Nephrin Protein Research Articles

The role of vitamin D receptor agonist on podocyte injury induced by high glucose

Abstract Background The effects of vitamin D receptor (VDR) agonist paricalcitol on the podocyte injury induced by high glucose (HG) were investigated in conditioned immortalized mouse podocytes (MPC-5). Methods (1) Grouped according to different glucose concentrations: normal group (NG): 5.6 mmol/L glucose; HG stimulation group: 25 mmol/L glucose (25HG); high osmotic control group (NG+M): 5.6 mmol/L glucose + 19.4 mmol/L D-mannitol. The expression levels of VDR, podocyte marker proteins podocin, nephrin and mesenchymal marker proteins α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMP9) in MPC-5 were measured, respectively. (2) Effect of VDR agonist-paricalcitol on podocyte epithelial-mesenchymal transition (EMT) induced by HG: cultured podocytes are divided into NG group, NG with dimethylsulfoxide (DMSO) group (NG+D), NG with paricalcitol (0.1 μmol/L) group (NG+P), HG group, HG with DMSO group (HG+D), and HG with paricalcitol (0.1 μmol/L) group (HG+P). The expression levels of VDR, podocyte marker proteins, marker proteins of mesenchymal cells, and the albumin flow in each group were then detected. Results (1) Under HG conditions, the expressions of VDR, podocin, and nephrin were decreased, while the expressions of α-SMA and MMP9 were increased (all P < 0.05). After administration of paricalcitol, the levels of VDR, podocin, and nephrin were increased, while the expressions of α-SMA and MMP9 were decreased compared with the control groups (all P < 0.05). (2) The results of albumin flow showed that the albumin flow of podocytes increased under the condition of HG, while it decreased after the treatment of paricalcitol. Conclusion The podocyte injury induced by HG could be partly rescued by Paricalcitol.

Parthenolide regulates DNMT1-mediated methylation of VDR promoter to relieve podocyte damage in mice with diabetic nephropathy

Current evidence showed that parthenolide (PN) has strong anti-inflammatory activity, but its effect on diabetic nephropathy (DN) is still unclear. In this research, high glucose (HG)-induced MPC5 cells were incubated with 5, 10 or 20 μM PN, and we found that PN incubation improved HG-induced MPC5 cells viability and apoptosis, ROS level, LDH activity and the secretion of inflammatory factors (IL-6, TNF-α and IL-1β), and inhibited the expression of DNMT1 protein, and promoted the expression of VDR, p-AKT, nephrin and podocin proteins. Lentivirus-mediated VDR overexpression vector (LV-VDR) transfection had the same effect, and LV-DNMT1 or si-VDR transfection or 100 nM AKT inhibitor MK2206 incubation reversed the effect of PN on cell functions. Research on the mechanism found that PN reduced the level of VDR methylation by reducing the enrichment of DNTM1 in the VDR promoter region under high glucose condition. In vivo, C57BL/6 mice were injected with streptozotocin (STZ) intraperitoneally to construct a DN mouse model, and 5mg/kg PN was administered intraperitoneally every other day. The results showed that PN treatment improved glomerular hypertrophy and renal fibrosis in STZ-induced mice. In general, PN regulated DNTM1-mediated VDR methylation, activated AKT, and alleviated DN.

Effects of Qidi Tangshen granules and their separate prescriptions on podocytes in mice with diabetic nephropathy

Effects of Qidi Tangshen granules and their separate prescriptions on podocytes in mice with diabetic nephropathy

Identification of Nephrin gene variants in Indian children associated with Steroid sensitive and Steroid resistant nephrotic syndrome

Identification of Nephrin gene variants in Indian children associated with Steroid sensitive and Steroid resistant nephrotic syndrome

Atractylodin inhibits fructose-induced human podocyte hypermotility via anti-oxidant to down-regulate TRPC6/p-CaMK4 signaling

Atractylodin inhibits fructose-induced human podocyte hypermotility via anti-oxidant to down-regulate TRPC6/p-CaMK4 signaling

Triptolide Alleviates Podocyte Epithelial-Mesenchymal Transition via Kindlin-2 and EMT-Related TGF-β/Smad Signaling Pathway in Diabetic Kidney Disease.

