MiR-455-5p ameliorates HG-induced apoptosis,oxidative stress and inflammatory via targeting SOCS3 in retinal pigment epithelial cells

Diabetic retinopathy (DR) remains the leading cause of blindness in adults with diabetes mellitus. Numerous microRNAs (miRNAs) have been identified to modulate the pathogenesis of DR. The main purpose of this study was to evaluate the potential roles of miR-455-5p in high glucose (HG)-treated retinal pigment epithelial (RPE) cells and underlying mechanisms. Our present investigation discovered that the expression of miR-455-5p was apparently downregulated in ARPE-19 cells stimulated with HG. In addition, forced expression of miR-455-5p markedly enhanced cell viability and restrained HG-induced apoptosis accompanied by decreased BCL2-associated X protein (Bax)/B-cell leukemia/lymphoma 2 (Bcl-2) ratio and expression of apoptotic marker cleaved caspase-3 during HG challenged. Subsequently, augmentation of miR-455-5p remarkably alleviated HG-triggered oxidative stress injury as reflected by decreased the production of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) content as well as NADPH oxidase 4 expression, concomitant with enhanced the activities of superoxide dismutase, catalase, and GPX stimulated with HG. Furthermore, enforced expression of miR-455-5p effectively ameliorated HG-stimulated inflammatory response as exemplified by repressing the secretion of inflammatory cytokines interleukin 1β (IL-1β), IL-6, and tumour necrosis factor-α in ARPE-19 cells challenged by HG. Most importantly, we successfully identified suppressor of cytokine signaling 3 (SOCS3) as a direct target gene of miR-455-5p, and miR-455-5p negatively regulated the expression of SOCS3. Mechanistically, restoration of SOCS3 abrogated the beneficial effects of miR-455-5p on apoptosis, accumulation of ROS, and inflammatory factors production in response to HG. Taken together, these findings demonstrated that miR-455-5p relieved HG-induced damage through repressing apoptosis, oxidant stress, and inflammatory response by targeting SOCS3. The study gives evidence that miR-455-5p may serve as a new potential therapeutic agent for DR treatment.     

Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte

Diabetic nephropathy (DN) remains one of the severe complications associated with diabetes mellitus. It is worthwhile to uncover the underlying mechanisms of clinical benefits of human urine-derived stem cells (hUSCs) in the treatment of DN. At present, the clinical benefits associated with hUSCs in the treatment of DN remains unclear. Hence, our study aims to investigate protective effect of hUSC exosome along with microRNA-16-5p (miR-16-5p) on podocytes in DN via vascular endothelial growth factor A (VEGFA). Initially, miR-16-5p was predicated to target VEGFA based on data retrieved from several bioinformatics databases. Notably, dual-luciferase report gene assay provided further verification confirming the prediction. Moreover, our results demonstrated that high glucose (HG) stimulation could inhibit miR-16-5p and promote VEGFA in human podocytes (HPDCs). miR-16-5p in hUSCs was transferred through the exosome pathway to HG-treated HPDCs. The viability and apoptosis rate of podocytes after HG treatment together with expression of the related factors were subsequently determined. The results indicated that miR-16-5p secreted by hUSCs could improve podocyte injury induced by HG. In addition, VEGA silencing could also ameliorate HG-induced podocyte injury. Finally, hUSC exosomes containing overexpressed miR-16-5p were injected into diabetic rats via tail vein, followed by qualification of miR-16-5p and observation on the changes of podocytes, which revealed that overexpressed miR-16-5p in hUSCs conferred protective effects on HPDCs in diabetic rats. Taken together, the present study revealed that overexpressed miR-16-5p in hUSC exosomes could protect HPDCs induced by HG and suppress VEGFA expression and podocytic apoptosis, providing fresh insights for novel treatment of DN.     

Astragaloside IV, a novel antioxidant, prevents glucose-induced podocyte apoptosis in vitro and in vivo

Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 μg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg(-1)·d(-1)) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation.     

