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Sestrin2 is identified as a stress-induced protein and could functionate in many aspects. In our study, we investigated the latent impact of Sestrin2 on podocyte injury and its molecular mechanism in vivo and in vitro in diabetic kidney disease (DKD). Sestrin2 was low-expressed in renal biopsies from individuals with DKD, the glomeruli from diabetic mice, and mouse podocytes exposed to high glucose (HG). Sestrin2 overexpression ameliorated HG-induced phenotypic alterations, apoptosis, and oxidative stress in conditionally immortalized mouse podocytes and modulated the activity of Thrombospondin-1 (TSP-1)/transforming growth factor (TGF-β1)/Smad3 pathway in podocytes. Moreover, TSP-1 inhibitor LSKL or TGF-β blocker Pirfenidone arrested podocyte injury induced by HG. Streptozotocin (STZ) was employed to render equivalent diabetes in B6-TgN (CMV-Sestrin2) (TgN) and wild-type (WT) control mice. Sestrin2 alleviated increased levels of 24‐h urinary protein, blood urea nitrogen, serum creatinine and triglyceride, and urine 8-OHdG in diabetic mice. Podocyte phenotypic alterations, increased expression of apoptosis-associated proteins and podocyte loss were observed in WT but not in diabetic TgN mice, as well as oxidative stress. Additionally, TSP-1/TGF-β1/Smad3 signaling pathway was also suppressed in glomeruli of diabetic TgN mice. Thus, Sestrin2 mitigates podocyte injury in DKD via orchestrating TSP-1/TGF-β1/Smad3 pathway, underlining Sestrin2 as a promising therapeutic target for DKD.

The transforming growth factor-β1 (TGF-β1)/connective tissue growth factor (CTGF) pathway plays an important role in the pathogenesis and progression of chronic kidney disease. Oxidative stress is also involved in TGF-β1 signalling. Sirtuin 1 (Sirt1) exerts a number of pleiotropic effects, protecting against renal disease, including inhibiting fibrosis and oxidative metabolism. In this study, we investigated the role of the Sirt1 activator, SRT1720, in unilateral ureteral obstruction (UUO)-induced tubulointerstitial fibrosis and aimed to determine whether this role depends on the inhibition of oxidative stress and the TGF-β1/CTGF pathway. Renal fibrosis was induced by UUO in CD1 mice. SRT1720 (100 mg/kg) was administered by intraperitoneal injection for 3 days prior to UUO and this was continued for 7 days following UUO. Histological changes were examined by Masson's trichrome staining. The expression of fibrosis-related factors was evaluated by immunohistochemistry, western blot analysis and RT-qPCR. Apoptosis was also examined. We also examined the superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPx) and reduced glutathione (GSH) levels. UUO induced renal fibrosis and apoptosis and decreased Sirt1 expression. The administration of SRT1720 increased the Sirt1 levels and partially attenuated UUO-induced renal fibrosis and apoptosis. Furthermore, SRT1720 attenuated the levels of oxidative stress (it decreased the MDA levels, and increased the SOD, GPx and GSH levels), which suggests that it protected the cells against ROS-induced damage. Moreover, SRT1720 effectively inhibited the levels of TGF-β1/CTGF induced by UUO. On the whole, these findings indicate that the Sirt1 activator, SRT1720, exerts protective effects against UUO-induced tubulointerstitial fibrosis. The mechanisms of action of SRT1720 may include, at least in part, the suppression of renal oxidative stress and the TGF-β1/CTGF signalling pathway. The Sirt1 activator may therefore be prove to be a potent therapeutic agent for the treatment of fibrotic kidney disease.

Thioredoxin-interacting protein (TXNIP), is identified as an inhibitor of the thiol oxidoreductase thioredoxin that acts endogenously, and is increased by high glucose (HG). In this study, we investigated the potential function of TXNIP on apoptosis of podocytes and its potential mechanism in vivo and in vitro in diabetic nephropathy (DN). TXNIP silencing attenuated HG-induced apoptosis and obliterated the activation of signaling pathways of mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) in conditionally immortalized mouse podocytes. Furthermore, the Raptor and Rictor shRNAs, mTOR specific inhibitor KU-0063794 and p38 MAPK inhibitor SB203580 were used to assess the role of mTOR or p38 MAPK pathway on podocyte apoptosis induced by HG. The Rictor and Raptor shRNAs and KU-0063794 appeared to reduce HG-induced apoptosis in podocytes. Simultaneously, SB203580 could also restrain HG-induced apoptosis in podocytes. Streptozotocin rendered equivalent diabetes in TXNIP-/- (TKO) and wild-type (WT) control mice. TXNIP deficiency mitigated renal injury in diabetic mice. Additionally, TXNIP deficiency also descended the apoptosis-related protein and Nox4 levels, the mTOR signaling activation and the p38 MAPK phosphorylation in podocytes of diabetic mice. All these data indicate that TXNIP deficiency may mitigate apoptosis of podocytes by inhibiting p38 MAPK or mTOR signaling pathway in DN, underlining TXNIP as a putative target for therapy.

