The Prophylactic Effects Of Cyclosporine A On Autoimmunological And Inflammatory Injuries To The Kidneys In The Streptozotocin-induced Diabetic Rat

Part 1 Preventive effect of cyclosporine A on diabetic nephropathy in a STZ-induced diabetic ratObjective Diabetic nephropathy is one of the most common chronic complications and the major cause of advanced renal failure. It is commonly accepted for the time being that therapeutic principles for diabetic nephropathy are described as following:the controls of hyperglycemia, blood pressure and diet as well as renal function protection. Renal function would decline when clinical proteinuria appears in the diabetics. It has been estimated that more than 20% of diabetic patients with clinical proteinuria would develop end-stage nephropathy, which would require renal dialysis or kidney transplantation. In the current study ,it is found by pathology that the infiltrations of lymphocytes and macrophages in renal tissue have shown a new evidence for the cyto-autoimmune injuries to the kidneys in diabetic nephropathy. Besides these, however, Inflammatory processes are also involved in the pathogenesis of the diabetic nephropathy. The evidences for this suggestion is based upon our molecular study of several cytokines, such as MCP-1,RANTES and NF-KB. Furthermore, Preventive efforts were also made to evaluate if cyclosporine A had a prophylactic effects on the kidney protection in the STZ-induced diabetic rats. Methods The diabetic models of rats( Sprague-Dawleys) were established by intravenous injection of streptozotocin(50mg/kg) through the tail. Then the diabetic rats were randomly divided into 8 groups, AMN, AMI, BML, BMM, BMH, AML ,AMM and AMH groups while the control rats were also randomly dividedinto 4 groups, NN, NL, NM and NH group to which Cyclosporine A was administrated at a various doses of lmg/kg/d,4 mg/kg/d and 8mg/kg/d respectively. Eight weeks later, serum glucose ,BUN, creatinine and microalbumin in the urine for 24 hours were evaluated. Kidney pathology was investigated and ultramicroscopic structure was observed . Fibrosis degree in the kidneys was asessed by Image-pro plu image analysis system. Results: There were a plenty of evidence for the obvious fibrosis while the ratio of kidney /body weight, BUN and creatinine in the serum as well as urinary microalbumin in AMN group were increased. Moreover, the projections of foot-cells were mixed together, with the widened basal membrabce, and evident apoptosis of some cells. In AMI group, however , the ratio of the kidney /body weight, renal ultramicroscopic structure, creatinine and microalbumin in the serum had no change much (P>0.05) while both kidney fibrosis and BUN were reduced significantly (P<0.05) .In CsA groups, both kidney fibrosis , BUN in the serum and microalbumin in the urine for 24 hours were decreased (P<0.05) while the ratio of kidney /body weight and serum creatinine had no statistic change. Renal ultramicroscopic structures were arranged regularly in middle and high- dosage CsA groups while destroyed obviously in low-dosage CsA groups .Compared with AMI group, renal fibrosis was reduced significantly in CsA group (P<0.05) . Conclusion: 1. autoimmune injuries to the kidneys in STZ- induced diabetic rat were found at the time of 8 weeks. 2. It is by insulin injection that the fibrosis may be relieved at least in part while CsA seems to be better than insulin for the kidney protection.3.The injuries to the kidney could be protected by the administration of CsA in the STZ-induced diabetic rats .4. CsA itself had no any evidence to show the sideeffects on the kidney in our study.Part two The preventive effect of CsA on renal lesions in STZ-induced diabetic rats through Immunological pathogenesisObjectives: Although diabetic nephropathy is currently believed to be one of non-immune disease, more and more evidence and experimental results have already shown that immunocytes may be involved in the pathogenesis of diabetic nephropathy. The infiltrations of lymphocytes and macrophages could induce fibrosis of inherent nephrocytes in non-immune nephropathy. The aim of our study is to investigate the mechanism of kidney pathogenesis by the intervention with CsA in STZ-induced diabetic rats. Methods: The diabetic models of rats( Sprague-Dawleys) were established by intravenous injection of streptozotocin(50mg/kg) through the tail. Then the diabetic rats were randomly divided into 8 groups, AMN,AMI,BML,BMM,BMH,AML ,AMM and AMH groups while the control rats were NN group to which Cyclosporine A was administrated at a various doses of lmg/kg/d,4 mg/kg/d and 8mg/kg/d respectively..Subunit classification of T lymphocytes in peripheral blood was detected by flow cytometry. The concentration of immunoglobin in serum of rat was determined by enzyme linked immunosorbent assay (ELISA). The infiltration of CD3+T lymphocytes and deposition of IgA, IgG in the renal tissues were evaluated by immunohistochemistry and immunofluorescence technique. Results: The amount of CD3+T lymphocytes in peripheral blood and the ratio of CD4+/CD8+ were obviously decreased in AMN group(P<0.01), The infiltrations of CD3+ cells and the deposition of IgA, IgG in the renal tissues were also more obvious than the other groups(P<0.01). While there were no significant difference of the above parameters between AMI and AMN group(P>0.05). in CsA -intervented groups ,The ratio of CD4+/CD8+ was significantly increased (P<0.01)while the infiltrations of CD3+ cells and the depositions of IgA, IgG in the renal tissues were significant attenuated (P<0.01), futhermre ,CD3+T lymphocytes count in peripheral blood was decreased in different degrees, especially in BML andBMH groups(P<0.05). Conclusions: l.The deregulation of immunologic function does exist in STZ-induced diabetic rats. Autoimmunology abnormities such as lymphocyte infiltrations and immunoglobulin depositions might be one of the most important pathogenesis of diabetic nephropathy. 2. no significant effect of Insulin treatment on immune functions was found in STZ-induced diabetic rats. 3. CsA shows the protective effect in STZ-induced diabetic rats though autoimmune interventionPart three The prophylactic effects of CsA on the kidney injuries in STZ-induced diabetic ratsObjective: The aim is to investigate the pathogenesis of protective effect of CsA on diabetic nephropathy by molecular study of MCP-1, RANTES and NF-KB. Methods: The expression and activation of nuclear factor-kappaB p65 protein were detected by immunohistochemistry.The mRNA expressions of MCP-land RANTES was detected by semi-quantitive reverse transcript-polymerase chain reaction(RT-PCR), and protein expressions of MCP-land RANTES was detected by western blot and immunohistochemistry .Results: The over-actived of NF-kappa B p65 and the over -expression of MCP-1 and RANTES were foundin AMN group (all P<0.01). These over expression can be significantly inhibited in AMI group(P<0.05)and CsA- intervented group (P<0.01).There was no significant differences of the mRNA and protein expressions of MCP-1 and RANTES between AMI group and AMN group(P>0.05). The activation of NF-kappa Bp65 protein were lower in CsA- intervented group than in AMI group (P<0.01).Conclusions: 1. the over activation of NF-kB and higher expressions of Chemotatic factor MCP-1 and RANTES maybe involved in the pathogenesis for DN. 2. insulin treatment can inhibit NF-kB activation to some extent, but has no effect on over-expressions of MCP-1 and RANTES 3. over -activation of NF-kB and over expressions of MCP-1 and RANTES could be inhibited by CsA treatment, which might be one of preventive mechanisms for renal lesions in STZ-induced diabetic rats.