Diabetes-induced chronic kidney diseases are widespread and decrease the quality of life for millions of affected individuals in China. To date, no therapies effectively alleviate these conditions. Triptolide, a traditionally used Chinese medicine, has shown promise in treating renal diseases. Here, the study aimed to decipher the exact mechanism by which it functions. It was hypothesized that triptolide might prevent the epithelial-mesenchymal transition (EMT) of podocytes by activating the kindlin-2 and TGF-β/Smad pathways. Triptolide or telmisartan was intragastrically administered to 9-week-old db/db and dm/dm mice with diabetic nephropathy (DN) for 12weeks. In addition, biochemical parameters and body weight were detected. WT-1, nephrin, podocin, E-cadherin, and α-SMA were determined by immunohistochemistry in the renal tissues of treated mice. Protein and mRNA expression of podocyte EMT markers, kindlin-2 and TGF-β/Smad, were analyzed to elucidate the underlying mechanism. It was observed that triptolide treatment relieved structural injuries and functional variations in diabetic mice. It also increased the protein and mRNA levels of nephrin, podocin, and E-cadherin and decreased the expression of α-SMA in diabetic mice. The protein and mRNA expressions of TGF-β1, p-SMAD3, and kindlin-2 decreased in diabetic kidneys following triptolide treatment. The findings demonstrated that triptolide might protect podocytes during DN by inhibiting podocyte EMT through inactivation of kindlin-2, combined with the downregulation of P-SMAD3 in the TGF-β/Smad signaling pathway.

Alpha-1 Acid Glycoprotein and Podocin mRNA as Novel Biomarkers for Early Glomerular Injury in Obese Children.

Introduction: Obesity, which is a serious problem in children, has a negative impact on many organs, including kidneys, and obesity-related glomerulopathy (ORG) is an increasingly common cause of ESKD (end-stage kidney disease) in adults. Early-detected and -treated glomerular lesions are reversible, so it is important to find a useful marker of early damage. The study aimed to evaluate the albumin-to-creatinine ratio (ACR), urinary alpha-1-acid glycoprotein (α1-AGP), and mRNA of podocyte-specific proteins as indicators of glomerular injury and their relationship with the degree of obesity and metabolic disorders. Materials and Methods: A total of 125 obese children and 33 healthy peers were enrolled. Patients were divided into two groups, depending on SDS BMI values. ACR, α1-AGP, mRNA expression of nephrin, synaptopodin, podocin, and C2AP protein in urine sediment were measured. Results: ACR values did not differ between groups and were within the normal range. α1-AGP and mRNA expression were significantly higher in obese children compared with controls. mRNA expression of the remaining podocyte proteins was similar in both groups. No significant differences concerning all examined parameters were found depending on the degree of obesity. There was a positive significant correlation between α1-AGP and ACR. Conclusions: Increased α1-AGP before the onset of albuminuria suggests its usefulness as a biomarker of early glomerular damage in obese children. An increased podocin mRNA expression also indicates podocyte damage and may be linked to ORG development. The lack of increase in expression of other podocyte proteins suggests that podocin mRNA may be a more specific and sensitive biomarker. The degree of obesity has no impact on the tested parameters, but further studies are needed to confirm it.

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

Modified Huangqi Chifeng Decoction Attenuates Proteinuria by Reducing Podocyte Injury in a Rat Model of Immunoglobulin a Nephropathy.

