Polydatin impairs mitochondria fitness and ameliorates podocyte injury by suppressing Drp1 expression

Polydatin (PD), a resveratrol glycoside, has been shown to protect renal function in diabetic nephropathy (DN), but the underlying molecular mechanism remains unclear. This study demonstrates that PD stabilize the mitochondrial morphology and attenuate mitochondrial malfunction in both KKAy mice and in hyperglycemia (HG)‐induced MPC5 cells. We use Western blot analysis to demonstrate that PD reversed podocyte apoptosis induced by HG via suppressing dynamin‐related protein 1 (Drp1). This effect may depend on the ability of PD to inhibit the generation of cellular reactive oxygen species (ROS). In conclusion, we demonstrate that PD may be therapeutically useful in DN, and that, podocyte apoptosis induced by HG can be reversed by PD through suppressing Drp1 expression.     

Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.     

Carbon monoxide alleviates senescence in diabetic nephropathy by improving autophagy

ObjectivesSenescence, characterized by permanent cycle arrest, plays an important role in diabetic nephropathy (DN). However, the mechanism of renal senescence is still unclear, and the treatment targeting it remains to be further explored.Materials and MethodsThe DN mice were induced by HFD and STZ, and 3 types of renal cells were treated with high glucose (HG) to establish in vitro model. Senescence‐related and autophagy‐related markers were detected by qRT‐PCR and Western blot. Further, autophagy inhibitors and co‐immunoprecipitation were used to clarify the mechanism of CO. Additionally, the specific relationship between autophagy and senescence was explored by immunofluorescence triple co‐localization and ELISA.ResultsWe unravelled that senescence occurred in vivo and in vitro, which could be reversed by CO. Mechanistically, we demonstrated that CO inhibited the dysfunction of autophagy in DN mice partly through dissociating Beclin‐1‐Bcl‐2 complex. Further results showed that autophagy inhibitors blocked the improvement of CO on senescence. In addition, the data revealed that autophagy regulated the degradation of senescence‐related secretory phenotype (SASP) including Il‐1β, Il‐6, Tgf‐β and Vegf.ConclusionsThese results suggested that CO protects DN mice from renal senescence and function loss via improving autophagy partly mediated by dissociating Beclin‐1‐Bcl‐2 complex, which is possibly ascribed to the degradation of SASP. These findings bring new ideas for the prevention and treatment of DN and the regulation of senescence.     

Klotho attenuates diabetic nephropathy in db/db mice and ameliorates high glucose-induced injury of human renal glomerular endothelial cells

Glomerular endothelial cell injury plays an important role in the development and progression of diabetic nephropathy (DN). The expression and function of klotho in glomerular endothelial cells remain unclear. Thus, this study aimed to investigate the expression and the functional role of klotho in DN progression in mice and in high glucose (HG)-induced cell injury of human renal glomerular endothelial cells (HRGECs) and the underlying mechanism. In this study, HRGECs were cultured with media containing HG to induce endothelial cell injury and db/db mice were used as DN model mice. Klotho was overexpressed or knocked down in HRECs to evaluate its role in HG-induced HRGECs injury. klotho-overexpressing adenovirus (rAAV-klotho) was injected into db/db mice via the tail vein to further validate the protective effect of klotho in DN. Decreased klotho expression was observed in DN patients, DN mice, and HG-exposed HRGECs. Furthermore, klotho overexpression significantly abolished the HG-induced HRGECs injury and activation of Wnt/β-catenin pathway and RAAS. In contrast, klotho knockdown exerted the opposite effects. Moreover, klotho attenuated diabetic nephropathy in db/db mice, which was also associated with inhibition of the Wnt/β-catenin pathway and RAAS. In conclusion, klotho attenuates DN in db/db mice and ameliorates HG-induced injury of HRGECs.     

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.     

Effect of loganin on experimental diabetic nephropathy

Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Loganin, an iridoid glucoside compound was isolated from Cornus officinalis Sieb. et Zucc. This study was conducted to investigate the efficacy of loganin on DN and to elucidate the potential mechanism. High glucose (HG) stimulated cultured human renal proximal tubular epithelial cells (HK-2) analyzed CTGF expression by Western blotting and investigated whether extracellular signal-regulated kinase (ERK) signaling pathway was involved. Streptozotocin (STZ)-induced experimental DN, randomized to receive intragastric (i.g.) of loganin. Renal tissue, blood and urine samples were collected to determine and analyze. In vitro study, loganin reduced CTGF excretion in HG-induced HK-2 cells through the ERK signaling pathway. In vivo study, I.g. of loganin 5 mg/kg or 10 mg/kg significantly ameliorated renal function and increased body weight. Meanwhile, loganin reduced renal CTGF expression by immunohistochemical staining, reduced serum levels of CTGF. Besides, there were no significant differences in blood sugar levels between the loganin groups compared to the STZ-treated group. Furthermore, loganin ameliorated renal pathology. These results suggested that loganin exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using loganin to treat DN.     

