Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan.
Br J Pharmacol. 2010 Aug;160(8):2055-68. doi: 10.1111/j.1476-5381.2010.00860.x.
Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells.
Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors.
NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA.
Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.
缝隙连接在调节细胞表型和确定各种损伤后细胞存活方面发挥重要作用。在这里，我们研究了缝隙连接在氨基糖苷诱导的肾小管细胞损伤中的作用。
通过免疫荧光染色、Western blot 和染料转移测定，比较了两种肾小管上皮细胞系 NRK-E52 和 LLC-PK1 的缝隙连接蛋白表达和功能。通过 WST 测定评估暴露于氨基糖苷后的细胞活力。通过转染缝隙连接蛋白连接蛋白 43（Cx43）、使用 Cx43 siRNA 和缝隙连接抑制剂来调节缝隙连接。
NRK-E52 细胞表达丰富的 Cx43 并通过缝隙连接细胞间通讯（GJIC）功能连接。暴露于氨基糖苷、G418 和 Hygromycin 增加了 NRK-E52 细胞中 Cx43 的磷酸化和 GJIC。这些氨基糖苷还降低了细胞活力，而缝隙连接抑制剂和 Cx43 siRNA 可预防这种降低。LLC-PK1 细胞缝隙连接缺陷且对氨基糖苷诱导的细胞毒性具有抗性。野生型 Cx43 的过表达使 LLC-PK1 细胞转变为对药物敏感的表型。缝隙连接抑制剂α-甘草次酸（α-GA）激活了 NRK-E52 细胞中的 Akt。抑制 Akt 通路增强了 G418 的细胞毒性，并消除了α-GA 的保护作用。此外，α-GA 还显著减弱了庆大霉素诱导的 NRK-E52 细胞的细胞毒性。
缝隙连接有助于氨基糖苷的细胞毒性作用。调节缝隙连接可能是预防和治疗氨基糖苷诱导的肾小管细胞损伤的一种有前途的方法。