Oxidative stress is an important contributory factor resulting the development of kidney injury in patients with diabetes. Numerous in vitro and in vivo studies have suggested that anthocyanins, natural phenols commonly existing in numerous fruits and vegetables, exhibit important anti-oxidative, anti-inflammatory and antihyperlipidemic effects; however, their effects and underlying mechanisms on diabetic nephropathy (DN) have not yet been fully determined. In the present study, the regulation of apoptosis metabolism and antioxidative effects exhibited by anthocyanins [grape seed procyanidin (GSPE) and cyanidin-3-O-β-glucoside chloride (C3G)] were investigated, and the molecular mechanism underlying this process was investigated in vivo and in vitro. GSPE administration was revealed to suppress renal cell apoptosis, as well as suppress the expression of Bcl-2 in diabetic mouse kidneys. Furthermore, GSPE administration was demonstrated to suppress the expression of thioredoxin interacting protein (TXNIP), in addition to enhancing p38 mitogen-activation protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) oxidase activity in diabetic mouse kidneys. In vitro experiments using HK-2 cells revealed that C3G suppressed the generation of HG-mediated reactive oxygen species, cellular apoptosis, the expression of cleaved caspase-3 and the Bax/Bcl-2 ratio; and enhanced the expression of cytochrome c released from mitochondria. Furthermore, treatment with C3G was revealed to suppress the expression of TXNIP, in addition to the phosphorylation of p38 MAPK and ERK1/2 oxidase activity in HK-2 cells under HG conditions. In addition, treatment with C3G was revealed to attenuate the HG-induced suppression of the biological activity of thioredoxin, and to enhance the expression of thioredoxin 2 in HK-2 cells under HG conditions. In conclusion, the present study demonstrated that anthocyanins may exhibit protective effects against HG-induced renal injury in DN via antioxidant activity.

Tumor necrosis factor-α (TNF-α) is suggested to induce mitochondrial dysfunction and apoptosis of renal tubular epithelial cells that possibly exacerbates renal function in chronic kidney disease (CKD). Here we investigated whether suppressor of cytokine signaling-1 (SOCS-1), an inhibitor of cytokine signaling, was involved in TNF-α-induced human renal tubular epithelial cells (HKCs) oxidative stress and apoptosis. TNF-α promoted the protein and mRNA expression of SOCS-1 in a time and dose dependent manner, along with increased cell apoptosis and activation of apoptosis signal regulating kinase-1(ASK1) in HKCs. Furthermore, overexpression of SOCS-1 in HKCs reduced TNF-α-mediated oxidative stress and apoptosis. Meanwhile, We also found that overexpression of SOCS-1 could regulate the activity of JAK/STAT signaling pathway. In addition, a specific JAK2 inhibitor, AG490, that both attenuated TNF-α-induced oxidative stress, also reduced apoptosis. Taken together, overexpression of SOCS-1 prevented TNF-α-mediated cell oxidative stress and apoptosis may be via suppression of JAK/STAT signaling pathway activation in HKCs.

Yunzhuo Ren,* Chunyang Du,* Li Yan, Jingying Wei, Haijiang Wu, Yonghong Shi, Huijun Duan Department of Pathology, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China *These authors contributed equally to this work Abstract: Transforming growth factor-β1 (TGF-β1) plays an important role in the pathogenesis and progression of chronic kidney disease. Connective tissue growth factor (CTGF) is a critical fibrogenic mediator of TGF-β1. Mammalian sirtuin 1 (Sirt1) is reported to attenuate renal fibrosis by inhibiting the TGF-β1 pathway. This study was designed to detect whether the delivery of CTGF siRNA in vivo directly ameliorates renal fibrosis. Furthermore, the relationship with Sirt1 underlying the protective effect of CTGF siRNA on interstitial fibrosis and apoptosis was explored. Here, we report that the expressions of CTGF and TGF-β1 were increased while Sirt1 expression and activity were both dramatically decreased in mouse kidneys with unilateral ureteral obstruction. Recombinant human TGF-β1 treatment in HK-2 cells increased CTGF levels and remarkably decreased Sirt1 levels and was accompanied by apoptosis and release of fibrosis-related factors. Recombinant human CTGF stimulation also directly induced apoptosis and fibrosis. The CTGF siRNA plasmid ameliorated tubular cell apoptosis and tubulointerstitial fibrosis, but did not affect Sirt1 expression and activity both in vivo and in vitro. Furthermore, overexpression of Sirt1 abolished TGF-β1-induced cell apoptosis and fibrosis, while Sirt1 overexpression suppressed CTGF expression via stimulation by TGF-β1. This study provides evidence that treatment strategies involving the delivery of siRNA targeting potentially therapeutic transgenes may be efficacious. Our results suggest that the decrease in Sirt1 is associated with the upregulated expression of CTGF in renal fibrosis, and may aid in the design of new therapies for the prevention of renal fibrosis. Keywords: ureteral obstruction, Sirt1, CTGF, fibrosis, apoptosis