A bioinformatics and network pharmacology approach to the mechanisms of action of Shenxiao decoction for the treatment of diabetic nephropathy

BackgroundThe epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is the main pathological alteration in diabetic nephropathy (DN). Traditional Chinese medicine (TCM) has been used for the treatment of DN in clinical practice and has been proven to be effective.PurposeThis aim of this study was to shed light on the efficacy of Shenxiao decoction (SXD) on the EMT of renal tubular epithelial cells and the molecular mechanisms of SXD in mice with DN, as well as on the high glucose (HG)- and TGF-β1-induced EMT of NRK-52E and HK-2 cells.Study design and methodsA bioinformatics and network pharmacology method were utilized to construct the active ingredient-target networks of SXD that were responsible for the beneficial effects against DN. The effects of RUNX3 were validated in HG- and TGF-β1-induced EMT processes in NRK-52E and HK-2 cells.ResultsBioinformatics analysis revealed that 122 matching targets were closely associated with the regulation of cell migration and the AGE-RAGE signaling pathway in diabetic complications. The results also revealed that, relative to the mice with DN, the mice in the treatment group had an improved general state and reduced blood glucose levels. The degradation of renal function was ameliorated by SXD. Moreover, the protective effects of SXD were also observed on renal structural changes. Furthermore, SXD suppressed the activation of the transforming growth factor (TGF)-β1/Smad pathway and upregulated the RUNX3 and E-cadherin levels and downregulated the extracellular matrix (ECM) protein levels in mice with DN. SXD was further found to prevent the HG- and TGF-β1-induced EMT processes in NRK-52E and HK-2 cells. Additionally, the overexpression of RUNX3 markedly inhibited the EMT and TGF-β1/Smad pathway induced by HG and TGF-β1 in NRK-52E and HK-2 cells.ConclusionTaken together, these results suggest that SXD maybe alleviate EMT in DN via the inhibition of the TGF-β1/Smad/RUNX3 signaling pathway under hyperglycemic conditions.     

Orientin Protects Podocytes from High Glucose Induced Apoptosis through Mitophagy

Diabetic nephropathy (DN) is one of the serious complications of diabetes mellitus. Orientin, a major bioactive constituent of Fenugreek, has been reported to possess antihyperglycemic properties. However, its effects on DN remain unclear. Therefore, we explored the protective effect of orientin on podocytes. Here, we assessed cell viability and toxicity, level of autophagy, mitochondrial morphological changes, and podocyte apoptosis. The results indicated that high glucose (HG) induced podocyte apoptosis as well as mitochondrial injury can be partially blocked by orientin. The results showed that orientin could repair autophagy disorder induced by HG, while 3‐methyladenine (3‐MA) reversed the protection of orientin. Our study demonstrated the possibility of treating DN with orientin.     

AMPK agonist alleviate renal tubulointerstitial fibrosis via activating mitophagy in high fat and streptozotocin induced diabetic mice

Renal tubulointerstitial fibrosis was a crucial pathological feature of diabetic nephropathy (DN), and renal tubular injury might associate with abnormal mitophagy. In this study, we investigated the effects and molecular mechanisms of AMPK agonist metformin on mitophagy and cellular injury in renal tubular cell under diabetic condition. The high fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetic mice model and HK-2 cells were used in this study. Metformin was administered in the drinking water (200 mg/kg/d) for 24 weeks. Renal tubulointerstitial lesions, oxidative stress and some indicators of mitophagy (e.g., LC3II, Pink1, and Parkin) were examined both in renal tissue and HK-2 cells. Additionally, compound C (an AMPK inhibitor) and Pink1 siRNA were applied to explore the molecular regulation mechanism of metformin on mitophagy. We found that the expression of p-AMPK, Pink1, Parkin, LC3II, and Atg5 in renal tissue of diabetic mice was decreased obviously. Metformin reduced the levels of serum creatinine, urine protein, and attenuated renal oxidative injury and fibrosis in HFD/STZ induced diabetic mice. In addition, Metformin reversed mitophagy dysfunction and the over-expression of NLRP3. In vitro pretreatment of HK-2 cells with AMPK inhibitor compound C or Pink1 siRNA negated the beneficial effects of metformin. Furthermore, we noted that metformin activated p-AMPK and promoted the translocation of Pink1 from the cytoplasm to mitochondria, then promoted the occurrence of mitophagy in HK-2 cells under HG/HFA ambience. Our results suggested for the first time that AMPK agonist metformin ameliorated renal oxidative stress and tubulointerstitial fibrosis in HFD/STZ-induced diabetic mice via activating mitophagy through a p-AMPK-Pink1-Parkin pathway.Subject terms: Diabetes complications, End-stage renal disease, Experimental models of disease     

