Nephrin Expression Research Articles

Low-dose lipopolysaccharide inducing continuous and obvious increase in urinary protein in hyperglycemic rats and the underlying mechanism.

Proteinuria is an important hallmark of diabetic nephropathy models, however it takes a long time for the proteinuria and is not stable. Therefore, low-dose lipopolysaccharide (LPS) was investigated in this work to induce rapid and stable proteinuria in hyperglycemic rats and the underlying mechanism was studied. Hyperglycemia rats was induced by high-fat feeding combined with intraperitoneal injection of streptozotocin (STZ). After 21 days, the model rats received a subinjury dose of 0.8 mg / kg LPS intraperitoneally (i.p.). We detected related biochemical indexes at different time periods after LPS injection and examined the expression of glomerular podocyte-associated proteins. Simultaneously, we measured expression of inflammatory factors, apoptotic proteins and albumin (ALB) in the renal cortex and renal medulla, respectively. PAS (Periodic Acid Schiff) staining was used to observe renal pathology. After LPS injection, urinary microalbumin (umALB) increased significantly and lasted longer. The expression of Nephrin, Podocin and necroptosis factor kappa B (NF-κB) in rennal cortex and Interleukin 18 (IL-18), Caspase-1, NF-κB and ALB in the renal medulla was significantly changed. Pathologically, the glomerular basement membrane was observed to be significantly thickened, the renal tubules were dilated, and the epithelial cells fell off in a circle. LPS promoted the continuous increase in urinary microalbumin in hyperglycemic rats, which was related to the damage to the glomerular basement membrane and renal tubular epithelial cells and to the inflammatory reaction in the kidney involved in NF-κB signaling, and this pathological damage can help to establish a stable model of diabetic nephropathy with increased proteinuria.

Shenkang protects renal function in diabetic rats by preserving nephrin expression

BackgroundShenkang injection has been used clinically to lower creatinine levels. This study explored the mechanism of Shenkang injection on protecting kidney function from hyperglycemia-mediated damage.MethodsThis study utilized a STreptoZotocin (STZ)-induced rat model of diabetes. In total, 60 rats were randomized into either the control group (n = 15) injected with vehicle or treatment group (n = 45) injected with STZ to induce hyperglycemia. Eight weeks after diabetes onset, diabetic rats were further randomized to receive different treatments for 4 consecutive weeks, including vehicle (diabetic nephropathy group, n = 15), Shenkang (n = 15), or Valsartan (n = 15). At 12 weeks, a series of urine and blood measures were examined and damage to the kidney tissue was examined using histology. Expression of nephrin and transforming growth factor-β1 (TGF-β1) were characterized using immunohistochemistry and Western blot.ResultsCompared to the control group, rats in the diabetic nephropathy group showed significant kidney damage demonstrated by high kidneyindex, high levels of urinary albumin, albumin/creatinine ratio (ACR), blood urea nitrogen as well as histological evidence. Shenkang injection significantly improved kidney function in the diabetic rats by decreasing kidney index, ACR, and serum creatinine. Shenkang treatment also mitigated kidney damage, improved nephrin expression, and decreased TGF-β1 expression in the kidneys.ConclusionsShenkang treatment protected renal function in diabetic rats by increasing nephrin expression, which protects diabetic rats from hyperglycemia-mediated kidney damage.

Morin hydrate promotes nephrin expression through modulation of Notch1-Snail signalling pathway in diabetic rats

Reduction in nephrin expression is usually observed in patients with diabetic nephropathy, and there is evidence that Notch1/Snail signalling pathway is essential for the expression of nephrin. This study investigated the role of morin hydrate (MH) on Notch1/Snail signalling pathway in the kidney of type-2 diabetic rats (T2D) exposed to diesel exhaust particles (DEP). The male wistar rats (n ​= ​40) were group into 8 groups: Group 1-Control; Group 2-DEP (0.5 ​mg/kg); Group 3-type-2 diabetic rat (T2D); Group 4-T2D ​+ ​DEP (0.5 ​mg/kg); Group 5-DEP (0.5 ​mg/kg) ​+ ​MH (30 ​mg/kg); Group 6-T2D ​+ ​MH (30 ​mg/kg); Group 7-T2D ​+ ​DEP 0.5 ​mg/kg ​+ ​MH 30 ​mg/kg; and Group 8-MH (30 ​mg/kg) only. Type-2 diabetes was induced in the rats through a one-time administration of STZ at 45 ​mg/kg after 14 days treatment with fructose solution at 20% w/v. The type-2 diabetic and non-diabetic rats were exposed to DEP at 0.5 ​mg/kg through nasal instillation every 48 ​h for 14 days. MH (30 ​mg/kg) was given orally every 24 ​h for 15 days. MH is a naturally occurring flavonoid chemical found in a variety of plants, and has been discovered to have a number of biological actions, including antioxidant, anti-inflammatory, and anticancer effects. The effect of MH on kidney function indices and electrolytes, and its modulatory role on Notch1/Snail signalling pathway were determined. In silico studies on binding affinity of MH with some proteins in this pathway was conducted and the electronic behavior of MH was predicted using DFT calculation. The results show that MH oral therapy ameliorates nephrotoxicity and protects the podocytes in T2D rats and T2D rats exposed to DEP. It decreased the serum levels of creatinine, urea and total protein, while also decreasing the levels of renal sodium and potassium ions and bicarbonate. MH increased mRNA expression of nephrin by modulating Notch1/Snail signalling pathway, and these results were supported by the molecular docking studies. This study suggests that MH promote glomerular filtration in T2D exposed to DEP.

