草酸钙晶体引起肾小管上皮细胞紧密连接损伤与TRPV5变化的调节机制

The regulatory mechanism of Ca Ox crystal-induced TJs injury and TRPV5 in renal tubular epithelial cells

目的探讨草酸钙晶体黏附肾小管上皮引起紧密连接(TJs)损伤与TRPV5变化的调节机制。方法利用一水草酸钙晶体(COM)暴露犬肾小管上皮细胞(MDCK)及TRPV5稳定过表达HEK-293细胞,2 mmol/L EGTA处理构建细胞TJs损伤模型;采用Western blot、荧光免疫、偏光显微镜等手段,分别检测TJs主要结构蛋白、Na^+-K^+-ATPase、TRPV5、晶体黏附量等指标,评估草酸钙晶体引起肾小管上皮细胞TJs损伤及TRPV5表达的变化。结果 COM呈下调MDCK细胞TJs蛋白Occludin和ZO-1表达水平(P<0.01)。TJs损害后,COM黏附增加(P<0.05);Na^+-K^+-ATPase介导TJs修复,在1 min时表达最强,随着TJs修复完成而逐步减弱,晶体黏附力也逐渐减弱(P<0.01)。COM与TRPV5稳转HEK-293细胞株共培养,下调TRPV5总蛋白及膜蛋白表达水平(P<0.01)。结论草酸钙晶体引起肾小管上皮细胞之间的TJs受损,促进草酸钙晶体黏附;细胞极性破坏,TRPV5膜蛋白转运失调,最终引起TRPV5膜蛋白表达减少。尿钙增加,负反馈激发Na^+-K^+-ATPase表达使TJs进入修复环节;随着TJs的自我修复,草酸钙晶体黏附减少。

Objective To investigate the regulatory mechanism of CaOx crystal induced tight junctions （TJs） injury and TRPV5 in renal tubular epithelial cells. Methods Calcium oxalate monohydrate （COM） exposure was performed on canine renal tubular epithelial cells （MDCK） and HEK - 293 ceils with stable over - expression of TRPV5. EGTA was administrated for TJs injury model. Western blot, immunofluorescence, polarizing microscope and other indica- tors were applied to assess TJs protein, Na ＋ - K ＋ - ATPase, TRPV5 and crystal adhesion etc. Results COM significant- ly down - regulated Occludin and ZO - 1 protein expression in MDCK cells （ P 〈 0. 01 ）. The TJs injury significantly increased the adhesion ability of COM to MDCK cells （P 〈 0. 05 ）. The expression of Na＋ - K ＋ - ATPase induced TJs re- covery and reached the highest level at lmin. COM inhibited the expression of TRPV5 membrane protein and total protein in HEK - 293 cells （P 〈 0. 01 ）. Conclusion CaOx can induce the TJs injury in renal tubular epithelial cells, and pro- mote the adhesion of CaOx crystals. When the cell polarity is destroyed, the TRPV5 membrane protein gets transportation problem and thus the expression of TRPV5 membrane protein decreases. With the increase of urinary calcium, the nega- tive feedback from Na ＋ - K ＋ - ATPase stimulates the repairmen of TJs. With the self - repairmen of TJs, CaOx crystal adhesion to cells decreases.