Klotho Hypermethylation In Kidney Biopsy Tissue And Peripheral Blood Mononuclear Cells In Chronic Kidney Disease Patients By Pyrosequencing

Part I Klotho hypermethylation in chronic kidney disease patients by pyrosequencingBackgroundDNA methylation is an epigenetic regulation of gene expression. Pyrosequencing is a qualitative and quantitative analysis technology of DNA sequences with high accuracy. Expression of Klotho is decreased in chronic kidney disease(CKD) patients and mice, its mechanism is still not fully understood, the Klotho gene promoter hypermethylation may play a role in it.The purpose of this part of our study was to analyze Klotho gene promoter hypermethylation status in patients with chronic kidney disease by pyrosequencing.MethodCKD patients admitted to Shanghai Zhongshan hospital between April2012and September2012for renal biopsy and with informed consent were included in the study. Patients with diabetes, autoimmune diseases, chronic liver disease,active infection,malignant tumor, smoking and those who had used corticosteroids or immunosuppressants were excluded. Klotho hypermethylation in kidney biopsy tissue and peripheral blood mononuclear cells was detected by pyrosequencing.The result was compared to47nephrectomy specimens and PBMC of48healthy volunteers.ResultsThere were24males and23females among47CKD patients and the mean value of their age,24hour urine protein and eGFR was42.89±13.91,1.94(IQR0.85-3.48) g/24h and31.30(IQR13.59-60.52) ml/min/1.73m2respectively. There were25males and22females among kidney biopsy tissue controls and the mean value of their age,24hour urine protein and eGFR was48.23±9.35,0.03(IQR0.00-0.19)g/24h and79.70(IQR70.74-101.35) ml/min/1.73m2respectively and those of healthy volunteers was36.84±10.09,0.01(IQRO.00-0.10) g/24h and101.09(IQR91.55-115.34) ml/min/1.73m2respectively, male to female ratio is1:1.Among47CKD patients’ kidney biopsy tissue, Klotho gene promoter CpG islands hypermethylation was detected in36cases (76.6%) and Klotho gene promoter CpG islands hypermethylation was not detected in47controls. Klotho hypermethylation level of kidney biopsy tissue in CKD petients was significantly higher than controls(17.04±6.42vs.9.34±2.43%, P<0.001). Among47CKD patients’PBMC, Klotho gene promoter CpG islands hypermethylation was detected in28cases(59.6%) and Klotho gene promoter CpG islands hypermethylation was not detected in48controls. Klotho hypermethylation level of PBMC in CKD petients was significantly higher than healthy controls(14.19±5.86vs.6.90±2.39%, P<0.001).ConclusionKlotho hypermethylation levels in kidney biopsy tissue and PBMC in CKD petients were significantly higher than controls, suggesting hypermethylation was probably one of the mechanisms for low expression of Klotho in CKD patients. Part Ⅱ Assessment of Klotho hypermethylation of kidney biopsy tissue by Klotho methylation level of peripheral blood mononuclear cells in chronic kidney disease patientsBackgroundSome researches showed the methylation level of PBMC in patients with tumor could be applied as biomarker for methylation level of tumor tissue. However, the association between them might be different due to the different detection methods, the target DNA fragment and the type of tumor. This section of our study was designed to explore the evaluation role of promoter methylation rate of Klotho gene in peripheral blood mononuclear cells (PBMC) to the promoter hypermethylation of Klotho gene in the kidney tissue in patients with chronic kidney disease.MethodCKD patients admitted to Shanghai Zhongshan hospital between April2012and September2012for renal biopsy and with informed consent were included in the study. Patients with diabetes, autoimmune diseases, chronic liver disease, active infection, malignant tumor, smoking and those who had used corticosteroids or immunosuppressants were excluded. Klotho gene promoter hypermethylation was detected by pyrosequencing. The percentage of area of tubular atrophy and interstitial fibrosis in renal cortex was evaluated by two pathologists.ResultsThere were24males and23females among47CKD patients and the mean value of their age,24hour