Research On The Role Of γ-GCS In The Interstitial Fibrosis Of Lupus Nephritis

Lupus nephritis is one of the most common complication of SLE, and is also a common secondary glomerulonephritis, is characterized by renal interstitial damage to glomerular and blood vessels. Later may develop glomerulosclerosis, fibrous crescents, tubular atrop hy and interstitial fibrosis. Develop end-stage renal interstitial fibrosis, leading to kidney failure. Kidney disease is one of the most important factors affecting long-term prognosis of SLE affecting, thus research on the Renal interstitial fibrosis in the pathogenesis of SLE is an important aspect of treatment.Current studies suggest that TGF-β1 plays an important role in renal interstitial fibrosis, which can stimulate excessive repair of renal inflammation, tubular epithelial transdifferentiation and promote glomerular mesangial cells and endothelial cell proliferation and extracellular matrix (extracellular matrix , ECM) synthesis and accumulation, to promote glomerular sclerosis and renal interstitial fibrosis. Studies have also shown that TGF-β1 in the LN patients with renal tubular epithelial cells and is simply the expression of fibroblasts was significantly higher, is an important stimulating factor of LN renal interstitial fibrosis process. It induced fibrosis by promoting ECM synthesis and inhibit its degradation to achieve. Plasminogen activator is a protease which can activate plasminogen into plasmin protease. and maintain matrix formation and degradation of fibrinolysis in a kind of dynamic balance. Plasminogen activator inhibitor(Pal-l) is the most effective physiological inhibitor of Plasminogen activator in the blood, which combined with the activator to suppress plasminogen activator. TGF-β1 can effectively stimulate the Pal-l expression in renal tissue, thus inhibiting the degradation of ECM, and facilitating its deposition in renal interstitial. This process is mediated by ROS completed. In the pathology of lupus nephritis, immune injury ally with Inflammation, resulting the damage to kidney of various oxidation factorrenal, like a nitrogen oxide and lipid peroxides.and eventually developed into renal interstitial fibrosis in irreversible damage. Glutathione (GSH) is one of the most important factors of anti-oxidation mechanism in the body, can effectively confront the role of oxidative damage factor.SLE patients for the latest reported levels of GSH were significantly lower than the control group, and is closely related with disease activity, thus renal tissue GSH level is an important factor in the development of renal damage to renal interstitial fibrosis.and GSH can also inhibit TGF-β-1-induced expression of Pal-l to promote ECM degradation through and ROS, to promote ECM degradation. Oxidative damage and other stimuli can be adjusted to varying degrees of renal tissue GSH concentrations. An important part of the regulation, is the transcription regulation of GSH synthesis rate-limiting enzyme-γ-glutamyl cysteine synthetase (γ-GCS), and thus regulate the concentration of intracellular GSH, promote kidney injury and repair or protect renal tissue against oxidative damage.To sum up, in the LN patients with a lower level of GSH in vivo represents the reduced antioxidant capacity ,Various oxidation factor within the circulation direct cause kidney damage,And mediated the role of induced fibrosis by TGF-β1. Study of GSH levels on the regulation of transcription factors ofγ-GCS is a new entry point to LN Mechanism of renal interstitial fibrosis.Objective:1.Comparison to normal mice to observe the level of the mouse model of lupus renal fibrosis in mice MRL/1pr factor TGF-β1 transcription and protein expression levels change.2.Comparison to normal mice to observe the expression of GSH and GSH synthesis rate-limiting enzymeγ-GCS in mouse kidney, to assess the lupus mouse kidney tissue antioxidant capacity changes. Explore MRL/1pr mouse renal tissue GSH andγ-GCS expression in the renal interstitial fibrosis. 3.Construction ofγ-GCS-pEGFP-c1 eukaryotic expression plasmid, cultured primary murine renal tubular epithelial cells in lupus. To make early preparations for follow-up study ofγ-GCS in renal tubular epithelial cells.Method:1.Grouping Matched group:16-week-old SPF level C57BL / 6 female mice;LN model group: 16 week old female mice SPF level MRL/1pr;Ten for each. To observe the Pathology by tissue staining. In the kidney tissue of MRL/lpr lupus mice and control mice, mRNA expression of TGF-β1 andγ-GCS were detected by Quantitative real-time PCR and the protein expression were detected by immunohistochemistry and westernbloting, respectively. Using colorimetric method detected tissue GSH level of renal.Cloned fragment sequence ofγ-GCS by PCR and recombined into the eukaryotic expression vector of PEGFP-c1, Cultured renal tubular epithelial cells by enzymatic digestionThe datum shown with mean±SD( x±s), were analyzed by LSD-t using SPSS 13.0. The difference was considered when P <0.05.Result:1. Pathological changes: It can be seen under light microscope HE stain that group part of the lupus mice renal tubular epithelial cells shrivel and fall off, lumen expansion, and small tubular epithelial cells, fibrosis, focal interstitial fibrosis in the small pipe, vacuolization, and protein tube.2. Fluorescence quantitative PCR results take a fixed concentration of cDNA as a template ,to establish the concentration gradient in order to fabricate the standard curve ofγ-GCS, TGF-β1 and GAPDH,using semi-quantitative method to compare the differentially expressions ofγ-GCS and TGF-β1 in C57BL / 6 mice and MRL/1pr lupus renal tissues mice. and mRNA expression levels ofγ-GCS was decreased compared to normal mice, the mRNA expression levels of TGF-β1 increased, the difference was statistically significant, (p <0.05). Lupus mouse renal tissue GSH levels decreased significantly (p <0.05).3. he results of dyeing showed that TGF-β1 staining mainly in the cytoplasm of renal tubular epithelial cells express higher. The renal tubular epithelial cells in MRL/1pr mouse express higher than in normal mice (p <0.05). Western blot analysis of the results of the use of gray-scale scan Experimental results show thatγ-GCS protein in renal tissue of lupus mice expressed decreased (p <0.05).4. Constructedγ-GCS-pEGFP-c1 eukaryotic expression vector successfully. Sequencing results of the gene sequence agreed withγ-GCSmRNA in the sequence. Cultured primary renal tubular epithelial cells successfully. Early cells are mostly spindle or round spindle. It can be observed that the nuclei ,which acted as the larger multilateral cobblestone-like, was light blue staining by hematoxylin stained.Conclusions:LN renal tissue, the increasing expression of TGF-β1 caused by inflammation stimulated reduced the expression of theγ-GCS, thereby reducing GSH synthesis, the oxidation - antioxidant system unbalanced in kidney tissue of LN patients. A large number of ROS, which produced in kidney tissue, mediated the fibrosis-promoting of fibrogenic cytokine TGF-β1 in renal of a certain extent, and accelerated the process of renal interstitial fibrosis. The study provides new guidance ideas for the future treatment and study of lupus nephritis, and has laid a foundation for further research on the pathogenesis of lupus nephritis. Constructedγ-GCS-pEGFP-c1 eukaryotic expression vector and cultured primary renal tubular epithelial cells successfully. In renal interstitial fibrosis in renal tubular epithelial cells, the role of the preparation of earlier stage done. To further study of the effect of theγ-GCS in renal interstitial fibrosis and in renal tubular epithelial cells, the preparation was done.