Nestin protects mouse podocytes against high glucose-induced apoptosis by a Cdk5-dependent mechanism

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate hyperglycemia‐induced podocyte apoptosis remain poorly understood. Recent findings indicate that the disruption of the cytoskeleton is related to the podocyte apoptosis. In the present study, we investigated the involvement of nestin, an important cytoskeleton‐associated class VI intermediate filament (IF) protein, in the high glucose (HG)‐induced podocyte apoptosis. Our data showed that HG decreased the expression level of nestin, either mRNA or protein, in a time‐dependent manner in cultured podocytes. Also, through knockdown of nestin expression by miRNA interference, the HG‐induced podocyte apoptotic rate was significantly increased. The expression of cleaved caspase‐3 was also markedly elevated. Considering that nestin is a substrate of cyclin‐dependent kinase 5 (Cdk5), we further assessed the expression of Cdk5 in HG‐treated podocytes. The results showed that HG stimulation increased the protein and mRNA expression of Cdk5 in a time‐dependent manner in cultured mouse podocytes. The protein activator of Cdk5, p35, was also increased in a time‐dependent manner by HG stimulation, and downregulation of Cdk5 by miRNA interference attenuated the nestin reduction in HG‐treated podocytes; the HG‐induced podocyte apoptosis, the increased cleaved caspase‐3 expression and the Bax/Bcl‐2 ratio were all effectively attenuated. These data suggested that nestin, which is dependent on Cdk5 regulation, plays a cytoprotective role in HG‐induced podocyte apoptosis. J. Cell. Biochem. 113: 3186–3196, 2012. © 2012 Wiley Periodicals, Inc.     

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress,mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1^S616, mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.     

Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy

Thioredoxin-interacting protein (TXNIP) is induced by high glucose (HG), whereupon it acts to inhibit thioredoxin, thereby promoting oxidative stress. We have found that TXNIP knockdown in human renal tubular cells helped prevent the epithelial-to-mesenchymal transition (EMT). Here, we studied the potential effect of TXNIP on podocyte phenotypic alterations in diabetic nephropathy (DN) in vivo and in vitro. In conditionally immortalized mouse podocytes under HG conditions, knocking down TXNIP disrupted EMT, reactive oxygen species (ROS) production, and mammalian target of rapamycin (mTOR) pathway activation. Further, Raptor short hairpin RNA (shRNA), Rictor shRNA, and mTOR specific inhibitor KU-0063794 were used to assess if the mTOR signal pathway is involved in HG-induced EMT in podocytes. We found that Raptor shRNA, Rictor shRNA, and KU-0063794 could all restrain HG-induced EMT and ROS production in podocytes. In addition, antioxidant Tempol or N-acetylcysteine presented a prohibitive effect on HG-induced EMT in podocytes. Streptozotocin was utilized to render equally diabetic in wild-type (WT) control and TXNIP ^−/− (TKO) mice. Diabetes did not increase levels of 24-hr urinary protein, serum creatinine, blood urea nitrogen, and triglyceride in TXNIP ^−/− mice. Podocyte phenotypic alterations and podocyte loss were detected in WT but not in TKO diabetic mice. Oxidative stress was also suppressed in diabetic TKO mice relative to WT controls. Also, TXNIP deficiency suppresses the activation of mTOR in glomeruli of streptozotocin-induced diabetic mice. Moreover, TXNIP expression, mTOR activation, Nox1, and Nox4 could be detected in renal biopsy tissues of patients with DN. This suggests that decreased TXNIP could ameliorate phenotypic alterations of podocytes via inhibition of mTOR in DN, highlighting TXNIP as a promising therapeutic target.     

The Ras GTPase-activating-like protein IQGAP1 is downregulated in human diabetic nephropathy and associated with ERK1/2 pathway activation

Podocyte injury may contribute to the pathogenesis of diabetic nephropathy (DN), but the underlying mechanism of hyperglycemia induced podocyte damage is not fully understood. The Ras GTPase-activating-like protein IQGAP1 is associated to the slit diaphragm proteins and the actin cytoskeleton in podocyte. Here, we studied IQGAP1 expression alterations in human DN biopsies and extracellular signal-regulated kinase (ERK)-dependent pathways of IQGAP1 expression in podocyte under high glucose (HG) media. In vivo, analysis of renal biopsies from patients with DN revealed a significant reduction in IQGAP1 expression compared to controls. In vitro, IQGAP1 mRNA and protein expression were observed to decline under HG media at 48 h. But phosphorylation of ERK1/2 was activated under HG media at 24 h and 48 h. However, HG-induced downregulation of IQGAP1 protein was attenuated by specific ERK1/2 activation inhibitor PD98059. Taken together, these results highlight the importance of IQGAP1 in DN, and suggest that IQGAP1 expression in podocyte under HG media is modulated by the ERK1/2 pathway, which may lead to the future development of therapies targeting IQGAP1 dysfunction in podocytes in DN.     

Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy

Numerous studies have shown that the NALP3 inflammasome plays an important role in various immune and inflammatory diseases. However, whether the NALP3 inflammasome is involved in the pathogenesis of diabetic nephropathy (DN) is unclear. In our study, we confirmed that high glucose (HG) concentrations induced NALP3 inflammasome activation both in vivo and in vitro. Blocking NALP3 inflammasome activation by NALP3/ASC shRNA and caspase-1 inhibition prevented IL-1β production and eventually attenuated podocyte and glomerular injury under HG conditions. We also found that thioredoxin (TRX)-interacting protein (TXNIP), which is a pro-oxidative stress and pro-inflammatory factor, activated NALP3 inflammasome by interacting with NALP3 in HG-exposed podocytes. Knocking down TXNIP impeded NALP3 inflammasome activation and alleviated podocyte injury caused by HG. In summary, the NALP3 inflammasome mediates podocyte and glomerular injury in DN, moreover, TXNIP participates in the formation and activation of the NALP3 inflammasome in podocytes during DN, which represents a novel mechanism of podocyte and glomerular injury under diabetic conditions.     

AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.     

Low expression of miR-186-5p regulates cell apoptosis by targeting toll-like receptor 3 in high glucose–induced cardiomyocytes

To investigate the effect and mechanism of microRNA-186-5p (miR-186-5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR-186-5p and toll-like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG-treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR-186-5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR-186-5p expression was downregulated in HG-treated cardiomyocytes and its overexpression reversed HG-induced apoptosis and cleaved caspase-3 protein expression. Moreover, TLR3 was indicated as a target of miR-186-5p and regulated by miR-186-5p. Knockdown of TLR3 suppressed HG-induced apoptosis and cleaved caspase-3 protein expression. Besides, restoration of TLR3 ablated the effect of miR-186-5p on cell apoptosis. Collectively, miR-186-5p attenuated HG-induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.     

circLRP6 regulates high glucose-induced proliferation,oxidative stress,ECM accumulation,and inflammation in mesangial cells

Aberrant regulation in mesangial cell proliferation, extracellular matrix (ECM) accumulation, oxidative stress, and inflammation under hyperglycemic condition contributes significantly to the occurrence and development of diabetic nephropathy (DN). However, the mechanisms underlying the hyperglycemia-induced dysregulations have not been clearly elucidated. Here, we reported that high mobility group box 1 (HMGB1) was highly elevated in high glucose (HG)-treated mesangial cells, and induced the phosphorylation, nuclear translocation, and DNA binding activity of NF-κB via toll-like receptor 4 (TLR4). Function assays showed that inhibition of HMGB1 mitigated HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via TLR4/NF-κB pathway. Increasing evidence has shown that circRNA, a large class of noncoding RNAs, functions by binding with miRNAs and terminating regulation of their target genes. We further investigated whether HMGB1 is involved in circRNA–miRNA–mRNA regulatory network. First, HMGB1 was identified and confirmed to be the target of miR-205, and miR-205 played a protective role against HG-induced cell injure via targeting HMGB1. Then circLRP6 was found to be upregulated in HG-treated mesangial cells, and regulate HG-induced mesangial cell injure via sponging miR-205. Besides, overexpression of miR-205 or knockdown of circLRP6 inhibited the NF-κB signaling pathway. Collectively, these data suggest that circLRP6 regulates HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via sponging miR-205, upregulating HMGB1 and activating TLR4/NF-κB pathway. These findings provide a better understanding for the pathogenesis of DN.     

Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway

Dysregulation of apoptotic and autophagic function are characterized as the main pathogeneses of diabetic nephropathy (DN). It has been reported that Karyopherin Alpha 2 (KPNA2) contributes to apoptosis and autophagy in various cells, but its role in DN development remains unknown. The purpose of present study was to explore the function and underling mechanisms of KPNA2 in development of DN. In this study, 30 mM high glucose (HG)-evoked podocytes were used as DN model. The expression of KPNA2 was detected by qRT-PCR and Western blot assays. The cell viability was tested by CCK-8 kit, the apoptosis was measured using flow cytometry assay, the apoptotic and the autophagy related genes was detected by Western blot. Our results indicated that KPNA2 was significantly increased after HG stimulation. Knockdown of KPNA2 inhibited apoptosis, and promoted cell viability and autophagy in HG-treated podocytes. In addition, silencing of KPNA2 deactivated mTORC1/p70S6K pathway activation via regulating SLC1A5. Further results demonstrated that activating mTORC1/p70S6K pathway strongly ameliorated the effect of KPNA2 on cell viability, apoptosis and autophagy. Therefore, our study suggested that knockdown of KPNA2 rescued HG-induced injury via blocking activation of mTORC1/p70S6K pathway by mediating SLC1A5.     

