Podocyte Adhesion Research Articles

Mechanosensitive Differentiation of Human iPS Cell-Derived Podocytes.

Stem cell fate decisions, including proliferation, differentiation, morphological changes, and viability, are impacted by microenvironmental cues such as physical and biochemical signals. However, the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study, we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion, differentiation, and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically, hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering, disease modeling, and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses.

Mathematical Modeling of Podocyte Adhesion Highlights the Role of Cell Contractility and Fluid Shear Stress on Kidney Function

Mathematical Modeling of Podocyte Adhesion Highlights the Role of Cell Contractility and Fluid Shear Stress on Kidney Function

FilGAP controls cell-extracellular matrix adhesion and process formation of kidney podocytes.

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes invitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.

Role of the Ste20-like kinase SLK in podocyte adhesion.

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients.

Proteinuria is a common and important clinical manifestation of chronic kidney disease (CKD) and an independent risk factor for the progression of kidney disease. As a component of the glomerular filtration barrier (GFB), podocyte plays a key role in the pathogenesis of glomerular diseases and proteinuria. However, the pathophysiology of glomerular diseases associated with mitochondrial function is incompletely understood. Here, we identified three novel mutations in MTX2, encoding a membrane protein in mitochondria, associated with multisystem manifestations including nephrotic proteinuria and kidney injury in two Chinese patients. Conditional podocyte-specific Mtx2 knockout (Pod-Mtx2-KO) mice present a series of podocyte and glomerular abnormalities from 8 weeks to old age, including microalbuminuria, glomerular mesangial hyperplasia, fusion and effacement of foot process. MTX2 deficiency impaired podocyte functions in vitro, manifested by reductions of adhesion, migration and endocytosis, which were further restored by overexpression of MTX2. Moreover, MTX2 defects led to abnormal mitochondrial structure and dysfunction, evidenced with defects of complex I and III, increased production of reactive oxygen species (ROS), and decreased protein levels of Sam50-CHCHD3-Mitofilin axis in the mitochondrial intermembrane space bridging (MIB) complex which is responsible for maintaining mitochondrial cristae morphology. Collectively, these findings reveal that the normal expression of MTX2 in glomerulus plays an important role in the adhesion, migration, endocytosis, proliferation and other physiological functions of podocytes, which may be realized by maintaining the morphological structure and function of mitochondria. Abnormal expression of MTX2 can lead to mitochondrial dysfunction and structural abnormalities by Sam50-CHCHD3-Mitofilin axis in podocyte, which further induces podocyte injury, glomerular lesions and proteinuria.

Influence of Podocyte Injury on the Development of Class IV Lupus Nephritis.

In the kidneys, Systemic Lupus Erythematosus leads to Lupus Nephritis (LN), a form of glomerulonephritis. There is evidence that patients with LN may present activation of specific pathways for podocyte injury. This injury can occur through different mechanisms such as loss of podocyte adhesion to the glomerular basement membrane, cell death or dedifferentiation. Podocyturia with consequent podocytopenia has been described in some nephropathies such as LN, highlighting the importance of studying podocyte injuries in this condition. Evaluating in situ morphological characteristics of podocytes becomes relevant for a better understanding of the processes involved in their pathogenesis. This study investigated podocytes in different classes of LN in renal biopsies performed by the Kidney Research Center at the Federal University of Triângulo Mineiro. Twenty control cases and 29 biopsy cases diagnosed with LN were selected, divided according to the histopathological classes of the disease. Podocyte density was assessed through immunohistochemistry for Wilms tumor 1 protein and the evaluation of foot process effacement was performed by transmission electron microscopy. Podocyte density was lower in the LN and this reduction was observed in all analyzed classes when compared to the control group. More foot process effacement was observed in the LN group, with more effacement in classes I/II and class IV compared to the control group. The class IV group showed more foot process effacement than the class III group and presented higher proteinuria levels compared to the classes I/II group. A strong, positive, and significant correlation was observed between the activity index and foot process effacement in the class IV group. Podocytes play an important role in the development of LN, and possibly, injuries to these cells are more closely related to the inflammatory/diffuse proliferative cellular process developed in class IV LN.

The Role of α3β1 Integrin Modulation on Fabry Disease Podocyte Injury and Kidney Impairment.

