The Effects Of RhALR On Renal Expressions Of Collgen-Ⅳ, MMP-9,TIMP-1 In Rats After 5/6 Renal Nephrectomy

Objective: Chronic renal failure (CRF), as a progressive injury of kidney and worsening of renal function, which is attributed to kinds of etiology,has posed great threats to patients. Fibrosis can be the main represent on renal pathological examination. Its pathological changes are glomerular sclerosis (GS) and renal interstitial fibrosis, which recognized as consequences of the tissue repair against gradually chronic injury. GS is characrerized by the progressive accumulation of extracellular matrix (ECM) components. Imbalance between remodeling of synthesis and degradation of ECM leads to over-accumulation of ECM and precedes the development of glomerular sclerosis. The crucial element of ECM is Collgen-Ⅳwith its accumulating along glomerular basement membrane. Therefore Col-Ⅳis widely thought to be hallmark of GS, according to considerable scientific literature home and abroad. For long time, reseachers focus their eyes on increased synthesis, while decreased degradation of ECM also be found play an very important role recent years. There are two main groups of ECM degradation of hydrolysis enzymes: the serine protease (plasmimogen system/plasminogen activator inhibitors, PAs/PAIs) and the matrix metalloproteinases (MMPs). The MMPs, initially secreted as proenzymes that are proteolytically activated, is a predominant group of ECM degradation of zinc-dependent protein hydrolysis enzymes. Recent studies have indicated that MMPs are expressed in glomeruli (predominantly in mesangial and epithelial cells) and in tubuli (mainly in proximal tubular cells). These proteins are also expressed by monocytes/macrophages, granulocytes and activated lymphocytes. Among the MMPs expressed in the kidney, the gelatinases MMP-9 is one of the most important, which exerts fuction over hydrolysis of Col-Ⅳ. Tissue inhibitor of metalloproteinases (TIMP) is the specific inhibitor of MMPs. Four members of the TIMP family have been characterised so far, designatedas TIMP-1, TIMP-2, TIMP-3, and TIMP-4. TIMP-1 and TIMP-2 were the most extensively studied. Activities of MMP-9 are inhibited by TIMP-1 through formation of non-covalent 1:1 complexes with MMP-9. We can say that TIMP-1 plays a key role in maintaining the balance between ECM deposition and degradation. With down regulation of MMP-9 and/or upregulation of TIMP-1, degradation of ECM is inhibited as well as glomerular sclerosis step by step. The abundance of TIMP-1 in kidneys was increased significantly in most experimental models and several human renal diseases, and the degree of TIMP-1 increase was associated with the extent of fibrosis.Augmenter of liver regeneration (ALR), as a thermostabilitied and non-special cytokine which acts on promoting liver regeneration, was discovered and cloned from ablactated rat livers by Hagiya, 1994. The recent studies of ALR greatly in liver have showed its fuctions of enhancing liver regeneration, reversing liver fibrosis,controlling liver-resident natural (NK) cells and stimulating DNA synthesis of liver cancer cells. It is conceivable that ALR of functions biological diversity followed many discoveries of its expression in tissues like liver,kidney, thymus gland, testis, etc. Researches promoted by Wang Aimin indicated that recombinant human augmenter of liver regeneration (rhALR) act on guarding hepar against fibrosis by mechanism of extracellular matrix degradation pathways, which derived from the evidence that the up-regulating of expression of hepatic stellate cell MMP-9 and the down-regulating of expression of TIMP-1 exposed to rhALR.ALR was also found to be able to inhibit expression of Collgen-Ⅰ, Collgen-Ⅲand TIMP-1 in liver. In terms of kidney, it has been shown that ALR is an influence factor in experimental models of acute renal failure (ARF), nephritis and renal turbular epithelial cell apoptosis by gentamycin. In deed, ALR directly or indirectly involved with the process of nephritis pathology and stimulating DNA synthesis in renal turbular cell under acute renal epithelial injury.However, the effects of ALR on CRF have not been studied enough home and abroad. To research rhALR on expression of MMP-9/TIMP-1 that are responsible for changes of Col-Ⅳproduction,either by a mechanism of MMPs that protect liver against fibrosis,or by MMPs family associated with renal fibrosis, finally release