The Expression Of NGAL In Renal Tissue Of Rats Following Unilateral Ureteral Obstruction

Reserch backgroundThe reserch shows that there is close relation between renal interstitial disease and the progression of chronic kidney failure. Renal interstitial fibrosis is a common feature of progressive renal diseases. It is a very hot pot to study the pathological process and mechanism of renal interstitial disease and search a new target for delaying and blocking renal the progression of renal fibrosis. A great many cytokines and biotic activators participate the genesis and development of renal interstitial fibrosis. All regulatory factors were connected through the biological signal-transducting mechanism, forming the consummate regulatory feedback system to regulate the pathological and physiological processes of organism.The generation and development of renal interstitial fibrosis are complicated. The main course include the fibroblast's activation, generation and phenotype transition, making it transform into myofibroblast, which can secrete a-smooth muscel actin. So that the generation of extracellular matrix(ECM) increaes, and the balance of ECM's production and degradation collapse with the regulation of mutiple cytokines and inflammatory cell infiltration etc, resulting in the accumulation of ECM and the progression of renal interstitial fibrosis. There are multiple cytokines participating the pathological process, including the promoting factors such as transforming growth factor beta-1(TGF-β1), connective tissue growth factor(CTGF), platelet-derived growth factor(PDGF) and the inhibiting factors like hepatocyte growth factor(HGF), decrin, and bone morphogenetic protein-7(BMP-7) etc. TGF-β1 is considered as the most important promoting fibrosis factor of the mutiple promoting cytokines, and most researchers tried to block the progression of Renal interstitial fibrosis by restrainning the TGF-β1. But it needs a lot of works to make use of TGF-β1 for clinical patients. Besides the inhibition of TGF-β1 may result in inflammation and death. So it's important to find new targets for supressing the development of RIF without hurting the physiologic function of organism.Matrix metalloproteinases(MMPs) are very important enzymes degrding ECM. MMPs'functions include ECM degration and activating other MMPs causing cascade effects. MMPs were secreted into ECM in form of proenzyme and turn into bioactive MMPs educing biological activity by taking off propeptide with reactivator's effect. Tissue inhibitors of metalloproteinase(TIMPs) can restrain proenzyme and enzyme activity of MMPs and play important roles in regulating the production and degradation of ECM. Renal interstitial fibrosis is mainly caused by accumulation interstitial collagen of typeⅠ,Ⅱ,ⅢandⅣ, glucoprotein and proteoglycan. Imbalance of MMPs and TIMPs is the key point of progression of renal interstitial fibrosis, while MMP-9 and TIMP-1 is the most important pair. TIMP-1 can combine MMP-9 precurosor turning into stable complex and hinder its self-activation. Meanwehile TIMP-1 can retrain activated MMP-9's activity by irreversibly combining its active center with Zn2+by ratio of one by one. However the activity of MMP-9 directly influences the progression of renal intersitial fibrosis. There are functional disorders of MMPs/TIMPs in the progression of renal interstitial fibrosis caused by various origins of diseases. There might be different outcomes using different methods, but there is a common tendency which is the hypo-expression of the TIMPs with or without the lower activity of MMPs. Therefore it is demanded to look for better activators for MMPs or restrainers for TIMPs which might be a new method to prevent and cure renal fibrosis.Neutrophil gelatinase-associated lipocalin, short for NGAL, also called lipocalin 2, is a new member of the lipocalin family, which was found as a scecreted protein from neutrophilic leukocyte by K jeldsen in 1993. Besides the common structure of hydrophobic group that the lipocalin family have, it still has the nomadic hydrosulfide group, which can combine MMP-9 precurosor and other molecules, establishing foundation for its special biological contribution. NGAL protein expressed in many organs in human body, such as bronchus, stomach, small intestinum, kidney, prostate and thymas gland. NGAL protein can educe its multiple biological effects by participating immune response reaction, the formation and reparation of renal tubular epithelial cells, and generation and progression of tumors.It is discovered that NGAL protein plays roles in regulating MMP-9 and TIMP-1.NGAL can combine MMP-9 protecting it from degradation. MMP-9 protein is ususlly stored in cells in form of MMP-9 precurosor, and activated after the series of N-apical being cut when secreted into extracelluar enviroment. MMP-9 precurosor usually exsit in four forms:monomer, homodimer, heterodimer of NGAL protein and MMP-9 precurosor connected by intermolecular disulfide bond, and the ternary complex combined with TIMP-1 by heterodimer. MMP-9 precurosor could still be activated in the ternary complex but get lower activity compared with the