Podocyte dysfunction plays a crucial role in renal injury and is identified as a key contributor to proteinuria in Fabry disease (FD), primarily impacting glomerular filtration function (GFF). The α3β1 integrins are important for podocyte adhesion to the glomerular basement membrane, and disturbances in these integrins can lead to podocyte injury. Therefore, this study aimed to assess the effects of chloroquine (CQ) on podocytes, as this drug can be used to obtain an in vitro condition analogous to the FD. Murine podocytes were employed in our experiments. The results revealed a dose-dependent reduction in cell viability. CQ at a sub-lethal concentration (1.0 µg/mL) induced lysosomal accumulation significantly (p < 0.0001). Morphological changes were evident through scanning electron microscopy and immunofluorescence, highlighting alterations in F-actin and nucleus morphology. No significant changes were observed in the gene expression of α3β1 integrins via RT-qPCR. Protein expression of α3 integrin was evaluated with Western Blotting and immunofluorescence, demonstrating its lower detection in podocytes exposed to CQ. Our findings propose a novel in vitro model for exploring secondary Fabry nephropathy, indicating a modulation of α3β1 integrin and morphological alterations in podocytes under the influence of CQ.

Regulation of Podocyte Adhesion by SEL1L-HRD1 ERAD via Integrin

Regulation of Podocyte Adhesion by SEL1L-HRD1 ERAD via Integrin

SuPAR and WT1 modify the adhesion of podocytes and are related to proteinuria in class IV lupus nephritis

IntroductionLupus nephritis (LN) affects up to 60 % of the patients with Systemic Lupus Erythematosus (SLE) and renal damage progression is associated with proteinuria, caused in part by the integrity of the glomerular basement membrane (GBM) and by podocyte injury. The soluble urokinase plasminogen activator receptor (suPAR) and Wilms Tumor 1 (WT1) have been related to podocyte effacement and consequently with proteinuria which raises questions about its pathogenic role in LN. ObjectiveDefine whether suPAR levels and WT1 expression influence in podocyte anchorage destabilization in LN class IV. Materials and methodsThis is a cross-sectional study of cases and controls. We studied patients with SLE without renal involvement (n = 12), SLE and LN class IV with proteinuria ≤0.5 g/24 h (n = 12), LN class IV with proteinuria ≥0.5 g/24 h (n = 12) and compared them with renal tissue control (CR) (n = 12) and control sera (CS) (n = 12). The CR was integrated by cadaveric samples without SLE or renal involvement and the CS was integrated by healthy participants. The expression and cellular localization of WT1, urokinase-type plasminogen activator receptor (uPAR), ac-α-tubulin, vimentin, and β3-integrin was assessed by immunohistochemistry (IHC). The concentration of suPAR in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). ResultsIn patients with LN, the activation of anchoring proteins was increased, such as podocyte β3-integrin, as well as the acetylation of alpha-acetyl-tubulin and uPAR, in contrast to the decrease in vimentin; interestingly, the cellular localization of WT1 was cytoplasmic and the number of podocytes per glomerulus decreased. The concentrations of suPAR was increased in patients with LN. ConclusionThe destabilization of podocyte anchorage modulated by β3-integrin activation, and tubulin acetylation, associated with decreased WT1 cytoplasmic expression, and increased suPAR levels could be involved in kidney damage in patients with LN class IV.

