N-乙酰半胱氨酸对膜性肾病大鼠肾脏的保护作用
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摘要
目的探讨内质网应激和氧化应激是否参与膜性肾病(MN)发病以及N-乙酰半胱氨酸(NAC)是否通过相应的拮抗发挥对MN大鼠肾脏的保护作用。方法采用阳离子化牛血清白蛋白制造MN模型,随机分为4组:正常组(Control组)、模型组(MN组)、NAC 100 mg/kg治疗组(NAC1组),NAC 200mg/kg(NAC2组)。分别在正式免疫第1、2、3、4周末处死大鼠,检测各组大鼠24 h尿蛋白,血肌酐、尿素氮、白蛋白;免疫荧光、光镜、电镜观察大鼠肾组织病理变化,TUNEL法检测肾脏固有细胞凋亡,免疫组化法检测肾组织肾母细胞瘤蛋白-1(WT-1)、糖调节蛋白78(GRP78)、肿瘤坏死因子受体相关因子2(TRAF2)、半胱氨酸蛋白酶12(Caspase12)的蛋白表达,采用WST法检测血浆中的超氧化物歧化(SOD),TBA法检测血浆丙二醛(MDA)表达。结果与正常组比较,模型组24 h尿蛋白、血肌酐、血尿素氮明显升高(P <0.05),白蛋白明显降低(P <0.05),肾组织IgG和上皮下电子致密物沉积明显增多,基底膜明显增厚,肾脏固有细胞凋亡增加(P <0.05),肾组织中GRP78、TRAF2、Caspase12蛋白表达增加(P <0.05),WT-1蛋白表达减少(P <0.05),血浆MDA表达增加(P <0.05),血浆SOD表达减少(P <0.05);与模型组比较,NAC治疗后24 h尿蛋白、血肌酐、血尿素氮明显降低(P <0.05),白蛋白明显升高(P <0.05),IgG沉积及上皮下电子致密物沉积减少,肾脏固有细胞凋亡减少(P <0.05),肾组织中GRP78、TRAF2、Caspase12蛋白表达减少(P <0.05),WT-1蛋白表达增加(P <0.05),血浆MDA表达减少(P <0.05),血浆SOD表达增加(P <0.05),而NAC1组与NAC2组两组间差异无统计学意义(P <0.05)。结论在此大鼠MN模型中,有内质网应激和氧化应激的参与;NAC能降低全身氧化应激水平,进而可能减少免疫复合物在肾脏沉积导致的损害;同时减弱肾脏多种细胞内质网应激,减少细胞凋亡(特别是足细胞),从而起到降低尿蛋白,改善肾功能等肾脏保护作用。
Objective To investigate whether endoplasmic reticulum stress and oxidative stress areinvolved in membranous nephropathy(MN)and whether their antagonist N-acetylcysteine(NAC)protects thekidneys of rats with MN. Methods The MN model was established by using cationized bovine serum albumin andwas randomly divided into normal group,model group(MN group),NAC 100 mg/kg treatment group(NAC1 group)and NAC 200 mg/kg treatment group(NAC2 group). Rats were sacrificed at the end of 1 st,2 nd,3 rd,and4 th week,meanwhile,24-hour urinary protein,blood urea nitrogen,serum creatinine,and serum albumin weremeasured in each group. Pathological changes of kidney tissue were observed by immunofluorescence,light microscopy,and electron microscopy. TUNEL method was used to detect the apoptosis of renal cells and immunohistochemistry to detect glycomodulin 78(GRP78),tumor necrosis factor receptor-related factor 2(TRAF2),Caspase 12 and Wilms tumour 1(WT-1)in kidney. Superoxide dismutase(SOD)in plasma was detected by WSTmethod,and malondialdehyde(MDA)expression by TBA method. Results Compared with those in normal group,24-hour urinary protein,serum creatinine,blood urea nitrogen in MN group increased significantly(P < 0.05),but serum albumin decreased sharply(P < 0.05). Deposition of IgG and subepithelial electron dense deposits inkidney tissue significantly increased,basement membrane obviously thickened,the apoptosis of renal cells was lower,the protein expression of GRP78,TRAF2,Caspase12 increased in renal tissue(P < 0.05),the proteinexpression of WT-1 decreased(P < 0.05),the level of plasma MDA was higher(P<0.05). However,the level ofplasma SOD was lower(P < 0.05). Compared with those in MN group,24-hour urinary protein,serum creatinine,blood urea nitrogen in NAC treatment group decreased significantly(P < 0.05)but serum albumin decreased sharply(P < 0.05). Deposition of IgG and subepithelial electron dense deposits in kidney tissue significantly decreased,basement membrane obviously thined,the apoptosis of renal cells was higher,the protein expression of GRP78,TRAF2,Caspase12 reduced in renal tissue(P < 0.05),the protein expression of WT-1 increased(P < 0.05),thelevel of plasma MDA was lower(P < 0.05). However,the level of plasma SOD was higher(P < 0.05),whilethere was no difference between NAC1 group and NAC2 group. Conclusions In this model of MN of rat,there isinvolvement of endoplasmic reticulum stress and oxidative stress. NAC can reduce the level of systemic oxidativestress,which may reduce the kidney damage by inhibition of immune reaction it can decrease the endoplasmic reticulum stress in various cells of the kidney and reduce apoptosis(especially podocytes),thus to reduce urinaryprotein and protect kidney function.