monomer type. However TIMP-1 of the ternary complex can combine other MMPs protein by its nomadic N apical structural domain, turning into quaternary complex. When MMP-9 precurosor of the quaternary complex is activated, it has much more activity than that of the ternary one. Thus NGAL protein apparently has the ability of regulating the complex mentioned above, which can counteract the supression of TIMP-1 on MMP-9's activity. It might be useful in obseving NGAL's expression in the model of renal interstitial fibrosis to investigate its mechanism of action in RIF and look for new ideas of curing RIF.Researchers found that the expression of NGAL protein strenthened in proximal tubules in the patients with acute drug induced interstitial nephritis compared with that of normal persons. The expression was negative correlated with the damage of interstitium, which meant that NGAL educed protective effects in the actue interstitial damage, but the spicific mechanism hadn't been identified, speculating that NGAL might be effective in the RIF progression. Jaya Mishra found that NGAL therapy could decrease the pathological damage in tubular epithelial cells significantly and the proximal tubules increased by creating the acute ischemic-reperfusion injury model of mouse, speculating that NGAL might induce interstial cells transforming into epithelial cells, contributing to the rebirth of renal epithelial cells.The research of NGAL protein at present mostly focus on its effect on the mechanism of tumors such as breast cancer and etc, and as an early detecting cytokine in the acute renal failure caused by renal ischamic-reperfusion injury, kidney transplantation, cardiopulmonary bypass and etc. There are few reaseaches on its effects in renal interstitial fibrosis, however the investigations mentioned above proved that NGAL might play roles in the progression in RIF. So it's necessary to investigate its expression in RIF model and find new methods to prevent and cure RIF.Objective1. To observe the expression of NGAL, MMP-9 and TIMP-1 in renal tissues of rats following unilateral uretral obstuction(UUO), and investigate the correlation of cytokines above in the progression of renal interstitial fibrosis.2. To investigate the mechanism of action for NGAL in the progression of RIF.Methods1. Forty-eight male Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group and an unilateral uretral obstruction group. UUO model was induced by ligating the left ureter of rats. Six rats in each group were sacrificed on the 1st,4th,7th and 14th day after UUO.2,Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of NGAL, MMP-9, TIMP-1 and FN were detected by immunohistochemical staining of ELiVisionTMplus.3,Analysis of data with SPSS 13.0 software.Results1. On the 1st day, there was little expasion in the distal tubules and little edema in the interstitium. On the 4th day there was renal tubular ectasia, scattered leukocyte and macrophage, interstitial cells were partialy denaturated in the UUO group. When the 7th day came, there was severe edema in interstitium and more infiltration of inflammatory cells and proliferation of fibrocyte. necrosis of interstitial cells in renal tubule and an increase of fiber in interstitial substance. Till the 14th day, there was asystematic thickening and shrinkage of renal tubular basement membrane, and the fibrosis was obvious. Injure exponent of interstitial substance and the extent of nephritic fibrosis in UUO group were much higher than that in SOR group at different time point respectively(P<0.05).2. Results of immunohistochemistry for FN indicated that:there was only a little expression of FN in renal glomeruli, tubular basement membrane and renal interstitium. While in the UUO group the expression of FN became higher and higher as obstructed time went(P<0.05). And the positive degree was much higher in UUO group than that of SOR group at each time point respectively(P<0.05).3. NGAL expressed in the proximal tubules in renal tissue in SOR group. Its expression expanded and strengthened in the early stage of RIF(on the 1st and 4th day after UUO), however it decreased as time went(on the 7th and 14th day), still higher than that of SOR group(P<0.05).4. MMP-9 expressed in the proximal tubular epithelial cells in renal tissue in SOR group. The expression of MMP-9 strengthened in the UUO group, rising in the early stage(from 1st to 7th day) and decreasing after 7th day(P<0.05).5. There was little expression of TIMP-1 in SOR group. After obstruction the expression of TIMP-1 gradually increased as time went, and the positive degree was higher in the UUO group than that of SOR group(P<0.05).ConclusionThe expression of NGAL increased on the early stage after obstruction indicated that it played roles in the early stage of renal interstitial fibrosis. There was apparent. negative correlation between NGAL positive degree and the injury index of interstitium and fibrosis degree meaning that it might restrain the progression of RIF. There was positive correlation between NGAL and the ratio of MMP-9 and TIMP-1 indicating that NGAL delayed the progression of RIF by regulating the balance of MMP-9/TIMP-1.