Key Findings
Diabetic nephropathy (DN) is a major complication in patients with either type 1 or type 2 diabetes mellitus. Accumulating data indicate that excessive oxidative stress (OS) and aberrant dynamics are the primary factors responsible for tubule damage in DN. Hyperglycemia-induced ROS activates p38 mitogen-activated protein kinase(MAPK), which induces phosphorylation of transcriptional factors, altered expression of genes, and production of fibronectin in mesangial cells, resulting in DN. Transforming growth factor β1 (TGF- β1) exerts biological activities through MAPK cascades in certain cell lines, especially through p38 MAPK in human mesangial cells. Kidney injury would be improved by decreasing the activation of the ROS/p38 MAPK/TGF-β1 signal pathway. The results of the present study concluded that high glucose induced OS and glucose toxicity of HK-2 cells. ALA/LA exerts protective effects in vitro through inhibition of ROS generation, down regulation of the activation of the p38MAPK pathway and the expression of TGF-β1 in HK-2 cells. ALA/LA treatment was also shown to inhibit cell death, thereby suggesting that ALA/LA protects HK-2 cells against high glucose induced cytotoxicity. ALA/LA can protect HK-2 cells from oxidative stress induced by high glucose not only by scavenging intracellular ROS, but also by enhancing endogenous antioxidant defense systems including the antioxidant enzyme defense system and the GSH system. The results confirmed that ALA/LA treatment reduced high glucose-induced activation of p38, and SB203580 (p38 inhibitor) (partially reduced ROS production. Taken together, these results demonstrate that ALAL/LA decreased the apoptosis induced by high glucose through the ROS/p38 signal pathway. In conclusion, high glucose induces OS and apoptosis of HK-2 cells. The therapeutic actions of anti-oxidants in vitro are directly related to controlling the activities of intracellular signaling pathways associated with OS, in which the p38MAPK pathways occupy the key positions in DN at the early stage. The potential mechanisms by which ALA/LA exerts protective effects against in glucose toxicity in HK-2 cells in vitro are through inhibition of ROS generation, down regulating the activation of the p38MAPK pathway, and the expression of TGF-β1.
ABSTRACT:
BACKGROUND: Growing evidence indicates that oxidative stress (OS) plays a pivotal role in Diabetic nephropathy (DN). In a previous study we demonstrated that ALA/LA protected HK-2 cells against high glucose-induced cytotoxicity. So we aimed to establish the glucose injury model of HK-2 cells and investigate the beneficial effects of ALA/LA on high glucose-induced excessive production of TGF-β1 and the possible mechanisms mediating the effects. METHODS: The expression of OS markers in high glucose-induced HK-2 cells treated with ALA/LA., including the antioxidant enzymes and reactive oxygen species (ROS) production, as well as the apoptosis rate were assayed by ELISA and flow cytometry. The p38/ transforming growth factor β1 (TGF-β1) signal pathway were measured by real-time RT-PCR and western blot. RESULTS: The modeling condition of glucose toxicity on HK-2 cells was at the glucose concentration of 40.9 mM. ALA/LA can significantly increase the activities of antioxidant enzymes and decrease ROS production stimulated by high glucose. The study also found that ALA/LA caused a decrease in the apoptosis rate and TGF-β1 level of HK-2 cells under high glucose stress through the ROS/p38 pathway. CONCLUSIONS: ALA/LA exerts protective effects in vitro through inhibition of ROS generation, down regulation of the activation of the p38MAPK pathway and the expression of TGF-β1 in HK-2 cells.
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