marker genes regarding pluripotency and podocytes were compared at different timepoints during differentiation of hiPSCs into podocytes and compared to human immortalized podocytes. For example, synaptopodin gene expression increased during differentiation and was highest in terminally differentiated hiPSC-podocytes. Phenotypical characterization showed a distinct network of filopodia in wild-type hiPSCs-derived podocytes. Differentiated cells from healthy controls were compared to patient-specific podocytes holding an INF2 mutation, generated with the same protocol. Patient-specific cells showed no distinct net of foot processes and a fragmented actin cytoskeleton. Untreated wild-type cells mostly represented classification type-A actin whereas untreated INF2 mutated cells mostly showed type-B and -C actin filaments. First experiments indicated that treatment of INF2 mutated cells with Y-27 632 lead to an even more impaired phenotype whereas stimulation with SDH improved cytoskeleton rearrangements with up to 20% of type-A actin. The small acting modulating agent Bis-T did not alter the actin fibers in INF2 mutated hiPSC-podocytes but increased total fluorescence of cortactin. Tacrolimus had no effect. CONCLUSION Podocytes could be generated from hiPSCs reprogrammed from a patient's skin punch biopsy and enabled to study phenotype alterations of patient-specific podocytes holding an INF2 mutation ex vivo with the potential to test medications and find new potential therapeutic targets in the future.

MO064: Expression Studies on Magi2 in Different FSGS Models

Abstract BACKGROUND AND AIMS MAGI2 is a tight junction protein and member of the membrane-associated guanylate kinase (MAGUK) family. In the glomerulus, it is exclusively expressed in podocytes and interacts with the slit diaphragm protein nephrin. Recent studies and podocyte-specific MAGI2 knockout mice have demonstrated the essential role of this protein in the glomerulus. The aim of this work was to study the expression of MAGI2 in different focal segmental glomerulosclerosis (FSGS) mouse models, in the GlomAssay, in a FSGS-like zebrafish model, and in human biopsies of patients suffering from minimal change disease and FSGS. Additionally, using CRISPR/Cas9 knockout of magi2 and nephrin in zebrafish, we investigated the influence of both proteins on each other’s localization. METHOD We used nephrotoxic serum (NTS) as well as uninephrectomy DOCA/salt-induced hypertension as murine models. The podocyte-specific nitroreductase/metronidazole zebrafish model was used to induce FSGS-like disease in zebrafish upon partial podocyte-depletion. MAGI2 expression was evaluated in kidney biopsies of patients affected by minimal change disease (MCD), primary and secondary FSGS. Slides were imaged using confocal laser scanning and super-resolution 3D-structured illumination microscopy (3D-SIM). Filtration slit density (FSD) was measured on human biopsies by PEMP (podocyte exact morphology measurement procedure). MAGI2/nephrin ratio measurements were performed with a customized macro for the ImageJ-based platform FIJI. Magi2 expression was studied in the GlomAssay. To evaluate the interaction between magi2 and nephrin in the zebrafish model, the respective proteins were knocked-out using CRISPR/Cas9. The knockout was validated by RT–PCR and the expression of these two proteins in both lines was analyzed by immunofluorescence staining. Proteinuria was assessed using the excretion of eGFP-tagged vitamin D-binding protein from the vasculature and reuptake in proximal tubule cells. RESULTS MAGI2 was expressed in a linear pattern along the filtration slit and colocalized with nephrin in all three species investigated (human, mouse and zebrafish) (3D-SIM in Fig. 1). MAGI2 was significantly downregulated in NTS-induced nephritis, as well as in uninephrectomized and DOCA/salt-treated mice. In the GlomAssay, Magi2 was significantly downregulated in dedifferentiated podocytes after 6 days in cell culture due to podocyte dedifferentiation. A reduction of MAGI2 was found in human biopsies of patients suffering from primary FSGS, but not in MCD and secondary FSGS, respectively. In addition, we found that magi2 was downregulated in zebrafish larvae using our FSGS-like zebrafish model. When we examined the effect of nephrin on magi2 expression and vice versa, we found that magi2 expression was unchanged in nephrin-KO larvae, whereas nephrin expression was significantly reduced in MAGI2a-KO larvae. CONCLUSION Here, we have shown that the expression of MAGI2 in the glomerulus of different species such as zebrafish, mice and humans is associated with the expression of nephrin. By using knockout larvae, we showed that nephrin expression is dependent on Magi2 and not vice versa. Since the expression of Magi2 seems to be specifically regulated depending on the podocytopathy investigated, we propose a role for MAGI2 to distinguish between different glomerulopathies.

