【作者】 李萍；

【导师】 刘伏友；

【作者基本信息】 中南大学 ， 内科学， 2007， 硕士

【摘要】 目的：观察螺内酯、依那普利及其联用对单侧输尿管梗阻（UUO）大鼠肾间质纤维化的影响，探讨其可能的作用机制。方法：30只SD雄性大鼠随机分为5组，假手术组、UUO模型组、UUO+螺内酯组(50mg．kg^-1．d^-1)、UUO+依那普利组(31mg．kg^-1．d^-1)、UUO+螺内酯(50mg．kg^-1．d^-1)+依那普利组(31mg．kg^-1．d^-1)，术前1天起各组大鼠给予相应药物混悬液或生理盐水2ml灌胃，术后14天处死大鼠，取左肾组织行HE、Masson染色观察肾脏病理改变，并用免疫组化的方法检测肾皮质TGF-β₁及FN表达；Western Blot法检测ColⅠ蛋白表达；检测各组大鼠血清肾功能、电解质生化指标。结果：（1）肾脏病理改变：①HE染色：UUO术后14天，部分肾小管明显扩张，上皮细胞变矮变平；部分肾小管萎缩，管腔缩小，闭合；肾间质炎症细胞浸润，间质面积增宽，纤维化程度明显增强。②Masson染色：运用图象分析法对肾间质纤维化改变进行半定量分析，各组蓝染面积百分比，分别为假手术组2.72±0.82，UUO组9.98±1.76，螺内酯组6.06±1.77，依那普利组6.48±1.24，两者联用组5.47±1.09；与假手术组比较，UUO组纤维化程度明显增加，差异具有统计学意义（P＜0.05）；各药物干预组与UUO组比较，纤维化程度明显减轻，其差异具有统计学意义（P＜0.05）；而各干预组之间纤维化程度两两比较，无统计学意义（P＞0.05）。以UUO组表达最强，两药联用组表达最弱。（2）免疫组织化学染色：运用图象分析法对肾小管间质TGF-β₁、FN进行半定量分析。①TGF-β₁的表达：各组TGF-β₁在肾小管间质阳性表达百分比分别为：假手术组5.45±2.35，UUO组31.58±5.86，螺内酯组16.73±2.58，依那普利组16.20±2.36，两者联用组14.41±2.50，除螺内酯组与依那普利组之间的比较无统计学差异（P＞0.05）外，其它各组之间的比较均有统计学意义（P＜0.05），以UUO组表达最强，两药联用组表达最弱。②FN的表达：各组FN在肾小管间质的表达百分比分别为：假手术组3.82±0.92，UUO组17.06±2.08，螺内酯组8.46±1.64，依那普利组6.95±1.67，两者联用组7.64±1.20，与假手术组比较，UUO组FN的表达明显增加（P＜0.05）；各药物干预组与UUO组比较，FN的表达明显下调（P＜0.05），各药物干预组之间的比较无统计学差异（P＞0.05），以依那普利组表达最弱。③相关分析：各大鼠肾小管间质TGF-β₁表达与FN的表达呈显著正相关（r=0.893，P＜0.05），与Masson染色纤维化面积呈显著正相关（r=0.787，P＜0.05）；各大鼠肾小管间质FN的表达与Masson染色纤维化面积呈显著正相关（r=0.805，P＞0.05）。（3）Western Blot显示，UUO术后14天，假手术组ColⅠ蛋白的表达很少，UUO及各药物干预组ColⅠ蛋白表达明显增高（p＜0.05）；与UUO组比较，各药物干预组ColⅠ蛋白表达明显下调，以两药联用组表达最弱（4）血生化指标：血钾浓度，UUO组及各药物干预与假手术组之间比较，差异无统计学意义（P＞0.05），而两药联用与UUO组相比较，血钾浓度明显升高（P＜0.05）。血钠浓度，与假手术组相比较，两药联用组明显下降（P＜0.05）。血肌酐浓度，与假手术组相比较，UUO组及两药联用组肌酐比明显升高（P＜0.05），血尿素氮浓度，各实验组两两比较，无统计学差异（P＞0.05）。结论：1单侧输尿管结扎（UUO）可以快速建立肾间质纤维化动物模型。2 TGF-β₁、FN和ColⅠ的表达增多与肾间质纤维化发生密切相关。3螺内酯通过下调肾脏TGF-β₁的高表达，减少FN和ColⅠ在间质的沉积，阻止肾间质纤维化进展。其抗纤维化作用与ACEI相似。4醛固酮受体拮抗剂与ACEI联用对减轻肾间质纤维化具有协同作用。5两药联用的同时增加了血钾增高、肾功能减退的危险性，联用时须监测肾功能、血电解质。更多还原

