摘要
目的:探讨单侧输尿管结扎模型(UUO)幼年大鼠肾间质纤维化过程中局部微血管损伤趋势及其特征分子血小板反应蛋白(TSP-1)与血管内皮生长因子(VEGF)组织定位表达和时相表达的趋势、潜在的病理意义,并评价用血管紧张素转换酶抑制剂(ACEI)苯那普利和血管紧张素Ⅱ受体拮抗剂(ARB)氯沙坦联合干预的效应。
方法:采用单侧输尿管结扎的方法制备实验性梗阻性肾小管间质损伤模型(UUO模型);3-4周龄幼年雄性Wistar大鼠96只,分为正常对照组(32只)、UUO模型组(32只)、UUO模型治疗组(32只);于实验第1周、第2周、第3周、第4周每组各取8只大鼠,取病变肾脏石蜡包埋并切片;HE染色评价肾组织常规病理变化,免疫组化方法检测大鼠肾组织CD34、TSP-1、VEGF的表达,探讨各组大鼠CD34、VEGF、TSP-1肾组织定位表达和时相表达的趋势,以及干预的影响。用SPSS13.0统计软件进行统计学处理。
结果:①各组大鼠肾组织石蜡切片经HE染色,光镜下观察肾组织常规病理,结果提示:对照组肾脏结构正常,肾小管排列紧密、整齐,小管基底膜光滑、连续,小管间质区无炎性细胞浸润。UUO模型组术后第1周,肾间质水肿,远端小管扩张,肾小球结构正常;术后第2周,肾间质炎性细胞局灶或弥漫性浸润,肾小管轻度扩张;术后第3周,肾间质炎性细胞弥漫性浸润,局灶性加重,肾小管不同程度扩张,小管细胞空泡变形、基底膜增厚断裂,小管间质区面积增大,肾间质轻度纤维化;术后第4周,肾小管间质病变继续加重,肾间质炎性细胞仍呈弥漫性浸润,肾小管大量塌陷,残存小管细胞基底膜明显断裂、萎缩,肾间质明显纤维化。干预组与模型组同时间比较,肾小管间质病变程度明显减低(P<0.05)。②免疫组化染色: CD34在肾小球毛细血管袢和肾小管周围毛细血管内皮细胞中表达,其免疫组化染色半定量分析:第1、2、3、4、周分别为:对照组:2.59±0.72、2.89±0.64、2.44±0.44、2.82±0.51;模型组:2.12±0.31、1.74±0.16、1.43±1.19、1.09±0.15;干预组:2.78±0.28、2.37±0.38、2.09±0.28、1.71±0.19。随试验时间的延长,UUO模型组肾组织中CD34表达量及面积显著递减(P<0.01),干预组与模型组相比这种趋势较缓(P<0.05)。VEGF在肾小管上皮细胞胞浆中表达,其免疫组化染色半定量分析:第1、2、3、4、周分别为:对照组:2.97±0.853.21±0.57、3.11±0.76、3.04±0.55;模型组:2.52±0.91、1.76±0.66、1.39±0.71、1.05±0.69;干预组:2.69±0.94、2.46±0.45、2.21±0.40、2.00±0.61。随试验时间的延长,UUO模型组肾组织中VEGF表达量及面积第1周变化不显著(P>0.05)第2、3、4周显著递减(P<0.01),干预组下降趋势不显著(P>0.05)。TSP-1蛋白主要表达于肾小球毛细血管袢、系膜基质、肾小管间质,其免疫组化染色半定量分析:第1、2、3、4、周分别为:对照组:1.86±0.30、1.81±0.26、1.81±0.18、1.86±0.23;模型组:2.02±0.27、3.46±0.28、2.84±0.42、2.93±0.25;干预组:2.19±0.19、3.06±0.27、2.60±0.27、2.69±0.22。随试验时间的延长,UUO模型组肾组织中TSP-1表达量及面积显著增加(P<0.01),其中以第2周达高峰,第3、4周这种趋势渐缓,但仍明显高于对照组,干预组这种变化趋势明显减轻。
结论:在幼年大鼠梗阻性肾小管间质纤维化进展中,存在着肾间质微血管的损伤,表现为肾小管周围毛细血管随肾小管间质纤维化同步减少直至消失。这种损伤可能与VEGF和TSP-1对肾小管周围毛细血管作用失衡有关。早期联合应用ACEI和ARB可能通过抑制AngⅡ调节VEGF和TSP-1的表达而减轻肾间质微血管的损伤,进一步延缓肾小管间质纤维化的进程。
Objective: To investigate the expressing tide and potential pothogenic role of thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) in microvascular injury on early stage tubulointerstitial lesions in young rats with Unilateral Ureteral Obstruction (UUO). Meanwhile, to evaluate the interfering effects with angiotensin coverting enzyme inhibitor(ACEI) Benazepril and angiotensinⅡtype 1 recepter angonist(ARB) Losartan.