MO044: Cellular mechanism of the exceptional dominant transmission in NPHS2-associated glomerulopathy

Abstract BACKGROUND AND AIMS Mutations of NPHS2 cause autosomal recessive steroid-resistant nephrotic syndrome (SRNS). The encoded podocin, a key component of the slit diaphragm, homo-oligomerizes through two C-terminal oligomerization sites. We formerly found that specific podocin variants may exert a dominant negative effect against R229Q podocin variant secondary to an altered oligomerization [1, 2]. Recently, two families with autosomal dominant focal segmental glomerulosclerosis were found to carry a de novo heterozygous NPHS2 last-exon frameshift mutations affecting the second oligomerization site (either c.989_992delTGTC, p.L330Pfs*17 or c.988_989delCT, p.L330Vfs*15) [3]. We hypothesized that these podocin variants exert a dominant negative effect against wild type (WT) podocin. METHODS We coexpressed eGFP-tagged WT podocin with either hemagglutinin-tagged (HA) WT, p.L330Pfs*17 or p.L330Vfs*15 podocin variants in cultured podocytes. Plasma membrane was stained with CF405M-conjugated wheat germ agglutinin (WGA), and immunohistochemical staining was performed with primary anti-HA and secondary AlexaFluor568-conjugated antibodies. All analysis was done in a blinded fashion. The membrane-targeting of podocin was assessed by the ratio of the WGA-labeled perimembranous area that colocalized with podocin (Mander's overlap coefficients, MOC) and the colocalization of the coexpressed variants were estimated as Pearson correlation coefficients (PCC) by FiJi software on confocal microscopic images. MOC and PCC were compared by Holmes–Bonferroni adjusted Wilcoxon signed-rank test. RESULTS The frameshift podocin variants were not membranous in monoexpression [MOC: HA-p.L330Pfs*17 (n = 26): 0.08 (0.03–0.22) median (interquartile ranges); HA-p.L330Vfs*15 (n = 24): 0.07 (0.02–0.16)]. The GFP-tagged WT podocin strongly colocalized with the HA-tagged WT or frameshift podocin variants [PCC: WT-eGFP + HA-WT (n = 19): 0.64 (0.49–0.71); WT-eGFP + HA-L330Pfs*17 (n = 17): 0.54 (0.35–0.58); WT-eGFP + HA-L330Vfs*15 (n = 17): 0.44 (0.29–0.66)]. The GFP-WT podocin was retained in intracellular compartments in the presence of any of the two frameshift podocin variants [MOCWT(WT) = 0.58 (0.46–0.69)], MOCWT(L330Pfs*17) = 0.01 (0.002–0.029), P = 2x10-6 versus WT(WT); MOCWT(L330Vfs*15) = 0.003 (0.001–0.013), P = 2 x 10−6 versus WT(WT)]. CONCLUSIONS The dominant podocin variants retain WT podocin in intracellular compartments explaining the exceptional dominant transmission of NPHS2-associated glomerulopathy. This is in accordance with our former results showing the primary role of C-terminal hetero-oligomerization in exerting interallelic interactions of NPHS2 alleles. FUNDING MTA-SE Lendulet Research Grant (LP2015-11/2015), OTKA KH125566 and K135798, and ÚNKP-20–3-I-SE-29 from the source of the National Research, Development and Innovation Fund.

