Prognosis Of Patients With Chronic Renal Diseases And The Mechanism Of Vascular Calcification

Kidney disease and renal cell carcinoma are common chronic diseases of kidney and are global diseases that affect human health.The prevalence of chronic kidney disease in China was 10.8%and that of end-stage renal disease was 0.03%,with increasing year by year.Renal cell carcinoma is one of the most common malignant tumors of the urinary system,30%of renal cell carcinoma patients have metastasis at the time of diagnosis,and the treatment effect is poor.The damage of kidney intrinsic cells is the common morbidity basis and the common morbidity mechanism of chronic kidney diseases,including inflammation,oxidative stress,autoimmune abnormalities and so on.Renal inherent cells have been exposed to various mutagenic active substances and toxicity of metabolites in urine,make the body produce and accumulate a large number of reactive oxygen species?ROS?,high levels of ROS can attack the mitochondrial DNA?mt DNA?led to the fracture,the integrity mt DNA,affected by respiratory chain related downstream gene transcription and translation process,the respiratory chain disorders increased ROS levels further oxidation products piled up and aggravating mt DNA mutation,form a vicious circle,cause chronic kidney disease.The occurrence and prognosis of chronic kidney diseases?renal cell carcinoma and CKD?are different attribute of the different genes and proteins of oxidative stress damage.Patients with renal cancer are mainly treated by surgery,not sensitive to radiotherapy and chemotherapy,and have a poor prognosis.And is because of the glomerular filtration function will be reduced for patients with chronic kidney disease?CKD?and appear a series of water,electrolyte and acid-base balance disorders and abnormal hormone secretion,decrease urinary phosphorus excretion,bone adjust abnormal hormone secretion increase osteoclast activity,bone phosphorus released into the blood,and then appear hyperphosphatemia,causing membrane in vascular smooth muscle cells?VSMCs?transdifferentiation,namely VSMCs osteogenesis sample change.Osteogenic transformation of VSMCs induces and accelerates calcium and phosphorus crystal deposition in the vascular wall to form vascular calcification.Metabolic acidosis is a common clinical manifestation of chronic kidney disease.Acidosis activates osteoclast-mediated bone calcium release,promotes physical and chemical dissolution of bone,and inhibits bone formation through osteoblasts.The pathogenesis of mesenchymal calcification is similar to bone formation.So,presumably,acidosis effects on bone can be extrapolated to the blood vessels in the membrane calcification in vitro recent experimental data to support this view,showed that p H lower can prevent calcium deposits in VSMCs but acidosis inhibiting calcium deposition mechanism is unclear in view of this,we proposed to investigate the mt DNA D-loop area single nucleotide polymorphisms in the role of chronic kidney disease and prognosis,and determined the acidosis in function and mechanism of the membrane calcification in vivo and in vitro.Part one:Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with the kidney survival time in chronic kidney disease patientsObjective:To investigate the association of SNPs in the D-loop of mt DNA with the kidney survival of CKD.Methods:The D-loop region of mt DNA was sequenced for 119 CKD patients from the inpatient of the Fourth Hospital of Hebei Medical University.The Kaplan-Meier method was used to identify disease outcome-associated SNPs in the D-loop of CKD patients.The Cox proportional hazards model was used to identify risk factors for the kidney survival of CKD.Results:In the present study,we identified 20 SNPs with a frequency higher than 5%and assessed the relationship of these SNPs with kidney survival time in CKD patients,a SNP of 146 was identified by log-rank test for statistically significant prediction of the kidney survival time.In an overall multivariate analysis,allele 146 was identified as an independent predictor of kidney survival time in CKD patients.The survival time of