JAK/STAT信号转导途径在糖尿病肾病肾小管上皮细胞中的表达

Expression of JAK/STAT pathway in proximal tubular opithelial cells in hyperglycemia induced nephropathy

目的:观察Janus酪氨酸蛋白激酶/信号转导子和转录激活子(JAK/STAT)信号转导途径在糖尿病肾病肾小管上皮细胞中的表达。方法:将体外培养的人肾近曲小管上皮细胞株(HKC)随机分为高糖组(30mmol/L)、低糖组(5.5mmol/L)和低糖+甘露醇组(糖5.5mmol/L+甘露醇24.5mmol/L)。采用酶联免疫吸附法(ELISA)测定HKC细胞上清液中转化生长因子-β1(TGF-β1)和型胶原的分泌;蛋白质免疫印迹法(Western blotting)检测平滑肌肌动蛋白(α-SMA)、E-钙黏素(E-cadherin)及信号蛋白STAT1、STAT3、磷酸化-STAT1(p-STAT1)和p-STAT3的表达;逆转录-聚合酶链反应(RT-PCR)检测TGF-β1mRNA表达。结果:与低糖组比较,高糖组不同时间点细胞培养上清液中TGF-β1、型胶原分泌明显增加(P均<0.01);HKC中p-STAT1和p-STAT3的蛋白及TGF-β1mRNA表达自6h起明显增加,至48h达最高,72h有所回落,而STAT1和STAT3在各时间点差异无显著性;HKC中α-SMA表达随刺激时间延长明显升高,E-cadherin表达明显下调(P均<0.01)。结论:JAK参与高糖诱导的HKC转分化,并刺激TGF-β1和细胞外基质的分泌。

Objective： To investigate the role of Janus kinase/signal transducers and activators of transcription （JAK/STAT） pathway activation in hyperglycemia induced transdifferentiation in renal proximal tubular epithelial cells. Methods： Cultured human kidney cells （HKC） were randomly divided into three groups： low glucose group （LG, 5.5 mmol/L）, high glucose group （HG, 30 mmol/L）, and low glucase＋ mannitol group （LG, 5.5 mmol/L ＋M, 24.5 mmol/L）. Immunoprecipitation and Western blotting analysis were used to determine the expression of tryosine phosphorylated JAK 2 （p -JAK2）. The protein expression of STAT1, STAT3, phospho -STAT1 （p- STAT1） and p -STAT3 and expression of α-smooth muscle actin （α-SMA）, E-cadherin were assessed with Western blotting. The contents of transforming growth factors, transforming growth factor β1 （TGF - β1）, fibronectin and type Ⅰ collagen in the supernatants of the cultured HKC were determined by enzyme -linked immunoadsorbent assay （ELISA）. TGF -β1 mRNA was assayed by reverse transcription and polymerase chain reaction （RT -PCR）. Results： Compared with LG control group, the expression of JAK2, p- STAT1, p -STAT3 and TGF- β1 mRNA was significantly increased in HG group at 6 hours, reaching its peak value at 48 hours, and it began to fall at 72 hours. Meanwhile, the contents of TGF -β1 and collagen I in the supernatants and the expression of α- SMA were increased （both P〈0.01）, the expression of E -cadherin was decreased （P〈0. 01）. The expression of JAK2, p - STAT1, p - STAT3 and TGF - β1 mRNA and the levels of TGF - β1 were significantly increased （all P〈 0. 01）. Conclusion ： Activation of JAK/STAT signaling pathway may be involved in the hyperglycemia induced transdifferentiation of HKC and overproduction of TGF - β1 and extracellular matrix proteins in HKCs.