FC015: High Salt Environment Disrupts the Regulatory Effect of Podocin on the Nephrin-Nephrin Distance

Abstract BACKGROUND AND AIMS The podocin encoding NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. Podocin homooligomerizes through C-terminal helical regions [1] and binds nephrin in the glomerular slit diaphragm [2]. We formerly showed that podocin regulates the distance between two neighbouring nephrin molecules in cis, i.e. the shortest dimension of the glomerular pore (3.5-4nm) [3, 4]. High sodium diet has been shown to promote glomerular injury and proteinuria [5]. We aimed to assess the effect of high salt environment on the nephrin-nephrin distance in the presence and absence of podocin. METHODS To generate extracellularly tagged nephrin constructs, cDNA sequences encoding YPet or mCherry were inserted in the human wild type nephrin cDNA (YPet-N1 plasmid) replacing the c.3136-3162 nucleotides. Thus, the p.Glu1046-Pro1054 residues of the fibronectin domain, located in the juxtamembraneous extracellular part of nephrin was replaced with either YPet or mCherry. HEK-293 cells were co-transfected with both nephrin constructs with or without wild type HA-tagged podocin construct (pLEX-MCS plasmid). To measure the effect of salt overload, DMEM was supplemented with either 25 mM NaCl or 50 mM mannitol as osmotic control 24-h prior to the FRET measurements. The fluorescence decay curves measured by time-correlated single photon counting (TCSPC) (Chronos BH, ISS Inc.) were decomposed into lifetime components and the longest lifetime population—most suitable for long-range FRET—was used for further calculations. Nonparametric tests were used for statistical analysis. RESULTS High salt environment did not influence the nephrin-nephrin distance in the absence of podocin. In accordance with our previous results, podocin decreased the nephrin-nephrin distance in physiological salt environment, as reflected by an increased FRET efficiency between the extracellular YPet and mCherry tags. High salt environment (+25 mM) completely disrupted this effect. Mannitol of similar osmotic concentration did not modify the effect of podocin (Figure 1). CONCLUSION High salt environment increases the distance between the nephrin molecules in cis by disrupting the organizing effect of podocin. The deleterious effect of high salt is not explained by an increased osmotic pressure.

MO219: Are C4D, Nephrın and WT1 Expressıons İmportant ın the Dıfferentıal Dıagnosıs between Focal Segmental Glomerulosclerosıs and Mınımal Change Dısease?

Abstract BACKGROUND AND AIMS Focal Segmental Glomerulosclerosis (FSGS) and Minimal Change Disease (MCD) are most common seen glomerulonephritis. Differential diagnosis between FSGS and MCD by light microscopy (LM) cannot be made in some times. In this situation, electron microscopy (EM) is needed for differential diagnosis. Rarely, EM may not be sufficient for differential diagnosis. There are needed new markers for differential diagnose between FSGS and MCD. The aim of this study is to determine which of the glomerular C4d, nephrine staining is specific and sensitive for FSGS and MCD. C4d, Recent experimental data show that complement activation plays a pathogenic role in the development of FSGS. Therefore, C4d staining in the sclerotic focus of the glomeruli in FSGS patients may have an important differential diagnosis. Nephrine is the main component of the slit diaphragm that connects the foot protrusions of the podocytes. Epithelial cell and podocyte dysfunction occurs as a result of defects in the genes encoding these key proteins expressed in GBM. Wilms’ tumour suppressor gene (WT1) is a transcription factor that plays very important role in the embryonal development of the genital organs and kidney. WT-1 is expressed in podocytes and is an important transcription factor for podocyte function. METHOD This is a retrospective study, comprised of patients diagnosed as FSGS, MCD and the ones who diagnosed as having FSGS or MCD couldnot be differentiated. The patients were divided into three groups. Group 1: FSGS patients, Group 2: patients with indefinite diagnosis; either FSGS or MCD and Group 3: MCD patients. All biopsy materials were stained with C4d, WT1 and nephrine. The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of C4d, WT1 and nephrine were evaluated for the diagnosis of both FSGS and MCD. RESULTS C4d staining in group 1 was significantly higher compared with group 2 and group 3 (P < .05). Nephrine was shown significantly more staining in group 3 patients than group 1 (P < .05). C4d was found to be associated with the presence of global or segmental sclerosis in the glomeruli, adhesion to the Bowman's capsule (P < .05). The specificity and sensitivity of C4d in FSGS patients were 85%, 73.9%, while it was 26.1% and 15.4% in MCD. Specificity and sensitivity of nephrine were 87%, 69% in MCD patients compared with 30.8% and 13% in FSGS (Fig. 1). PPV and NPV values of C4d and nephrine in FSGS and MCD were shown in Figure 2. CONCLUSION C4d and nephrine have predictive diagnostic value for FSGS and MCD respectively. C4d and nephrine are more specific and sensitive to FSGS and MCD, respectively. However, there is a need for randomized studies with larger study groups

