ABSTRACT

The elevated intracellular Ca²⁺ and oxidative stress are well-reported mechanisms behind renal tubular epithelial injury initiated by various insults. Given that TRPV4 and connexin43 (Cx43) channels are activated by a wide range of stimuli and regulate both intracellular Ca²⁺ and redox status, we speculated an involvement of these channels in renal tubular cell injury. Here, we tested this possibility and explored the potential underlying mechanisms. Our results demonstrated that exposure of renal tubular epithelial cells to aminoglycoside G418 led to cell death, which was attenuated by both TRPV4 and gap junction (Gj) inhibitor. Activation of TRPV4 caused cell damage, which was associated with an early increase in Cx43 expression and function. Inhibition of Cx43 with chemical inhibitor or siRNA largely prevented TRPV4 activation-induced cell damage. Further analysis revealed that TRPV4 agonists elicited a rise in intracellular Ca²⁺ and caused a Ca²⁺-dependent elevation in TXNIP (a negative regulator of the antioxidant thioredoxin). In the presence of Gj inhibitor, however, these effects of TRPV4 were largely prevented. The depletion of intracellular Ca²⁺ with Ca²⁺ chelator BAPTA-AM or downregulation of TXNIP with siRNA significantly alleviated TRPV4 activation-initiated cell injury. Collectively, our results point to a critical involvement of TRPV4/Cx43 channel interaction in renal tubular cell injury through mechanisms involving a synergetic induction of intracellular Ca²⁺ and oxidative stress. Channel interactions could be an important mechanism underlying cell injury. Targeting channels could have therapeutic potential for the treatment of acute tubular cell injury.

摘要

升高的细胞内Ca ²⁺和氧化应激是由各种损伤引发的肾小管上皮损伤背后的机制。鉴于TRPV4和连接蛋白43（Cx43）通道可被多种刺激激活，并调节细胞内Ca ²⁺和氧化还原状态，我们推测这些通道参与了肾小管细胞损伤。在这里，我们测试了这种可能性，并探讨了潜在的潜在机制。我们的结果表明，肾小管上皮细胞暴露于氨基糖苷G418会导致细胞死亡，这被TRPV4和间隙连接（Gj）抑制剂减弱。TRPV4的激活引起细胞损伤，这与Cx43表达和功能的早期增加有关。用化学抑制剂或siRNA抑制Cx43很大程度上防止了TRPV4激活诱导的细胞损伤。进一步的分析表明，TRPV4激动剂引起细胞内Ca ²⁺升高并引起Ca ²⁺依赖性升高的TXNIP（抗氧化剂硫氧还蛋白的负调节剂）。然而，在Gj抑制剂的存在下，TRPV4的这些作用被很大程度上阻止。用Ca ²⁺螯合剂BAPTA-AM耗尽细胞内Ca ²⁺或用siRNA降低TXNIP的表达显着减轻了TRPV4激活引发的细胞损伤。总体而言，我们的研究结果表明，TRPV4 / Cx43通道相互作用通过涉及细胞内Ca ²⁺协同诱导和氧化应激的机制在肾小管细胞损伤中至关重要。通道相互作用可能是细胞损伤的重要机制。靶向通道可能具有治疗急性小管细胞损伤的治疗潜力。