Berberine regulates mesangial cell proliferation and cell cycle to attenuate diabetic nephropathy through the PI3K/Akt/AS160/GLUT1 signalling pathway

High glucose (HG) is one of the basic factors of diabetic nephropathy (DN), which leads to high morbidity and disability. During DN, the expression of glomerular glucose transporter 1 (GLUT1) increases, but the relationship between HG and GLUT1 is unclear. Glomerular mesangial cells (GMCs) have multiple roles in HG‐induced DN. Here, we report prominent glomerular dysfunction, especially GMC abnormalities, in DN mice, which is closely related to GLUT1 alteration. In vivo studies have shown that BBR can alleviate pathological changes and abnormal renal function indicators of DN mice. In vitro, BBR (30, 60 and 90 μmol/L) not only increased the proportion of G1 phase cells but also reduced the proportion of S phase cells under HG conditions at different times. BBR (60 μmol/L) significantly reduced the expression of PI3K‐p85, p‐Akt, p‐AS160, membrane‐bound GLUT1 and cyclin D1, but had almost no effect on total protein. Furthermore, BBR significantly declined the glucose uptake and retarded cyclin D1‐mediated GMC cell cycle arrest in the G1 phase. This study demonstrated that BBR can inhibit the development of DN, which may be due to BBR inhibiting the PI3K/Akt/AS160/GLUT1 signalling pathway to regulate HG‐induced abnormal GMC proliferation and the cell cycle, supporting BBR as a potential therapeutic drug for DN.     

Benzene and the genotoxicity of its metabolites. II. The effect of the route of administration on the micronuclei and bone marrow depression in mouse bone marrow cells

Benzene (880 mg/kg) and 4 of its metabolites, i.e., phenol (265 mg/kg), hydroquinone (80 mg/kg), catechol (40 mg/kg), and p-benzoquinone (5-20 mg/kg) have been tested for their capability to induce micronuclei in bone marrow cells of male mice after oral administration or intraperitoneal injection. Oral administration of benzene shows more activity than intraperitoneal injection, whereas the metabolites show more activity if administered by the latter method. The respective genotoxic strengths of the benzene metabolites are the following: hydroquinone much greater than phenol greater than catechol = p-benzoquinone. This last is active when administered orally.     

二苯乙烯苷对糖尿病肾病大鼠氧化应激反应相关因子的影响

目的：探讨二苯乙烯苷（TSG）对糖尿病肾病（DN）大鼠氧化应激效应和肾功能的影响。方法：将雄性大鼠随机分为正常对照 组、DN 大鼠模型组和TSG 治疗组,采用腹腔注射链脲佐菌素（60 mg/kg）建立DN大鼠模型,给药8 周后所有大鼠称体重、肾重。 并且通过腹腔采血的方式,收集各组大鼠的血液,观察、测量各组大鼠血清中相应的生化指标,然后运用比色法测定各组大鼠血 清中氧化应激相关因子活性和含量。结果：TSG能够有效的增强肾脏对血肌酐、尿素氮的清除率,从而减轻由高血糖引起的肾脏 损伤,并且能够显著降低DN大鼠血液中NO、NOS含量,提高T-SOD、CAT 活力值。结论：二苯乙烯苷能够改善DN 大鼠血清中 相应的生化指标,有效抑制DN大鼠肾脏的氧化应激反应,对DN具有显著的治疗作用。     

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.     

Lipophagy deficiency exacerbates ectopic lipid accumulation and tubular cells injury in diabetic nephropathy

Autophagy-mediated lipotoxicity plays a critical role in the progression of diabetic nephropathy (DN), but the precise mechanism is not fully understood. Whether lipophagy, a selective type of autophagy participates in renal ectopic lipid deposition (ELD) and lipotoxicity in the kidney of DN is unknown. Here, decreased lipophagy, increased ELD and lipotoxcity were observed in tubular cells of patients with DN, which were accompanied with reduced expression of AdipoR1 and p-AMPK. Similar results were found in db/db mice, these changes were reversed by AdipoRon, an adiponectin receptor activator that promotes autophagy. Additionally, a significantly decreased level of lipophagy was observed in HK-2 cells, a human proximal tubular cell line treated with high glucose, which was consistent with increased lipid deposition, apoptosis and fibrosis, while were partially alleviated by AdipoRon. However, these effects were abolished by pretreatment with ULK1 inhibitor SBI-0206965, autophagy inhibitor chloroquine and enhanced by AMPK activator AICAR. These data suggested by the first time that autophagy-mediated lipophagy deficiency plays a critical role in the ELD and lipid-related renal injury of DN.Subject terms: Chronic kidney disease, Diabetes complications     

