他克莫司对阿霉素肾病大鼠肾组织氧化应激及klotho蛋白表达的影响
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摘要
目的观察他克莫司对阿霉素肾病大鼠肾组织氧化应激及klotho蛋白表达的影响。方法将雄性Wistar大鼠随机分为对照组(NC组)、阿霉素肾病模型组(ADR组)、奥美沙坦酯治疗组(OLM组)、他克莫司治疗组(FK506组),每组15只。其中ADR组、OLM组、FK506组均给予一次性尾静脉注射阿霉素5 mg/kg构建阿霉素肾病模型。造模成功后,OLM组、FK506组分别连续12周给予奥美沙坦酯10 mg/(kg·d)、他克莫司0.5 mg/(kg·d)灌胃,NC组及ADR组分别给予等量生理盐水灌胃。分别于实验第4、8、12周末检测各组大鼠24 h尿蛋白;每组分别于实验第4、8、12周末各处死5只大鼠,取肾组织,测定其超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,苏木精-伊红染色观察其形态学变化,实时荧光定量PCR(q-PCR)检测klotho mRNA的表达,Wes-tern blot检测klotho蛋白的表达。结果 ADR组、OLM组及FK506组大鼠各时间点24 h尿蛋白含量均高于NC组,OLM组和FK506组较ADR组降低,FK506组较OLM组降低,差异均有显著性(F=126.969～781.905,P<0.05)。ADR组、OLM组及FK506组大鼠各时间点肾组织中SOD活性均较NC组降低,OLM组和FK506组较ADR组升高,FK506组较OLM组升高,差异均有统计学意义(F=125.782～467.589,P<0.05)。ADR组、OLM组及FK506组大鼠各时间点肾组织中MDA含量均较NC组升高,OLM组及FK506组较ADR组降低,FK506组较OLM组降低,差异均有统计学意义(F=52.782～162.589,P<0.05)。ADR组、OLM组及FK506组大鼠各时间点肾组织中klotho的表达均较NC组降低,OLM组及FK506组较ADR组升高,FK506组较OLM组升高,差异具有统计学意义(F=116.671～609.513,P<0.05)。结论阿霉素肾病大鼠肾脏组织klotho表达降低,他克莫司相对于奥美沙坦酯可以明显提高阿霉素肾病大鼠肾脏组织中klotho的表达,减少肾脏氧化应激损伤。
Objective To investigate the effect of tacrolimus on oxidative stress and klotho protein expression in renal tissue in rats with adriamycin nephropathy. Methods A total of 60 male Wistar rats were randomly divided into normal control group(NC group), adriamycin nephropathy group(ADR group), olmesartan medoxomil treatment group(OLM group), and tacrolimus treatment group(FK506 group), with 15 rats in each group. The rats in the ADR group, the OLM group, and the FK506 group were given one-time injection of 5 mg/kg adriamycin via the caudal vein to establish a model of adriamycin nephropathy. After modeling, the rats in the OLM group and the FK506 group were given olmesartan medoxomil(10 mg·kg~(-1)·d~(-1)) and tacrolimus(0.5 mg/kg~(-1)·d~(-1)), respectively, by gavage for 12 consecutive weeks, and those in the NC group and the ADR group were given an equal volume of normal saline by gavage. 24 h urinary protein was measured at the end of weeks 4, 8, and 12. Five rats in each group were sacrificed at the end of weeks 4, 8, and 12, and renal tissue was collected to measure superoxide dismutase(SOD) activity and malondialdehyde(MDA) content; hematoxylin and eosin staining was used to observe morphological changes, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of klotho. Results At all time points, the ADR group, the OLM group, and the FK506 group had significantly higher 24 h urinary protein than the NC group; the OLM group and the FK506 group had significantly lower 24 h urinary protein than the ADR group; the FK506 group had significantly lower 24 h urinary protein than the OLM group(F=126.969-781.905,P<0.05). At all time points, the ADR group, the OLM group, and the FK506 group had significantly lo-wer SOD activity in renal tissue than the NC group; the OLM group and the FK506 group had significantly higher SOD activity than the ADR group;the FK506 group had significantly higher SOD activity than the OLM group(F=125.782-467.589,P<0.05).At all time points,the ADR group,the OLM group,and the FK506 group had significantly higher MDA content in renal tissue than the NC group;the OLM group and the FK506 group had significantly lower MDA content than the ADR group;the FK506 group had significantly lower MDA content than the OLM group(F=52.782-162.589,P<0.05).At all time points,the ADR group,the OLM group,and the FK506 group had significantly lower expression of klotho in renal tissue than the NC group;the OLM group and the FK506 group had significantly higher expression than the ADR group;the FK506 group had significantly higher expression than the OLM group(F=116.671-609.513,P<0.05). Conclusion There is a reduction in the expression of klotho in renal tissue in rats with adriamycin nephropathy.Compared with olmesartan medoxomil,tacrolimus can significantly increase the expression of klotho in renal tissue and reduce oxidative stress injury in the kidney in rats with adriamycin nephropathy.