Huangqi decoction ameliorates kidney injury in db/db mice by regulating the BMP/Smad signaling pathway

PurposeThis study aims to investigate the mechanism underlying the beneficial effects of Huangqi decoction (HQD) on Diabetic kidney disease (DKD) in diabetic db/db mice.MethodsEight-week-old male diabetic db/db mice were randomly divided into four groups: Model (1% CMC), HQD-L (0.12 g/kg), HQD-M (0.36 g/kg), and HQD-H (1.08 g/kg) groups. Non-diabetic db/m mice were served as the control group. These mice received HQD treatment for 8 weeks. After treatment, the kidney function, histopathology, micro-assay, and protein expression levels were assessed.ResultsHQD treatment improved the albumin/creatine ratio (ACR) and 24 h urinary albumin excretion, prevented the pathological phenotypes of increased glomerular volume, widened mesangial areas, the of mesangial matrix proliferation, foot process effacement, decreased nephrin expression and reduced number of podocytes. Expression profiling analysis revealed global transcriptional changes that predicted related functions, diseases and pathways. HQD treatment activated protein expressions of BMP2, BMP7, BMPR2, and active-Rap1, while inhibiting Smad1 and phospho-ERK. In addition, HQD was associated with improvements in lipid deposition in the kidneys of db/db mice.ConclusionHQD ameliorated the progression of DKD in db/db mice by regulating BMP transcription and downstream targets, inhibiting the phosphorylation of ERK and the expression of Smad1, promoting Rap1 binding to GTP, and regulating the lipid metabolism. These findings provide a potential therapeutic approach for treating DKD.

Protective Effect of Astragaloside IV against Cadmium-Induced Damage on Mouse Renal Podocytes (MPC5).

In this study, we investigated the protective effect of Astragaloside IV (Ast) on mouse podocytes and its possible mechanism of action by constructing a cadmium-induced mouse renal podocytes model. We investigated the effects of cadmium (Cd) toxicity on cell number, morphology, the mitochondrial status of subcellular organelles, protein and gene levels, and the protective effects of Ast by constructing a model of Cd-induced damage to mouse renal podocytes (MPC5) and giving Ast protection at the same time. The results showed that exposure of MPC5 cells to CdCl2 culture medium containing 6.25 μM concentration acted with low cell mortality, but the mortality of MPC5 cells increased with the prolongation of cadmium exposure time. Given Ast, the death rate in the low dose group (12.5 μM) was significantly reduced, while the death rate in the medium dose group (25 μM) was extremely significantly reduced. In comparison to the control group, the Cd-exposed group exhibited a significant increase of 166.7% in malondialdehyde (MDA) content and a significant decrease of 17.1% in SOD activity. The mitochondrial membrane potential was also reduced to varying degrees. However, in the Ast-protected group compared to the Cd-exposed group, the MDA content significantly decreased by 20.8%, the SOD activity decreased by 7.14%, and the mitochondrial membrane potential showed a significant increase. Fluorescence staining of mitochondrial membrane potential indicated that Cd exposure caused mitochondrial apoptosis. In the 12-h cadmium-exposed group, the protein expression of Nephrin in mice significantly decreased by 33.4%. However, the expression of the Desmin protein significantly increased by 67.8%, and the expression of the autophagy protein LC3-II significantly increased by 55.5%. Meanwhile, the expression of PINK1, a mitochondrial autophagy pathway protein, was significantly increased in the 12 h and 24 h cadmium exposure groups. The mRNA level of PINK1 was significantly increased, and that of Parkin was decreased in the 48 h cadmium exposure group. Compared to the Cd-exposed group, the Ast group showed more significant improvements in the expression of podocyte structure, functional proteins, and mitochondrial autophagy pathway proteins. The immunological assay of mitochondrial autophagic pathway proteins further indicated that Cd-induced damage to MPC5 cells might be associated with the dysregulation of mitochondrial autophagy.

Ang-(1-7) attenuates podocyte injury induced by high glucose in vitro.