Modified Huangqi Chifeng decoction (MHCD) has been used to reduce proteinuria in immunoglobulin A nephropathy (IgAN) for many years. Previously, we have demonstrated its protective role in glomerular mesangial cells. Podocyte injury, another key factor associated with proteinuria in IgAN, has also attracted increasing attention. However, whether MHCD can reduce proteinuria by protecting podocytes remains unclear. The present study aimed to investigate the protective effects of MHCD against podocyte injury in a rat model of IgAN. To establish the IgAN model, rats were administered bovine serum albumin, carbon tetrachloride, and lipopolysaccharide. MHCD in three doses or telmisartan was administered once daily for 8 weeks (n = 10 rats/group). Rats with IgAN developed proteinuria at week 6, which worsened over time until drug intervention. After drug intervention, MHCD reduced proteinuria and had no effect on liver and kidney function. Furthermore, MHCD alleviated renal pathological lesions, hyperplasia of mesangial cells, mesangial matrix expansion, and podocyte foot process fusion. Western blot analysis revealed that MHCD increased the expression of the podocyte-associated proteins nephrin and podocalyxin. Additionally, we stained podocyte nuclei with an antibody for Wilms’ tumor protein one and found that MHCD increased the podocyte number in rats with IgAN. In conclusion, these results demonstrate that MHCD attenuates proteinuria by reducing podocyte injury.

Wogonin protects glomerular podocytes by targeting Bcl-2-mediated autophagy and apoptosis in diabetic kidney disease

Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg−1 · d−1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

Sarsasapogenin restores podocyte autophagy in diabetic nephropathy by targeting GSK3β signaling pathway

Sarsasapogenin restores podocyte autophagy in diabetic nephropathy by targeting GSK3β signaling pathway

Atractylodis rhizoma water extract attenuates fructose-induced glomerular injury in rats through anti-oxidation to inhibit TRPC6/p-CaMK4 signaling

Atractylodis rhizoma water extract attenuates fructose-induced glomerular injury in rats through anti-oxidation to inhibit TRPC6/p-CaMK4 signaling

Salvianolic acid A attenuates steroid resistant nephrotic syndrome through suPAR/uPAR-αvβ3 signaling Inhibition

Salvianolic acid A attenuates steroid resistant nephrotic syndrome through suPAR/uPAR-αvβ3 signaling Inhibition

The Effects of Rhubarb for the Treatment of Diabetic Nephropathy in Animals: A Systematic Review and Meta-analysis.

Background: Rhubarb, also known as Da Huang, is a traditional Chinese medicine, and it was often used as a laxative in the past. Recently, multiple studies have applied rhubarb to treat diabetic nephropathy (DN). Anthraquinones, including emodin and rhein, have been extracted from rhubarb and used to explore the effective components and possible mechanisms of rhubarb for DN. Evaluating the efficacy of rhubarb may provide a scientific reference for the clinical application of rhubarb for the treatment of DN. Objective: 1) To evaluate the efficacy of rhubarb in the treatment of DN; 2) To identify the most effective ingredient of rhubarb for DN; 3) To explore the specific mechanism of rhubarb in treating DN. Methods: Data sources: related studies were identified by searching Cochrane Library, Ovid-EMBASE, PubMed, SinoMed, WanFang, VIP, CNKI, and other Chinese magazines.Assessment and analysis: SYRCLE’s risk of bias tool for animal studies was used to assess the quality of articles. The meta-analysis was performed in accordance with the Cochrane Handbook for Systematic Reviews of Interventions. Data analysis adopted RevMan 5.3 and STATA 12.0 software.This study was published in the register with PROSPERO, number CRD42020204701. Results: Aggregated data were collected from 27 eligible studies. The results illustrated an intense improvement in the following outcomes in rhubarb-treated animals with DN (p < 0.05): blood glucose, serum creatinine (Scr), blood urea nitrogen (BUN), albumin creatinine ratio (ACR), urine protein (UP), urinary albumin excretion (UAE), renal index (two kidneys weight/body weight, KW/BW), tubulointerstitial injury index (TII), transforming growth factor-beta1 (TGF-β1) mRNA and protein, alpha-smooth muscle actin (α-SMA) protein, and E-cadherin (E-cad) protein. Of these, DN animals with rhubarb exhibited a significantly higher level of E-cad protein. In addition, the level of the other outcomes mentioned above decreased significantly, while there was no significant association between the intervention and nephrin protein (p > 0.05). Conclusion: This systematic review and meta-analysis demonstrated that rhubarb has a positive therapeutic effect on animals with DN, which may provide confidence and some theoretical reference for clinical application to a certain extent.