MiR-874 alleviates renal injury and inflammatory response in diabetic nephropathy through targeting toll-like receptor-4

Diabetic nephropathy (DN) is a kind of diabetic complication with capillary damage, and its pathogenesis remains obscure. Recently, microRNAs have been identified as diagnostic biomarkers in various diseases including DN. Toll-like receptor 4 (TLR4) contributes to inflammation, and it has been implicated in diabetes pathophysiology. This study was designed to investigate the role of miR-874 and TLR4 in a streptozotocin (STZ)-induced DN rat model and glucose-induced mouse podocyte model. In the current study, we reported that miR-874 was markedly downregulated in DN rats and glucose-induced mouse podocytes compared with the corresponding control groups with the activation of TLR4. In addition, we observed that overexpression of miR-874 was able to alleviate renal injury in DN rats. The cell counting kit (CCK-8) assay and 5-Ethynyl-2′-deoxyuridine (EdU) assay demonstrated that glucose simulation significantly inhibited podocyte proliferation and induced cell apoptosis, which can be reversed by miR-874 mimics significantly. Notably, miR-874 overexpression dramatically attenuated the inflammatory response, indicated by the decreased levels of interleukin-6, L-1β, and tumor necrosis factor α (TNF-α). Finally, the binding correlation between miR-874 and TLR4 was confirmed by carrying out dual-luciferase reporter assay in our study. It was found that overexpression of miR-874 depressed TLR4 levels in podocytes. These findings implied for the first time that the overexpression of miR-874 repressed glucose-triggered podocyte injury through targeting TLR4 and suggested that miR-874/TLR4 axis might represent a pathological mechanism of DN.     

AKAP1 mediates high glucose-induced mitochondrial fission through the phosphorylation of Drp1 in podocytes

Increasing evidence suggests that mitochondrial dysfunction plays a critical role in the development of diabetic kidney disease (DKD), however, its specific pathomechanism remains unclear. A-kinase anchoring protein (AKAP) 1 is a scaffold protein in the AKAP family that is involved in mitochondrial fission and fusion. Here, we show that rats with streptozotocin (STZ)-induced diabetes developed podocyte damage accompanied by AKAP1 overexpression and that AKAP1 closely interacted with the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). At the molecular level, high glucose (HG) promoted podocyte injury and Drp1 phosphorylation at Ser637 as proven by decreased mitochondrial membrane potential, elevated reactive oxygen species generation, reduced adenosine triphosphate synthesis, and increased podocyte apoptosis. Furthermore, the AKAP1 knockdown protected HG-induced podocyte injury and suppressed HG-induced Drp1 phosphorylation at Ser637. AKAP1 overexpression aggravated HG-induced mitochondrial fragmentation and podocyte apoptosis. The coimmunoprecipitation assay showed that HG-induced Drp1 interacted with AKAP1, revealing that AKAP1 could recruit Drp1 from the cytoplasm under HG stimulation. Subsequently, we detected the effect of drp1 phosphorylation on Ser637 by transferring several different Drp1 mutants. We demonstrated that activated AKAP1 promoted Drp1 phosphorylation at Ser637, which promoted the transposition of Drp1 to the surface of the mitochondria and accounts for mitochondrial dysfunction events. These findings indicate that AKAP1 is the main pathogenic factor in the development and progression of HG-induced podocyte injury through the destruction of mitochondrial dynamic homeostasis by regulating Drp1 phosphorylation in human podocytes.     

MDM2 is implicated in high‐glucose‐induced podocyte mitotic catastrophe via Notch1 signalling

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in ‘post‐mitosis’ state and unable to regenerate. Re‐entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi‐nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up‐regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p‐H3. Genetic deletion of MDM2 partly reversed HG‐induced mitotic phase re‐entering of podocytes. Moreover, HG‐induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin‐3a, an inhibitor of MDM2‐p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG‐mediated podocyte MC. In conclusion, high glucose up‐regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG‐induced MC in podocytes.     