MO074: SGLT2 Inhibition Promotes Intrinsic Kidney Regeneration by Cells of the Renin Lineage

Abstract BACKGROUND AND AIMS With chronic kidney disease (CKD) prevalence rapidly increasing, the need for novel therapies is rising. Sodium glucose co-transporter-2 (SGLT2) inhibitors were originally developed to treat hyperglycemia in patients with type 2 diabetes mellitus. Clinical trials with the SGLT2 inhibitor empagliflozin revealed a marked attenuation of the slope of kidney function decline, also in patients with non-diabetic CKD. The exact mechanism of this kidney sparing effect still remains to be clarified. Interestingly, cells of renin lineage (CoRL), residing in the juxtaglomerular apparatus to regulate blood pressure and fluid balance, have been demonstrated to harbor a stem cell like potential. CoRL have the ability to replenish glomerular cell number by dedifferentiating, migrating and subsequently replacing various glomerular cell types in different kidney injury mouse models. Considering that empagliflozin treatment affects renin plasma levels and electrolyte balance in patients, we hypothesized that empagliflozin could have an effect on CoRL-induced glomerular regeneration. METHOD Experiments were performed in a Ren1cre; tdTomato lineage-trace mouse strain that expresses a tomato fluorescent label in all cells derived from renin lineage. Two kidney injury mouse models were applied; bilateral ischemia reperfusion injury (bIRI) and 5/6 nephrectomy (5/6NTx). Empagliflozin (10 mg/kg) was administered daily via oral gavage for 14 days. Subsequently, mice were sacrificed and kidneys were harvested for histological analysis. RESULTS In both the bIRI and 5/6NTx model, empagliflozin intake led to an increase (>2-fold) of CoRL found in the intraglomerular regions compared with vehicle control littermates. These CoRL seemed to selectively differentiate towards different glomerular cell types per model: bIRI combined with empagliflozin administration resulted in an increase of claudin- (10-fold) and integrin-α8- (1.5-fold) tomato double positive cells, suggesting favored differentiation from CoRL to respectively a parietal epithelial or mesangial cell type. In contrast, in the empagliflozin treated 5/6NTx model, an increase (1.5-fold) in tomato-podocyn double positive cells was observed, implying more restocking of glomerular podocytes by CoRL in this model. CONCLUSION SGLT2 inhibition by empagliflozin treatment leads to increased CoRL-mediated intrinsic regeneration potential and provides the kidney with different replenished cell types in different kidney disease models. Our findings demonstrate a novel mechanism via which SGLT2 inhibition might protect against kidney injury.

Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy.

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3′ of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

Expression of ALMS1 in podocytes: possible role in filtration function

Previously, we identified Alstrom Syndrome 1 (ALMS1) as an interacting partner of NKCC2 in the Thick Ascending limb of the loop of Henle (TAL). Mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by progressive metabolic alterations that include obesity, hypertension, and chronic kidney disease (CDK). SNPs in ALMS1 are associated with lower GFR in GWAS. However, role of ALMS1 in glomerular filtration has not been studied. We hypothesized that ALMS1 is expressed in glomerular podocytes where it may play a role in podocyte filtration function. To begin studying this, we localized ALMS1 expression to podocytes by immunofluorescence labeling of Actinin‐4, abundantly expressed in podocytes, and ALMS1. Confocal imaging showed that ALMS1 is co‐localized with Actinin‐4 in rat podocytes. In addition, ALMS1 was also expressed in an immortalized mouse podocyte cell line, as was Actinin‐4 and nephrin, as measured by Western blot and immunofluorescence microscopy. To determine if ALMS1 plays a role in podocyte pathology we measured nephrin expression in ALMS1 KO rats. These rats were generated in the Dahl SS genetic background and are hypertensive on a normal salt diet. We found that nephrin expression in renal cortical lysates was decreased by 61±08% (p<0.05, n=7) in ALMS1 KO rats. To study if ALMS1 deletion could play a role in filtration barrier function we measured albumin/creatinine ratio in urine samples from 14‐16 weeks old wild type (WT) and ALMS1 KO rats. In ALMS1 KO rats, urinary albumin excretion was higher than control rats (WT: 4±2; ALMS1 KO: 26±6, n=6, p<0.05). Taken together, our data show that ALMS1 is expressed in rat and mouse podocytes where it may play a role in filtration barrier function by maintaining podocyte biology.

