| Objective: With the increase of the living standards of human and aging of population,the incidence of diabetes mellitus is going up year by year,followed with rising of number of the patients with diabetic nephropathy(DN). DN, which has become one of the main etiological factors resulting in renal failure.It is very important to clarify the pathogenesis in the molecular and genic level.Mesangial cells play an important role in maintaining structure and function of the glomerulus and in the pathogenesis of glomerular diseases. The hypertrophia of mesangial cells and the accumulation of mesangial extracellular matrix are primary events, those are importent events leading to the progression to glomerulosclerosis .In 1998,Miyata et al. isolated a new gene from human mesangial cells termed'megsin',which is a new mesangium- predominant gene in human,rats,mice and belongs to serpin. Furthermore ,recent analysis of various experiments and tissue revealed megsin is up-regulated predominantly in human kidney diseases. Human IgA nephropathy and diabetic nephropathy and rat anti-Thy-1 nephritis lesions are manifested as proliferation of mesangial cells and mesangial matrix or amplification, their pathogenic mechanism and the extent of lesions with the expression level of megsin are related. The analysis show that the expression of megsin probably mediated through the extracellular matrix deposition and cell life cycle involved in the process of glomerular sclerosis. The study discovered varying degrees of mesangial matrix accumulation, mesangial cell proliferation and amplification of immune complex deposition in the models (over-expression megsin cDNA transgenic mice) , and discoverd that even though megsin expresses in almost all organizations, but the pathogenicity of over-expressing megsin transgenic mice is limited to the glomerular.we presume over-expressed megsin may lead to mesangial dysfunction, destroying the treatment to immune complexes, and increasing in mesangial matrix by breaking the balance of matrix degradation.The cell cycle abnormalities in Diabetes could activate the occurrence of diabetic nephropathy (DN) and regulate the progression of the disease. p27kip1 gene can regulate the cell cycle and inhibit cell division and mainly combinate to cyclins to inhibit cyclin-cyclin depedent kinase( CDKs). p27kip1 almost can inhibit all cyclin-CDKs complex kinase activity, and play a negative effect on cell cycle regulation. Study shows that, p27kip1 play an important role for the hypertrophia of mesangial cells as well as renal hypertrophy in glomerular diseases and regulate cell cycle as a CDK inhibitor. Under normal circumstance, p27kip1 express increaing in the G0/G1 ,when cells entered the S phase, expressing decreased, if the expression of p27 is up-regulated due to some stimulus ,then the cell cycle will arrest in G1 phase, and leading to cell hypertrophy. Studies have shown that the expression level of P27 protein is regulated by different factors in glomerular mesangial cells. The analysis suggest that P27 play an important role in the regulation of mesangial cells proliferation. P27 is structural expression in the normal quiescent mesangial cells, and the proliferation of mesangial cells correlate with the expression level of p27 in Vivo. Due to the important role of P27 in the regulation of mesangial cell proliferation ,many study will also make it as the target of inhibitting mesangial cell proliferation.And we could inhibit the mesangial cell proliferation or hypertrophy by increasing or inhibitting the expression level of P27 through some direct or indirect interventions.Methods: 60 healthy male unilateral nephrectomy CD-1 mice were randomly divided to 4 groups.One of 4 groups was regarded as normal control group(A),then the others received a single intraperitoneal injection of STZ(dissolved in 0.1mol/L citrate buffer, pH 4.5)at a dose of 150mg/kg Wt. The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L randomly and urinary glucose (+++)～(++++) after 72 hours of STZ injection. Then megsin plasmid, megsin-siRNA plasmid and control plasmid via the tail vein of mice were severally injected to diabetic control group (B), megsin plasmid group (C) and megsin-siRNA group (D). All mice were allowed free access to food and water during the experiment. After 1,2 and 12 weeks, serum creatinine(Scr) and 24 hours urinary protein were measured, then five mice were sacrificed for each group respectively, kidney mass/body mass were measured. The renal cortical tissues were obtained and used for light microscopy. The expression of megsin, type IV collagen and P27 in renal cortex was measured with immunohistochemical method and Western-Blot. The results were semiquantitatively analyzed with an image-processing system.Results: 1. General state of health: The mice of group B,C and D all presented polydipsia, polyuria, polyphagia and progressive weight loss after 4 or 5 days of STZ injection. There is no significant difference in three groups. 2. Kidney mass/body mass ratio ,UP and Scr were higher in group B and C than in group A and D at 12 weeks (P<0.01),group C in those were higher than groupB(P<0.01).3.light microscopy:at 1week the structure of glomerulus were nomal,at 2, 12 weeks,glomerular volume became larger. The glomerular volume of groupB,C,D became larger, mesangial cells proliferation, extracellular matrix increased.The number of mesangial cells were more in group B and C than group A and D.4. Immunohistochemical analysis showed that the expression of megsin and type IV collagen in renal cortex was obviously increased in group B and C than group A and D(P<0.01),and the expression of those was higher in group D than group A(P<0.01).At 1 week and 2weeks, the expression of P27 in renal cortex was obviously increased in group B and C than group A and D (P<0.01),and was higher in group D than in group A,but at 12 weeks there was no significant difference(P>0.01)between group B and group D .5.Western-Blot analysis showed that at 1,2weeks the IOD of P27 in renal cortex was obviously increased in groupB and C than group A and D(P<0.01),and and was higher in group D than in group A,but at 12weeks there was no significant difference (P> 0.01)between group B and group D.Conclusion: 1.The expression of megsin enhanced in diabetic mice,that consisting with mesangial cells hyperplasia, renal hypertrophy and renal dysfunction, and suggesting that megsin may play a role at the occurrence and development of diabetic nephropathy.2.The expression of P27 was significantly increased in over-expressed megsin diabetic mice ,and the renal mesangial cells proliferation, glomerular hypertrophy, renal dysfunction were significantly increased in over-expressed megsin diabetic mice than in diabetic mice, those suggested that megsin promoted glomerular hypertrophy by up-regulating the expression of P27, and may be involved in the one of process in diabetic nephropathy.3.The expression of P27 has been declined in the renal tissue of lower-expressed megsin diabetic mice, that be confirmed by the transfection of megsin siRNA,and the glomerular hypertrophy and the renal dysfunction have been reduced, those suggested that the inhibition of megsin is an effective means for prevention and treatment in early diabetic nephropathy ,and providing a scientific basis for the exploring of a new target for diabetic nephropathy.
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