The incidence of diabetic nephropathy (DN) is gradually increasing worldwide. Podocyte injury, such as podocyte apoptosis and loss of the slit diaphragm (SD)-specific markers are early pathogenic features of DN. The cultured mouse podocytes were separated into a high glucose-treated (HG, 30mM) group to mimic DN in vitro, a low glucose-treated (LG, 5mM) group as a control and HG+ angiotensin-(1-7)(Ang-(1-7)) and HG+Ang-(1-7) + D-Ala7-Ang-(1-7) (A779, Ang-(1-7)/Mas receptor antagonist) experimental groups. The Cell Counting Kit-8 (CCK-8) method and flow cytometry was used to detect podocyte activity and podocyte apoptosis respectively. The expression of angiotensin type 1 receptor (AT1R), Mas receptor (MasR) and podocyte-specific markers were examined by q-PCR and Western blot, respectively. The results showed that the decrease in podocyte activity; the increase in podocyte apoptosis; the decreased mRNA and protein expression of nephrin, podocin, WT-1 and MasR; and the upregulated expression of AT1R induced by HG could be reversed by Ang-(1-7). However, these effects were blocked by A779. The possible mechanisms of the Ang-(1-7)-mediated effect depended on MasR. In addition, the protective effect of Ang-(1-7) on podocyte activity was dose-dependent and most obvious at 10 µM. A779 had the greatest antagonistic action against Ang-(1-7) at a concentration of 10 μM. This study reveals that binding of Ang-(1-7) to its specific receptor MasR may counteract the effects of Ang II mediated by AT1R to significantly attenuate podocyte injury induced by high glucose. Ang-(1-7)/MasR targeting in podocytes may be a therapeutic approach to attenuate renal injury in DN.

#6693 INCREASED INFILTRATION OF LRG1+VCAN+LY6CHIGH MACROPHAGES ARE ESSENTIAL CONTRIBUTORS TO KIDNEY INJURY IN ADR MICE

Abstract Background and Aims The ADR mouse model is a well-established rodent model of FSGS that can manifest with albuminuria. Resident macrophages are present in the tubulointerstitium of the kidney, and circulating macrophages can be recruited to the kidney in disease states. However, the presence of macrophages in glomeruli and the role of macrophages in podocyte injury in ADR mouse models remain unknown. Method Single-cell RNA sequencing comprehensively characterized the expression of macrophages in mice glomeruli. Eight-week-old male BALB/c mice were used to induce ADR model. Immunofluorescence was used to detect the changes of renal macrophages after injected with ADR at different time points. Transwell co-culture system were used to detect the effect of Lrg1+Vcan+Ly6Chigh macrophages on MPCs. HCH6-1,the FPR1 inhibitor were used to restrain ANXA1-FPR1 pathway. Results Totally 506 macrophages were sequenced and Lrg1+Vcan+Ly6Chigh macrophages were significantly increased in ADR induced nephrotic mice glomeruli (P<0.01). The ligand receptor relationship between podocytes and macrophages were changed(P<0.01, means>1). Immunofluorescence demonstrated that macrophages are present in ADR glomeruli(P<0.05). However, the temporal and spatial changes of macrophages in ADR glomeruli still need to be proved by further experiments. After co-culture of Lrg1+Vcan+Ly6Chighmacrophages and MPCs, the expression of nephrin in MPCs decreased(P<0.05), which proved that macrophages can cause podocyte injury. In ADR group, the ANXA1-FPR1 signaling pathway was activated, and renal Lrg1+Vcan+Ly6Chighmacrophage infiltration, albuminuria and renal injury were reduced in ADR+HCH6-1gruop (all P<0.05). Conclusion Lrg1+Vcan+Ly6Chighmacrophages are recruited into mice glomeruli by activation of ANXA1-FPR1 signaling pathway and cause the damage of podocyte.

#4313 AN ANTI-FIBROTIC GENE ATTENUATES DIABETIC KIDNEY INJURY IN DB/DB MICE BY INHIBITING FIBROSIS VIA EPITHELIAL-MESENCHYMAL TRANSITION PATHWAY

Abstract Background and Aims Diabetic nephropathy (DN) is a major cause of mortality in patients with diabetes and chronic kidney disease, but there is a lack of effective therapeutic drugs for this disease. The development and progression of DN is influenced by fibrosis. transforming growth factor (TGF)-β1 is a key cytokine involved in fibrosis in many different organ systems. Anti-fibrotic gene (Anti-F) is a TGF-β/Smad signaling. Here we examined the therapeutic effect of Anti-F in a model of DN using db/db mice with streptozotocin (STZ) treatment. Method The db/db mice were divided into five groups; db/m+ (wild type), db/db+saline, db/db+STZ, db/db+STZ+CMV-Anti-F, and db/db+STZ+TGF-β-Anti-F. STZ was peritoneally injected for five consecutive days (50mg/kg) and Anti-F (40μg/head) was peritoneally administered once every two weeks. Mice were sacrificed four months after STZ injection. Results The Anti-F with CMV and TGF-β promoter administration markedly alleviated metabolic syndrome assessed by obesity and hyperglycemia, and renal dysfunction assessed by renal overweight and albuminuria in db/db+STZ mice. The administration obviously mitigated glomerular damage in diabetic mice, as reflected by the reduction of increased mesangial expansion and the expression of nephrin and podocin in db/db+STZ mice. Additionally, renal interstitial fibrosis was also significantly inhibited by Anti-F through suppressing epithelial-mesenchymal transition (EMT) signaling including α-SMA, Twist, and Snail, as well as inflammation reflected by IL-1β, MMP-2, and MMP-9 in diabetic mice. Conclusion Anti-F attenuated the development of DN in db/db mice with type 2 diabetes. The protective effect was associated with decreased inflammation and subsequent attenuation of EMT-mediated renal fibrosis. Thus, this study suggests that targeting the Anti-F could be considered as a novel therapeutic approach for preventing the progression of DN.