Objective To investigate whether endoplasmic reticulum stress and oxidative stress areinvolved in membranous nephropathy(MN)and whether their antagonist N-acetylcysteine(NAC)protects thekidneys of rats with MN. Methods The MN model was established by using cationized bovine serum albumin andwas randomly divided into normal group,model group(MN group),NAC 100 mg/kg treatment group(NAC1 group)and NAC 200 mg/kg treatment group(NAC2 group). Rats were sacrificed at the end of 1 st,2 nd,3 rd,and4 th week,meanwhile,24-hour urinary protein,blood urea nitrogen,serum creatinine,and serum albumin weremeasured in each group. Pathological changes of kidney tissue were observed by immunofluorescence,light microscopy,and electron microscopy. TUNEL method was used to detect the apoptosis of renal cells and immunohistochemistry to detect glycomodulin 78(GRP78),tumor necrosis factor receptor-related factor 2(TRAF2),Caspase 12 and Wilms tumour 1(WT-1)in kidney. Superoxide dismutase(SOD)in plasma was detected by WSTmethod,and malondialdehyde(MDA)expression by TBA method. Results Compared with those in normal group,24-hour urinary protein,serum creatinine,blood urea nitrogen in MN group increased significantly(P < 0.05),but serum albumin decreased sharply(P < 0.05). Deposition of IgG and subepithelial electron dense deposits inkidney tissue significantly increased,basement membrane obviously thickened,the apoptosis of renal cells was lower,the protein expression of GRP78,TRAF2,Caspase12 increased in renal tissue(P < 0.05),the proteinexpression of WT-1 decreased(P < 0.05),the level of plasma MDA was higher(P<0.05). However,the level ofplasma SOD was lower(P < 0.05). Compared with those in MN group,24-hour urinary protein,serum creatinine,blood urea nitrogen in NAC treatment group decreased significantly(P < 0.05)but serum albumin decreased sharply(P < 0.05). Deposition of IgG and subepithelial electron dense deposits in kidney tissue significantly decreased,basement membrane obviously thined,the apoptosis of renal cells was higher,the protein expression of GRP78,TRAF2,Caspase12 reduced in renal tissue(P < 0.05),the protein expression of WT-1 increased(P < 0.05),thelevel of plasma MDA was lower(P < 0.05). However,the level of plasma SOD was higher(P < 0.05),whilethere was no difference between NAC1 group and NAC2 group. Conclusions In this model of MN of rat,there isinvolvement of endoplasmic reticulum stress and oxidative stress. NAC can reduce the level of systemic oxidativestress,which may reduce the kidney damage by inhibition of immune reaction it can decrease the endoplasmic reticulum stress in various cells of the kidney and reduce apoptosis(especially podocytes),thus to reduce urinaryprotein and protect kidney function.
引文
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[13]WANG Z,LI Y F,HAN X Y.Kidney protection effect of ginsenoside re and its underlying mechanisms on Cisplatin-induced kidney injury[J].Cell Physiol Biochem,2018,48(5):2219-2229.
[14]BURLAKA I,NILSSON L M,SCOTT L,et al.Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease[J].Kidney Int,2016,90(1):135-148.
[15]梁劲松,宋文林,吴艳.α-酮酸联合低蛋白饮食治疗糖尿病肾病对机体氧化应激及电解质水平的影响[J].实用医学杂志,2017,33(16):2717-2720.
[16]KRATA N,ZAGOZDZON R,FORONCEWICZ B.Oxidative stress in kidney diseases:the cause or the consequence?[J].Arch Immunol Ther Exp(Warsz),2018,66(3):211-220.
[17]DAEHN I,CASALENA G,ZHANG T,et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis[J].The Journal of clinical investigation,2014,124(4):1608-1621.
[18]ZHANG Q,LIU J,CHEN S,et al.Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress[J].Apoptosis,2016,21(4):432-442.
[19]TONG Q,WU L,GAO Q,et al.pparβ/δagonist provides neuroprotection by suppression of ire1α-caspase-12-mediated endoplasmic reticulum stress pathway in the rotenone rat model of Parkinson′s disease[J].Mol Neurobiol,2016,53(6):3822-3831.