Renal tubular epithelial cells (RTEC) apoptosis, which plays a key role in the pathogenesis and progression of diabetic nephropathy (DN), is believed to be contributive to the hyperglycemia-induced kidney failure, though the exact mechanisms remain elusive. In this study, we investigated how inhibition of c-Src/p38 MAPK pathway would affect RTEC apoptosis. The c-Src inhibitor PP2 i.p. administered every other day for 8 weeks to diabetic db/db mice significantly reduced their kidney weights, daily urinary volumes, blood glucose, blood urea nitrogen, serum creatinine, triglyceride and urine albumin excretion, whereas deactivation of c-Src and p38 MAPK were also observed, along with decreases in both Bax/Bcl-2 ratio and cleaved caspase-3 level in the kidneys. In vitro, exposure of HK-2 cells (a human RTEC line), to high glucose (HG) promoted phosphorylation of c-Src and p38 MAPK, and subsequently, as revealed by western blotting, TUNEL assay and flow cytometry, increased cell death, which can be inhibited by PP2. Especially, a specific p38 MAPK inhibitor, SB203580, that both attenuated HG-induced c-Src activation and abrogated the expression of PPARγ and CHOP, also reduced apoptosis. Taken together, PP2 inhibits c-Src and therefore reduces apoptosis in RTEC, which at least in part, is due to suppressed p38 MAPK activation in diabetic kidney.

Oxidative stress is implicated in the pathogenesis of diabetic kidney injury. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge reactive oxygen species (ROS). Here, we investigated the effect and molecular mechanism of mitochondria-targeted antioxidant peptide SS-31 on injuries in diabetic kidneys and mouse mesangial cells (MMCs) exposed to high-glucose (HG) ambience. CD-1 mice underwent uninephrectomy and streptozotocin treatment prior to receiving daily intraperitoneal injection of SS-31 for 8 wk. The diabetic mice treated with SS-31 had alleviated proteinuria, urinary 8-hydroxy-2-deoxyguanosine level, glomerular hypertrophy, and accumulation of renal fibronectin and collagen IV. SS-31 attenuated renal cell apoptosis and expression of Bax and reversed the expression of Bcl-2 in diabetic mice kidneys. Furthermore, SS-31 inhibited expression of transforming-growth factor (TGF)-β1, Nox4, and thioredoxin-interacting protein (TXNIP), as well as activation of p38 MAPK and CREB and NADPH oxidase activity in diabetic kidneys. In vitro experiments using MMCs revealed that SS-31 inhibited HG-mediated ROS generation, apoptosis, expression of cleaved caspase-3, Bax/Bcl-2 ratio, and cytochrome c (cyt c) release from mitochondria. SS-31 normalized mitochondrial potential (ΔΨm) and ATP alterations, and inhibited the expression of TGF-β1, Nox4, and TXNIP, as well as activation of p38 MAPK and CREB and NADPH oxidase activity in MMCs under HG conditions. SS-31 treatment also could reverse the reduction of thioredoxin (TRX) biologic activity and upregulate expression of thioredoxin 2 (TRX2) in MMCs under HG conditions. In conclusion, this study demonstrates a protective effect of SS-31 against HG-induced renal injury via an antioxidant mechanism in diabetic nephropathy.

Non-coding RNA (ncRNA) comprises a substantial portion of primary transcripts that are generated by genomic transcription, but are not translated into protein. The possible functions of these once considered ‘junk’ molecules have incited considerable interest and new insights have emerged. The two major members of ncRNAs, namely micro RNA (miRNA) and long non-coding RNA (lncRNA), have important regulatory roles in gene expression and many important physiological processes, which has recently been extended to programmed cell death. The previous paradigm of programmed cell death only by apoptosis has recently expanded to include modalities of regulated necrosis (RN), and particularly necroptosis. However, most research efforts in this field have been on protein regulators, leaving the role of ncRNAs largely unexplored. In this review, we discuss important findings concerning miRNAs and lncRNAs that modulate apoptosis and RN pathways, as well as the miRNA–lncRNA interactions that affect cell death regulation.