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

ABSTRACTNephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Autophagic Deficiency and Dedifferentiation in Podocytes of Mice Lacking Acid Ceramidase (Asah1) Gene

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain with a podocyte‐specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Asah1fl/fl/Podocre, Asah1 is a gene code of mouse acid ceramidase). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether autophagic deficiency and associated dedifferentiation of podocytes contribute to the pathogenesis of podocytopathy in Asah1fl/fl/Podocre mice. By Western blot analysis and immunofluorescent staining, we demonstrated the significant reduction of epithelial markers (ZO‐1 and P‐cadherin) and remarkable elevation of mesenchymal markers (α‐SMA and FSP‐1) in podocytes lack of Asah1 gene in vivo and in vitro. Also, we found that Asah1 gene knockout induced autophagic deficiency in podocytes, which were evidenced by autophagosome accumulation and decreased lysosome‐autophagosome interaction in these cells. Given the vital role of transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and interaction with other vehicles, we tested whether this lysosomal channel is implicated in autophagic deficiency in podocytes lacking Asah1 gene. As a selective TRPML1 channel agonist, ML‐SA5 enhanced lysosome‐autophagosome interaction in WT/WT podocytes, but only had partial effects on this interaction in Asah1fl/fl/Podocre podocytes. Autophagosome accumulation was also attenuated by ML‐SA5 in podocytes lacking Asah1 gene. On the contrary, ML‐SI1 as a TRPML1 channel inhibitor impaired autophagic flux in WT/WT podocytes and amplified autophagic deficiency in Asah1fl/fl/Podocre podocytes. The enhancements of lysosome trafficking and lysosome‐autophagosome interaction by ML‐SA5 were blocked by dynein inhibitors, EHNA and ciliobrevin D, indicating the contribution of dynein to lysosome movement in response to TRPML1 channel‐mediated Ca2+ release. Pathologically, Asah1 gene deletion‐induced podocyte dedifferentiation, reduction of slit diaphragm proteins, and proteinuria were inhibited by ML‐SA5 but enhanced by ML‐SI1. Based on these results, we conclude that Asah1 gene deletion induces autophagic deficiency and dedifferentiation in podocytes through blockade of lysosomal TRPML1 channel, thereby leading to the development of podocytopathy and nephrotic syndrome in Asah1fl/fl/Podocre mice.

Application of Scanning Ion Conductance Microscopy in the Studies of Podocytes Morphological Changes

High‐resolution imaging of glomerular podocytes morphology is becoming an essential and helpful tool for studying their role in renal disease progression. Electron microscopy (EM) is commonly used to analyze the effacement of the podocytes’ cell foot processes and the glomerular filtration barrier integrity loss. However, EM does not allow studying living samples and requires multiple complex procedures for sample preparation. Here, we propose and demonstrate the successful application of Scanning Ion Conductance Microscopy (SICM) for studying morphological changes of cultured podocytes and freshly isolated glomeruli.SICM provides a high‐resolution (comparable to EM) non‐optical imaging of cell topography using hopping probe microscopy principles and new dynamic reconstruction software. This technique can be used for analysis of cell membrane morphology and different functional characteristics, such as cell volume, membrane potentials, single ion‐channel currents, and other parameters. The major advantage of the current method is that it can be applied to live samples with complex morphology, such as intact glomeruli, under physiologically relevant liquid conditions.We have established and demonstrated a successful application of the SICM method in the live human glomerulus and provided a proof of principle for future dynamic analysis of membrane morphology and varied functional parameters in the living glomerulus. As a proof of principle, we applied this method for estimation the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. Using topographical quantification of the length and irregular shape of the podocyte foot processes, we showed an absence of interdigitated foot processes on the selected region of the glomerular slit diaphragm in isolated T2DN glomeruli.In addition to the application of SICM with freshly isolated glomeruli, we used this approach to visualize and quantify foot process dynamics in cultured human podocytes during pharmacological applications. Thus, we showed that destroying the F‐actin with cytochalasin D completely stopped the migration of the podocyte filopodium through the remodeling of the actin cytoskeleton. Here, we further applied SICM to detect morphological changes in human podocytes after activation of Protease‐activated receptor‐1 (PAR1) by acute application of agonist TFLLR‐NH2. A series of experiments revealed that PAR1 activation led to retraction of the lamellipodium and a significant decrease in cell surface area (+51±46 vs. ‐15±8 µm2, 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05).In summary, SICM has numerous advantages and has excellent potential for precise quantitative analyses of the morphological changes in the glomerulus and cultured podocytes. Moreover, it opens the possibility of performing patch‐clamp recordings on the specific parts of the podocyte cell body and advances our understanding of the function and mechanism of the glomerulus filtration barrier.