#5649 HEPARANASE OVEREXPRESSION MODIFIES PODOCYTE MOTILITY

Abstract Background and Aims Heparanase is an endo-β-d-glucuronidase that degrades heparan sulfate side chains, which are essential components of extracellular matrices and cell surfaces, including in the kidney filtration apparatus, the glomerulus. Upregulation of heparanase in glomerular diseases (GDs) was demonstrated in experimental models of GDs and in humans. Using transgenic mice overexpressing the human heparanase gene, we recently showed that heparanase reduced proteinuria and preserved the glomerular filtration barrier (GFB) after Adriamycin-induced injury. Furthermore, heparanase improved survival of key GFB cells, the podocytes and enhanced their autophagic flux both in vitro and in vivo. Here, we aimed to study the potential role of heparanase on podocyte locomotion and adhesion. Methods We utilized immortalized human podocyte cell line AB8/13 (a kind gift of Prof. Moin Saleem, Bristol, UK) as an experimental platform. Cells were infected with pLenti6/V5-DEST carrying the heparanase gene construct (H) or control empty vector (V) and were allowed to differentiate for 14 days. To induce injury, differentiated podocytes were treated with Adriamycin (0.5 μg/mL), mimicking Adriamycin nephropathy, an established experimental model for progressive proteinuric GDs. For readouts, we used scratch assay, RT-qPCR, Western blot (WB) and immunofluorescence (IF) analyses. Results The in vitro scratch assay showed that differentiated (H) podocytes migrated at a slower rate compared with control (V) podocytes, both at baseline and after a 12-hour treatment with Adriamycin. Hence, we hypothesized that heparanase may alter podocyte adhesion properties. Indeed, IF staining of differentiated (H) podocytes exhibited an increase in focal contacts containing vinculin compared with control (V) cells treated with Adriamycin. Of note, we observed no differences in vinculin abundance by WB, suggesting that heparanase mediates the active form of vinculin. Moreover, among the integrins, we noticed temporal upregulation of ITGB1 transcription, as early as two hours after Adriamycin treatment, in (H) compared with control (V) podocytes (P = 0.032). Conclusions Collectively, our results suggest that in response to Adriamycin injury, constitutive heparanase overexpression could stabilize podocyte focal adhesions and control motility. Although further research is needed, improving the anchoring of podocytes over the basement membrane may provide an additional cellular mechanism by which heparanase conferred protection against Adriamycin toxicity.

A high-impact FN1 variant correlates with fibronectin-mediated glomerulopathy via decreased binding to collagen type IV

The glomerular basement membrane (GBM) consists of laminins, collagen IV, nidogens, and fibronectin and is essential for filtration barrier integrity in the kidney. Critically, structural and functional abnormalities in the GBM are involved in chronic kidney disease (CKD) occurrence and development. Fibronectin is encoded by FN1 and is essential for podocyte-podocyte and podocyte-matrix interactions. However, disrupted or disordered fibronectin occurs in many kidney diseases. In this study, we identified a novel mutation (c.3415G>A) in FN1 that causes glomerular fibronectin-specific deposition in a gain-of-function manner, that may be associated with thin basement membrane nephropathy (TBMN) and expand the spectrum of phenotypes seen in glomerulopathy with fibronectin deposits (GFND). Our studies confirmed this variant increased fibronectin's ability to bind to integrin, thereby maintaining podocyte adhesion. Also, we hypothesised that TBMN arose as the fibronectin variant exhibited a decreased capacity to bind COL4A3/4. Our study is the first to identify and link this novel pathogenic mutation (c.3415G>A) in FN1 to GFND as well as TBMN, which may broaden the phenotype and mutation spectrums of the FN1 gene. We believe our data will positively impact genetic counselling and prenatal diagnostics for GFND with TBMN and other associated conditions that may be commonly benign conditions in humans, and may not require proteinuria-lowering treatments or renal biopsy.

Complementary Nck1/2 Signaling in Podocytes Controls α Actinin-4-Mediated Actin Organization, Adhesion, and Basement Membrane Composition.

Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

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.

Basement membrane defects in CD151-associated glomerular disease

BackgroundCD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3β1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease.MethodsNext-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9.ResultsWe report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence.ConclusionsOur results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Generation of endoplasmic reticulum stress-dependent reactive oxygen species mediates TGF-β1-induced podocyte migration.

Podocyte migration results in proteinuria and glomerulonephropathy. Transforming growth factor-β1 (TGF-β1), endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) can mediate podocyte migration; however, the crosstalk between them is unclear. This study determined the relationships between these factors. ER stress biomarkers (GRP78, p-eIF2α or CHOP), intracellular ROS generation, integrin-β3 and cell adhesion and migration were studied in a treatment of experiment using TGF-β1 with and without the ER stress inhibitors: 4-phenylbutyric acid (4-PBA, a chemical chaperone), salubrinal (an eIF2α dephosphorylation inhibitor) and N-acetylcysteine (NAC, an antioxidant). ER stress biomarkers (p-eIF2α/eIF2α and GRP78), ROS generation and intergrin-β3 expression increased after TGF-β1 treatment. NAC down-regulated the expression of GRP78 after TGF-β1 treatment. 4-PBA attenuated TGF-β1-induced p-eIF2α/eIF2α, CHOP, ROS generation and intergrin-β3 expression. However, salubrinal did not inhibit TGF-β1-induced p-eIF2α/eIF2α, CHOP, ROS generation or integrin-β3 expression. NAC abrogated TGF-β1-induced integrin-β3 expression. At 24h after treatment with TGF-β1, podocyte adhesion and migration increased. Furthermore, NAC, 4-PBA and an anti-interin-β3 antibody attenuated TGF-β1-induced podocyte adhesion and migration. This study demonstrated that TGF-β1-induced ER stress potentiates the generation of intracellular ROS to a high degree through the PERK/eIF2α/CHOP pathway. This intracellular ROS then mediates integrin-β3 expression, which regulates podocyte migration.