Objective To investigate the effect of tacrolimus on oxidative stress and klotho protein expression in renal tissue in rats with adriamycin nephropathy. Methods A total of 60 male Wistar rats were randomly divided into normal control group(NC group), adriamycin nephropathy group(ADR group), olmesartan medoxomil treatment group(OLM group), and tacrolimus treatment group(FK506 group), with 15 rats in each group. The rats in the ADR group, the OLM group, and the FK506 group were given one-time injection of 5 mg/kg adriamycin via the caudal vein to establish a model of adriamycin nephropathy. After modeling, the rats in the OLM group and the FK506 group were given olmesartan medoxomil(10 mg·kg~(-1)·d~(-1)) and tacrolimus(0.5 mg/kg~(-1)·d~(-1)), respectively, by gavage for 12 consecutive weeks, and those in the NC group and the ADR group were given an equal volume of normal saline by gavage. 24 h urinary protein was measured at the end of weeks 4, 8, and 12. Five rats in each group were sacrificed at the end of weeks 4, 8, and 12, and renal tissue was collected to measure superoxide dismutase(SOD) activity and malondialdehyde(MDA) content; hematoxylin and eosin staining was used to observe morphological changes, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of klotho. Results At all time points, the ADR group, the OLM group, and the FK506 group had significantly higher 24 h urinary protein than the NC group; the OLM group and the FK506 group had significantly lower 24 h urinary protein than the ADR group; the FK506 group had significantly lower 24 h urinary protein than the OLM group(F=126.969-781.905,P<0.05). At all time points, the ADR group, the OLM group, and the FK506 group had significantly lo-wer SOD activity in renal tissue than the NC group; the OLM group and the FK506 group had significantly higher SOD activity than the ADR group;the FK506 group had significantly higher SOD activity than the OLM group(F=125.782-467.589,P<0.05).At all time points,the ADR group,the OLM group,and the FK506 group had significantly higher MDA content in renal tissue than the NC group;the OLM group and the FK506 group had significantly lower MDA content than the ADR group;the FK506 group had significantly lower MDA content than the OLM group(F=52.782-162.589,P<0.05).At all time points,the ADR group,the OLM group,and the FK506 group had significantly lower expression of klotho in renal tissue than the NC group;the OLM group and the FK506 group had significantly higher expression than the ADR group;the FK506 group had significantly higher expression than the OLM group(F=116.671-609.513,P<0.05). Conclusion There is a reduction in the expression of klotho in renal tissue in rats with adriamycin nephropathy.Compared with olmesartan medoxomil,tacrolimus can significantly increase the expression of klotho in renal tissue and reduce oxidative stress injury in the kidney in rats with adriamycin nephropathy.
引文
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[6] 张勇,张蓓,宁华英,等.单次尾静脉注射法阿霉素大鼠肾病模型的建立[J].中国实验动物学报,2013,21(1):1-4,107.
[7] QI X M,WANG J,XU X X,et al.FK506 reduces albuminuria through improving podocyte nephrin and podocin expression in diabetic rats[J].Inflammation Research,2016,65(2):103-114.
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[11] LIM S C,LIU J J,SUBRAMANIAM T,et al.Elevated circulating alpha-klotho by angiotensin Ⅱ receptor blocker losartan is associated with reduction of albuminuria in type 2 diabetic patients[J].Journal of the Renin-Angiotensin-Aldosterone System,2014,15(4):487-490.
[12] JIN J,JIN L,LIM S W,et al.Klotho deficiency aggravates tacrolimus-induced renal injury via the phosphatidylinositol 3-kinase-Akt-forkhead box protein O pathway[J].American Journal of Nephrology,2016,43(5):357-365.
[13] YAMAMOTO M,CLARK J D,PASTOR J V,et al.Regulation of oxidative stress by the anti-aging hormone Klotho[J].Journal of Biological Chemistry,2005,280(45):38029-38034.
[14] 贺明,陆利民,姚泰.肾素-血管紧张素系统在调节肾脏活动和慢性肾功能不全中的作用[J].生理科学进展,2004,35(2):188-192.
[15] 武晓碧,汪晓霞,张希尧,等.缬沙坦对糖尿病大鼠klotho蛋白及氧化应激影响的研究[J].中国糖尿病杂志,2017,25(8):733-736.
[16] SHEN Xiujin,JIANG Hong,YING Meike,et al.Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models[J].Scientific Reports,2016,6(6):32087-32116.
[17] HAUGEN E N,CROATT A J,NATH K A.Angiotensin Ⅱ induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro[J].Kidney International,2000,58(1):144-152.
[18] 常洁,姜宗培,张海燕,等.NADPH氧化酶介导血管紧张素Ⅱ诱导的腹膜间皮细胞转分化及细胞外基质积聚[J].中华肾脏病杂志,2007,23(5):328-333.
[19] YOON H E,GHEE J Y,PIAO S,et al.Angiotensin Ⅱ bloc-kade upregulates the expression of Klotho,the anti-ageing gene,in an experimental model of chronic cyclosporine nephropathy[J].Nephrology Dialysis Transplantation,2011,26(3):800-813.
