磷酸二酯酶5在肾脏纤维化中的保护作用

Phosphodiesterase 5 Inhibitors Attenuate Unilateral Ureteral Obstruction-Induced Renal Fibrosis

【目的】探究磷酸二酯酶5(PDE5)抑制剂西地那非(SIL)或LW1646对单侧输尿管结扎(UUO)诱导的肾间质纤维化的保护作用。【方法】C57BL/6雄性小鼠被随机分为假手术Sham(n=6)、7UUO(n=6)、7UUO+SIL(n=6)或7UUO+LW1646(n=6)组,术前1h给予SIL或LW1646、或溶剂灌胃给药,连续给药7 d后处死小鼠,收集梗阻侧肾脏标本。对肾组织进行H&E和Masson’s染色,使用免疫印迹和RT-qPCR检测肾脏中的纤维化、内质网应激、自噬、凋亡相关蛋白表达水平以及纤维化相关基因表达水平。使用人近端小管上皮细胞(HK-2)给予TGF-β148 h,检测cGMP对内质网应激和纤维化相关蛋白水平的影响。使用衣霉素孵育HK-2细胞24 h,检测PDE5抑制剂对内质网应激、纤维化相关蛋白、自噬和凋亡蛋白水平的影响。【结果】在行UUO术后7 d小鼠体质量相对于假手术组显著下降(P<0.0001),梗阻侧肾脏可见明显肾小管扩张、肾间质炎症细胞浸润,TGF-β1蛋白和mRNA水平、内质网应激、自噬及凋亡相关蛋白表达水平显著升高(P<0.05)。而给予SIL或LW1646后小鼠梗阻侧肾脏管腔扩张、炎症细胞浸润被缓解。肾脏TGF-β1的蛋白及mRNA表达水平被不同程度下调,PDE5抑制剂显著下调内质网应激、自噬和凋亡水平。在HK2细胞中,TGF-β1诱导的Fibronectin蛋白以及BiP蛋白表达水平显著升高,在cGMP的作用下可被部分逆转;衣霉素诱导的Fibronectin、BiP蛋白水平上调可被SIL或LW1646逆转,同时自噬和凋亡的异常水平也被PDE5抑制剂改变。【结论】PDE5抑制剂可以通过改善肾脏组织细胞内质网应激、自噬水平,以及抗凋亡等途径和缓解纤维化进展,同剂量的LW1646在对缓解内质网应激降低细胞外基质蛋白的表达水平的效果优于SIL。

【Objective】To investigate whether phosphodiesterase(PDE)5 inhibitors sildenafil(SIL)or LW1646 pre⁃vented renal interstitial fibrosis induced by unilateral ureteral obstruction(UUO).【Methods】Male C57BL/6 mice were ran⁃domly divided into four groups(n=6),namely the Sham group,7UUO group,7UUO+SIL group and 7UUO+LW1646 group.Sildenafil(SIL)or LW1646,or vehicle was administered 1 hour before surgery,and the mice were continuously treated once daily(i.g.,50 mg/kg)for 7 days.The obstructed kidneys were harvested on day 7.Hematoxylin-eosin(HE)and Masson’s staining was used to examine renal histology.Immunoblotting and RT-qPCR were used to detect the expres⁃sion levels of protein and mRNA for fibrosis,apoptosis,endoplasmic reticulum(ER)stress,autophagy,and pro-fibrotic factors.Human proximal tubule epithelial cells(HK-2)were treated with TGF-β1 for 48 hours or tunicamycin for 24 hours,respectively,to evaluate whether cyclic guanosine monophosphate(cGMP)or PDE 5 inhibitors prevents ER stress and pro-fibrotic responses.【Results】At the 7 th days after UUO,the body weight of the mice showed a significant decrease(P<0.0001)compared with that in the sham group.The obstructed kidneys showed a significant tubular dilation and inter⁃stitial inflammation.The levels of protein and mRNA expression in apoptosis,ER stress,autophagy-related protein and pro-fibrotic factors were also markedly increased in UUO mice(P<0.05).In contrast,SIL or LW 1646 treatment was asso⁃ciated with attenuated tubular dilation,infiltration of inflammatory cells and collagen content in the obstructed kidney of the mice.The protein and mRNA expression levels of renal TGF-β1 were markedly decreased,and the protein expression levels of apoptosis,endoplasmic reticulum stress,and autophagy markers were also significantly downregulated by PDE 5 inhibitors.In HK-2 cells,TGF-β1 induced increased expression levels of fibronectin and BiP,which was at least partial⁃ly reversed by cGMP,a product of PDE inhibition.Additionally,PDE 5 inhibitors were found to modulate aberrant levels of autophagy and apoptosis.【Conclusion】In conclusion,PDE 5 inhibitors,in particular,LW 1646,can alleviate the progres⁃sion of fibrosis by improving ER stress,apoptosis and autophagy as well as downregulating protein and mRNA expression of TGF-β1.