urine protein and eGFR was42.89±13.91,1.94(IQR0.85-3.48) g/24h and31.30(IQR13.59-60.52) ml/min/1.73m2respectively.The promoter methylation rates of the Klotho gene in the PBMC of CKD patients showed significantly positive association with that in the kidney biopsy tissue (r=0.811, P <0.001). The promoter methylation rates of Klotho gene in kidney biopsy tissue and PBMC of patients appeared significantly negative association with eGFR, respectively (r=-0.827, P<0.001; r=-0.626, P<0.001). The promoter methylation rates of Klotho gene in the kidney biopsy tissue and PBMC of CKD patients were found significantly positive association with tubulointerstitial fibrosis area (r=0.865, P<0.001; r=0.748, P<0.001) and no significant association with24-hour urine protein.ConclusionThe promoter methylation status of Klotho gene in PBMC showed positive association with that in the kidney biopsy tissue of patients with chronic kidney disease.It could be used as a non-invasive evaluation index for the promoter methylation status of Klotho gene in kidney tissue. The promoter methylation of Klotho gene was associated with decrease of eGFR and tubulointerstitial fibrosis. Further studies will be required to explore the association of promoter methylation of Klotho gene with the progression of CKD. Part Ⅲ Relationship between Klotho methylation level of peripheral blood mononuclear cells and clinical-pathological injury in IgA nephropathy patientsBackgroundIgA nephropathy is the most common primary chronic glomerulonephritis.Our previous study showed that Klotho gene promoter methylation rates were significantly increased in peripheral blood mononuclear cells (PBMC) of patients with chronic kidney disease and it showed significantly positive association with that of kidney tissue,which suggesting it could be used as a non-invasive evaluation index for the promoter methylation status of Klotho gene in kidney tissue.This study was performed to investigate the relationship between promoter methylation status of Klotho gene in PBMC and clinical-pathological injury in patients with IgA nephropathy.MethodPatients admitted to Shanghai Zhongshan hospital between August2012and February2013for renal biopsy and diagnosed as primary IgA nephropathy and with informed consent were included in the study.Klotho hypermethylation was examined by pyrosequencing. We assessed the glomerular, tubulointerstitial and vascular lesions according to semiquantitative Katafuchi score system. Then we analyzed the relationship between promoter methylation status of Klotho gene and the clinical-pathological injury of IgA nephropathy.ResultsThere were35males and53females among86IgA nephropathy patients and the mean value of their age, eGFR was35.77±11.0and86.41ml/min/1.73m2(IQR66.85-104.34ml/min/1.73m2). The promoter methylation rate of the Klotho gene in PBMC of patients with eGFR≥60was significantly lower than that of patients with eGFR<60(5.38±1.42%vs.6.46±1.98%, P<0.05).Analyzed by Single factor correlation analysis,the eGFR of IgA nephropathy patients showed significantly negative association with male, age,mean arterial blood pressure(MAP) and promoter methylation rate of the Klotho gene in PBMC (p=-0.216, P<0.05; r=-0.469, P<0.001;r=-0.331, P<0.01; r=-0.281, P<0.01). We did further analysis by Multiple regression analysis and saw eGFR was negatively associated with age and promoter methylation rate of the Klotho gene in PBMC(P<0.01; P<0.05) and not associated with male and MAP. The promoter methylation rate of the Klotho gene in PBMC of patients with IgA nephropathy showed significantly positive association with renal interstitial fibrosis (ρ=0.273,P<0.05) and no association with index of global ghomerulosclerosis,segmental lesions, ghomerular proliferation, tubular atrophy, interstitial inflammatory cell infiltration, arterillar thickening and arterillar hyalinosis.ConclusionRise of promoter methylation rate of Klotho gene in PBMC was independent risk factors for the decrease of eGFR and it was associated with the aggravation of renal interstitial fibrosis.The higher of promoter methylation rate, the more severe of renal interstitial fibrosis.