MO081THE INFLUENCE OF VITAMIN D3 ON PODOCYTE DIFFERENTIATION IN SITU AND IN VITRO

Abstract Background and Aims Dedifferentiation of podocytes affects their complex 3 D morphology and is the main initiator for the development of chronic kidney disease (CKD). Unfortunately, there is no causal therapy for CKD until today. Thus, inadequate and late treatment lead to end-stage renal disease which subsequently makes renal replacement therapy inevitable. To address this, new treatment options are of high significance for CKD patients. Recently, vitamin D3 (VitD) became a promising candidate, but it is controversially discussed. In the present study, we investigated the influence of VitD on podocyte differentiation and the related pathways in situ and in vitro. Method We combined a podocyte dedifferentiation model (GlomAssay) with an automated imaging procedure (Aquifer Imaging Machine). We analyzed cultured glomeruli from transgenic mice expressing cyan-fluorescent protein (CFP) under the control of the nephrin promoter which were treated with VitD and its` analogue (calcipotriol). In this model, the decreasing CFP fluorescence is as a read out for podocyte (de)differentiation. Additionally, VitD-, calcipotriol- and VitD receptor (VDR) inhibitor (PS121912)-treated glomeruli were investigated by RNA-Seq and LC-MS/MS to reveal the molecular effects of VitD on podocyte differentiation. Furthermore, we treated cultured murine podocytes with VitD, calcipotriol and PS121912 to elucidate the morphological and molecular changes by immunofluorescence staining, RT-qPCR and Western blot. Results VitD- and calcipotriol-treated glomeruli showed a significantly higher intensity of CFP fluorescence after 9 days, indicating higher level of nephrin compared to the control. This was verified by RT-qPCR and Western blot for nephrin and CFP. Additionally, we found an upregulation of VDR in VitD- and calcipotriol-treated glomeruli compared to controls. By transcriptomic and proteomic analysis, we identified molecular patterns that are specific for the different treated groups. Thus, we observed differential gene expression in VitD- and Wnt-signaling pathway as well as regulated genes that are essential for the actin cytoskeleton, focal adhesion formation and the slit membrane. Beside this, cultured podocytes showed a significant upregulation of the slit membrane protein nephrin, VDR and CYP24A1 by VitD. This is accompanied by an altered morphology of the podocytes due to a reorganization of the actin cytoskeleton. Conclusion Our results show that VitD influences podocyte differentiation in situ and in vitro by the regulation of specific signaling pathways.

