| Object:It is believed that interstitial fibrosis is the common final pathway leading to end stage renal failure. One of the most prominent pathologic characteristics of renal fibrosis is the accumulation of myofibroblasts, which are thought to be the main source of the increased extracellular matrix (ECM) deposition. Recent studies suggest that tubular epithelial-myofibroblast transdifferentiation (TEMT) is one of the important mechanisms of renal interstitial fibrosis. TEMT can be induced efficiently in cultured tubular epithelial cells by some cytokines such as TGF-(. TNF-α overexpression has been reported in the kidney with interstitial fibrosis, but whether the high expression of TNF-α is related to TEMT remains unclear. Aristolochic acid (AA), a nephrotoxic agent, can injure directly renal tubular epithelial cells, inducing transdifferentiation, necrosis and apoptosis of these cells,thus resulting in renal interstitial fibrosis. It's reported that TEMT induced by AA may be the prominent reason of renal interstitial fibrosis (RIF). It's found out that TGF-βand bFGF can induce transdifferentiation of renal tubular epithelial cell separately. The function of other cytokine remain unknown yet. It's still in experiment period to prevent and treat RIF using western medicine and has not been applied to clinic. The study of using Chinese herb to prevent and treat RIF was rarely reported, especially of combination Chinese herbs. Human kidney cell (HKC) was used to examine whether TNF-α can induce TEMT and increase accumulation of fibronectin and type I collagen, and whether there is a synergism between TNF-α and TGF-β in inducing TEMT in vitro. Renal interstitial fibrosis was induced in rats by injecting pure AA into peritoneum to examine the effect of TEMT during the process of interstitial fibrosis, and the expression of TNF-α during the TEMT. Unilateral ureteral obstruction (UUO) model was also made to examine the preventing and treating effect of Chinese herbs on renal interstitial fibrosis.Methods:This experiment was composed of three parts. In the first part,HKC cells were divided into four groups: 1.Serum free as control. 2. treated with TGF-((8ng/ml ) as positive control. 3. treated with TNF-α (1, 5, 50,100ng/ml). 4. treated with TGF-( (8ng/ml) plus TNF-α(50ng/ml, 100ng/ml), respectively. The cells were incubated for 48 hours. Transdifferentiation of HKC was assessed by examining morphologic change and expression of α-smooth muscle actin (α-SMA), E-cadherin, cytokeratin (CK) and vimentin (Vim). The fibronectin and type I collagen in culture supernatant of HKC quantitatively detected morphological, indirect immunofluorescence, immunohistochemistry and ELISA methods. In the second part, Wister rats were divided into two groups. 1. AA group (n=30): AA(5mg·kg-1·d-1) intraperitoneal injected (ip) for sixteen weeks 2. Control group (n=5): normal saline (2ml·d-1), ip, for sixteen weeks. Every six rats at weeks 8, 12, 16, 20, 24 in AA group and five rats at week 16 in control group were sacrificed. The blood, urine and renal tissue specimen were taken for renal function measurements, including 24 hours urine protein and urine β2 microglobulin (β2-MG), and pathological studies including tubulo-interstitial area calculation by computor. In the third part, 32 Wister rats were divided into four groups. 8 rats as pseudoperation group, other 24 rats were used to induce Unilateral Ureteral Obstruction model. In therapeutic group, rats were treated by gavage with different dosage of Chinese herbs (4.0g·kg-1·d-1 and 2.0g·kg-1·d-1). Rats were killed after three weeks, the kidneys were taken out for examination of the pathological changes and expression of α-SMA, Vim and CK in renal interstitial fibrosis. Specific value of area with immunohistochemistry in renal epithelial cells and total area in renal epithelial cells were quantitatively analysed.Results:In the first part, HKC cultured with TNF-α at 50, 100 ng/ml became elongated and fibroblast-like in shape, while the control HKC <... |
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