Novel Role of Dual Oxidase 2 as a Mediator of Podocyte Injury in the Diabetic Environment

Glomerular injury is a prominent pathological feature of diabetic nephropathy (DN). In glomerular epithelial cells, or podocytes, hyperglycemia alters slit diaphragm proteins and causes foot process effacement, apoptosis and cell detachment. Oxidative stress has emerged as an important pathogenic mechanism in the development of glomerular injury in DN. However, the mechanisms by which these factors exert their action remain poorly understood.We provide evidence that the NADPH oxidase of the Nox family, Dual oxidase 2 (Duox2), is present in cultured glomerular cells, including podocytes. Exposure of cultured podocytes to high concentrations of glucose (HG) elicited a rapid upregulation of Duox2 protein expression. Inhibition of Duox2 with specific siRNA prevented the HG‐induced increase in intracellular reactive oxygen species (ROS) generation and hydrogen peroxide (H2O2) production in cultured podocytes.In additional experiments, we established a functional link between Duox2‐derived ROS generation and podocyte injury in response to HG. Impairment of Duox2 function nearly abolished HG‐mediated DNA fragmentation, apoptosis and decrease in/reorganization of slit diaphragm protein expression in podocytes, indicating that Duox2 is required for the deleterious effects of glucose in podocytes.Our study demonstrates, for the first time, that Duox2 is responsible for increased ROS generation and subsequent alteration of podocyte function in response to HG. Our work serves as proof of concept to demonstrate the utility of targeting Duox2 as a future therapeutic intervention to reduce diabetes‐mediated glomerular lesions.

A Novel Approach to Assess Podocyte Damage at Single Cell Level

Diseases affecting podocytes in kidney glomeruli are heterogeneous with respect to triggers, molecular mechanisms, and speed of progression. Frequently, healthy and diseased podocytes are present in parallel. Single cell technology is ideally suited to capture such heterogeneity including temporal resolution, which can be achieved by trajectory analysis. However, trajectories require large numbers of cells, which are difficult to obtain for rare cell populations such as podocytes. Here, we propose a novel analysis approach for single‐cell data in rare cell types, which relies on a podocyte disease score (PDS) to monitor the health status of individual cells. PDS is based on integrative meta‐analysis of published and our own bulk and single‐cell transcriptomic data of glomeruli and primary podocytes obtained from a multitude (n=37) of mouse models featuring podocyte damage. Among others, podocyte damage triggers integrated in the analysis include diabetes, ageing, mutations in slit diaphragm proteins, alterations of transcription factors, and adriamycin‐induced podocytopathy. In a rank‐based approach, a geneset was derived, whose expression quantitatively predicts podocyte damage irrespective of the trigger and at single‐cell resolution. Our PDS is robust, correlates with albuminuria in mice and humans, and allows for aligning disease states across independent and novel datasets. Applying PDS to published and novel glomerular single‐nucleus RNAseq data from mouse models recapitulated established molecular pathways relevant to the models, and identified common and model‐specific pathways related to podocyte damage. As such, Eph‐ephrin signaling was identified as a pathway that is universally altered upon podocyte damage at transcriptional level, and was confirmed by STED microscopy on the protein level in two mouse models. In conclusion, PDS is a valid and versatile method to assess podocyte damage at single‐cell level and allows to characterize cellular processes in podocytes in mice and humans.