Methods: 96 young male Wistar rats at the age of 2-3 weeks were randomly divided into 3 groups, the control group (n=32),UUO untreated group(n=32),UUO treated group with ACEI and ARB (n=32). At the time points that treated for 1,2,3,4 weeks, eight rats were sacrificed in each group and the kidneys were obtained to evaluate the renal tissue morphologic changes through HE staining. At the same time, we detected and evaluated the protein expression of TSP-1, VEGF and CD34 in tubulointerstitial areas by immunohistochemical ways, as well as the effect of treatment. All the data were analyzed by statistics software SPSS13.0.
Results:①Our results showed renal tissue normal in control group. And in UUO untreated group, we observed some significant morphologic changes in tubulointerstitial areas, that include the interstitial edema and the distal tubular dilation at 1st week after UUO. It was showed obviously at week 2, such as more dilated tubuli; generous inflammatory cells which were infiltrated into renal tissue especially in tubulointerstitial regions; foam degenerated tubuler cells, thicken and breaked tubular basement membrane and broadened tubulointerstitial area. With the experimental session continuing ,pathological changes in tubulointerstitial region were becoming more and more serious in untreated group after UUO. Compared with control group,there were significant different in each time points (weeks 1,2,3 and 4). The extent of tubulointerstitial pathological changes in treated group is more alleviative than the untreated group(P<0.05), but still more serious than the control group(P<0.01).②Immunohistochemical staining indicated that in control group CD34 was expressed in the peritubular capillary epiothelial cells. Semi-quantitative value of CD34 expression was, control group: 2.59±0.72、2.89±0.64、2.44±0.44、2.82±0.51;model untreated group:2.12±0.31、1.74±0.16、1.43±1.19、1.09±0.15;treated group:2.78±0.28、2.37±0.38、2.09±0.28、1.71±0.19 at time point of weeks 1, 2,3 and 4 respectively. The decrease of this expression was remarkable in the untreated group (P<0.01), and was moderate in the treated group (P<0.05) in the time-course experiment. TSP-1 proteins were expressed in the wall of Bowman's capsule and VEGF was expressed in the tubular epithelial cells as well as podocytes. Semi-quantitative value of the expression of VEGF was, control group:2.97±0.853.21±0.57、3.11±0.76、3.04±0.55;model untreated group:2.52±0.91、1.76±0.66、1.39±0.71、1.05±0.69;treated group:2.69±0.94、2.46±0.45、2.21±0.40、2.00±0.61 at weeks 1, 2, 3 and 4 respectively. Compared with control group, the expression of VEGF changed moderately (P>0.05) in model group at 1st week, but decreased remarkably at week 2, 3, 4 (P<0.01). However, this decrease was not significant in the treated group (P>0.05). The expression of TSP-1 was mainly located in the Bowman's capsule, mesangial area, glomergular capillary loops and tubulointerstitial region. Semi-quantitative value of TSP-1 was, control group:1.86±0.30、1.81±0.26、1.81±0.18、1.86±0.23;model untreated group:2.02±0.27、3.46±0.28、2.84±0.42、2.93±0.25;treated group:2.19±0.19、3.06±0.27、2.60±0.27、2.69±0.22 at week 1, 2, 3 and 4 respectively. As time going on, this espression was increased significantly in model untreated group (P<0.01). The peak time is the second week and the increase rate is slow at the 3rd and 4th week. All the marking protein expressions are weaker in treated group.
Conclusion: Our results have shown that peritubular capillary lesions were observed in tubulointerstitial fibrosis in young UUO rats, and the abnormal expression of VEGF and TSP-1 proteins may participate in this lesion. The interfering effects of ACEI and ARB could ameliorate tubulointerstitial fibrosis by regulating the expression of VEGF and TSP-1 in obstructive renal tissues.
引文
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