#3345 EFFECTS OF CONDITIONED MEDIUMS COLLECTED FROM 2D OR 3D CULTURED MSCS ON KIDNEY FUNCTIONS OF RATS WITH AUTOIMMUNE DIABETES INDUCED WITH STREPTOZOCIN

Abstract Background and Aims The main mechanism of therapeutic action of mesenchymal stem cells (MSCs) could be resulted from their secretome content rather than their direct differentiation into damaged cells [1, 2]. Preconditioning of MSCs with different strategies like culturing in 3 dimensional (3D) culture scaffolds could improve their therapeutic potential [1]. Based on our previous study [1] in which the concentrations of total protein and specifically VEGF and IL-4 in conditioned mediums (CMs) collected from MSCs cultured in 3D culture conditions were increased compared to one of 2D, the aim of this study is comparative analysis of effects of CMs obtained from MSCs incubated under 2D and 3D culture conditions on kidney functions of rats with autoimmune type I diabetes (aT1D) induced with streptozocin (STZ). Method Isolation and characterization of the umbilical cord MSCs and the following analysis of concentrations of total protein and paracrine factors in the 2D-CMs and 3D-CMs were performed as it was mentioned [1]. Sprague-Dawley rats were treated with 20 mg/kg STZ for 5 consecutive days to induce T1D and 12 doses of CMs were intraperitoneally introduced for 4 weeks in accordance with the timeline defined in Figure 1. Therapeutic effects of CMs were comparatively investigated by biochemical, physical, histopathological and immunohistochemical analysis. Results At the end of the 2nd week, urinary albumin/creatinine (u-Alb/Cr) ratio and creatinine removal rate (Crr) of diabetes were significantly aggravated compared to control group (p = 0,0001 and p = 0,13 respectively). At the end of the 7th week, u-Alb/Cr ratios of both treatment groups were significantly improved compared to one of diabetes group (p = 0,029 vs p = 0,002). There was no statistical difference among all groups for Crr values. In the post treatment period u-Alb/Cr ratio was exacerbated (p = 0,014) while Crr value was not changed compared to pre-treatment period (p = 0,713). There was no statistical change between the pre- and post-treatment values of u-Alb/Cr ratio and Crr for both treatment groups. Renal mass index of control group was lower than the ones of experimental groups, while weight percent change from pre- to post-treatment period of control group was statistically higher than the ones of experimental groups (p = 0,0001). However, there was no statistical difference for both parameters among the experimental groups (Figure 2). Histopathological evaluations showed that cortical tubular damage (CTD) was significantly ameliorated in only D+3D-CM group compared to diabetes group (p = 0,012), while there was no statistical change in glomerular fibrosis in both treatment groups (Figure 3.1 and 3.2 respectively). Immunohistochemical analysis indicated that nephrin expression was insignificantly increased in both treatment groups compared to diabetes group (Figure 3.3). Preliminary evaluation of thicknesses of glomerular basal membrane and pedicel with transmission electron microscopy (TEM) showed no clear difference among all the groups (Figure 4). Conclusion CM obtained from MSCs in 3D culture conditions could partially ameliorate kidney functions of aT1D by improving CTD and expression of nephrin which is crucial for the integrity of glomerular filtration barrier.

Fasudil compensates podocyte injury via CaMK4/Rho GTPases signal and actin cytoskeleton-dependent activation of YAP in MRL/lpr mice