[20] DEVARAJ S,SYED B,CHIEN A,et al.Validation of an immunoassay for soluble klotho protein decreased levels in diabetes and increased levels in chronic kidney disease[J].American Journal of Clinical Pathology,2012,137(3):479-485.
[21] SENGUPTA A,MOLKENTIN J D,PAIK J H,et al.FoxO transcription factors promote cardiomyocyte survival upon induction of oxidative stress[J].The Journal of Biological Che-mistry,2011,286(9):7468-7478.
[22] GREER E L,BRUNET A.FOXO transcription factors at the interface between longevity and tumor suppression[J].Oncogene,2005,24(50):7410-7425.
[2] KURO-O M,MATSUMURA Y,AIZAWA H,et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing[J].Nature,1997,390(6655):45-51.
[3] MITANI H,ISHIZAKA N,AIZAWA T,et al.In vivo klotho gene transfer ameliorates angiotensin Ⅱ-induced renal damage[J].Hypertension,2002,39(4):838-843.
[4] 徐辰.KLOTHO基因介导钙磷代谢与慢性肾脏病关系的研究进展[J].国际泌尿系统杂志,2012,32(5):692-695.
[5] 卢晓梅,马玲,于艳秋.奥美沙坦酯对慢性心力衰竭小鼠肾脏氧化应激的作用[J].中华肾脏病杂志,2011,27(3):190-193.
[6] 张勇,张蓓,宁华英,等.单次尾静脉注射法阿霉素大鼠肾病模型的建立[J].中国实验动物学报,2013,21(1):1-4,107.
[7] QI X M,WANG J,XU X X,et al.FK506 reduces albuminuria through improving podocyte nephrin and podocin expression in diabetic rats[J].Inflammation Research,2016,65(2):103-114.
[8] PAVENSTADT H,KRIZ W,KRETZLER M.Cell biology of the glomerular podocyte[J].Physiological Reviews,2003,83(1):253-307.
[9] VALKO M,RHODES C J,MONCOL J,et al.Free radicals,metals and antioxidants in oxidative stress-induced cancer[J].Chemico-Biological Interactions,2006,160(1):1-40.
[10] XIAO Nengming,ZHANG Yanming,ZHENG Quan,et al.Klotho is a serum factor related to human aging[J].Chinese Medical Journal,2004,117(5):742-747.
[11] LIM S C,LIU J J,SUBRAMANIAM T,et al.Elevated circulating alpha-klotho by angiotensin Ⅱ receptor blocker losartan is associated with reduction of albuminuria in type 2 diabetic patients[J].Journal of the Renin-Angiotensin-Aldosterone System,2014,15(4):487-490.
[12] JIN J,JIN L,LIM S W,et al.Klotho deficiency aggravates tacrolimus-induced renal injury via the phosphatidylinositol 3-kinase-Akt-forkhead box protein O pathway[J].American Journal of Nephrology,2016,43(5):357-365.
[13] YAMAMOTO M,CLARK J D,PASTOR J V,et al.Regulation of oxidative stress by the anti-aging hormone Klotho[J].Journal of Biological Chemistry,2005,280(45):38029-38034.
[14] 贺明,陆利民,姚泰.肾素-血管紧张素系统在调节肾脏活动和慢性肾功能不全中的作用[J].生理科学进展,2004,35(2):188-192.
[15] 武晓碧,汪晓霞,张希尧,等.缬沙坦对糖尿病大鼠klotho蛋白及氧化应激影响的研究[J].中国糖尿病杂志,2017,25(8):733-736.
[16] SHEN Xiujin,JIANG Hong,YING Meike,et al.Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models[J].Scientific Reports,2016,6(6):32087-32116.
[17] HAUGEN E N,CROATT A J,NATH K A.Angiotensin Ⅱ induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro[J].Kidney International,2000,58(1):144-152.
[18] 常洁,姜宗培,张海燕,等.NADPH氧化酶介导血管紧张素Ⅱ诱导的腹膜间皮细胞转分化及细胞外基质积聚[J].中华肾脏病杂志,2007,23(5):328-333.
[19] YOON H E,GHEE J Y,PIAO S,et al.Angiotensin Ⅱ bloc-kade upregulates the expression of Klotho,the anti-ageing gene,in an experimental model of chronic cyclosporine nephropathy[J].Nephrology Dialysis Transplantation,2011,26(3):800-813.
[20] DEVARAJ S,SYED B,CHIEN A,et al.Validation of an immunoassay for soluble klotho protein decreased levels in diabetes and increased levels in chronic kidney disease[J].American Journal of Clinical Pathology,2012,137(3):479-485.
[21] SENGUPTA A,MOLKENTIN J D,PAIK J H,et al.FoxO transcription factors promote cardiomyocyte survival upon induction of oxidative stress[J].The Journal of Biological Che-mistry,2011,286(9):7468-7478.
[22] GREER E L,BRUNET A.FOXO transcription factors at the interface between longevity and tumor suppression[J].Oncogene,2005,24(50):7410-7425.