The Role of miRNAs in Podocyte Injury in Diabetic Nephropathy: Mechanisms and Clinical Applications.

The anti-aging gene, klotho, has been identified as a multi-functional humoral factor and is implicated in multiple biological processes. However, the effects of klotho on podocyte injury in diabetic nephropathy are poorly understood. Thus, the current study aims to investigate the renoprotective effects of klotho against podocyte injury in diabetic nephropathy. We examined lipid accumulation and klotho expression in the kidneys of diabetic patients and animals. We stimulated cultured mouse podocytes with palmitate to induce lipotoxicity-mediated podocyte injury with or without recombinant klotho. Klotho level was decreased in podocytes of lipid-accumulated obese diabetic kidneys and palmitate-treated mouse podocytes. Palmitate-treated podocytes showed increased apoptosis, intracellular ROS, ER stress, inflammation, and fibrosis, and these were significantly attenuated by klotho administration. Klotho treatment restored palmitate-induced downregulation of the antioxidant molecules, Nrf2, Keap1, and SOD1. Klotho inhibited the phosphorylation of FOXO3a, promoted its nuclear translocation, and then upregulated MnSOD expression. In addition, klotho administration attenuated palmitate-induced cytoskeleton changes, decreased nephrin expression, and increased TRPC6 expression, eventually improving podocyte albumin permeability. These results suggest that klotho administration prevents palmitate-induced functional and morphological podocyte injuries, and this may indicate that klotho is a potential therapeutic agent for the treatment of podocyte injury in obese diabetic nephropathy.

The anti-aging gene, klotho, has been identified as a multi-functional humoral factor and is implicated in multiple biological processes. However, the effects of klotho on podocyte injury in diabetic nephropathy are poorly understood. Thus, the current study aims to investigate the renoprotective effects of klotho against podocyte injury in diabetic nephropathy. We examined lipid accumulation and klotho expression in the kidneys of diabetic patients and animals. We stimulated cultured mouse podocytes with palmitate to induce lipotoxicity-mediated podocyte injury with or without recombinant klotho. Klotho level was decreased in podocytes of lipid-accumulated obese diabetic kidneys and palmitate-treated mouse podocytes. Palmitate-treated podocytes showed increased apoptosis, intracellular ROS, ER stress, inflammation, and fibrosis, and these were significantly attenuated by klotho administration. Klotho treatment restored palmitate-induced downregulation of the antioxidant molecules, Nrf2, Keap1, and SOD1. Klotho inhibited the phosphorylation of FOXO3a, promoted its nuclear translocation, and then upregulated MnSOD expression. In addition, klotho administration attenuated palmitate-induced cytoskeleton changes, decreased nephrin expression, and increased TRPC6 expression, eventually improving podocyte albumin permeability. These results suggest that klotho administration prevents palmitate-induced functional and morphological podocyte injuries, and this may indicate that klotho is a potential therapeutic agent for the treatment of podocyte injury in obese diabetic nephropathy.

Diabetic nephropathy is one of the major complications of diabetes mellitus and is the leading cause of end-stage renal disease worldwide. Podocyte injury contributes to the development of diabetic nephropathy. However, the molecules that regulate podocyte injury in diabetic nephropathy have not been fully clarified. MicroRNAs (miRNAs) are small non-coding RNAs that can inhibit the translation of target messenger RNAs. Previous reports have described alteration of the expression levels of many miRNAs in cultured podocyte cells stimulated with a high glucose concentration and podocytes in rodent models of diabetic nephropathy. The associations between podocyte injury and miRNA expression levels in blood, urine, and kidney in patients with diabetic nephropathy have also been reported. Moreover, modulation of the expression of several miRNAs has been shown to have protective effects against podocyte injury in diabetic nephropathy in cultured podocyte cells in vitro and in rodent models of diabetic nephropathy in vivo. Therefore, this review focuses on miRNAs in podocyte injury in diabetic nephropathy, with regard to their potential as biomarkers and miRNA modulation as a therapeutic option.

