Netrin-1对高糖诱导的肾小管上皮细胞损伤的影响

目的:研究netrin-1对高糖诱导的肾小管上皮细胞损伤的影响。方法:用高糖培养人肾小管上皮HK-2细胞,real-time PCR和Western blot测定细胞中netrin-1的表达水平。用过表达netrin-1慢病毒感染HK-2细胞,检测其对高糖环境下HK-2细胞中netrin-1表达的影响。流式细胞术测定细胞凋亡,Western blot测定cleavedcaspase-3蛋白水平,2,4-二硝基苯肼法检测培养液中乳酸脱氢酶(LDH)活性,硫代巴比妥酸法检测培养液中丙二醛(MDA)含量,ELISA法测定培养液中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)含量。结果:高糖处理后的HK-2细胞中netrin-1的mRNA和蛋白水平均明显下调(P <0.05)。过表达netrin-1慢病毒能够上调高糖环境下HK-2细胞中netrin-1的表达水平(P <0.05)。高糖可促进肾小管上皮细胞分泌IL-1β和TNF-α,提高细胞培养液中LDH和MDA的水平(P <0.05),使肾小管上皮细胞的caspase-3活化并诱导细胞凋亡;上调netrin-1的HK-2细胞经高糖诱导后,细胞分泌的IL-1β和TNF-α减少,细胞中活化的caspase-3蛋白水平降低,培养液中LDH和MDA水平降低,细胞凋亡减少(P <0.05)。结论:上调netrin-1减轻高糖诱导的肾小管上皮细胞氧化损伤和炎性损伤,减少细胞凋亡。

Effect of netrin-1 on high glucose induced injury of renal tubular epithe-lial cells

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose. METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose. Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells. HK-2 cells were infected with netrin-1-over-expressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed. The apoptosis rate was analyzed by flow cytometry. The protein level of cleaved caspase-3 was determined by Western blot. lactate dehydrogenase (LDH) activity in the culture medium was measured by 2,4-binitrobenzene hydrazine method. The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA. RESULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05). Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose. High glucose promoted the secretion of IL-1β and TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05). After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1β and TNF-α secretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05). CONCLUSION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.