Study On Traditional Chinese Medicine Syndromes Of Diabetic Nephropathy Based On Klotho Gene Polymorphism

Diabetic Nephropathy(DN) is one of the most serious microvascular complications of Diabetes Mellitus(DM). It is believed that genetic background plays an important role in the occurrence and development of DN.Therefore, illuminating DN and its risk factors from the perspective of genetics are significant in the diagnosis and treatment of diseases.ObjectiveThe relations between Klotho gene and the risk of T2 DM, the risk of DN,TCM symptoms and syndrome of DN patients are investigated. The susceptible gene types are screened, in order to prevent and treat potential high-risk patients as soon as possible. According to the research results of first part of the study, the efficacy and safety of TCM in the treatment of DN are further assessed systematically.Methods1. In this study, 178 cases of Han population in Hubei Province are collected and analyzed, including 123 cases of patient group(56 cases ofT2 DM, 67 cases of DN) and 55 cases of healthy control group. The general information of the cases is collected and the cases are surveyed by TCM semiotics. The blood genome DNA is extraced by centrifugal column method.The Klotho Gene information and the series of promoter region G-395 A and exon regions of F352 V and C370 S are obtained from NCBI. Single Nucleotide Polymorphism(SNP) of these loci is detected by PCR and direct sequencing technique. The genotype and allele distribution characteristics of Klotho SNP in T2 DM and DN patients are analyzed by SPSS software. The relations between Klotho SNP and DM, DN are discussed. The relations between the Klotho SNP of DN patients and TCM symptoms and syndrome are studied further.2. The used electronic databases contain foreign databases such as Pub Med, EMBASE, Cochrane Library, and Chinese databases such as CNKI,Sino Med, Wanfang Data, CQVIP. The clinical research of ’Warming Yang and Benefiting Qi, Promoting Blood Circulation and Inducing Diuresis’ in treatment of DN are collected comprehensively. The literature retrieval ended on April 1, 2016, and the meta analysis of approriate literature is processed by Rev Man5.3 software.Results1. In Klotho gene G-395 A locus, three kinds of genotypes are found, and in F352 V and C370 S loci, only two kinds of genotypes are found.2. There is no statistical difference between cases and controls of G-395 A genotype and allele distribution frequency(X2=1.197, P=0.274; X2=1.083P=0.298). Between T2 DM and DN, there is statistical difference(X2=5.016,P=0.025,OR=2.475,95%CI:1.108~5.528;X2=5.872,P=0.015,OR=2.404,95%CI:1.166~4.956). There is no relations between DN patients with G-395 A genotype frequency and Mogensen staging(Z=0.123, P>0.05). The results of logistic regression analysis show that there are statistically relations between DN and A allele, history of hypertension, diabetes, Hb A1c(OR=1.774、2.198、1.735、1.306;95%CI:1.195-2.635、1.330-3.632、1.183-2.548、1.022-1.671).3. There is significant difference among three kinds of TCM syndrome type in cases of G-395 A genotype and allele frequency distribution(X2=8.700,P=0.013; X2=6.591 P=0.037). The distribution of GA+AA genotype and A allele frequency shows that Yin-Yang deficiency type < Qi-Yin deficiency type< spleen-kidney yang deficiency type. Compared the distributions of each two groups of genotype GA+AA, there was significant difference between Qi-Yin deficiency group and spleen-kidney yang deficiency group(X2=6.551 P=0.01),and between Yin-Yang deficiency group and spleen-kidney yang deficiency(X2=6.866, P<0.01), but there was no statistical significance between Qi-Yin deficiency group and Yin-Yang deficiency group(X2=0.015, P>0.05).Compared the distributions of gene frequencies of each two groups of genotype G-395 A, there was significant difference between Qi-Yin deficiency group and spleen-kidney yang deficiency group(X2=4.73, P<0.05), and between Yin-Yang deficiency group and spleen-kidney yang deficiency(X2=4.86,P<0.05), but there was no statistical significance between Qi-Yin deficiency group and Yin-Yang deficiency group(P>0.05).4. There is statiscally significant between the DN patients’ edema levels of different genotypes and different alleles(Z=2.951, P = 0.003 Z=2.549, P=0.011). There is no significance between the G-395 A polymorphism and symptom levels of weakness, soreness and weakness of waist and knees,inappetence and abdominal distension(P > 0.05).5. The cases of randomized controlled trial totaled 16 and the cases of DN patients totaled 1107. The results of Meta analysis showed that compared to western medicine control group, the experimental group of integrated traditional Chinese medicine and western medicine, based on ’Warming Yang and Benefiting Qi, Promoting Blood Circulation and Inducing Diuresis’,performs better in the significant efficiency and total efficiency of the treatment of DN(RR=1.97 95%CI 1.50~2.59, Z=4.86,P<0.00001;RR=1.48,95%CI 1.35 ~ 1.62, Z=8.58, P<0.00001), in the decrease of the serum creatinine and urea nitrogen(MD=-32.33, 95%CI-58.20 ~-6.47, Z=2.45,P=0.01; MD=-2.20, 95%CI-4.13~-0.26, Z=2.22, P=0.03), in the increase of high density lipoprotein(MD=0.16, 95%CI 0.09~0.23, Z=4.33, P<0.0001), in the decrease of cholesterol, triglyceride, low density lipoprotein(MD=-0.82,95%CI-1.28 ~-0.36, Z=3.49, P=0.0005; MD=-0.40, 95%CI-0.53 ~-0.26,Z=5.95, P<0.0001; MD=-0.49, 95%CI-0.76~-0.21, Z=3.46, P=0.0005), in the decrease of urinary protein(MD=-0.46, 95%I-0.61~-0.31, Z=6.01, P <0.000 01), in the control of fasting blood glucose and the improvement of Hb A1c(MD=-0.34, 95%CI-0.57 ~-0.12, Z=3.00, P = 0.003; MD=-0.31,95%CI-0.50 ~-0.12, Z=3.15, P = 0.002), and performs the same in the decrease of the levels of tumor necrosis factor alpha(P = 0.33).Conclusion1. Three genotypes of G-395 A have well distribution characteristics in the Han population of Hubei region, but the polymorphisms of F352 V and C370 S are relatively rare.2. In this subject, the relation between the G-395 A polymorphism and T2 DM risk is not found. The polymorphism of Klotho G-395 A is not relatedto the severity of DN. The apperance of A allele may increase the risk of renal damage in patients with T2 DM, and may be one of the independent risk factors for the kidney complications of T2 DM patients of Chinese Han population in Hubei Province. And miscellaneous type GA and homozygous mutant AA genotype may be genetic susceptibility risk genotypes of DN. At the same time, the Hb A1 c, the clinical course of DM and the medical history of hypertension may be the independent risk factors of T2 DM.3. GA and AA genotypes may be the susceptible genotype of DN patients with spleen-kidney yang deficiency. The DN patients, carrying the A allele,may be more easy to transfer to spleen-kidney yang deficiency. A allele may be a risk factor for DN of spleen-kidney yang deficiency.4. GA, AA genotypes may be susceptibility genotype for DN patients with a severe degree of edema. The DN patients, carrying the A allele, may be more likely to appear edema.5. Compared to western medicine control group, the experimental group of integrated traditional Chinese medicine and western medicine, based on’Warming Yang and Benefiting Qi, Promoting Blood Circulation and Inducing Diuresis’, can lower the serum creatinine, urea nitrogen, reduce urinary protein,regulate the abnormal lipid metabolism, improve blood glucose control, but has little improvement of the inflammatory state. The integrated traditional Chinese and Western medicine performs better in safety and deserve to be popularized in clinical application.