两肾一夹大鼠高血压模型肾组织可溶性表氧化物水解酶表达及作用

Expression and role of soluble epoxide hydrolase in renal tissue of two kidneys one clip hypertension rats model

目的:初步探讨可溶性表氧化物水解酶(soluble epoxide hydrolase,sEH)在肾血管性高血压肾组织中的表达及其作用。方法:采用两肾一夹高血压大鼠模型,雄性SD大鼠分为两组,假手术(sham)组(n=8)和两肾一夹(two kidney one clip,2K1C)组(n=8),术前及术后每10天测定大鼠血压并收集24 h尿液,观察40 d,用自动生化分析仪测定血钠、肌酐及尿蛋白,用放射免疫法测定血浆和肾组织内肾素活性和血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)含量,免疫组织化学染色和Western blotting的方法检测肾组织的sEH、过氧化物酶体增殖激活受体γ(peroxi-some proliferator-activated receptor-γ,PPARγ)表达情况,六铵银染色观察肾形态学变化。结果:2K1C组大鼠术后10d血压(124.04±6.79)mmHg(1 mmHg=0.133kPa),比sham组(106.70±7.71)mmHg显著升高,P<0.001;术后30 d尿蛋白排泄量(292.33±20.53)mg/d,显著高于同期sham组(206.81±37.61)mg/d,P=0.005,这种差异并一直持续到观察结束;术后40 d,2K1C组与sham组比较的具体值为:肾素活性在血浆为(132.90±31.22)vs.(50.00±13.66)ng/(L.h),P=0.03,在钳夹(左)肾组织为(324.90±56.66)vs.(128.40±36.88)ng/(g.h),P=0.01,右肾为(345.10±42.68)vs.(103.00±19.87)ng/(g.h),P<0.001;AngⅡ浓度在血浆为(10 470.00±1 760.00)vs.(4 810.00±1 164.00)ng/L,P=0.02,在钳夹(左)肾组织为(2 094.00±372.20)vs.(735.90±154.40)ng/g,P=0.005,右肾(1 774.00±206.60)vs.(648.10±217.90)ng/g,P=0.002;Western blotting示肾皮质sEH表达量(sEH/β-actin)左肾为(1.73±0.12)vs.(0.33±0.08),P<0.001,右肾为(0.70±0.05)vs.(0.43±0.09),P=0.04;肾皮质内PPARγ的表达量(PPARγ/β-actin)左肾为(0.89±0.11)vs.(0.17±0.05),P=0.002,右肾为(0.56±0.07)vs.(0.27±0.07),P=0.04。免疫组织化学染色定量分析2K1C皮质光密度高于sham组,sham组和2K1C组髓质的平均光密度都分别高于皮质。肾病理显示钳夹肾出现局灶肾小球硬化和肾小管萎缩,对侧肾出现代偿性肾小球肥大和肾小管扩张。结论:sEH可能在肾血管性高血压的发生发展中发挥重要作用,其作用机制可能与肾素-血管紧张素-醛固酮系统以及PPARγ有关。

Objective：To investigate renal expression of soluble epoxide hydrolase（sEH） in 2-kidney-1-clip rats and explore the role of sEH in renal arterial stenosis hypertensive development.Methods： Hypertensive models were established in Sprague-Dawly rats by chronic partial occlusion of left renal artery.In the study,16 male Sprague-Dawly rats were randomized into sham operation group and 2-kidney-1-lip（2K1C） group（n=8,each group）,and were observed for 40 days.Before operation and every ten days after operation,systolic blood pressure（SBP） was measured and twenty-four-hour urine was collec-ted.At the end of the observation,the blood and kidneys were harvested.The serum Na,24-hour urine protein excretion were measured.Renin activity and angiotensinⅡconcentrition in plasm and renal tissue were evaluated by radioimmunoassay（RIA）.The expression of sEH,peroxisome proliferator-activated receptor-γ（PPARγ） in kidneys were assessed by immunohistochemistry and Western blotting.Histology was analysed after kidney sections were stained by Grocott-Gomori methenamine-silver nitrate.Results： After surgery,the systolic blood pressure in 2K1C group gradually became higher than that in sham group.Urine protein excretion was statistically increased in 2K1C group on the 30 th and 40 th days,while serum sodium was of no significant difference from those of the two groups.Renin-angiotensin system in both clipped and nonclipped kidneys were also invoked by the 2K1C surgery.Both sEH and PPARγ were upregulated in renocortex and renomedulla in 2K1C group.The two groups were compared： in SBP,on the 10 th day,（106.70±7.71） vs.（124.04±6.79） mmHg,P0.001,and on the 40 th day,（107.80±10.01） vs.（150.40±11.76） mmHg,P0.001;Urine protein excretion,on the 30 th day,（206.81±37.61）vs.（292.33±20.53）mg/d,P=0.005;Serum sodium,（179.76±29.20） vs.（157.72±51.00）mmol/L,P=0.44;Renin activity[plasm（50.00±13.66） vs.（132.90±31.22）ng/（L·h）,P=0.03;clipped kidney（128.40±36.88）vs.（324.90±56.66）ng/（g·h）,P=0.01;nonclipped kidney（103.00±19.87）vs.（345.10±42.68）ng/（g·h）,P0.001];AngⅡ[plasm（4 810.00±1 164.00）vs.（10 470.00±1 760.00） ng/L,P=0.02,clipped kidney（735.90±154.40）vs.（2 094.00±372.20）ng/g,P=0.005,nonclipped kidney（648.10±217.90）vs.（1 774.00±206.60）ng/g,P=0.002];the expression of sEH（sEH/β-actin） in renocortex [clipped kidney（0.33±0.08）vs.（1.73±0.12）,P0.001,nonclipped kidney（0.43±0.09）vs.（0.70±0.05）,P=0.04];the expression of PPARγ（PPARγ/β-actin） in renocortex [clipped kidney（0.17±0.05） vs.（0.89±0.11）,P=0.002,and nonclipped kidney（0.27±0.07） vs.（0.56±0.07）,P=0.04].Clipped kidney showed more severe glomerulosclerosis and tubular atrophy in 2K1C group than in sham group.Conclusion： sEH probably plays an important role in the development of hypertension in the rat models of renovascular hypertension.The activation of PPAR-γ and RAAS by renal arterial stenosis are associated with sEH upregulation,suggesting that they might regulate sEH expression and take part in hypertensive development.