关键词:  线粒体DNA, 实时PCR, 肾脏疾病, 生物标志物, 微囊泡

Abstract:

ABSTRACT Objective: To establish a method to quantitate mitochondrial DNA (mtDNA) and to explore its potential as biomarker of kidney disease. Methodology:A specific mtDNA fragment was amplified by PCR and cloned to a T vector plasmid.This construct was used for generating a standard curve that relates copy nubmers with Ct values.The producibility and sensitivity were optimized using the mtDNA construct. The preliminary study to examine mtDNA content in the serum samples or urine microvesicles from the patients with focal segmental glomerulosclerosis (FSGS) was performed. The mtDNA content in the microvesicles from human immortalized podocytes treated with or without puromycin aminonucleosides (PAN) was also detected. Results: (1) The mtDNA copy nubmer assay by SYBR Green I-based real-time PCR was successfully set up with excellent reproducibility and sensitivity. (2) With this method, the serum levels of 10 FSGS patients and 6 healthy controls were examined, and there were significant lower serum mtDNA levels in FSGS patients compared with that in controls (P <0.05). (3) The copy numbers of MV mtDNA in a given volume of urine samples from 5 FSGS patients and 5 healthy controls were also measured, respectively, and there was higher in patients with FSGS than that in controls; meanwhile the MV content in the urine samples of the FSGS patients was also higher than that of controls. (4) The mtDNA content in the microvesicles from human immortalized podocytes treated with or without PAN was compared, and the mtDNA content was lower in the MV of PAN-treated podocytes than that of untreated cells; in addition, the intracellular mtDNA content in PAN-treated podocytes was also lower. Conclusion:This method should be able to find its use in exploring the potential of mtDNA as a biomarker for kidney diseases.

Key words: mitochondrial DNA, real-time PCR, kidney diseases, biomarker microvesicles