| Part 1: The correlation study of urolithiasis gene polymorphismsObjective: Through the research of the relationship between urolithiasis patients and clinical data and the single nucleotide polymorphisms of genes associated with urinary calculi,to identify the susceptibility factors and susceptibility gene sites of urinary calculi,and to further confirm the molecular pathogenesis of urolithiasis.Methods: A case-control study was carried out.A total of 502 patients with stones in hospitalized patients were selected.And 500 healthy normal subjects matched with stone patients were screened from the physical examination center as the control group.The general conditions and clinical test indexes of each group were recorded.Four genes that cause urinary calcium elevation associated with the formation of kidney stones in the genome-wide association study(GWAS)were selected: TRPV5,CLDN14,KLOTHO,and DGKH.12 SNPs of the four genes were screened though the Hapmap database and haploview4.2 software.Illumina high-throughput second-generation sequencing methods were used to genotype of the 12 SNPs.The representative of each genotype was analyzed by the law of genetic balance of Hardy-Weinberg(HWE).Logistic regression was used to calculate odds ratio(OR)and 95% confidence interval(CI)to analyze the relationship between the genotype and susceptibility of urolithiasis,and the homozygous genotype for the common allele of each SNP was used as the reference in the logistic regression analyses.urinary tract stones.Results: 1.There were significant differences in body mass index and blood glucose between the two groups,the stone group was significantly higher than the control group(P<0.05).More patients in the stones group were overweight(body mass index >24),and their blood glucose level was higher than normal(6.726.72±1.76>5.9),while the body mass index and blood glucose levels in control group were in the normal range.2.The frequency of TRPV5,CLDN14,KLOTHO,and DGKH genotypes all met the Hardy-Weinberg(HWE)equilibrium law in the control group(P>0.05).(1)When we compared the genotypes of the three polymorphic sites of the TRPV5 gene,we found that only the rs4252418 polymorphism was significantly associated with the risk of urolithiasis.Compared with the AA homozygous genotype carrier,the OR value of urolithiasis in the AG heterozygous genotype carriers was 0.010(95% CI=0.007 to 0.015,P=0.000).(2)The rs397703 polymorphism in the three polymorphic loci of the KLOTHO gene was significantly associated with the risk of urolithiasis.Compared with CC homozygous genotype carriers,the TT homozygote genotype and the CT heterozygote genotype carriers had a lower OR 0.387(95% CI = 0.013-0.036,P = 0.000).(3)When comparing the genotypes of the three polymorphic loci of the CLDN14 genotype,we found that the rs219780 polymorphism was significantly associated with the risk of urinary tract calculi,as compared with the GG homozygous genotype carriers.The polymorphism of the urinary calculi in the AG heterozygous genotype carriers was 0.438(95%CI=0.316,1.832,P=0.045),which was significantly associated with the risk of urinary tract calculi.(4)When the genotypes of the three polymorphic sites of DGKH genotype frequencies were analyzed and compared,we did not find any polymorphism sites that were significantly associated with the risk of urolithiasis.Conclusions: 1.Compared with clinical indexes,body mass index and blood glucose in stone group were significantly higher than those in healthy control group;2.TRPV5 gene rs4252418 polymorphism may be significantly associated with the risk of urinary tract calculi.Patients with AA homozygous genotypes were more likely to develop urinary tract stones;3.KLOTHO gene rs397703 polymorphism was significantly associated with the risk of urinary tract stones.CC homozygous genotype carriers had a higher risk of developing urinary tract stones.4.CLDN14 genotype rs219780 polymorphism loci is significantly associated with the risk of urinary tract stones,GG homozygous genotype carriers have a higher risk of urinary tract stones;5.Compared to DGKH genotypes No risk associated with the occurrence of urinary tract stones was found at the site.Part 2: Stone-related gene-gene,gene-environment interaction studiesObjective: By studing the important social(environmental)factors and gene-gene relationships that affect urolithiasis,we will analyze gene-gene,gene-environment interactions and discuss the interaction of genes and environmental factors in the pathogenesis of urolithiasis.Found a combination of genetic and environmental factors that significantly increase the risk of