FC 037NEUTROPHILS PLAY A KEY ROLE IN THE INITIATION OF GLOMERULAR HEMATURIA IN A POSTINFECTIOUS IGAN EXPERIMENTAL MODEL

Abstract Background and Aims Hematuria is a common finding in patients with IgA nephropathy (IgAN), occurring mainly after upper respiratory tract infections. Hematuria can lead to acute kidney injury and chronic loss of renal function in IgAN. However, the mechanisms involved in egression of erythrocytes from the glomerular capillaries into the urinary space are unknown. To answer this question, we developed an infection with Streptococcus pneumoniae (SP) in a humanized experimental IgAN model (α1KICD89tg mice) that resembles the pathological and clinical findings of disease (IgA1 and soluble CD89 mesangial deposits, complement activation, proteinuria and hematuria). Method α1KICD89tg mice (12 weeks old) received an intranasal instillation of SP (107 bacteria). Blood, urine and renal samples were obtained during 1 month after induction of respiratory infection. The presence of SP in lungs from these mice was confirmed by microbiological analysis. Hematuria was quantified in the urinary sediment and renal function was determined by biochemical analysis. Renal histological characteristics were evaluated by hematoxylin/eosin, masson's trichrome and PAS staining. IgA glomerular deposits, activation of complement system and infiltration of proinflammatory cells was examined by immunohistochemistry or immunofluorescence. Circulating leukocyte populations were studied on a hemocytometer. Renal inflammatory cytokines, metalloproteases, as well as markers of tubular and glomerular damage were determined in kidneys by RT-PCR and western-blot. To further validate the role of neutrophils in this pathological setting, we selective depleted these cells through a single injection of anti-Ly6G mAb (200 µg/kg i.p). Results SP-intranasal instillation in α1KICD89tg mice increased hematuria, microalbuminuria and proteinuria, peaking at 48h after induction of the respiratory infection. SP instillation caused disruption of the glomerular basement membrane, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin, as well as higher glomerular accumulation of IgA and proteins of complement system (C3, MBL). Hematuria intensity was positively correlated with the presence of interstitial F4/80+ macrophages, matrix metalloproteinase 9 (MMP-9), inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, CCL-2, CCL5 and CX3CL1/CX3CR1) as well as p65 NF-κB activation. Hematuria was negatively correlated with anti-inflammatory IL-10 mRNA expression, Factor H levels and collagen IV content. Notably, SP infection induced expression of the tubular injury markers N-GAL and KIM-1. Increased peripheral neutrophils levels were observed in the SP-infected α1KICD89tg mice. Mechanistically, anti-Ly6G-mediated neutrophil depletion reduced SP-mediated hematuria, proteinuria and albuminuria, prevented loss of synaptopodin and nephrin, decreased renal inflammation and MMP-9 expression in α1KICD89tg mice Conclusion In a humanized mouse model of IgAN, hematuria bouts following respiratory tract infections are caused by a neutrophil-mediated alteration of the glomerular filtration barrier (podocyte damage, complement deposits and loss of Collagen IV). These findings may help to unveil novel potential therapeutic approaches to combat one of the key elements in the progression of IgAN and related conditions.

MO625INHIBITION OF SGLT-2 CANNOT RESCUE NEPHRIN EXPRESSION IN DIABETIC NEPHROPATHY

Abstract Background and Aims Early glomerular damage in diabetes is induced by high blood glucose level (hyperglycemia) which affects the glomerular filtration barrier and leads to proteinuria. Podocyte-specific proteins like the transmembrane protein nephrin form the slit diaphragm that is important for proper function of the glomerular filtration barrier. Sodium-glucose co-transporter 2 (SGLT2) specific proteins are involved in glucose reabsorption in the kidney and maintain the normal glucose level in the blood. Recent studies showed remarkable success of SGLT2-inhibition in patients with diabetic nephropathy. Therefore we wanted to study if hyperglycemia induced reduction of nephrin expression is affected bySGLT-2 inhibition. Therefore we induced hyperglycemia in zebrafish larvae by knockdown of Pancreatic duodenal homeobox 1 (Pdx1) transcription factor and treated zebrafish larvae with Empagliflozin. In parallel we treated Bl/6 mice with streptozotozin and treated them with Empagliflozin. We then analyzed nephrin expression in both model systems. Method Zebrafish is an ideal model to study glomerular diseases, because the zebrafish larvae develops a pronephros with high homology to the human glomerulus. In order to inhibit SLGT-2 after Pdx1-knockdown, we treated both control and diabetic zebrafish larvae with 10µM Empagliflozin from 1dpf to 5dpf. We used a zebrafish line that expresses a fluorescent Vitamin D binding plasma protein Tg(l-fabp:DBP:eGFP) to measure proteinuria by measuring the GFP signal in both retinal and glomerular vessels of 120 hpf larvae. Immunohistochemistry against nephrin was performed using a specific zebrafish antibody. Results Conclusion Despite the promising effects of SLGT-2 inhibitor treatment in patients with diabetic nephropathy the early effects on nephrin expression are not addressed and remain an unchanged problem by this novel treatment option.