kidney in the CKD patients with 146C was significantly shorter than that of kidney in CKD patients with 146T?relative risk,2.336;95%CI,1.319-3.923;P=0.001?.Conclusions:SNPs in the D-loop can predict the kidney survival of CKD patients.Analysis of genetic polymorphisms in the mitochondrial D-loop can help to identify CKD patient subgroup at high risk of a poor disease outcome.Part two:Single nucleotide polymorphisms in the D-loop region of outcomeObjective:To investigate the relationship between single nucleotide polymorphisms in the D-loop region of mitochondrial DNA and renal cell carcinoma outcome.Methods:The D-loop region of mt DNA was sequenced for 75 patients with Renal cell carcinoma from the inpatient of the Fourth Hospital of Hebei Medical University.The Kaplan-Meier method was used to identify disease outcome-associated SNPs in the D-loop of CKD patients.The Cox proportional hazards model was used to identify risk factors for the kidney survival of CKD.Results:A total of 75 patients were enrolled in this study.A review was conducted every six months over a five–year period.None of these patients was lost to follow-up in five years.None of these patients received adjuvant chemotherapy or radiation therapy following RCC resection.The relationship between the data collected during the 5-year follow–up and patients'clinical characteristics was analyzed using the Kaplan–Meier method and the log-rank test.Sex and TNM classification were not statistically significant predictors of postoperative survival time;however,age and tumor size were correlated with survival time in these patients?Table 1?.The survival rates of patients with age?55 years was higher than those with age>55 years.Tumor size showed an association with survival rates when patients with a tumor diameter?5cm were compared to those with a tumor diameter<5cm.These data demonstrate that age and size of the tumor are good predictors of RCC outcome.In our previous study,we identified 14 SNPs with a frequency higher than 5%and investigated the association of theses SNPs with the risk of RCC without considering relationships between these SNPs and disease outcome.Recently,we assessed the association of these SNPs with outcome of these RCC patients.RCC patients were divided into two groups on the basis of their genotype at each SNP site,the post-operational survival curve was plotted using the Kaplan-Meier method for all RCC patients at these sites.A dramatic difference in survival rate was found for allele 262?Fig.1?.The rare allele262T was associated with a significantly shorter?P<0.001?length of survival?Fig.1?.The allele 262 also belong to RCC risk associated SNPs we identified before.We used multivariate analysis by the Cox proportional hazards model with the prediction factors,including one SNP,age and tumor size.As shown in Table 2,the 262 alleles,age and tumor size were identified as independent predictors for RCC outcome.The length of survival for patients with the rare allele 262T genotype was significantly less than that for patients with the frequent allele 262C?relative risk,2.136,95%CI,1.863-2.449;P<0.001?at the 262 site.These data demonstrated the strong prediction power of nucleotide 262 on outcome for RCC patients.Conclusions:SNPs in the mt DNA D-loop were found to be independent prognostic markers for RCC outcome.The analysis of genetic polymorphisms in the D-loop might help to identify patient subgroup at high risk for a disease outcome,thereby helping to refine therapeutic decisions in RCC patients.Part three:Extracellular acidosis suppresses calcification of vascular calcium channelsObjective:To determine the roles and mechanism of acidosis on medial calcification in vitro and in vivo.Methods:in vivo experiment:renal failure was induced by?5/6?nephrectomy,standard two-step operation,and vascular calcification was induced by calcitriol.The rats were randomly divided into four experimental groups:sham operation group?n=6,as control group?,5/6Nx+carrier+distilled water group?n=5?,5/6Nx+calcitriol 600 ng/kg/day?n=5?,5/6Nx+calcitriol 600 ng/kg/day+acidosis group?n=5,0.28mmol/L NH₄Cl concentration in drinking