| Objective In order to explore the molecular basis of the early phase of interstitial inflammation and fibrosis in the rat model of protein-overload proteinuria, the expression of five candidate cytokines are investigated in the present study. They include thrombospondin-1 (TSP-1), transforming growth factor beta-1(TGF- 3 l),connective tissue growth factor (CTGF), Urokinase-type plasminogen activator(uPA) and plasminogen activator inhibitor(PAI-1). The other objective is to probe into the interfering effects of angiotensin converting enzyme inhibitor-benazepril and kerine on the five cytokines mentioned above in the process of glomerlosclerosic and tubulointerstitial fibrosis lesion in rats with protein-overload proteinuria. Methods The rat model of protein-overload proteinuria was adopted to investigate the relationship between proteinuria and acute tubulointerstitial injury. 75 Wistar rats weighing 80-110g at 3-4 weeks old were chosen in this experiment, of which 45 rats are used as the model group, 30 rats as control group .Those in model group were given 1.0g bovine serum albumin (BSA) dissolved in saline intraperitoneal (i.p.) injections daily; those in control group were injected daily with an equivalent volume of saline. The nephritic model was established six days after the injections, which was proved by the blood parameters and the renal pathologic changes. After every three days, 10 rats (5in model group and 5 in control group) were sacrificed . Afterwards, the 45 rats left were further randomly divided into three groups?untreated group, benazepril and kerine treated group ,control group, with 15 rars in each group. Treated by benazriple and kerine for 3 weeks, after every week, 15 rats (5 untreated, 5 treated and 5 control) were killed. Right at the time of their death the kidney in each rat was bivalved. One section was stored for immunohischemical single and double staining to detect the protein expression of TSP-1,TGF- 3 1 and uPA. The other section was stored for in situ hybridization to detect the mRNA expression of CTGF and PAI-1. At the same time, 24-hour proteinuria prior to their death and blood parameters were measured. After being treated by benazriple and kerine ,rats were killed to detect the efficiency of treatment.Results In contrast to control group ,the quantity of 24-hour proteinuria of model groups increased significantly on the third day ( 36.6±15.19mg/24h vs 7.16±1.42mg/24h), peaked on the ninth day ( 131.10±18.3mg/24h vs 7.31± 1.71 mg/24h), stayed high level on 30th day (104.29±21.77mg/24h vs 6.51± 0.66mg/24h). The change of blood parameters wasn't significantly evident. TSP-1 protein expression started increasing on the third day (2.42±0.26 vs 2.12 ±0.24), peaked on the ninth day ( 3.39±0.41 vs 2.06±0.69), remained at high level till the 30th day (2.71±0.13 vs 1.97±0.39). TGF- 1 protein expression started on the sixth day (2.63 ±0.14 vs 1.70±0.56),peaked on the ninth day (2.89±0.1 2 vs 1.63±0.42), remained at high level till 30th day (2.75 ±0.22 vs 1.65±0.39). CTGF mRNA expression started increasing on the sixth day (3.55±0.13 vs 1.98±0.24),peaked on the ninth day (3.98±0.35 vs 2.01± 0.16),remained at high level till on 30th day (3.36±0.12 vs 1.94±0.15). The change tendency of expression of TSp-1 protein, TGF- 3 1 protein and CTGFmRNA was consistent with the quantity of proteinuria. TSP-1 protein expression was closely correlated with the expression of TGF- 1 protein and CTGF mRNA ( r1=0.842 r2=0.971) . PAI-1 mRNA expression began to increase on the third day (2.33±0.20 vs 1.75±0.44 ), peaked on the ninth day ( 3.76±0.18 vs 1.55±0.64 ), remained at high level till the 30th day ( 2.76±0.12 vs 1.47± 0.20 ). On the contrary, uPA protein expression decreased significantly on the ninth day (1.18±0.12 vs 1.53±0.30 ), still remained at low level with the development of tubulointerstitial fibrosis. TSP-1 protein expression was positively correlated to PAI-1 mRNA expression ( r=0.902) , while TSP-1 protein expression with uPA protein... |
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