Klotho inhibits PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy

ObjectiveThis work aims to elucidate the effect and the regulatory mechanisms of miR-205-5p on podocyte injury and oxidative stress in diabetic nephropathy.MethodsA mouse model of diabetic nephropathy was established. Fasting blood glucose, 24 hours urinary albumin, serum creatinine and blood urea nitrogen of mice were detected. H&E and Tunel staining of mice renal tissues were executed to detect histological changes and apoptosis. A cell model of diabetic nephropathy was constructed by inducing mouse podocytes with high glucose. The function of miR-205-5p on viability, apoptosis, and levels of malondialdehyde, superoxide dismutase and glutathione in the diabetic nephropathy cell model was evaluated by CCK-8 assay, Tunel staining and enzyme-linked immunosorbent assay. Binding of miR-205-5p and vascular endothelial growth factor A was verified by dual luciferase reporter gene assay. Rescue experiment was implemented on the diabetic nephropathy cell model to research whether miR-205-5p regulated diabetic nephropathy development by targeting vascular endothelial growth factor A. Quantitative reverse transcription-polymerase chain reaction and Western blot were for the detection of gene expression.ResultsThe increased fasting blood glucose, 24 hours urinary albumin, serum creatinine and blood urea nitrogen levels, the intensified apoptosis and injury, and the down-regulated miR-205-5p were observed in renal tissues. miR-205-5p relieved podocyte injury in diabetic nephropathy, as it increased cell viability, decreased cell apoptosis, reduced malondialdehyde, and elevated superoxide dismutase and glutathione in the diabetic nephropathy cell model. Vascular endothelial growth factor A was up-regulated in renal tissues of diabetic nephropathy mice, and directly suppressed by miR-205-5p. Vascular endothelial growth factor A up-regulation abolished the protection of miR-205-5p on the diabetic nephropathy cell model.ConclusionsmiR-205-5p might relieve podocyte injury in diabetic nephropathy by suppressing Vascular endothelial growth factor A. It might be a promising target for diabetic nephropathy treatment.

To explore the influence of long non-coding (lnc) RNA Gm15645 on the podocyte injury in mice with diabetic nephropathy. Male db/db mice (with Type 2 diabetes) with a genetic background of C57BLKs/J and db/m mice (healthy) born in littermates were randomly divided into three groups. db/db group was injected with lncRNAGm15645 shRNA lentivirus with a podocyte-specific marker NPHS2; db/db blank group was injected with saline, and db/db control group was injected withnon-sense lentivirus. The results of PAS staining, pathological changes of renal tissue, relative expression of GSK-3beta, and podocin expression were compared. lncRNAGm15 645 was overexpressed and podocin was down-regulated in the lentivirus overexpressed group. Mesangial cell proliferation, mesangial matrix hyperplasia, thickened basement membrane, widely fused foot process, and podocyte injury were observed by PAS staining. The expression of Gm15645 in the db/db group was significantly lower than that of the db/db blank group and db/db control group (P< 0.05), while the expression of podocin was higher (P< 0.05). Gm15645 was co-stained with podocin in renal tissue, and the target gene was GSK-3beta. lncRNAGm15645 may provide an early biomarker for the occurrence of podocyte injury in diabetic nephropathy. The mechanism may be related to the feedback regulation of GSK-3beta gene.

Objectives Diabetic nephropathy (DN) is the most common microvascular complication of diabetes mellitus. This study investigated the mechanism of triptolide (TP) in podocyte injury in DN. Methods DN mouse models were established by feeding with a high-fat diet and injecting with streptozocin and MPC5 podocyte injury models were induced by high-glucose (HG), followed by TP treatment. Fasting blood glucose and renal function indicators, such as 24 h urine albumin (UAlb), serum creatinine (SCr), blood urea nitrogen (BUN), and kidney/body weight ratio of mice were examined. H&E and TUNEL staining were performed for evaluating pathological changes and apoptosis in renal tissue. The podocyte markers, reactive oxygen species (ROS), oxidative stress (OS), serum inflammatory cytokines, nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway-related proteins, and pyroptosis were detected by Western blotting and corresponding kits. MPC5 cell viability and pyroptosis were evaluated by MTT and Hoechst 33342/PI double-fluorescence staining. Nrf2 inhibitor ML385 was used to verify the regulation of TP on Nrf2. Results TP improved renal function and histopathological injury of DN mice, alleviated podocytes injury, reduced OS and ROS by activating the Nrf2/heme oxygenase-1 (HO-1) pathway, and weakened pyroptosis by inhibiting the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome pathway. In vitro experiments further verified the inhibition of TP on OS and pyroptosis by mediating the Nrf2/HO-1 and NLRP3 inflammasome pathways. Inhibition of Nrf2 reversed the protective effect of TP on MPC5 cells. Conclusions Overall, TP alleviated podocyte injury in DN by inhibiting OS and pyroptosis via Nrf2/ROS/NLRP3 axis.