water?.The solution of NH₄Cl concentration0.28mmol/l was added into drinking water to induce 5/6nx metabolic acidosis.Blood was taken from the aorta without oxygen.The aorta was divided into three parts.The L-type calcium channel?LTCC??3 subunit and Runx2 in the cranial segment of the aorta were studied by means of von Kossa and immunohistochemistry.The middle part was used for the evaluation of calcification.The last part was for the quantification of LTCC?3 subunits and Runx2 m RNA.Creatinine and blood urea nitrogen concentrations were measured by spectrophotometry.p H and bicarbonate were determined by a selective electrode.The calcium content was determined by microplate reader.In Vitro Experiment:Rat VSMCs were obtained from the tunica media of an adult male Sprague Dawley rat thoracic aorta.The third-generation cells were identified for future experiments.Alizarin red staining was used to detect calcification of vascular smooth muscle cells.The calcium content of vascular smooth muscle cells was measured by calcium assay kit.The activity of ALP in vascular smooth muscle cells was determined by ALP activity assay kit.Intracellular calcium ion concentration in vascular smooth muscle cells was detected by calcium ion probe.The protein expression of LTCC?3 subunits and Runx2 in vascular smooth muscle cells was determined by immunohistochemical and western blotting,and the gene expression of those measured by RT-PCR.Results:No deaths were recorded during the first 10 days in any groups.At 16 days,one of the?1/6?rats in 5/6 Nx+CTR group died and one?1/10?of the rats in 5/6 Nx group died.No statistically significant differences between groups were seen for body weight,water and food consumption.Plasma creatinine was significantly?P<0.05?increased in all 5/6 Nx groups.Nephrectomy alone did not have an effect on the acid–base balance of the rats.Compared with non-acidotic groups,plasma p H and bicarbonate significantly?P<0.05?decreased in 5/6 Nx+CTR+AC group.Calcium content in 5/6Nx+CTR was higher than that in sham and 5/6 Nx?9.66±1.62 vs 2.33±0.42,2.41±0.41,P<0.05,respectively?.Received with NH₄Cl effectively decreased Calcium content?4.94±0.74,vs 9.66±1.62,P<0.05 vs 5/6 Nx+CTR?.Von Kossa-stained tissue sections of the aorta showed in Figure 2A,calcium deposits in rats treated with NH₄Cl was lower than those in 5/6 Nx+CTR.The calcium content of VSMCs in the?-GP+p H 7.1 group was lower than that in the?-GP+p H 7.4 group.The results of Alizarin red stain were in line with that of calcium content.The m RNA and protein levels of LTCC?3 subunit in VSMCs was lower in the?-GP+p H 7.1 group than that in?-GP+p H 7.4group?P<0.05?.Consistently,Ca²⁺influx is lower in?-GP+p H7.1 group than that in?-GP+p H7.4 group?P<0.05?.The effect of calcifying medium on Runx2,a vital marker of osteoblast differentiation,was markedly enhanced following?-GP treatment when the p H was 7.4,while this effect was inhibited by acidosis and in the presence of verapamil as well.ALP activity had the same trend,acidosis provided a significant inhibition on ALP activity,compared with cells grown in calcification medium with p H 7.4.Addition of verapamil into the calcification medium also inhibited the increase of ALP activity.LTCC?3 subunit and Runx2 expression were upregulated in 5/6 Nx+CTR group.There was a gradual decrease in LTCC?3 subunit and Runx2expression in 5/6 Nx+CTR+AC group.Immunoreactivity score?IRS?showed that LTCC?3 subunit and Runx2 IRS in 5/6 Nx+CTR group was higher than that in sham and 5/6 Nx+V group?LTCC?3 subunit IRS:6.20±1.64,vs2.16±0.75,P<0.05 vs sham;vs 3.00±1.00,P<0.05 vs 5/6 Nx+V;Runx2IRS:6.60±2.51,vs1.83±0.98,P<0.05 vs sham;vs2.60±0.55,P<0.05 vs 5/6Nx+V?.Acidosis significantly decreased LTCC?3 subunit and Runx2expression?LTCC?3 subunit,vs3.83±1.33,P<0.05 vs 5/6 Nx+CTR;Runx2,vs3.00±1.45,P<0.05 vs 5/6 Nx+CTR?.Conclusions:The study demonstrate that acidosis prevented the vascular medial calcification induced by calcitriol administrated to uremic through LTCC/Ca²⁺/Runx2 signaling pathway.The results could be useful for considering strategies for the prevention VC and oral sodium bicarbonate appropriately.