拟钙剂R568通过激活PLC信号通路减轻自发性高血压大鼠肾损伤

Calcimimetic R568 attenuates kidney injury in spontaneously hypertensive rats by activating PLC signaling pathway

目的:探讨钙敏感受体(calcium-sensing receptor,CaSR)激动剂R568对自发性高血压大鼠(spontaneously hypertensive rats,SHR)肾脏病变的影响。方法:以16周龄雄性SHR和Wistar Kyoto(WKY)大鼠(血压正常)为研究对象,实验分为WKY组、SHR+生理盐水(normal saline,NS)组及SHR+R568组。干预8周后(24周龄),使用无创血压仪器检测各组大鼠血压,接着处死大鼠,收集各组大鼠肾脏。采用苏木精-伊红、过碘酸-雪夫及Masson染色观察各组大鼠肾脏形态学改变;采用免疫组化检测各组大鼠肾脏组织中中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NAGL)、CD68和磷脂酶C(phospholipase C,PLC)的表达;采用免疫荧光染色检测各组大鼠肾脏组织中CaSR表达及巨噬细胞的极化类型;采用Western blotting和qRT-PCR检测CaSR、NAGL、Ⅲ型胶原蛋白、核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)、caspase-1、白细胞介素1β(interleukin-1β,IL-1β)及IL-18的表达。结果:与WKY组相比,SHR+NS组大鼠血压显著升高,肾组织中CaSR和PLC表达及M2型巨噬细胞数量显著减少,而CD68、NAGL和NLRP3炎症小体相关蛋白表达及M1型巨噬细胞数量显著增多(P<0.05);与SHR+NS组相比,R568干预8周后大鼠血压显著降低,大鼠肾组织中CaSR和PLC表达及M2型巨噬细胞数量显著增多,而CD68、NAGL和NLRP3炎症小体相关蛋白表达及M1型巨噬细胞数量显著减少(P<0.05)。结论:CaSR激活可通过激活PLC信号通路抑制NLRP3炎症小体活化,减少巨噬细胞向M1极化,促进其向M2极化,以降低血压并减轻高血压肾损伤。

AIM:To reveal the effect of calcium-sensing receptor(CaSR)agonist R568 on renal injury in spontaneously hypertensive rats(SHR).METHODS:Sixteen-week-old male SHR and Wistar Kyoto(WKY)rats were divided into WKY group,SHR+normal saline(NS)group,and SHR+R568 group.After 8 weeks of intervention(24weeks of age),blood pressure of the rats was measured using a non-invasive blood pressure instrument,and then the rats were executed and their kidneys were collected.Hematoxylin-eosin staining,periodic acid-Schiff staining and Masson staining were used to observe the morphological changes of the kidneys.The expression of neutrophil gelatinase-associated lipocalin(NAGL),CD68 and phospholipase C(PLC)in renal tissues were detected by immunohistochemistry.The expression of CaSR and the polarization type of macrophages in renal tissues were detected by immunofluorescence staining.Western blotting and qRT-PCR were used to detect the expression of CaSR,NAGL,collagen typeⅢ,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1,interleukin-1β(IL-1β)and IL-18.RESULTS:Compared with WKY group,rat blood pressure in SHR+NS group was significantly increased,the CaSR and PLC expression and the number of M2 macrophages in renal tissues were significantly decreased,and CD68,NAGL and NLRP3 inflammasome-associated protein expression and M1 macrophages number in renal tissues were significantly increased(P<0.05).Compared with SHR+NS group,rat blood pressure was significantly decreased,the CaSR and PLC expression and the number of M2 macrophages in renal tissues were significantly increased,and CD68,NAGL and NLRP3 inflammasomeassociated protein expression and M1 macrophages number in renal tissues were significantly decreased after 8 weeks of R568 intervention(P<0.05).CONCLUSION:Activation of CaSR inhibits NLRP3 inflammasome activation and reduces M1 macrophage polarization via activating PLC signaling pathway in SHR,thus decreasing blood pressure and attenuating hypertensive kidney injury.