Deposition of immune complexes in the glomerulus leads to irreversible renal damage in lupus nephritis (LN), of which podocyte malfunction arises earlier. Fasudil, the only Rho GTPases inhibitor approved in clinical settings, possesses well-established renoprotective actions; yet, no studies addressed the amelioration derived from fasudil in LN. To clarify, we investigated whether fasudil exerted renal remission in lupus-prone mice. In this study, fasudil (20mg/kg) was intraperitoneally administered to female MRL/lpr mice for 10weeks. We report that fasudil administration swept antibodies (anti-dsDNA) and attenuated systemic inflammatory response in MRL/lpr mice, accompanied by preserving podocyte ultrastructure and averting immune complex deposition. Mechanistically, it repressed the expression of CaMK4 in glomerulopathy by preserving nephrin and synaptopodin expression. And fasudil further blocked cytoskeletal breakage in the Rho GTPases-dependent action. Further analyses showed that beneficial actions of fasudil on the podocytes required intra-nuclear YAP activation underlying actin dynamics. In addition, in vitro assays revealed that fasudil normalized the motile imbalance by suppressing intracellular calcium enrichment, thereby contributing to the resistance of apoptosis in podocytes. Altogether, our findings suggest that the precise manners of crosstalks between cytoskeletal assembly and YAP activation underlying the upstream CaMK4/Rho GTPases signal in podocytes is a reliable target for podocytopathies treatment, and fasudil might serve as a promising therapeutic agent to compensate for the podocyte injury in LN.

Clemizole hydrochloride, a potent TRPC5 calcium channel inhibitor, prevents cisplatin-induced nephrotoxicity in Spraque-Dawley rats.

Cis-diamminedichloroplatinum (II) (cisplatin, Cis) is widely employed to treat several types of cancer. It has many important toxic side effects; one of the most important of which is nephrotoxicity. Clemizole hydrochloride (Clem) as the most potent inhibitor of TRPC5 channels was tested in an animal model of Cis-induced nephrotoxicity. Rats were divided into the following groups: control;Cis (8 mg/kg);Cis + 1 mg/kg Clem; Cis + 5 mg/kg Clem; Cis + 10 mg/kg Clem. Kidney injury was detected by histopathological and biochemical analysis. Urine urea nitrogen (UUN), creatinine, urine neutrophil gelatinase-associated lipocalin (NGAL), serum catalase (CAT), and malondialdehyde (MDA) levels were determined by enzyme-linked immunosorbent assay. Total antioxidant status (TAS) and total oxidant status (TOS)were studied using a colorimetric assay. Nephrin, synaptopodin, and Rac family small GTPase 1(RAC1) expressions were detected by Western blot analysis. Cis was found to induce histopathological alterations, including tubular degeneration, congestion, hemorrhage, hyaline casts, glomerular collapse, and apoptotic cell death. Clem at a doseof 1 and 5 mg/kgattenuated histopathological alterations. UUN, creatinine, and NGAL levels increased in the Cis-administered group, while all doses of Clem decreased in those. CAT and TAS levels decreased, whileTOS and oxidative stress index levels increased in the Cis-treated group. A dose of 1 and 5 mg Clemshowed antioxidant effects against oxidative stress. Cis induced lipid peroxidation by increasing MDA levels. All doses of Clem reduced MDA levels. Nephrin and synaptopodin expressions were decreased by Cis, and all doses of Clem increased that. All doses of Clem successfully depressed RAC1 expression. Clem showed a highly ameliorating effect on toxicity caused by Cis by blocking TRPC5 calcium channels.

Huangkui capsule alleviates doxorubicin-induced proteinuria via protecting against podocyte damage and inhibiting JAK/STAT signaling.

Huangkui capsule (HKC), a Chinese patent medicine, has been widely used in China as adjuvant therapy for chronic kidney disease (CKD). It displays superior anti-proteinuria efficacy than losartan in patients with CKD at stages 1-2, however, the mechanism of HKC alleviating proteinuria has not been well elucidated. This study aims to confirm the therapeutic effect and investigate associated underlying mechanism of HKC against proteinuria by in vivo and in vitro experiments. We established a doxorubicin (DOX) induced proteinuria mouse model to evaluate kidney function by biochemical markers measurement and to observe histopathological alterations by hematoxylin and eosin (H&E), Masson's trichrome and Periodic Acid-Schiff (PAS)-stained sections of renal, respectively. Moreover, the expressions of Nephrin and Podocin were measured by immunohistochemistry (IHC) and western blotting analysis to investigate podocyte damage. Furthermore, we established Mouse Podocyte Clone-5 (MPC-5) injury model to identify the active components of HKC against podocyte damage by detecting the expressions of Nephrin, Podocin, and ZO-1 proteins. At last, the key protein levels of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway were assessed by western blotting analysis to explore the underlying mechanism of HKC against proteinuria. Our results showed that HKC administration for three consecutive weeks dose-dependently ameliorated both renal function and histopathological damages, elevated the expressions of Nephrin and Podocin, the pivotal molecules maintaining filtration function of the podocyte, indicating the promising protective effect against podocyte injury under DOX exposure. Consistently, in vitro experiments showed HKC administration effectively reversed the abnormal expressions of Nephrin and Podocin in MPC-5 cells treated with DOX, suggesting its protective effect against podocyte injury to maintain filtration barrier integrity. In addition, Hibifolin was identified as the most active ingredients in HKC, which suppressed upstream JAK2/STAT3 and PI3K/Akt pathway phosphorylation to maintain the structural and functional integrity of podocyte filtration barrier. Of note, AG490, a selective JAK2 inhibitor, was used to further affirm the role of Hibifolin involving in regulation JAK2/STAT3. Our study suggested that HKC may protect podocytes via JAK2/STAT3 and PI3K/Akt pathway to display its effects of ameliorating proteinuria.

