Yunzhuo Ren

In Vivo Delivery of Gremlin siRNA Plasmid Reveals Therapeutic Potential against Diabetic Nephropathy by Recovering Bone Morphogenetic Protein-7

Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor-beta1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.

Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells.

Epithelial to mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both high glucose and transforming growth factor-β (TGF-β) are able to induce EMT in cell culture. In this study, we examined the role of the thioredoxin-interacting protein (TXNIP) on EMT induced by high glucose or TGF-β1 in HK-2 cells. EMT was assessed by the expression of α-smooth muscle actin (α-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. High glucose (30mM) was shown to induce EMT at 72h. This was blocked by knockdown of TXNIP or antioxidant NAC. Meanwhile, we also found that knockdown of TXNIP or antioxidant NAC inhibited high glucose-induced generation of reactive oxygen species (ROS), phosphorylation of p38 MAPK and ERK1/2 and expression of TGF-β1. HK-2 cells that were exposed to TGF-β1 (4ng/ml) also underwent EMT. The expression of TXNIP gene and protein was increased in HK-2 cells treated with TGF-β1. Transfection with TXNIP shRNA was able to attenuate TGF-β1 induced-EMT. These results suggested that knockdown of TXNIP antagonized high glucose-induced EMT by inhibiting ROS production, activation of p38 MAPK and ERK1/2, and expression of TGF-β1, highlighting TXNIP as a potential therapy target for diabetic nephropathy.

Attenuation of glomerular injury in diabetic mice with tert-butylhydroquinone through nuclear factor erythroid 2-related factor 2-dependent antioxidant gene activation.

Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a positive regulator of the expression of antioxidant genes. This study is aimed at examining the effect of tert-butylhydroquinone (tBHQ), an activator of Nrf2, on hyperglycemia-related diabetic nephropathy. Methods: CD-1 mice were induced with streptozotocin and treated with 1% tBHQ, or omitting it, in their diet for 12 weeks. Four and twelve weeks later, the levels of serum and glomerular malondialdehyde (MDA), blood glucose, kidney and body weights, and proteinuria were measured. The pathogenic process in the kidney was examined histologically and by transmission electron microscopy. The relative levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression and nuclear accumulation of Nrf2 in the glomeruli were determined by reverse transcription polymerase chain reaction and Western blot assays. Results: In the glomeruli of diabetic mice, treatment with tBHQ significantly reduced the levels of serum and glomerular MDA, kidney weight and proteinuria, decreased fibronectin accumulation and mitigated the pathogenic processes. It also enhanced Nrf2, HO-1 and γ-GCS expression and Nrf2 nuclear accumulation. Conclusions: tBHQ has beneficial effects on reducing hyperglycemia-induced kidney injury, which is associated with the enhanced expression of Nrf2, and its downstream antioxidant HO-1 and γ-GCS in the glomeruli of diabetic mice.

Mitochondria-targeted peptide SS-31 attenuates renal injury via an antioxidant effect in diabetic nephropathy.

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.

Notch pathway is involved in high glucose‐induced apoptosis in podocytes via Bcl‐2 and p53 pathways

Recent studies have shown that Notch pathway plays a key role in the pathogenesis of diabetic nephropathy (DN), however, the exact mechanisms remain elusive. Here we demonstrated that high glucose (HG) upregulated Notch pathway in podocytes accompanied with the alteration of Bcl‐2 and p53 pathways, subsequently leading to podocytes apoptosis. Inhibition of Notch pathway by chemical inhibitor or specific short hairpin RNA (shRNA) vector in podocytes prevented Bcl‐2‐ and p53‐dependent cell apoptosis. These findings suggest that Notch pathway mediates HG‐induced podocytes apoptosis via Bcl‐2 and p53 pathways. J. Cell. Biochem. 114: 1029–1038, 2013.

p38 MAPK pathway is involved in high glucose-induced thioredoxin interacting protein induction in mouse mesangial cells

Excessive reactive oxygen species (ROS) play a key role in the pathogenesis of diabetic nephropathy. The thioredoxin (TRX) system, a major thiol antioxidant system, regulates the reduction of intracellular ROS. Here we show that high glucose (HG) inhibits TRX ROS‐scavenging function through p38 mitogen‐activated protein kinase (MAPK)‐mediated induction of thioredoxin interacting protein (TXNIP) in mouse mesangial cells (MMCs). Knockdown of TXNIP in MMCs reversed HG‐induced reduction of TRX activity and inhibited HG‐induced activation of p38 MAPK and increased synthesis of TGF‐β1 and fibronectin. These data suggest that HG‐induced overexpression of TXNIP in MMCs, which may be via the p38 MAPK pathway.