[20]吴明松,罗素元,郑翔,等.内质网应激分子GRP78在不同时程吗啡依赖大鼠睾丸中的表达[J].实用医学杂志,2016,32(13):2108-2112.
[21]HANNA D,FLORENCE L,MARKUS J,et al.Early-childhood membranous nephropathy due to cationic bovine serum albumin[J].N Engl J Med,2011,364(22):2101-2110.
[2]XU X,WANG B A,CHEN N,et al.Long-term exposure to air pollution and increased risk of membranous nephropathy in China[J].J Am Soc Nephrol,2016,27(12):3739-3746.
[3]CATTRAN D C,BRENCHLEY P E.Membranous nephropathy:integrating basic science into improved clinical management[J].Kidney Int,2017,91(3):566-574.
[4]CYBULSKY A V.The intersecting roles of endoplasmic reticulum stress,ubiquitin-proteasome system,and autophagy in the pathogenesis of proteinuric kidney disease[J].Kidney Int,2013,84(1):25-33.
[5]SHI H,ZOU J,ZHANG T,et al.Protective effects of DHA-PCagainst vancomycin-induced nephrotoxicity through the inhibition of oxidative stress and apoptosis in BALB/c mice[J].Agric Food Chem,2018,66(2):475-484.
[6]YUANY,XU X,ZHAO C,et al.The roles of oxidative stress,endoplasmic reticulum stress,and autophagy inaldosterone/mineralocorticoid receptor-induced podocyte injury[J].Lab Invest,2015,95(12):1374-1386.
[7]PONTICELLI C,PASSERINI P.Management of idiopathic membranous nephropathy[J].Expert Opin Pharmacother,2010,11(13):2163-2175.
[8]Improving Global Outcomes(KDIGO)Glomerulonephritis Work Group.KDIGO clinical practice guidelinne for glomerulonephritis[J].Kidney Inter,2012,23(2):139-274.
[9]XIA Q,LIU C,ZHENG X.N-acetylcysteine ameliorates contrastinduced kidney injury in rats with unilateral hydronephrosis[J].Mol Med Rep,2018,17(2):2203-2210.
[10]XU C,DING W,YANG L,et al.Contributions of endoplasmic reticulum stress and reactive oxygen species to renal injury in aldosterone/salt-induced rats[J].Nephron Exp Nephrol,2014,126(1):25-32.
[11]NOGUEIRA G B,PUNARO G R,OLIVEIRA C S,et al.N-acetylcysteine protects against diabetic nephropathy through control of oxidative and nitrosative stress by recovery of nitric oxide in rats[J].Nitric Oxide,2018,18(78):22-31.
[12]彭素英,王玉洁,何宇,等.阳离子化牛血清白蛋白膜性肾病大鼠模型的建立与鉴定[J].山东医药,2016,56(48):36-38.
[13]WANG Z,LI Y F,HAN X Y.Kidney protection effect of ginsenoside re and its underlying mechanisms on Cisplatin-induced kidney injury[J].Cell Physiol Biochem,2018,48(5):2219-2229.
[14]BURLAKA I,NILSSON L M,SCOTT L,et al.Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease[J].Kidney Int,2016,90(1):135-148.
[15]梁劲松,宋文林,吴艳.α-酮酸联合低蛋白饮食治疗糖尿病肾病对机体氧化应激及电解质水平的影响[J].实用医学杂志,2017,33(16):2717-2720.
[16]KRATA N,ZAGOZDZON R,FORONCEWICZ B.Oxidative stress in kidney diseases:the cause or the consequence?[J].Arch Immunol Ther Exp(Warsz),2018,66(3):211-220.
[17]DAEHN I,CASALENA G,ZHANG T,et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis[J].The Journal of clinical investigation,2014,124(4):1608-1621.
[18]ZHANG Q,LIU J,CHEN S,et al.Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress[J].Apoptosis,2016,21(4):432-442.
[19]TONG Q,WU L,GAO Q,et al.pparβ/δagonist provides neuroprotection by suppression of ire1α-caspase-12-mediated endoplasmic reticulum stress pathway in the rotenone rat model of Parkinson′s disease[J].Mol Neurobiol,2016,53(6):3822-3831.
[20]吴明松,罗素元,郑翔,等.内质网应激分子GRP78在不同时程吗啡依赖大鼠睾丸中的表达[J].实用医学杂志,2016,32(13):2108-2112.
[21]HANNA D,FLORENCE L,MARKUS J,et al.Early-childhood membranous nephropathy due to cationic bovine serum albumin[J].N Engl J Med,2011,364(22):2101-2110.