Astragalus polysaccharide alleviates angiotensin II-induced glomerular podocyte dysfunction by inhibiting the expression of RARRES1 and LCN2.

Podocyte loss is a predictor of kidney disease development, including diabetic nephropathy. Astragalus polysaccharide (APS) was considered a renoprotective drug, whereas the mechanisms operated by APS on podocyte dysfunction are rarely mentioned. This study aims at the mechanistic underlying of APS on angiotensin II (Ang II)-induced podocyte dysfunction. Mouse glomerular podocytes MPC5 were induced with Ang II, the morphologic changes were observed and nephrin, desmin and Wilms' tumour protein-1 (WT-1) levels were determined. The MPC5 cells were treated with APS (50, 100 and 200 μg/mL) and transduced with retinoic acid receptor responder protein 1 (RARRES1) overexpression vectors. The expression of RARRES1, lipocalin-2 (LCN2), nephrin and desmin was tested, MPC5 cell viability and apoptosis were evaluated, and the levels of an endocytotic receptor megalin, Bcl-2, Bax, interleukin (IL)-6, IL-1β and tumour necrosis factor (TNF)-α were assessed. The binding of RARRES1 to LCN2 was predicted and verified. Mice were infused with Ang II to evaluate histopathological alterations and 24-h urinary albumin content. Ang II induction suppressed MPC5 cell viability, reduced the expression of nephrin, WT-1, megalin and Bcl-2, and augmented the expression of desmin, Bax, IL-6, IL-1β and TNF-α, which were significantly nullified by APS treatment. RARRES1 interacted with LCN2, and APS treatment inhibited RARRES1 and LCN2 expression in a dose-dependent manner, thereby alleviating Ang II-induced podocyte dysfunction. Ang II infusion in mice facilitated pathological alterations in renal tissues and increased urinary albumin content, which were attenuated after APS treatment. Overall, APS treatment alleviated Ang II-induced podocyte dysfunction by inhibiting RARRES1/LCN2 expression and blocked kidney injury development in vivo.

Decellularization of Mouse Kidneys to Generate an Extracellular Matrix Gel for Human Induced Pluripotent Stem Cell Derived Renal Organoids

Chronic Kidney Disease (CKD) is a major cause of morbidity and mortality characterized by progressive renal fibrosis, and in extreme cases, renal failure. Human CKD models that replicate the biological complexity of the kidney and CKD are lacking and will be invaluable in identifying drugs to revert and/or prevent fibrosis. To address this unmet need, we developed 3D renal organoids where human induced pluripotent stem cells (hiPSCs) were differentiated to renal progenitors within a renal extracellular matrix (rECM) gel, based on the premise that an rECM could recreate the renal niche to facilitate hiPSC-derived renal progenitor generation. We used mouse kidneys as a source of rECM and identified that superior detergent-mediated decellularization of mouse kidneys was achieved with a combination of 0.5% w/v Sodium Dodecyl Sulphate and 1% v/v Triton-X and mechanical agitation for 60 h. HiPSCs that underwent specification to become metanephric mesenchyme (MM) were subsequently cultured within the rECM gel and, notably, mesenchymal to epithelial transition (MET) was observed, as judged by expression of nephron markers K-cadherin, Nephrin and WT1. These data demonstrate a role for rECM gel in developing human renal organoids from hiPSCs, which will aid the further development of a human disease model for renal fibrosis.

Moshen granule ameliorates membranous nephropathy by blocking intrarenal renin-angiotensin system signalling via the Wnt1/β-catenin pathway