Suppressor of Cytokine Signaling-1 Ameliorates Expression of MCP-1 in Diabetic Nephropathy

Background: Janus kinase (JAK)/signal transducers and activators of transcription (STAT) contribute to diabetic nephropathy. Suppressor of cytokine signaling-1 (SOCS-1) is one of the negative feedback regulators of JAK/STAT signaling. This study investigated the effect of SOCS-1 on the JAK/STAT pathway and MCP-1 expression in diabetic nephropathy. Methods: Streptozotocin-induced diabetic mice received pEF-FLAG-I/mSOCS-1 plasmid or pEF-FLAG-I vector for 4 weeks and were compared with age-matched nondiabetic mice. Functional and pathologic markers, expression of monocyte chemoattractant protein-1 (MCP-1) and TGF-β1 and phosphorylation of STAT1 and STAT3 were assessed. The effect of SOCS-1 on the expression of MCP-1 in mesangial cells under high glucose conditions was also examined.Results: Urine albumin excretion and renal hypertrophy were alleviated in diabetic mice by overexpression of SOCS-1. The expression of TGF-β1 and MCP-1 and the activation of STAT1 and STAT3 were significantly inhibited in diabetic kidney by gene delivery of SOCS-1. In cultured mesangial cells, overexpression of SOCS-1 markedly suppressed high glucose-induced MCP-1 expression. Conclusions: This study suggests that SOCS-1 may attenuate renal damage by ameliorating MCP-1 expression and regulation of the phosphorylation of JAK/STAT in diabetic mice.

Knockdown of thioredoxin interacting protein attenuates high glucose-induced apoptosis and activation of ASK1 in mouse mesangial cells

Mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Here we show that thioredoxin interacting protein (TXNIP) is involved in high glucose (HG)‐induced mouse mesangial cell (MMC) apoptosis. HG induced activation of apoptosis signal regulating kinase‐1 (ASK1) in a time‐dependent manner in MMCs. Treatment with antioxidant, tempol, or knockdown of TXNIP in MMCs reduced HG‐mediated apoptosis, expression of cleaved caspase‐3, Bax/Bcl‐2 ratio and activation of ASK1. These data suggest that knockdown of TXNIP prevented HG‐induced cell apoptosis and activation of ASK1 may be via reduction of oxidative stress in MMCs.

Anthocyanins inhibit high-glucose-induced cholesterol accumulation and inflammation by activating LXRα pathway in HK-2 cells

The dysregulation of cholesterol metabolism and inflammation plays a significant role in the progression of diabetic nephropathy (DN). Anthocyanins are polyphenols widely distributed in food and exert various biological effects including antioxidative, anti-inflammatory, and antihyperlipidemic effects. However, it remains unclear whether anthocyanins are associated with DN, and the mechanisms involved in the reciprocal regulation of inflammation and cholesterol efflux are yet to be elucidated. In this study, we evaluated the regulation of cholesterol metabolism and the anti-inflammatory effects exerted by anthocyanins (cyanidin-3-O-β-glucoside chloride [C3G] or cyanidin chloride [Cy]) and investigated the underlying molecular mechanism of action using high-glucose (HG)-stimulated HK-2 cells. We found that anthocyanins enhanced cholesterol efflux and ABCA1 expression markedly in HK-2 cells. In addition, they increased peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptor alpha (LXRα) expression and decreased the HG-induced expression of the proinflammatory cytokines intercellular adhesion molecule-1 (ICAM1), monocyte chemoattractant protein-1 (MCP1), and transforming growth factor-β1 (TGFβ1), as well as NFκB activation. Incubation with the PPARα-specific inhibitor GW6471 and LXRα shRNA attenuated the anthocyanin-mediated promotion of ABCA1 expression and cholesterol efflux, suggesting that anthocyanins activated PPARα-LXRα-ABCA1-dependent cholesterol efflux in HK-2 cells. Moreover, the knockout of LXRα abrogated the anti-inflammatory effect of anthocyanins, whereas the PPARα antagonist GW6471 does not have this effect. Further investigations revealed that LXRα might interfere with anthocyanin-induced decreased ICAM1, MCP1, and TGFβ1 expression by reducing the nuclear translocation of NFκB. Collectively, these findings suggest that blocking cholesterol deposition and inhibiting the LXRα pathway-induced inflammatory response might be one of the main mechanisms by which anthocyanins exert their protective effects in DN.

Thioredoxin-interacting protein regulates lipid metabolism via Akt/mTOR pathway in diabetic kidney disease.

Abnormal lipid metabolism contributes to the renal lipid accumulation, which is associated with diabetic kidney disease, but its precise mechanism remains unclear. The growing evidence demonstrates that thioredoxin-interacting protein is involved in regulating cellular glucose and lipid metabolism. Here, we investigated the effects of thioredoxin-interacting protein on lipid accumulation in diabetic kidney disease. In contrast to the diabetic wild-type mice, the physical and biochemical parameters were improved in the diabetic thioredoxin-interacting protein knockout mice. The increased renal lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, and phosphorylated Akt and mTOR associated with diabetes in wild-type mice was attenuated in diabetic thioredoxin-interacting protein knockout mice. Furthermore, thioredoxin-interacting protein knockout significantly increased the expression of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 in diabetic kidneys. In vitro experiments, using HK-2 cells, revealed that knockdown of thioredoxin-interacting protein inhibited high glucose-mediated lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, as well as activation of Akt and mTOR. Moreover, knockdown of thioredoxin-interacting protein reversed high glucose-induced reduction of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 expression in HK-2 cells. Importantly, blockade of Akt/mTOR signaling pathway with LY294002, a specific PI3K inhibitor, replicated these effects of thioredoxin-interacting protein silencing. Taken together, these data suggest that thioredoxin-interacting protein deficiency alleviates diabetic renal lipid accumulation through regulation of Akt/mTOR pathway, thioredoxin-interacting protein may be a potential therapeutic target for diabetic kidney disease.