Association of Uremic Sarcopenia and Mitochondrial Copy Number and Its Clinical Correlates in Taiwanese Hemodialysis Patients

Research design and methods Subjects In this study, we shall recruit 200 patients on maintenance hemodialysis. In addition, 50 age- and sex-matched subjects act as controls. The HD patients have been on stable dialysis for at least 3 months HD patients are being treated three times weekly with standard bicarbonate dialysis (Na 138 mmol/L, HCO3 35 mmol/L, K 1.5 mmol/L, Ca 1.25 mmol/L, Mg 0.75 mmol/L) and by high-flux HD with synthetic membranes (dialysis filters surface area: 1.7 to 2.1 m2). All HD patients enroll in our study will be tested with DEXA and handgrip strength. Demographic data will be collected and laboratory examination are conducted as listed below.

Biochemical Determinations Blood samples for laboratory testing were drawn from the venous end of a vascular access at the beginning of the hemodialysis session and then stored at -80°C until time of analysis. The high sensitive C-reactive protein (hsCRP) levels were determined by a commercial immunoturbidimetric assay using a Hitachi autoanalyzer (model 7170). The detection limit and interval for CRP was 0.1 mg/L and 0.1-500.0 mg/L, respectively. The baseline serum albumin was measured by the bromocresol green method on a Hitachi autoanalyzer (model 7170). Serum levels of cholesterol, triglyceride, and low- and high-density lipoprotein cholesterol were determined by standard laboratory methods.

Determination of relative leucocyte mtDNA copy number The real-time PCR reaction was performed in triplicate for each reaction. The 10ul PCR reaction contains 1X TaqMan Universal PCR Master Mix buffer, 500nM of each primer, 200nM of TaqMan probe, and 0.2-2ng of total genomic DNA extract, PCR conditions are 2 min at 50℃, 10 min at 95℃, followed by 40 cycles of 15 s of denaturation at 95℃ and 60 s of annealing/extension at 60℃. Real-time quantitative analysis was performed on StepOne Real-Time PCR system(ABI). The primers and probes for real-time PCR are listed in Table 1. The PCR products that involve mitochondria DNA(MtDNA) and nuclear DNA.

Standard Curve:

Standard DNA solutions for the mitochondrial genome and the nuclear 18S rRNA dene (nDNA) were generated from PCR products cloned in a vector of pCR2.1-TOPO. Serial dilutions were made and RT-QPCR reactions were performed to construct the standard curve from Ct values and the number of copies of the standard plasmid DNA.