the development of GS. In term of the kidney,my work aimed to provide experimental theoretic basis for the prevention and treatment of CRD in the early stage.This study attempts to investigate the effects of rhALR on MMP-9, Col-Ⅳand TIMP-1 with 5/6 Nephrectomy Rats,finally stepping down the progression of GS.Methods: Sixty healthy male SD rats were randomly divided into three groups: sham operation group (S, n=20), CRF model group without treatment (C, n=20) and CRF model group treated with rhALR (rhALR, n=20). The S group was subjected to a sham operation while the C group and rhALR group gained 5/6 nephrectomize. All rats were kept in a temperature-even and humidity-controlled room for 12weeks, eating and drinking freely. 12 weeks later, the model of CRF were successfully established. The rats in group rhALR received a single intraperitoneal injection of rhALR at a dose of 200ug/kg/d, once daily by one week. At the end of the study, the rats were killed, their serum samples were collected for chemical examine and their renal tissue were collected for histological and molecular biology examination. Renal tissues stained with HE and Masson were observed of changes in glomerular pathology in ordinary light microscope. The expression of MMP-9, TIMP-1 and Col-Ⅳwere measured by immunocytochemistry, The expression of MMP-9, TIMP-1 were measured by Western blot. The results were semiquantitatively analyzed with an image-processing system. All the data were analysed by SPSS13.0 statistics software, P value<0.05 was considered to have statistical significance.Result: Chemical examine: Group C and group rhALR have high level of Scr and BUN than group S. While group rhALR have lower level of Scr and BUN compared with group C. Renal pathology: In group C, light microscopy showed glomerular compensatory hypertrophy, proliferation of glomerulus mesangial cells (GMCs), increased of the matrix, capillary collapse, focal segmental sclerosis, inflammatory cell infiltration and fibroplasia occured in renal interstitium. The group of S appeared as satisfactory morphology. The rhALR treatment reduced the renal damage compared with that in group C as above mentioned. Quantitative analysis showed that the glomerular sclerosis index (GSI) of group C was higher than that in the other groups (C 96.33±6.97 vs S 5.78±0.63, n=15, P<0.05; rhALR 58.56±3.71 vs S 5.78±0.63, n=15, P<0.05 respectively). The GSI of the rhALR rats treated with rhALR was notably attenuated when compared with that of the group C rats (C 96.33±6.97 vs rhALR 58.56±3.71, n=15, P<0.05).Immunohistochemistry: It has been shown that the positive staining for TIMP-1 and TIMP-1 was observed in cytoplasm of the mesangial cells, for Col-Ⅳwas seen along glomerular basement membrane (GBM). The level of Col-Ⅳand TIMP-1 in C and rhALR groups were more visible than that in S group (P<0.01). But expression level in group C were significantly higher in rhALR group (P<0.05). The expression of MMP-9 in group S was large in amount, while in C group it was markedly decreased, yet expression of MMP-9 of group rhALR was much more compared with group C.Western blot: Extract protein of each groups, the results of western blot showed that a small amount of Col-Ⅳand TIMP-1 expressed in group S. After received 5/6 nephrectomize for 12 weeks,protein expression of Col-Ⅳand TIMP-1 in C and rhALR group was higher than the S group, there were significant difference(P>0.05). The results showed that 5/6 nephrectomize can increase the over-accumulation of ECM. At the same time, compared with rhALR group and C group, rhALR group reduced significantly (P<0.05). On the other hand, the expression of MMP-9 was most in group S, falling sharply in group C. Under the treatment of rhALR ,the expression of MMP-9 in group rhALR with considerable recovery compared with group C (P<0.05). The trend was contrasted with the expression of Col-Ⅳand TIMP-1 protein in western blot.Conclusions: The rhALR acts as a factor contributing to decrease accumulation of Col-Ⅳas well as protect the kidney against GS by the up-regulating of expression of MMP-9 and the down-regulating of expression of TIMP-1 in renal tissues. Taken together, The study presented here opens the possibility of obtaining new insights on clinical treatment of CRF and understanding of rhALR function. We look forward to its bright prospect of further exploitation and its important actual significance in clinical use.