Objective Abnormal signaling pathways play a crucial role in the mechanisms of podocyte injury in diabetic nephropathy. They also affect the recovery of podocytes after islet transplantation (IT). However, the specific signaling abnormalities that affect the therapeutic effect of IT on podocytes remains unclear. The purpose of this study was to assess whether the RhoA/ROCK/NF-κB signaling pathway is related to podocyte restoration after IT. Methods A mouse model of diabetic nephropathy was established in vivo using streptozotocin. The mice were then subsequently reared for 4 weeks after islet transplantation to determine the effect of IT. Islet cells, CCG-1423 (RhoA Inhibitor), and fasudil (ROCK inhibitor) were then cocultured with podocytes in vitro to assess their protective effects on podocyte injury induced by high glucose (HG). Protein expression levels of RhoA, ROCK1, synaptopodin, IL-6, and MCP-1 in kidney tissues were then measured using immunohistochemistry and Western blotting techniques. Results Islet transplantation reduced the expression levels of RhoA/ROCK1 and that of related inflammatory factors such as IL-6 and MCP-1 in the kidney podocytes of diabetic nephropathy. In the same line, islet cells reduced the expression of RhoA, ROCK1, and pp65 in immortalized podocytes under high glucose (35.0 mmol/L glucose) conditions. Conclusions Islet transplantation can reverse podocyte injury in diabetes nephropathy by inhibiting the RhoA/ROCK1 signaling pathway. Islet cells have a strong protective effect on podocytes treated with high glucose (35.0 mmol/L glucose). Discovery of signaling pathways affecting podocyte recovery is helpful for individualized efficacy evaluation and targeted therapy of islet transplantation patients.

Endoplasmic reticulum stress (ER stress) plays an important role in podocyte injury in diabetic nephropathy. Wnt/β-catenin signaling modulates ER stress, yet the epigenetic regulation of β-catenin in ER stress and podocyte injury remains largely unknown. Herein, we tested the hypothesis that LINC00355 recruits EZH1 to the promoter region of CTNNBIP1 and trimethylates H3K4 to regulate ER-stress induced podocyte injury in DN. LINC00355 is upregulated in podocytes and correlates with renal function decline in DN patients. LINC00355 localizes in the nucleus and exerts biological functions by directly binding EZH1, which epigenetically targets CTNNBIP1 through repressive trimethylation of H3K4 and activates Wnt/β-catenin signaling and ER stress. Further, we provide mechanistic evidences that LINC00355 recruits EZH1 to the promoter region of CTNNBIP1 and regulates ER-stress induced podocyte injury in DN. Our data reveal a major role of LINC00355/EZH1/CTNNBIP1 network in triggering podocyte injury, providing new evidences for understanding the role of ER stress in DN.

BackgroundThe microRNA-30 family plays a critical role in the pathogenesis of podocyte injury. Cx43 plays an essential role in intercellular communication, which is essential for coordinated kidney function. This study was conducted to explore the function of microRNA-30s/Cx43 in podocyte injury in diabetic nephropathy (DN), both in vivo and in vitro.MethodsSD rats were given streptozotocin (STZ) injections to induce DN. Podocytes were incubated in the medium in the presence or absence of high glucose (HG). The effects of the microRNA-30/Cx43 axis on DN and its underlying mechanisms were investigated by TUNEL assay, PAS, immunohistochemical staining, immunofluorescence staining, Western blot, RT-qPCR, RNA interference, and luciferase reporter assay. Podocytes were transfected with microRNA-30 family mimics, microRNA-30 family inhibitors, Cx43 siRNA, and negative controls to detect the effect of the microRNA-30/Cx43 axis. MicroRNA-30 family mimic AAVs, and microRNA-30 family inhibitor AAVs applied to regulate microRNA-30 family expression in the kidneys of the STZ-induced DN model rats to reveal the underlying mechanisms of the microRNA-30/Cx43 axis in DN.ResultsMicroRNA-30 family member expression was downregulated in HG-treated podocytes and the glomeruli of STZ-induced DN rats. Luciferase reporter assays confirmed Cx43 is a directed target of microRNA-30s. The overexpression of microRNA-30 family members attenuated the HG-induced podocyte injury and protected against podocyte apoptosis and endoplasmic reticulum stress (ERS) both in vivo and in vitro. Also, silencing Cx43 expression eased podocyte apoptosis, injury, and ERS induced by a HG+microRNA-30 family inhibitor. Double-immunofluorescence staining assays proved the co-localization of caspase12 and Cx43.ConclusionsThe overexpression of microRNA-30 family members prevents HG-induced podocyte injury and attenuates ERS by modulating Cx43 expression. The microRNA-30/Cx43/ERS axis might be a potential therapeutic target to treat DN.