Salvianolate ameliorates oxidative stress and podocyte injury through modulation of NOX4 activity in db/db mice

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and NOX4 is up-regulated in podocytes in response to high glucose. In the present study, the effects of Salvianolate on DN and its underlying mechanisms were investigated in diabetic db/db mice and human podocytes. We confirmed that the Salvianolate administration exhibited similar beneficial effects as the NOX1/NOX4 inhibitor GKT137831 treated diabetic mice, as reflected by attenuated albuminuria, reduced podocyte loss and mesangial matrix accumulation. We further observed that Salvianolate attenuated the increase of Nox4 protein, NOX4-based NADPH oxidase activity and restored podocyte loss in the diabetic kidney. In human podocytes, NOX4 was predominantly localized to mitochondria and Sal B treatment blocked HG-induced mitochondrial NOX4 derived superoxide generation and thereby ameliorating podocyte apoptosis, which can be abrogated by AMPK knockdown. Therefore, our results suggest that Sal B possesses the reno-protective capabilities in part through AMPK-mediated control of NOX4 expression. Taken together, our results identify that Salvianolate could prevent glucose-induced oxidative podocyte injury through modulation of NOX4 activity in DN and have a novel therapeutic potential for DN.     

Pterostilbene and allopurinol reduce fructose-induced podocyte oxidative stress and inflammation via microRNA-377

High dietary fructose is an important causative factor in the development of metabolic syndrome-associated glomerular podocyte oxidative stress and injury. Here, we identified microRNA-377 (miR-377) as a biomarker of oxidative stress in renal cortex of fructose-fed rats, which correlated with podocyte injury and albuminuria in metabolic syndrome. Fructose feeding increased miR-377 expression, decreased superoxide dismutase (SOD) expression and activity, and caused O₂⁻ and H₂O₂ overproduction in kidney cortex or glomeruli of rats. This reactive oxygen species induction increased p38 MAPK phosphorylation and thioredoxin-interacting protein (TXNIP) expression and activated the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome to produce interleukin-1β in kidney glomeruli of fructose-fed rats. These pathological processes were further evaluated in cultured differentiated podocytes exposed to 5 mM fructose, or transfected with miR-377 mimic/inhibitor and TXNIP siRNA, or co-incubated with p38 MAPK inhibitor, demonstrating that miR-377 overexpression activates the O₂⁻/p38 MAPK/TXNIP/NLRP3 inflammasome pathway to promote oxidative stress and inflammation in fructose-induced podocyte injury. Antioxidants pterostilbene and allopurinol were found to ameliorate fructose-induced hyperuricemia, podocyte injury, and albuminuria in rats. More importantly, pterostilbene and allopurinol inhibited podocyte miR-377 overexpression to increase SOD1 and SOD2 levels and suppress the O₂⁻/p38 MAPK/TXNIP/NLRP3 inflammasome pathway activation in vivo and in vitro, consistent with the reduction of oxidative stress and inflammation. These findings suggest that miR-377 plays an important role in glomerular podocyte oxidative stress, inflammation, and injury driven by high fructose. Inhibition of miR-377 by antioxidants may be a promising therapeutic strategy for the prevention of metabolic syndrome-associated glomerular podocyte injury.     

Sinensetin ameliorates high glucose-induced diabetic nephropathy via enhancing autophagy in vitro and in vivo

Diabetic nephropathy (DN) affects around 40% of people with diabetes, the final outcome of which is end-stage renal disease. The deficiency of autophagy and excessive oxidative stress have been found to participate in the pathogenesis of DN. Sinensetin (SIN) has been proven to have strong antioxidant capability. However, the effect of SIN on DN has not been studied. We examined the effect of SIN on cell viability and autophagy in the podocyte cell line, MPC5 cells, treated with high glucose (HG). For in vivo studies, DN mice models were established by intraperitoneal injected with streptozotocin (40 mg/kg) for 5 consecutive days and fed with a 60% high-fat diet, and SIN was given (10, 20, and 40 mg/kg) for 8 weeks via intraperitoneal injection. The results showed that SIN could protect MPC5 cells against HG-induced damage and significantly improve the renal function of DN mice. Moreover, SIN remarkably restored the autophagy activity of MPC5 cells which was inhibited under HG conditions. Consistent with this, SIN efficiently improved autophagy in the kidney tissue of DN mice. In brief, our findings demonstrated the protective effect of SIN on DN via restoring the autophagic function, which might provide a basis for drug development.