A critical re-analysis of cases of post-transplantation recurrence in genetic nephrotic syndrome

BackgroundGenetic defects in podocyte proteins account for up to 30% of steroid-resistant nephrotic syndrome (SRNS) in the paediatric population. Most children with genetic SRNS are resistant to immunosuppression and at high risk of progression to stage 5 chronic kidney disease. Kidney transplantation is often the treatment of choice. The possibility of post-transplantation disease recurrence in genetic SRNS remains controversial, and poses fundamental questions about disease biology.MethodsWe critically evaluated the published cases of post-transplantation recurrence in genetic patients, particularly testing ‘mutations’ against the most recent population variant databases, in order to clarify the diagnoses, and compare the clinical courses and responses to therapy.ResultsBiallelic pathogenic variants in NPHS1 leading to a complete absence of nephrin were the most commonly reported and best understood instance of nephrotic syndrome occurring post-transplantation. This is an immune-mediated process driven by antibody production against the novel nephrin protein in the allograft. We also identified a number of plausible reported cases of post-transplantation recurrence involving pathogenic variants in NPHS2 (8 patients, biallelic), one in WT1 (monoallelic) and one in NUP93 (biallelic). However, the mechanism for recurrence in these cases remains unclear. Other instances of recurrence in genetic disease were difficult to interpret due to differing clinical criteria, inclusion of patients without true pathogenic variants or the influence of other factors on renal outcome.ConclusionsOverall, post-transplantation recurrence remains very rare in patients with genetic SRNS. It appears to occur later after transplantation than in other patients and usually responds well to plasmapheresis with a good renal outcome.

Store‐operated calcium entry mediated high glucose‐induced podocyte injury and mitochondrial impairment

Purpose Diabetic Nephropathy is one of the major complications of diabetes mellitus. Hyperglycemia is a known initiator of diabetes mellitus. Accumulating evidence suggests that podocyte injury is tightly associated with the onset and progression of diabetic nephropathy. However, the mechanisms underlying podocyte injury induced by hyperglycemia or high glucose (HG) is poorly understood. Store-operated calcium entry (SOCE) is a multifunctional signaling pathway in many cell types. However, its role in podocyte injury in the settings of diabetes is not known. The aim of the present study was to examine if SOCE mediated HG-induced podocyte injury by impairing mitochondrial function. Methods All experiments were carried out in cultured immortalized human podocytes. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and nephrin (a podocyte specific protein). Calcium imaging was used to analyze SOCE. TMRE fluorescence was used to probe the mitochondria membrane potential (MMP). Results HG (25mM) significantly increased podocyte Orai1 protein abundance for time periods ranging from 2 to 12 hours. This HG effect was dose dependent. Consistently, Ca2+ imaging experiments showed that HG treatment (25 mM for 12 hours) significantly enhanced podocyte SOCE. Furthermore, the abundance of nephrin protein decreased in podocytes after exposure to HG, indicating podocyte injury in the presence of ambient HG. However, the HG-induced decrease of nephrin was blunted by BTP2 (an SOCE inhibitor, 4 µM). Moreover, HG treatment (25 mM for 24 hours) decreased MMP, indicating damage of mitochondria by HG. The decrease of MMP was prevented by BTP2, suggesting the contribution of SOCE to the detrimental effect of HG treatment. Conclusion The present study suggests that upregulated SOCE contributes to HG-induced podocyte injury, possibly by impairing mitochondrial function.

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy.

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter's role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.