蛋白激酶D在急性肾损伤中的表达及作用研究

Expression and role of protein kinase D in acute kidney injury

目的探讨蛋白激酶D(PKD)在急性肾损伤(AKI)小鼠肾脏中的表达和影响。方法取27只C57/B6J小鼠随机分为3组:对照组、顺铂(Cisplatin)组和Cisplatin+CID755673(CID)组,腹腔注射顺铂诱导AKI模型,造模后72 h处死小鼠收集肾组织及血清。另取23只C57/B6J小鼠随机分为3组:假手术(Sham)组、双侧肾缺血再灌注(BIR)组和BIR+CID组,夹闭双侧肾蒂30 min诱导AKI模型,造模后24 h处死小鼠收集肾组织及血清。免疫组化染色检测肾组织PKD表达。测定小鼠血清肌酐和尿素氮(BUN)浓度,取肾脏组织行PAS染色,评估肾脏功能。实时定量PCR检测PKD、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤分子-1(KIM-1)、凋亡相关基因(Bax)、凋亡效应蛋白(caspase-3)、白细胞介素(IL)-1β、IL-6、单核细胞趋化蛋白-1(MCP-1)mRNA表达水平。免疫印迹法检测PKD、NGAL、KIM-1、Bax及活化的caspase3蛋白表达。结果顺铂处理及缺血/再灌注诱导后,AKI小鼠肾组织中PKD的表达较对照组显著增加(P<0.05)。抑制PKD显著加重顺铂和缺血/再灌注诱导的AKI小鼠的肾功能障碍和肾小管上皮细胞损伤,加重肾小管上皮细胞凋亡和炎症反应。结论PKD在AKI小鼠肾脏中显著上调,抑制PKD可显著加重肾损伤,为防治AKI提供了新的治疗靶点。

Objective To investigate the expression of protein kinase D(PKD)in the kidneys of mice with acute kidney injury(AKI)and its effect.Methods A total of 27 C57/B6J mice were divided into three groups:a Control group,a Cisplatin group and a Cisplatin+CID group.Then,an AKI model was induced by intraperitoneal injection of cisplatin in mice.The mice were sacrificed 72 h after modeling to collect renal tissue and serum.Meanwhile,another 23 C57/B6J mice were divided into three groups:a Sham group,a bilateral renal ischemia-reperfusion injury(BIR)group and a BIR+CID group.Then,an AKI model was established through clipping the bilateral renal roots of mice for 30 min,and renal tissues and serum were collected 24 h after modeling.The expression of PKD in the kidneys was detected by immunohistochemical staining.Renal function was evaluated through measurement of serum creatinine and blood urea nitrogen(BUN),as well as PAS staining.The mRNA expression of PKD,neutrophil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),apoptotic markers(Bax),interleukin(IL)-1β,IL-6 and monocyte chemoattractant protein-1(MCP1)was detected by real-time qPCR.The protein expression of PKD,NGAL,KIM-1,Bax and cleaved caspase3 was detected by Western blot.Results After cisplatin treatment or ischemia-reperfusion injury,the expression of PKD in the kidneys of AKI mice was remarkably elevated(P<0.05).Inhibition of PKD significantly aggregated the renal dysfunction and renal tubular epithelial damage in AKI mice after cisplatin treatment or ischemia-reperfusion injury,and aggregated the apoptosis of tubular epithelial cells and inflammation.Conclusions PKD is significantly up-regulated in mice with AKI,and inhibition of PKD significantly aggravats renal injury,which provides a new target for the prevention and treatment of AKI.