BackgroundMembranous nephropathy (MN) is one of the cardinal causes of nephrotic syndrome in adults, but an adequate treatment regimen is lacking. PurposeWe assessed the effect of Moshen granule (MSG) on patients with MN and cationic bovine serum albumin (CBSA)-induced rats. We further identified the bioactive components of MSG and revealed the underlying molecular mechanism of its renoprotective effects. MethodsWe determined the effect of MSG on patients with MN and CBSA-induced rats and its components on podocyte injury in zymosan-activated serum (ZAS)-elicited podocytes and revealed their regulatory mechanism on the Wnt/β-catenin/renin-angiotensin system (RAS) signalling axis. ResultsMSG treatment improved renal function and reduced proteinuria in MN patients and significantly reduced proteinuria and preserved the protein expression of podocin, nephrin, podocalyxin and synaptopodin in CBSA-induced MN rats. Mechanistically, MSG treatment significantly inhibited the protein expression of angiotensinogen, angiotensin converting enzyme and angiotensin II type 1 receptor, which was accompanied by inhibition of the protein expression of Wnt1 and β-catenin and its downstream gene products, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in CBSA-induced MN rats. We further identified 81 compounds, including astragaloside IV (AGS), calycosin, barleriside A and geniposidic acid, that preserve the podocyte-specific protein expression in ZAS-induced podocytes. Among these four compounds, AGS exhibited the strongest inhibitory effects on podocyte protein expression. AGS treatment significantly inhibited the protein expression of RAS components and Wnt1 and β-catenin and its downstream gene products in ZAS-induced podocytes. In contrast, the inhibitory effect of AGS on podocyte-specific proteins, β-catenin downstream gene products and RAS components was partially abolished in ZAS-induced podocytes treated with ICG-001 and β-catenin siRNA. ConclusionThis study first demonstrates that AGS mitigates podocyte injury by inhibiting the activation of RAS signalling via the Wnt1/β-catenin pathway by both pharmacological and genetic methods. Therefore, AGS might be considered a new β-catenin inhibitor that inhibits the Wnt1/β-catenin pathway to retard MN in patients.

Growth associated protein 43 deficiency promotes podocyte injury by activating the calmodulin/calcineurin pathway under hyperglycemia

Podocyte injury is a crucial factor in the pathogenesis of diabetic kidney disease (DKD), and finding potential therapeutic interventions that can mitigate podocyte injury holds significant clinical relevance. This study was to elucidate the role of growth associated protein-43(Gap43) in podocyte injury of high glucose (HG). We confirmed the expression of Gap43 in human glomerulus and found that Gap43 expression was downregulated in podocytes of patients with DKD and HG-treated podocytes in vitro. Gap43 knockdown in podocytes promoted podocyte apoptosis, increased migration ability and decreased nephrin expression, while overexpression of Gap43 markedly suppressed HG-induced injury. Moreover, the increased expression and activity of calcineurin (CaN) were also abrogated by overexpression Gap43 in HG. Pretreatment with a typical CaN inhibitor FK506 in Gap43 knockdown podocytes restored the injury. Mechanistically, co-immunoprecipitation experiments suggested that Gap43 could bind to calmodulin (CaM). Pull-down assay further demonstrated that Gap43 and CaM directly interacts with each other via amino acids 30–52 of Gap43 and amino acids 133–197 of CaM. In addition, we also identified Pax5 as potential transcription inhibitor factor mediating Gap43 expression. In conclusion, the study indicated that the Gap43/CaM-CaN pathway may be exploited as a promising therapeutic target for protecting against podocyte injury in high glucose.

TRIM29 promotes podocyte pyroptosis in diabetic nephropathy through the NF-kB/NLRP3 inflammasome pathway.

Diabetic nephropathy (DN) is one of the most common complications of diabetes. Gradual loss of podocytes is a sign of DN and pyroptosis mechanistically correlates with podocyte injury in DN; however, the mechanism(s) involved remain unknown. Here we reveal that TRIM29 is overexpressed in high glucose (HG)-treated murine podocytes cells and that TRIM29 silencing significantly inhibits podocyte damage due to HG treatment, as evidenced by lower desmin expression and greater nephrin expression. Additionally, flow cytometry analysis showed that TRIM29 silencing significantly inhibited HG treatment-induced pyroptosis, which was confirmed by immunoblotting for NLRP3, active Caspase-1, GSDMD-N, and phosphorylated NF-κB-p65. Conversely, overexpression of TRIM29 could trigger pyroptosis that was attenuated by NF-κB inhibition, indicating that TRIM29 promotes pyroptosis through the NF-κB pathway. Mechanistic studies revealed that TRIM29 interacts with IκBα to mediate its ubiquitination-dependent degradation, which in turn leads to NF-κB activation. Taken together, our data demonstrate that TRIM29 can promote podocyte pyroptosis by activating the NF-κB/NLRP3 pathway. Thus, TRIM29 represents a potentially novel therapeutic target that may also be clinically relevant in the management of DN.

Loss of S1P Lyase Expression in Human Podocytes Causes a Reduction in Nephrin Expression That Involves PKCδ Activation.

