非对称性二甲基精氨酸通过激活铁死亡促进小鼠早期肾损伤

Asymmetric dimethylarginine induces early kidney injury in mice through ferropotosis

目的:探讨内源性一氧化氮合酶(nitric oxide synthase,NOS)抑制物非对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)是否通过诱导细胞铁死亡促进小鼠早期肾损伤。方法:将16只8周龄SPF级C57BL/6小鼠随机分为对照组(n=8)和实验组(n=8),实验组小鼠正常饮食,每天灌胃给予小鼠ADMA(60 mg·kg^(-1)·d^(-1)),对照组小鼠正常饮食,每天灌胃给予等体积的生理盐水,实验周期为16周。采用口服葡萄糖耐量和胰岛素耐受实验检测血糖代谢;总胆固醇(total cholesterol,TC)和甘油三酯(triglyceride,TG)测定试剂盒(COD-PAP法和GPO-PAP法)以及高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)检测试剂盒(微板法)检测血清TC、TG和HDL-C水平;HE染色法观察肾组织的形态变化;ELISA法检测血清中ADMA及肾组织炎症因子白细胞介素6(interleukin-6,IL-6)和IL-1β含量;比色法检测肾组织的铁含量、脂质过氧化产物丙二醛(malonaldehyde,MDA)含量、血尿素氮(blood urea nitrogen,BUN)和一氧化氮(nitric oxide,NO)含量;通过WST-8法测定肾组织中超氧化物歧化酶(superoxide dismutase,SOD)活性;Western blot法检测肾组织中内皮型NOS(endothelial NOS,eNOS)、诱导型NOS(inducible NOS,iNOS)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、Bcl2、Bax和caspase-1的蛋白表达。结果:与正常对照组相比,ADMA处理组小鼠出现糖耐受和胰岛素耐受,血清TG和TC水平升高(P<0.01),HDL-C水平降低(P<0.05),即糖脂代谢异常。同时,ADMA直接诱导小鼠发生类似于慢性肾病(CKD)的早期肾损伤,如肾小球肥大、系膜细胞肥大和BUN增多(P<0.01)、促炎因子IL-6和IL-1β含量显著增多(P<0.01)等。此外,ADMA处理组小鼠肾组织的ADMA含量增多(P<0.01),eNOS蛋白表达和总NO含量降低,但iNOS蛋白表达水平则显著升高(P<0.01);同时还观察到肾脏组织铁超载,脂质过氧化产物MDA增多,抗氧化酶SOD活性和GPX4蛋白表达水平降低(P<0.01),Bcl2蛋白表达水平降低,Bax和caspase-1蛋白表达水平升高。结论:ADMA可能通过激活铁死亡促进小鼠早期肾损伤。

AIM:To investigate whether asymmetric dimethylarginine(ADMA),an endogenous nitric oxide synthase(NOS)inhibitor,induces early kidney injury in mice through ferroptosis.METHODS:Sixteen 8-week-old SPF C57BL/6 male mice were randomly assigned to control group(n=8)and experimental group(n=8).The experimental mice were orally administered with ADMA(60 mg·kg-1·d-1)daily for 16 weeks,while the control mice were orally admin‐istered with an equal volume of saline daily.After the treatment,glucose tolerance and insulin tolerance were measured in both groups.Serum samples were collected to measure ADMA levels,total cholesterol(TC),triglycerides(TG),and high-density lipoprotein cholesterol(HDL-C)levels.Blood urea nitrogen(BUN)levels were also determined.Kidney tis‐sue samples were collected to assess pathological morphology,interleukin-6(IL-6)and IL-1βlevels,iron content,nitric oxide(NO)levels,malondialdehyde(MDA)levels,superoxide dismutase(SOD)activity,and the protein expression of endothelial NOS(eNOS),inducible NOS(iNOS),glutathione peroxidase 4(GPX4),Bcl2,Bax and caspase-1.RE-SULTS:Compared with the control group,the ADMA-treated mice showed impaired glucose tolerance and insulin sensi‐tivity,indicating abnormal glucose metabolism.Serum TG and TC levels were elevated(P<0.01),while HDL-C level was decreased(P<0.05),indicating abnormal lipid metabolism.Additionally,ADMA directly induced early renal injury in mice resembling chronic kidney failure,characterized by glomerular hypertrophy,glomerular mesangial cell hypertro‐phy and increased BUN levels(P<0.01),as well as significantly elevated levels of pro-inflammatory cytokines IL-6 and IL-1β(P<0.01).Moreover,the ADMA-treated mice exhibited increased ADMA levels in kidney tissue(P<0.01),re‐duced expression of eNOS protein and total NO levels,but significantly increased expression of iNOS protein(P<0.01).Furthermore,kidney tissue analysis revealed iron overload,increased MDA levels,decreased SOD activity,decreased ex‐pression of GPX4 protein and anti-apoptotic protein Bcl2(P<0.01),and increased expression of apoptosis markers Bax and caspase-1 proteins(P<0.05 or P<0.01).CONCLUSION:ADMA may induce early kidney injury in mice through ferropotosis.