urinary calculi.Methods: Through literature search to find out the social(environmental)influencing factors affecting the occurrence of urolithiasis,questionnaires and data were collected from cases and controls,and the gene-gene and gene-environment interactions were analyzed by cross-analysis.Gene-gene and gene-environment interactions were discussed in the pathogenesis of urolithiasis.Results: 1.The interaction between TRPV5-Rs4252418 and CLDN14-Rs219780 was statistically significant(P<0.05):(1)The interaction between TRPV5-Rs4252418 and CLDN14-Rs219780 was S=1.19,indicating positive interaction between TRPV5 and CLDN14.There is a mutual increase in the risk of urinary tract stones.AP=0.23,indicating that there is a 23% risk of urinary tract calculi associated with the mutation of the two gene alleles is the interaction of the two genes.RERI = 0.20 indicates that the risk of occurrence of urinary tract calculi due to the interaction of the two genes is 0.20 times(OR = 0)of the unknown cause(OR = 1)(P < 0.05).(2)The interaction between TRPV5-rs4252418 and KLOTHO-Rs397703 is S=1.56,indicating that there is a positive interaction between the two,that is,TRPV5 and KLOTHO have an increased risk of urinary tract calcification.AP = 0.03,indicating that the two gene alleles are mutated with a 3% risk relative to the occurrence of urolithiasis as a two-gene interaction.A RERI of 0.12 indicates that the risk of urinary tract calculi due to the interaction of the two genes is 0.12 times(OR)less than the unknown cause(OR=1)(P<0.05).2.After TRPV5,CLDN14 and KLOTHO and drinking water quantity were tested by fork analysis,the interactions based on the additive model did not find statistical significance,but the interaction based on the multiplicative model had a statistical significance(ORint=0.65,P'=0.002);TRPV5-Rs4252418 and the amount of drinking water interacted with OR> 1,indicating that there was a positive interaction between the two,indicating that both mutations can increase the risk of urinary tract stones.Conclusions: 1.There is a positive interaction between TRPV5-Rs4252418 and CLDN14-Rs219780 in urinary tract calculi;2.The interaction between TRPV5-Rs4252418 and KLOTHO-Rs397703 increases the risk of urolithiasis;3.There is a positive interaction between TRPV5-Rs4252418 and drinking water,and both of them increase the risk of urinary tract calculi.Part 3: Expression of TRPV5 in kidneys of containing calcium stones and normal kidneys.Objective: To compare the expression of TRPV5,a cell membrane calcium channel,in kidneys of patients with calcium-containing kidney stones and normal stone-free persons,and to explore the mechanism of TRPV5 in the formation of calcium-containing kidney stones.Methods: Five patients with calcium-containing kidney stones and five stone-free persons were selected in our hospital before 2017.The stone group consisted of 3 males and 5 females,with an average age of 55.6±16.21 years,including 1 kidney cast stone and 4 multiple stones.The 24 H urinary calcium levels were higher than normal in the five patients with calcium-containing kidney stones(>300 mg/24 h for men and >250 mg for women),and analysis of stone composition in patients with stone confirmed calcium stones.A small amount of renal cortex tissue was taken during the surgery for experimental study.In the normal control group,5 stone free cases were kidney cancer patients with normal urinary calcium,including 2 males and 3 females,with an average age of 53.2±14.37 years old.There were no stones in the imaging examinations,no stones were found after surgery,and no other factors such as renal atrophy were observed.The normal cortical tissue was taken from the kidney during surgery.The differences in the expression of TRPV5 in renal tissues of calcium-containing kidney stones and normal stone-free persons were compared by immunohistochemistry and Western-blot.Results: Immunohistochemistry and Western-blot were performed on the renal tissues of the two groups respectively.The images were analyzed using Image-pro plus 6.0 and ImageJ image analysis software.The t-test results showed that the TRPV5 protein expression level was significantly lower in the calcium stone group than their in control group,immunohistochemistry(mean fluorescence intensity: t=4.198,p=0.001,P<0.01;mean cumulative fluorescence intensity: t = 2.963,p=0.013);Western-blot(gray value: t=4.951,p=0.008,P<0.01).Conclusion: The expression level of TRPV5 protein in renal tissue of patients with calcium-containing kidney stones is significantly lower than that in normal kidney tissue of stone free persons. |
PDF Full Text Request