| Objective: Diabetic nephropathy(DN)is one of the most common and serious chronic complication of diabetes, which has already been one of the main etiological factors of end-stage renal disease. In recent years, the morbidity of diabetic nephropathy increased year by year, which have brought the heavy financial and emotional burden to the society and family. A number of studies indicate that diabetic nephropathy is associated with activation of polyol pathway and protein kinase C, oxidative stress, renal hemodynamic changes, vasoactive substances, cytokines, susceptible gene and many other factors. However, the pathogenesis of diabetic nephropathy has not been studied clearly. Therefore, to explore the pathogenesis and control strategies of diabetic nephropathy are very important。Megsin (mesangial cell-predominant gene with a homology to serpin) is a mesangium-predominant gene,belongs to serpin(serine protease inhibitor). Megsin gene locates on human chromosome 18q21.3, about 20kb, including 8 exons and 7 introns. Studies confirmed that the expression of megsin is enhanced in diabetic nephropathy, which is consist with the degree of renal extracellular matrix accumulation, renal hypertrophy and renal dysfunction. These studies suggested that megsin plays an important role in the mechanism of the occurrence and development of diabetic nephropathy.In this study, we observed the relationship between diabetic nephropathy and megsin gene polymorphism of Chinese Hans lived in Hebei province by using polymerase chain reaction - restriction fragment length polymorphism. The result which would be helpful to provide the theoretical basis of molecular genetics for diagnosis, prevention and treatment of diabetic nephropathy. Subjects and methods: 158 patients with type 2 diabetes mellitus were recruited randomly from Nephrology department and Endocrinology department in the third hospital of Hebei medical university. These Patients were divided into two groups according to their urinary albumin excretion rate: 84 patients without nephropathy and 74 patients with diabetic nephropathy. The Patients in diabetic nephropathy group were divided into 3 groups according to their levels of urinary protein and serum creatinine:42 patients with microalbuminuria, 15 patients with clinical proteinuria and 17 patients with renal insufficiency. At the same time,80 people who had medical examination in our hospital were randomly selected and they were enrolled as normal control group. 3ml blood were taken from their cubital vein for extracting genomic DNA and detecting the biochemical indicators. Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) were used for detecting megsin gene C2093T polymorphism, At the same time ,the levels of cholesterol, triglycerides , HbA1c, serum creatinine, urea nitrogen, glucose and other biochemical markers were detected, blood pressure, height, weight were measured and body mass index (BMI) was counted. Chi-square analysis was used for testing Hardy-Weinberg equilibrium of megsin genotype frequency. All measurement data were testing for normality. Data of normal distribution was presented as mean±standard deviation (?x±s) and data of non-normal distribution was presented as median and interquartile range. One way analysis of variance (one way ANOVA) or non-parametric test was performed to compare measurement data between groups. Chi-square analysis was used to compare enumeration data between groups. Logistic regression analysis was performed to analyze risk factors of diabetic nephropathy in type 2 diabetes. All operations were carried out by the SPSS 17.0 statistical software. P value<0.05 was considered to be statistically significant.Result:1 Biochemical and clinical parameters among the groups Compared with the healthy control group, the levels of BMI, systolic blood pressure, diastolic blood pressure, serum creatinine, urea nitrogen, glucose, triglycerides, cholesterol were significantly higher in patients with type 2 diabetes(P<0.05 or 0.01). The levels of systolic blood pressure, diastolic blood pressure, serum creatinine, urea nitrogen in patients with diabetic nephropathy were significantly higher than those in diabetes without nephropathy(P<0.05 or 0.01). Compared with microalbuminuria group, systolic blood pressure, urine protein was significantly higher in clinical proteinuria group and renal insufficiency group. Compared with microalbuminuria group and clinical proteinuria group, the levels of disease duration, systolic blood pressure, diastolic blood pressure, serum creatinine, Urea nitrogen, urinary protein were significantly increased in renal insufficiency group(P<0.05 or 0.01).2 The results of electrophoresis and Hardy-Winberg equilibrium test of the genotypesPCR fragment of Megsin gene had a length of 256bp. After the reaction of PCR products with restriction endonuclease HaeⅢ, The results of electrophoresis on 8% polyacrylamide gel were the following three: genotype TT produces only one fragmengt of 256bp; genotype TT produces two fragmengts of 114bp and 142bp; genotype CT produces three fragmengts of 114bp, 142bp and 256bp. Each genotype of megsin were in Hdary-Weinbger equilibrium. The chi-square values of the three groups were 2.22, 3.34, 2.87, P values>0.05. The research shows that the subjects were representative.3 The distribution of genotypes and alleles in various groups There was no significant difference of megsin gene C2093T genotypes frequencies and allele frequencies between type 2 diabetic group and healthy control group. In the type 2 diabetic group, there was a significant difference of C2093T genotypes between DM group and diabetic nephropathy group. The frequency of CT genotype in diabetic nephropathy group was significantly higher than the frequency in the diabetic group (48.65% vs 30.95%, P<0.05). Difference of allele frequency was not significant. In diabetic nephropathy group, there was no significant difference of C2093T genotypes and alleles among microalbuminuria group, clinical proteinuria group and renal insufficiency group.。4 Biochemical and clinical parameters and the incidences of diabetic nephropathy among the three genotypes In the diabetic group, there was no significant of age, sex, disease duration, BMI, blood pressure, glucose, serum creatinine, urea nitrogen, triglycerides, cholesterol, HbA1C, urine protein among the three genotypes groups (P values> 0.05). In diabetic patients who carry genotype CT , the incidence of diabetic nephropathy was 58.06%, which was significantly higher than that in 25.00% patients who carry genotype TT (58.06%vs25.00%,χ2 = 4.410, P <0.05) and genotype CC (58.06%vs41.67%,χ2 = 3.84, P = 0.05) . The incidence of diabetic nephropathy in genotype CT is 4.145 times than that in genotype TT(OR = 4.145, 95%CI 1.024-16.855) and 1.938 times than that in genotype CC(OR = 1.938, 95%CI 0.997-3.770).5 The risk factors of diabetic nephropathy in type 2 diabetes Logistic regression analysis showed that megsin C2093T single nucleotide polymorphism, disease duration and systolic blood pressure were the independent risk factors of diabetic nephropathy in type 2 diabetes.Conclusion:1. megsin gene C2093T polymorphism exists in Hebei Han population.2.megsin gene C2093T polymorphism is associated with diabetic nephropathy in type 2 diabetes, genotype CT is probably a susceptibility gene of type 2 diabetic nephropathy... |
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