Sphingosine 1-phosphate (S1P) lyase (SPL, Sgpl1) is an ER-associated enzyme that irreversibly degrades the bioactive lipid, S1P, and thereby regulates multiple cellular functions attributed to S1P. Biallelic mutations in the human Sglp1 gene lead to a severe form of a particular steroid-resistant nephrotic syndrome, suggesting that the SPL is critically involved in maintaining the glomerular ultrafiltration barrier, which is mainly built by glomerular podocytes. In this study, we have investigated the molecular effects of SPL knockdown (kd) in human podocytes to better understand the mechanism underlying nephrotic syndrome in patients. A stable SPL-kd cell line of human podocytes was generated by the lentiviral shRNA transduction method and was characterized for reduced SPL mRNA and protein levels and increased S1P levels. This cell line was further studied for changes in those podocyte-specific proteins that are known to regulate the ultrafiltration barrier. We show here that SPL-kd leads to the downregulation of the nephrin protein and mRNA expression, as well as the Wilms tumor suppressor gene 1 (WT1), which is a key transcription factor regulating nephrin expression. Mechanistically, SPL-kd resulted in increased total cellular protein kinase C (PKC) activity, while the stable downregulation of PKCδ revealed increased nephrin expression. Furthermore, the pro-inflammatory cytokine, interleukin 6 (IL-6), also reduced WT1 and nephrin expression. In addition, IL-6 caused increased PKCδ Thr505 phosphorylation, suggesting enzyme activation. Altogether, these data demonstrate that nephrin is a critical factor downregulated by the loss of SPL, which may directly cause podocyte foot process effacement as observed in mice and humans, leading to albuminuria, a hallmark of nephrotic syndrome. Furthermore, our in vitro data suggest that PKCδ could represent a new possible pharmacological target for the treatment of a nephrotic syndrome induced by SPL mutations.

Dysregulation of Angiopoietin-like-4 Associated with Hyperlipidemia-induced Renal Injury by AMPK/ACC Pathway.

Angiopoietin-like protein 4 (Angptl4) is a glycoprotein that is involved in regulating lipid metabolism, which has been indicated as a link between hypertriglyceridemia and albuminuria in glomerulonephropathy. Deregulated lipid metabolism is increasingly recognized as an important risk factor of glomerulonephropathy. This study aimed to investigate the Angptl4 expression in renal tissue and podocyte under hyperlipidemia conditions and explore the potential molecular mechanisms. The role of Angptl4 in hyperlipidemia-induced glomerular disease and the detailed underlying mechanisms are unclear. This study sought new insights into this issue. We measured Angptl4 levels in the plasma and urine from patients with hyperlipidemia and healthy people. Rats were fed a high fat diet (HFD) to induce dyslipidemia model and the human podocytes were stimulated by palmitic acid as in vivo and in vitro experiments. The podocytes injury and the Angptl4 level in renal tissues were evaluated. Furthermore, the mechanism of Angptl4 on podocytes injury was investigated. The urinary Angptl4 level was gradually upregulated in both patients with hyperlipidaemia and high fat-diet-induced rats. HFD rats showed increased 24 h urinary protein and glomerular tuft area at week 12. The levels of nephrin and WT-1 were down-regulated, but the Angptl4 levels were markedly upregulated on the glomerular of rats on HFD. In the human podocytes, lipid accumulation accompanied by increases of Angptl4, but the expression of nephrin, WT-1, p-AMPKα and p-ACC was decreased after palmitic acid treatment. However, this injury effect was mediated by the aminoimidazole-4-carboxamide-1β-D-ribofuranoside (AICAR), activator of the low energy sensor AMPK/ACC signaling. This study was the first of its kind to show that podocyte damage induced by dyslipidemia could be associated with upregulated Angptl4 and that patients with hyperlipidemia might have relatively high urinary Angptl4 expression. The dysregulation of Angptl4 in the podocytes under hyperlipidemia is possibly carried out through AMPK/ACC signaling pathway.

Angiopoietin-like protein 3 deficiency combined with valsartan administration protects better against podocyte damage in streptozotocin-induced diabetic nephropathy mice.

Diabetic nephropathy (DN) is a common leading cause of end-stage renal disease (ESRD). Podocyte injury is a major pathogenesis of DN. Pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) is insufficient to fully prevent the development of ESRD. The present investigation aims to evaluate the protective function of valsartan, an angiotensin receptor blocker, alone and in combination with angiopoietin-like protein 3 (Angptl3) knockout against renal damage and podocyte injury in streptozotocin (STZ)-induced diabetic mice. The mice were divided into four groups: normal control group, STZ-induced DN group, valsartan+DN group (val, 100mg/kg, po), and Angptl3-/- + valsartan+DN group. Tests on kidney function, renal pathology, podocyte ultrastructure, podocyte apoptosis, reactive oxygen species (ROS) production, and autophagy were performed. The combined Angptl3 knockout/valsartan treatment significantly attenuated diabetes-induced renal pathological damage and improved podocyte ultrastructure compared with valsartan alone. The combined administration ameliorated glomerular injury by increasing nephrin, podocin, and CD2-associated protein (CD2AP) expression levels and inhibiting podocyte loss by apoptosis. Compared with valsartan alone, Angptl3-/- and valsartan combination therapy significantly improved the renal function, as demonstrated by decreasing levels of serum urea nitrogen, creatinine, and urinary albumin. Additionally, the combination treatment significantly activated autophagy and reduced the ROS production than valsartan alone. These findings highlight the role of valsartan to Angptl3 knockout could have much better outcome that opens the future for drugs that could inhibit Angptl3.