The role of filamins in mechanically stressed podocytes.

Glomerular hypertension induces mechanical load to podocytes, often resulting in podocyte detachment and the development of glomerulosclerosis. Although it is well known that podocytes are mechanosensitive, the mechanosensors and mechanotransducers are still unknown. Since filaminA, an actin-binding protein, is already described to be a mechanosensor and mechanotransducer, we hypothesized that filamins could be important for the outside-in signaling as well as the actin cytoskeleton of podocytes under mechanical stress. In this study, we demonstrate that filaminA is the main isoform of the filamin family that is expressed in cultured podocytes. Together with filamin B, filamin A was significantly up-regulated during mechanical stretch (3 days, 0.5Hz, and 5% extension). To study the role of filaminA in cultured podocytes under mechanical stress, filaminA was knocked down (FlnaKD) by specific siRNA. Additionally, we established a filaminA knockout podocyte cell line (FlnaKO) by CRISPR/Cas9. Knockdown and knockout of filaminA influenced the expression of synaptopodin, a podocyte-specific protein, focal adhesions as well as the morphology of the actin cytoskeleton. Moreover, the cell motility of FlnaKO podocytes was significantly increased. Since the knockout of filamin A has had no effect on cell adhesion of podocytes during mechanical stress, we simultaneously knocked down the expression of filaminA and B. Thereby, we observed a significant loss of podocytes during mechanical stress indicating a compensatory mechanism. Analyzing hypertensive mice kidneys as well as biopsies of patients suffering from diabetic nephropathy, we found an up-regulation of filaminA in podocytes in contrast to the control. In summary, filaminA and B mediate matrix-actin cytoskeleton interactions which are essential for the adaptation of cultured podocyte to mechanical stress.

Effects of Perfusion Pressures on Podocyte Loss in the Isolated Perfused Mouse Kidney.

Podocytes are lost in most glomerular diseases, leading to glomerulosclerosis and progressive kidney disease. It is generally assumed, that podocytes are exposed to the filtration flow and thus to significant shear forces driving their detachment from the glomerular basement membrane (GBM). In this context, foot process effacement has been proposed as potential adaptive response to increase adhesion of podocytes to the GBM. We have tested these hypotheses using optical clearing and high-resolution 3-dimensional morphometric analysis in the isolated perfused murine kidney. We investigated the dynamics of podocyte detachment at different perfusion pressures (50, 300 and more than 450 mmHg) in healthy young or old mice (20 vs. 71 weeks of age), or mice injected with anti-GBM serum to induce global foot process effacement. Results show that healthy podocytes in young mice are tightly attached onto the GBM and even supramaximal pressures did not cause significant detachment. Compared to young mice, in aged mice and mice with anti-GBM nephritis and foot process effacement, gradual progressive loss of podocytes had occurred already before perfusion. High perfusion pressures resulted in a relatively minor additional loss of podocytes in aged mice. In mice with anti-GBM nephritis significant additional podocyte loss occurred at this early time point when increasing perfusion pressures to 300 mmHg or higher. This work provides the first experimental evidence that podocytes are extraordinarily resistant to acutely increased perfusion pressures in an ex vivo isolated kidney perfusion model. Only in glomerular disease, significant numbers of injured podocytes detached following acute increases in perfusion pressure.

PODO: Trial Design: Phase 2 Study of PF-06730512 in Focal Segmental Glomerulosclerosis