【Abstract】 Objective: To investigate whether spironolactone, a mineralocorticoid receptor antagonist, alone or in combination with enalapril, an angiotensin converting enzyme inhibitor （ACEI）, ameliorates renal interstitial fibrosis in an experimental model of complete unilateral ureteral obstruction （UUO）.Methods: Thirty male SD rats were randomly assigned to five groups and were treated as follows: sham operation group （sham group）, UUO group, spironolactone treatment group (50mg.k^-1.d^-1), enalapril treatment group (31mg.k^-1.d^-1) and their combination group. Immunohistochemistry were performed to examine the expression of transforming growth factor beta 1（TGF-β₁） and fibronectin （FN） at the 14^th day in the kidney. The expression areas of TGF-β₁ and FN were quantitatively analyzed by image analysis. HE and Masson Trichrome staining were performed to investigate renal pathological changes and the degree of interstitial fibrosis. The fibrotic areas were also quantitatively analyzed by image analysis. The expression of collagen type I were measured by Western blot. The plasma concentration of sodium, potassium, creatinine and blood urea nitrogen was detected at the 14^th day after UUO.Results: In comparison with the sham group, the area of TGF-β₁ and FN deposition increased significantly in UUO group and three treatment groups （P<0.05）. The area of TGF-β₁ and FN deposition in three treatment groups were apparently less than those in UUO group（P<0.05）. The expression of TGF-β₁ in combination treatment group was significantly reduced compared with spironolactone treatment group （P<0.05） and elanapril treatment group （P<0.05）. There were no statistically significant difference between spironolactone treatment group and elanapril treatment group （P>0.05）. The expression of TGF-β₁ in tubules and interstitium was closely related to the expression of FN （r =0.893, P<0.05） and the fibrotic areas （r=0.787, P<0.05）.The expression of FN was also closely related to the fibrotic areas （r=0.805, P<0.05）. Western blot showed that in comparison with the sham group, the expression of collagen type I （Col I ） protein in UUO group and treatment groups were significantly upregulated （P<0.05）, Compared with UUO group, collagen type I protein were downregulated in treatment groups especially in combination treatment group （P<0.01）. There were no statistically significant difference between spironolactone treatment group and elanapril treatment group （P>0.05）. Compared with UUO group, the plasma concentration of potassium in combination treatment group was significantly increased （P<0.05）. Compared with sham group, the plasma concentration of sodium in combination treatment group was significantly decreased （P<0.05）. The plasma concentration of creatinine and blood urea nitrogen was significantly increased in UUO group （P<0.05） and in combination treatment group （P<0.05）.Conclusions:1. UUO is a ripe, well-characterized model of experimental renal disease culminating in tubulointerstitial fibrosis.2. The increase of FN and Col I were correlated with the increased expression of TGF-β₁ and the degree of interstitial lesions in obstructive nephropathy.3. Spironolactone may suppress fibrosis, at least in part via down-regulating TGF-β₁ expression and decrease accumulation of extracellular matrix include FN and Col I . Its anti-fibrosis effect may be similar to ACE inhibitor.4. concomitant use of spironolactone and ACE inhibitor had synergistic effect on extenuating tubulointerstitial fibrosis5. concomitant use of spironolactone and ACE inhibitor also added the risk of the upregulation of the plasma concentration of potassium and renal hypofunction, thus we should monitor plasma concentration of electrolytes and renal function.更多还原