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

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.

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.

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.

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.

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.

BackgroundDiabetic nephropathy (DN) is one of the principal complications of diabetes and podocyte injury plays an important role in the DN pathogenesis. Wnt/β-catenin signaling overactivation confers podocyte injury and promotes multiple types of renal disease. However, the underlying mechanism of Wnt/β-catenin signaling activation in DN progression has not been fully elucidated. Long noncoding RNA (lncRNA) is a large class of endogenous RNA molecules lacking functional code capacity and which participates in the pathogenesis of human disease, including DN.MethodA diabetes model was constructed by intraperitoneal injection of Streptozotocin in rats. The MPC5 cells were used to create the in vitro model. Western blot and Quantitative reverse-transcriptase-PCR were used to examine the expression of protein and mRNA. The migrated capacity was analyzed by Transwell migration assay. The cell viability was detected by CCK8.ResultsIn the present study, we revealed the association of lncRNA Maternally Expressed Gene 3 (MEG3) with aberrant activation of Wnt/β-catenin signaling and the role of MEG3/Wnt axis in podocyte injury. We found that high glucose (HG) treatment suppressed MEG3 expression in cultured podocytes, activated Wnt/β-catenin signaling and caused podocyte injury as indicated by the downregulation of podocyte-specific markers (podocin and synaptopodin) and the upregulation of snail1 and α-smooth muscle actin. Overexpression of MEG3 attenuated HG-induced podocyte injury by reducing Wnt/β-catenin activity, repressing cell migration, reactive oxygen species production and increasing the viability of podocytes. Furthermore, we provided evidences that restoration of Wnt/β-catenin signaling by specific agonist impeded the protective effect of MEG3 on podocyte injury. Current results demonstrated that MEG3/Wnt axis plays an important role in fostering podocyte injury and may serve as a potential therapeutic target for the treatment of DN.ConclusionlncRNA MEG3 ameliorates podocyte injury in DN via inactivating Wnt/β-catenin signaling.

Shenxiao decoction ameliorates podocyte injury in diabetic nephropathy via upregulating RUNX3 expression and inhibiting the JAK2/STAT3 signaling pathway.

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.

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

RETRACTED: LncRNA MALAT1/microRNA let-7f/KLF5 axis regulates podocyte injury in diabetic nephropathy

Purpose: The aim of present study was to explore the pharmacological mechanisms of Niaoduqing granules on the treatment of podocyte injury in diabetic nephropathy (DN) via network pharmacology and experimental validation. Methods: Active ingredients and related targets of Niaoduqing, as well as related genes of podocyte injury, proteinuria and DN, were obtained from public databases. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analysis were performed to investigate the potential mechanisms. High glucose (HG) -induced MPC5 cell injury model was treated with the major core active ingredients of Niaoduqing and used to validate the predicted targets and signaling pathways. Results: Totally, 16 potential therapeutic targets were identified by intersecting the targets of Niaoduqing and disease, in which 7 of them were considered as the core targets via PPI network analysis. KEGG enrichment analysis showed that AGE-RAGE signaling pathway was identified as the most crucial signaling pathway. The results of in vitro experiments revealed that the treatment of Niaoduqing active ingredients significantly protected MPC5 cells from HG-induced apoptosis. Moreover, Niaoduqing could significantly attenuate the HG-induced activation of AGE-RAGE signaling pathway, whereas inhibited the over-expression of VEGF-A, ICAM-1, PTGS-2 and ACE in HG-induced MPC5 cells. Conclusion: Niaoduqing might protect against podocyte injury in DN through regulating the activity of AGE/RAGE pathway and expression of multiple genes. Further clinical and animal experimental studies are necessary to confirm present findings.

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.

This work aimed to elucidate the mechanisms of Se@Tri-PTs in alleviating podocyte injury via network pharmacology and in vitro cellular assay. Selenized tripterine phytosomes (Se@Tri-PTs) have been confirmed to undertake synergistic and sensitized effects on inflammation, which may be curatively promising for diabetic nephropathy (DN). However, the mechanisms of Se@Tri-PTs in alleviating podocyte injury, a major contributor to DN, still remain unclear. The objective of the study was to find out the underlying mechanisms of Se@Tri-PTs in alleviating podocyte injury in diabetic nephropathy. The key components and targets of Tripterygium wilfordii (TW) significant for DN as well as the signaling pathways involved have been identified. A high glucose-induced podocyte injury model was established and verified by western blot. The protective concentration of Se@Tri-PTs was screened by CCK-8 assay. Podocytes cultured with high glucose were treated with Se@Tri-PTs under protective levels. The expression of key protective proteins, nephrin and desmin, in podocytes, was assayed by western blot. Further, autophagy- related proteins and factors, like NLRP3, Beclin-1, LC3II/LC3, P62, and SIRT1, were analyzed, which was followed by apoptosis detection. Network pharmacology revealed that several monomeric components of TW, especially Tri, act on DN through multiple targets and pathways, including the NLRP3-mediated inflammatory pathway. Se@Tri-PTs improved the viability of podocytes and alleviated their injury induced by high glucose at 5 μg/L or above. High-glucose induction promoted the expression of NLRP3 in podocytes, while a low concentration of Se@Tri-PTs suppressed the expression. A long-term exposure of high glucose significantly inhibited the autophagic activity of podocytes, as manifested by decreased Beclin-1 level, lower ratio of LC3 II/LC3 I, and up- regulation of P62. This abnormality was efficiently reversed by Se@Tri-PTs. Importantly, the expression of SIRT1 was up-regulated and podocyte apoptosis was reduced. Se@Tri-PTs can alleviate podocyte injury associated with DN by modulating NLRP3 expression through the pathway of SIRT1-mediated autophagy.

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.