肾康注射液对链脲佐菌素诱导的大鼠DN的作用研究

目的:研究肾康注射液(SKI)对糖尿病肾病(DN)的作用。方法:采用大鼠腹腔注射链脲佐菌素65 mg/kg体重建立DN大鼠模型,SKI高、中、低分别腹腔注射SKI 10 m L/kg,5 m L/kg,2.5 m L/kg,2次/天;正常组和模型组分别给予生理盐水5 m L/kg,2次/天;8周后,观察、测量相应生化和病理等指标。结果:SKI可明显增强DN大鼠肾脏对血肌酐和血尿素氮的清除率,显著降低血液中总胆固醇、甘油三脂和低密度脂蛋白的含量和升高血液中高密度脂蛋白含量,显著升高血清中T-SOD和CAT的活力,降低血清中NO及MDA含量和降低血清中NOS的活力,显著改善糖尿病引起的肾组织损伤。结论:SKI对治疗DN的治疗作用是肯定的其主要表现在改善血脂代谢紊乱,增强机体抗氧化应激能力及增强肾脏对肌酐和尿素氮的清除能力进而改善肾脏损伤。     

HNRNPA1-mediated exosomal sorting of miR-483-5p out of renal tubular epithelial cells promotes the progression of diabetic nephropathy-induced renal interstitial fibrosis

Diabetic nephropathy (DN) is a serious complication in type 1 and type 2 diabetes, and renal interstitial fibrosis plays a key role in DN progression. Here, we aimed to probe into the role and potential mechanism of miR-483-5p in DN-induced renal interstitial fibrosis. In this study, we corroborated that miR-483-5p expression was lessened in type 1 and type 2 diabetic mice kidney tissues and high glucose (HG)-stimulated tubular epithelial cells (TECs), and raised in the exosomes derived from renal tissues in type 1 and type 2 diabetic mice. miR-483-5p restrained the expressions of fibrosis-related genes in vitro and renal interstitial fibrosis in vivo. Mechanistically, miR-483-5p bound both TIMP2 and MAPK1, and TIMP2 and MAPK1 were bound up with the regulation of miR-483-5p on renal TECs under HG conditions. Importantly, HNRNPA1-mediated exosomal sorting transported cellular miR-483-5p out of TECs into the urine. Our results expounded that HNRNPA1-mediated exosomal sorting transported cellular miR-483-5p out of TECs into the urine, thus lessening the restraint of cellular miR-483-5p on MAPK1 and TIMP2 mRNAs, and ultimately boosting extracellular matrix deposition and the progression of DN-induced renal interstitial fibrosis.Subject terms: Cell biology, Molecular biology     

Tacrolimus ameliorates tubulointerstitial inflammation in diabetic nephropathy via inhibiting the NFATc1/TRPC6 pathway

Tubulointerstitial inflammation is crucial for the progression of diabetic nephropathy (DN), and tubular cells act as a driving force in the inflammatory cascade. Emerging data suggested that tacrolimus (TAC) ameliorates podocyte injury and macrophage infiltration in streptozotocin (STZ) mice. However, the effect of TAC on tubulointerstitial inflammation remains unknown. We found that albuminuria and tubulointerstitial damage improved in db/db mice treated with TAC. Macrophage infiltration and expression of IL‐6, TNF‐α, fibronectin, collagen 1 and cleaved caspase 3 were inhibited as well. In addition, the expression of nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) was up‐regulated in the kidneys of DN patients and correlated with tubular injury and inflammation. The expression of NFATc1 and TRPC6 also increased in the kidneys of db/db mice and HK‐2 cells with high glucose (HG), while TAC inhibited these effects. HG‐induced inflammatory markers and apoptosis were reversed by TAC and NFATc1 siRNA in HK‐2 cells, which was abolished by TRPC6 plasmid. Furthermore, HG‐induced TRPC6 expression was inhibited by NFATc1 siRNA, while NFATc1 nuclear translocation was inhibited by TAC, but was restored by TRPC6 plasmid in HK‐2 cells under HG conditions. These findings suggest that TAC ameliorates tubulointerstitial inflammation in DN through NFATc1/TRPC6 feedback loop.