Altered activities of long noncoding RNAs (lncRNAs) have been implicated in the regulation of microRNAs. microRNA-27a (miR-27a) upregulation has been shown to induce endoplasmic reticulum (ER) stress podocyte injury in diabetic nephropathy (DN). Herein, we aim to interrogate the mutually regulated network of miR-27a with long intergenic noncoding RNA 1619 (LINC01619) and the target gene. LINC01619 downregulation was found in human DN renal biopsy tissues and contributed to proteinuria and diminished renal function. LINC01619 was expressed in podocyte cytoplasm and involved in ER stress signaling pathway. LINC01619 exerted biological function by serving as a "sponge" for miR-27a, which negatively targeted forkhead box protein O1 (FOXO1) and activated ER stress. In diabetic rats and high-glucose cultured podocytes, LINC01619 triggered oxidative stress and podocyte injuries as demonstrated by increased apoptosis, diffuse podocyte foot process effacement, and decreased renal function. Innovation and Conclusion: This study demonstrates that LINC01619 functions as a competing endogenous RNA and regulates miR-27a/FOXO1-mediated ER stress and podocyte injury in DN. Antioxid. Redox Signal. 29, 355-376.

To observe the effect of high glucose stimulation on the expression of guanylate exchange factor NHX1 in mouse kidney cells, and to explore the role of NHX1 in high glucose-induced podocyte injury and its possible molecular mechanism. The expression of NHX1 in podocytes of diabetic nephropathy patients was observed by immunofluorescence staining and laser confocal microscopy. The immortalized podocytes were cultured in vitro, and the podocytes were stimulated with high glucose for 48 h. RT-PCR, Western blot and immunization were used. Fluorescence detection of mRNA and protein expression of NHX1 in podocytes stimulated by high glucose; Western blot, immunofluorescence and scratch assay were used to detect the expression of NHX1 and the expression of podocin, the activity of podocytes and the nuclear access of TRPV5. The transcription of the target gene downstream of TRPV5 was detected by RT-PCR. NHX1 expression was significantly decreased in podocytes and high glucose-stimulated podocytes of diabetic nephropathy patients (P<0.05). After silencing NHX1, podocyte marker protein podocin expression was significantly decreased and podocyte activity was increased. The nuclear translocation of TRPV5 increased, and the transcription of the target gene downstream of TRPV5 increased (P < 0.05). In contrast, the expression of overexpressed NHX1 group was significantly increased, the activity of podocytes was decreased, the nuclear access of TRPV5 was decreased, and the transcription of the target gene downstream of TRPV5 was decreased (P<0.05). NHX1 may reduce podocyte injury in diabetic nephropathy by inhibiting TRPV5 entry into the nucleus.

Sp1-mediated upregulation of Prdx6 expression prevents podocyte injury in diabetic nephropathy via mitigation of oxidative stress and ferroptosis

AimThis study utilized db/db mice and MPC5 cells induced by high glucose as experimental models to examine the protective mechanisms of the traditional Chinese medicine formula TangNaikang (TNK) in mitigating podocyte injury in diabetic nephropathy (DN).MethodsThe chemical constituents of TNK and TNK-containing serum were identified through UPLC-Q-TOF/MS. The underlying mechanism of TNK in treating DN was analyzed using network pharmacology. In vivo, following an 8-week intervention, db/db mice's serum biomarkers (TC, TG, HDL, LDL, AGEs, BUN, Scr, and β2-MG) were compared. H&E, PAS staining, and electron microscopy were used to perform a histopathological investigation on kidney sections. High glucose-induced MPC5 cells were treated with TNK-containing serum. Cellular viability was measured through a CCK-8 assay. The expression levels of podocyte-associated and PI3K/AKT pathway proteins in kidney tissues and MPC5 cells were determined by immunofluorescence, western blotting, and RT-qPCR analysis.ResultsThe UPLC-Q-TOF/MS results showed that the TNK formula consisted of 69 compounds, including flavonoids, triterpenoids, and lignans. TNK-containing serum was identified with 34 compounds including 9 TNK prototype components and 25 metabolites. TNK was found to be substantially linked with the PI3K/AKT pathway using network pharmacology. When compared to the model group, the TNK-H group mice had significantly improved serum lipid profiles as well as renal structural and functional profiles. Immunofluorescence and western blotting analyses indicated that TNK regulated the expression levels of the podocyte-associated (SYNPO, nephrin, CD2AP, and podocin) as well as PI3K/AKT pathway proteins (PI3K, AKT, SHIP2, IRS2, and GLUT4). These data were confirmed by RT-qPCR results. TNK-containing serum enhanced MPC5 cell viability via modulating the PI3K/AKT pathway and inhibiting SHIP2.ConclusionTNK ameliorates podocyte injury in DN and high glucose-induced MPC5 cells by modulating the SHIP2/PI3K/AKT pathway.

tBHQ attenuates podocyte injury in diabetic nephropathy by inhibiting NADPH oxidase-derived ROS generation via the Nrf2/HO-1 signalling pathway