TRPC6介导的钙离子紊乱通过激活STAT3促进肾小管上皮细胞损伤后炎症反应及肾间质纤维化

TRPC6-mediated calcium disorder promotes inflammatory response and renal interstitial fibrosis after renal tubular epithelial cell injury by activating STAT3

目的:采用动物及细胞实验探讨肾小管上皮细胞(TECs)中瞬时受体电位阳离子通道C亚族成员6(TRPC6)介导的钙离子(Ca^(2+))紊乱对肾脏纤维化过程中炎症反应及细胞外基质合成的影响及机制。方法:(1)在体实验:建立小鼠单侧输尿管梗阻(UUO)模型,分为假手术组及UUO模型组,分别向两组小鼠腹腔注射TRPC6抑制剂BTP2或溶媒,在手术后14 d取肾组织留检。通过激光共聚焦检测TECs内Ca^(2+)浓度;通过HE染色及Masson染色检测肾组织病理改变;Westernblot法检测TRPC6、纤维化相关蛋白I型胶原(COL1)、信号转导及转录激活因子3(STAT3)及p-STAT3的表达水平;real-timePCR法检测炎症因子白细胞介素1β(IL-1β)、IL-6及肿瘤坏死因子α(TNF-α)的mRNA水平。(2)体外实验:应用TGF-β刺激人肾脏近曲小管上皮细胞株HK-2,向HK-2细胞转染TRPC6质粒或TRPC6 siRNA,Westernblot法检测TRPC6、COL1、STAT3及p-STAT3的表达水平;real-timePCR法检测炎症因子IL-1β、IL-6及TNF-α的mRNA水平。结果:(1)UUO模型中,TECs内Ca^(2+)浓度较假手术组显著升高;腹腔注射BTP2可减轻肾小管损伤及肾脏炎症细胞浸润,显著降低肾组织炎症因子IL-1β、IL-6及TNF-α的mRNA水平,降低肾组织COL1及p-STAT3蛋白水平(P<0.05)。(2)体外实验中,TGF-β刺激下,HK-2细胞中炎症因子IL-1β、IL-6及TNF-α的mRNA水平和COL1表达增多,高表达TRPC6进一步增加炎症因子及COL1的表达,沉默TRPC6则降低炎症因子及COL1的表达(P<0.05)。结论:在UUO模型及体外培养的TECs实验中,TRPC6介导的Ca^(2+)紊乱通过促进STAT3磷酸化,加重肾脏炎症反应及肾间质纤维化水平。

AIM:To investigate the effect of calcium(Ca^(2+))disorder mediated by transient receptor potential cation channel subfamily C member 6(TRPC6)on renal inflammation and interstitial fibrosis in vivo and in vitro study.METHODS:(1)In vivo,unilateral ureteric obstruction(UUO)operation was performed in adult C57BL/6 mice and sham operation was performed in the control animals with the same procedure without ligation of the left ureter.The mice in each group were injected intraperitoneally with either BTP2(TRPC6 inhibitor)or vehicle.The mice(n=6)were sacrificed at 14 d after UUO and obstructed kidneys were harvested and the concentration of Ca^(2+)in renal tubular epithelial cells(TECs)were assessed by immunofluorescence.Kidney sections were prepared with HE and Masson staining.The mRNA levels of proinflammatory cytokines interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)were examined by real-time PCR and the protein levels of TRPC6,collagen type I(COL1),total signal transducer and activator of transcription 3(STAT3)and p-STAT3 were determined by Western blot.(2)In vitro,human proximal tubular epithelial HK-2 cells were transfected with TRPC6 plasmid or TRPC6 siRNA,and then treated with TGF-β.The mRNA levels of IL-1β,IL-6 and TNF-αwere examined by real-time PCR,and the protein levels of TRPC6,COL1,STAT3 and p-STAT3 were determined by Western blot.RESULTS:(1)The concentration of Ca^(2+)was significantly increased in TECs of UUO group compared with sham group.The mice injected with BTP2 showed alleviated renal structural destruction,reduced levels of IL-1β,IL-6,TNF-α,COL1 and p-STAT3 as compared with UUO group.(2)Following stimulation with TGF-β,silencing of TRPC6 in cultured HK-2 cells decreased cytokine(IL-1β,IL-6 and TNF-α)production,and protein levels of COL1 and p-STAT3 as compared with the control cells.Over-expressed TRPC6 aggravated TGF-β-induced inflammatory response and COL1 expression.CONCLUSION:In vivo and in vitro,Ca^(2+)disorder mediated by TRPC6 promotes STAT3 phosphorylation,thus increasing the level of renal inflammation and renal interstitial fibrosis.