| Tubulointerstitial fibrosis(TIF) is a common end-point pathway of many chronic renal diseases caused by inflammatory and non-inflammatory pathogenic factors and contributes to the permanent loss of renal function. How to clarify the mechanism and to find a more effective treatment of TIP is main aim of the present study. There is increasing evidence that transforming growth factor TGF-P plays an essential role in tubulointerstitial flbrosis process, but the precise cellular and molecular mechanisms of extracellular matrix (ECM) accumulation induced by TGF-P have not been clarified so far. Recent studies have showed that Smad proteins are intracellular mediators of transforming growth factor- P signaling. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. However, the relationship between Smad protein and its downstream target gene PAI-1 and TIMP-1 expressions and TIP process is unknown. So it is necessary to further clarify the role of TGF-p/Smad signaling pathway in the development of TIP and elaborate new therapeutic strategies aiming at slowing TIP development and preserving renal function.Hepatocyte growth -factor/scatter factor (HGF/SF) is a multifunctional cytokine with mitogenic, motogenic, and morphogenic activities for renal tubular cells. It is noteworthy that TGF-1 is a potent negative regulator for HGF gene expression in several types of cells. Hepatocyte growth factor can reverse the inhibitory effect TGF-P on tubular cell growth and branchingtubulogenesis. Previous studies have revealed HGF can suppress hepatic fibrosis and promote renal tubular regenertion following acute injury. It is not clear, however, whether HGF may ameliorate the progression of tubulointerstitial fibrosis.Methods:Unilateral ureteral obstruction (UUO) as an experimental model to induce tubulointerstitial fibrosis and primary culture of renal proximal tubular cells were established in our present study. With methods of reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, substrate zymography, in situ end labeling of fragmented DNA, immunohistochemistry, liposome-mediated gene transfer technique, ELISA and pathological examine, We carried out the following experiments to do this study:(l) Analysis of characteristics of TGF-pj and its downstream target genes (PAI-1,TIMP-1) mPvNA expression during the development of TIF in the UUO model, and the therapeutic mechanism of hepatocyte growth factor (HGF) against development of TIF. (2) Localization of Smad2,3,6,7 in normal rat kidney and investigation of the relationship between Smad protein expression and development of TIF in the kidney with UUO.(3) Exploration of effect Smad? gene transfer on TGF-pi-induced tubular cell cycle arrest, tubular apoptosis and FN synthesis.Results1. Expressions of TGF-1, PAI-1, TIMP-1 mRNA significantly increased at day 3 after UUO compared with these in sham-operated rats at same time point, reached the top at. day 7, and then decreased gradually after day 21. Changes of above-mentioned three genes expression were similar during progression of TIF. Hydroxyproline content in obstructed kidney also progressively increased, and reached the top at day 21.2. Compared with sham-operated rats at 21 day, TGF-pi, PAI-1,TIMP-1 mRNA expressions, hydroxyproline content, and tubular apoptosis markedly decreased in HGF treated rats, in contrast, tubular proliferation and MMP2,9 activities significantly increased.3. Immunohistochemistry staining and Western Blot studies indicated that Smad proteins 2,3,6,7 were detected in normal kidney. Smad2 and Smad3 mainly expressed in renal tubular cell including proximal, distal and collecting tubular cells, and Henle's loops, rarely in glomeruli, while Smad6 and Smad7 were presented in both the glomeruli and proximal renal tubular cell.4. E...
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