IntroductionFocal segmental glomerulosclerosis (FSGS) is characterized by proteinuria and a histologic pattern of glomerular lesions of diverse etiology that share features including glomerular scarring and podocyte foot process effacement. Roundabout guidance receptor 2 (ROBO2)/slit guidance ligand 2 (SLIT2) signaling destabilizes the slit diaphragm and reduces podocyte adhesion to the glomerular basement membrane (GBM). Preclinical studies suggest that inhibition of glomerular ROBO2/SLIT2 signaling can stabilize podocyte adhesion and reduce proteinuria. This clinical trial evaluates the preliminary efficacy and safety of ROBO2/SLIT2 inhibition with the ROBO2 fusion protein PF-06730512 in patients with FSGS.MethodsThe Study to Evaluate PF-06730512 in Adults With FSGS (PODO; ClinicalTrials.gov identifier NCT03448692), an open-label, phase 2a, multicenter trial in adults with FSGS, will enroll patients into 2 cohorts (n = 22 per cohort) to receive either high- or low-dose PF-06730512 (intravenous) every 2 weeks for 12 weeks. Key inclusion criteria include a confirmed biopsy diagnosis of FSGS, an estimated glomerular filtration rate (eGFR) ≥45 ml/min/1.73 m2 based on the Chronic Kidney Disease Epidemiology Collaboration formula (30–45 with a recent biopsy), and urinary protein-to-creatinine ratio (UPCR) >1.5 g/g. Key exclusion criteria include collapsing FSGS, serious/active infection, ≥50% tubulointerstitial fibrosis on biopsy, and organ transplantation. The primary endpoint is change from baseline to week 13 in UPCR; secondary endpoints include safety, changes in eGFR, and PF-06730512 serum concentration.ResultsThis ongoing trial will report the efficacy, safety, pharmacokinetics, and biomarker results of PF-06730512 for patients with FSGS.ConclusionFindings from this proof-of-concept study may support further development and evaluation of PF-06730512 to treat FSGS and warrant assessment in phase 3 clinical trials.

Bisphenol A impaired cell adhesion by altering the expression of adhesion and cytoskeleton proteins on human podocytes

Bisphenol A (BPA), a chemical -xenoestrogen- used in food containers is present in the urine of almost the entire population. Recently, several extensive population studies have proven a significant association between urinary excretion of BPA and albuminuria. The alteration of glomerular podocytes or "podocytopathy" is a common event in chronic albuminuric conditions. Since many podocytes recovered from patients' urine are viable, we hypothesized that BPA could impair podocyte adhesion capabilities. Using an in vitro adhesion assay, we observed that BPA impaired podocyte adhesion, an effect that was abrogated by Tamoxifen (an estrogen receptor blocker). Genomic and proteomic analyses revealed that BPA affected the expression of several podocyte cytoskeleton and adhesion proteins. Western blot and immunocytochemistry confirmed the alteration in the protein expression of tubulin, vimentin, podocin, cofilin-1, vinculin, E-cadherin, nephrin, VCAM-1, tenascin-C, and β-catenin. Moreover, we also found that BPA, while decreased podocyte nitric oxide production, it lead to overproduction of ion superoxide. In conclusion, our data show that BPA induced a novel type of podocytopathy characterizes by an impairment of podocyte adhesion, by altering the expression of adhesion and cytoskeleton proteins. Moreover, BPA diminished production of podocyte nitric oxide and induced the overproduction of oxygen-free metabolites. These data provide a mechanism by which BPA could participate in the pathogenesis and progression of renal diseases.

Loss of Roundabout Guidance Receptor 2 (Robo2) in Podocytes Protects Adult Mice from Glomerular Injury by Maintaining Podocyte Foot Process Structure

Roundabout guidance receptor 2 (ROBO2) plays an important role during early kidney development. ROBO2 is expressed in podocytes, inhibits nephrin-induced actin polymerization, down-regulates nonmuscle myosin IIA activity, and destabilizes kidney podocyte adhesion. However, the role of ROBO2 during kidney injury, particularly in mature podocytes, is not known. Herein, we report that loss of ROBO2 in podocytes [Robo2 conditional knockout (cKO) mouse] is protective from glomerular injuries. Ultrastructural analysis reveals that Robo2 cKO mice display less foot process effacement and better-preserved slit-diaphragm density compared with wild-type littermates injured by either protamine sulfate or nephrotoxic serum (NTS). The Robo2 cKO mice also develop less proteinuria after NTS injury. Further studies reveal that ROBO2 expression in podocytes is up-regulated after glomerular injury because its expression levels are higher in the glomeruli of NTS injured mice and passive Heymann membranous nephropathy rats. Moreover, the amount of ROBO2 in the glomeruli is also elevated in patients with membranous nephropathy. Finally, overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion. Taken together, these findings suggest that kidney injury increases glomerular ROBO2 expression that might compromise podocyte adhesion and, thus, loss of Robo2 in podocytes could protect from glomerular injury by enhancing podocyte adhesion that helps maintain foot process structure. Our findings also suggest that ROBO2 is a therapeutic target for podocyte injury and podocytopathy.