糖原合成激酶-3β/β-连环蛋白信号通路在贫铀致人肾近曲小管上皮细胞损伤中的作用

Abstract

Objective To investigate the effects of glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling on the human renal proximal tubular epithelial HK-2 cell injury induced by depleted uranium(DU), and provide a new enlightenment for the development of DU antidotes. Methods HK-2 cells were exposed to different concentrations of DU for 3-24 h, then the protein expressions of kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and nuclear β-catenin were detected by immunofluorescence staining. The protein expressions of p-GSK-3β(S9), GSK-3β and c-myc were detected by Western blot assay. HK-2 cells were transiently transfected by GSK-3β (KD) plasmid or treated by TDZD-8 to inhibit the activity of GSK-3β specifically. Other HK-2 cells were transiently transfected by β-catenin plasmid to overexpress the β-catenin protein. Results The percentages of KIM-1 and NGAL-positive cells increased with DU exposure time and concentrations from 300 and 600 μmol/L, and they were significantly higher than those of the blank control at 6-24 h of DU exposure (KIM-1-positive cells: t=11.06, 18.97, 30.49, P<0.05; t=6.79, 16.02, 85.45, P<0.05; NGAL-positive cells: t=11.78, 11.37, 34.29, P<0.05; t=7.34, 21.63, 36.84, P<0.05). In contrast, the ratio of p-GSK-3β(S9) to GSK-3β and percentage of nuclear β-catenin-positive cells were significantly higher than that of the blank control at 3-24 h of DU exposure (p-GSK-3β(S9)/GSK-3β: t=3.95, 4.69, 5.40, 3.34, P<0.05; nuclear β-catenin-positive cells: t=4.61, 6.52, 36.64, 14.93, P<0.05) with a maximum response at 9 h of DU exposure accompanied with corresponding increase of protein level of c-myc, a downstream target gene of β-catenin. Transient transfection of HK-2 cells with GSK-3β(KD) plasmid significantly inhibited the activity of GSK-3β (t=8.07, P<0.05) and reduced the DU-increased percentage of KIM-1-positive cells (t=24.77, P<0.05). Treatment cells with TDZD-8 inhibited the activity of GSK-3β and enhanced the percentage of nuclear β-catenin-positive cells, and it also significantly reduced the percentage of KIM-1-positive cells in HK-2 cells exposed to DU (t=6.25, 6.73, P<0.05). Moreover, overexpression of β-catenin significantly reduced DU-induced cell injury (t=7.48, P<0.05). Conclusions GSK-3β/β-catenin signaling plays a key role in regulating the DU-induced cytotoxicity of HK-2 cells. Inhibition of GSK-3β activity and overexpression of β-catenin can protect the HK-2 cells from DU-induced damage. Key words: Depleted uranium; GSK-3β; β-catenin; KIM-1; HK-2 cell