Role Of Mitochondrial DAMPs In Kidney Injury And The Potential Of Mitochondrial DNA As Biomarker Of Kidney Diseases

Objctive:Mitochondria are the organelles in eukaryotic cells that supply energy to the cells by producing ATP.In addition,mitochondria are involved in other cellular processes,such as apoptosis,oxidative stress and the maintenance of calcium homeostasis.In recent years,it has been shown that many components of mitochondria are damage associated molecular parttens(DAMPs),and can act on pattern related receptors(PRRs)of immune cells or parenchymal cells,thereby inducing inflammatory responses and tissue injury.Due to the facts that kidney infiltrating immune cells and parenchymal cells express various PRRs and that high levels of mitochondrial DAMPs are found in the circulation of patients with trauma or bum who often exhibit kidney injury,we investigate whether circulating mitochondrial DAMPs can induce kidney injury.In addition,we attempt to establish a method to quantitate mitochondrial DNA(mtDNA)to explore its potential as biomarker of kidney disease.Methodology:Part One:The role of mitochondrial DAMPs in kidney injury.Preparation of mitochondrial DAMPs:1)the specific fragment of mtDNA with a proven immune activity was prepared by PCR,2)rat liver mitochondrial debris soluble content(MTD)was prepared using sucrose gradient centrifugation followed by disruption by sonication.Animal experiments:mtDNA and MTD were injected by tail vein to Balb/c mice or Wistar rats;urine,blood,and renal and lung biopsies were collected at different time points after injection.Urine protein,serum creatinine,the ratio of peripheral blood neutrophils,and kidney or lung pathology were determined.In vitro cell experiments:cultured human immortalized podocyte were treated with mtDNA or MTD and p38 MAPK activity(phosphorylation)was examined by immunoblotting.Part Two:Development of SYBR Green I-based real-time PCR assay of mtDNA copy number and its application in kidney disease diagnosis.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 reproducibility and sensitivity were optimized using the mtDNA construct.Preliminary study to examine mtDNA content in the serum samples or urine microvesicles from the patients with focal segmental glomerulosclerosis(FSGS)was performed.We also examined the mtDNA content in the microvesicles from human immortalized podocytes treated with or without puromycin aminonucleosides(PAN).Results:Part One:1)Injected exogenous mtDNA was rapidly depleted within 3 h (?) after injection probably due to the presence of a large amount of DNases in circulation;Urinary albumin elevation could be observed in the mice;2)a small amount of MTD(from 1-5%of the liver)injection did not induce proteinuria in the mice,but slightly widened alveolar interstitium and caused infiltration of a small number of mononuclear cells in it;3)the injection of a larger amount of MTD(from 30%of the liver)caused acute inflammation,lung mild exudative changes,focal tubular brush border loss and instantaneous proteinuria in the rats;4)Both mtDNA and MTD were capable of activating p38 pathway in the human immortalized podocytes.Part Two: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,we examined the serum levels of 10 FSGS patients and 6 healthy controls and found that FSGS patients had significantly lower serum mtDNA levels compared with the controls(P<0.05),3)we also measured the copy numbers of MV mtDNA in a given volume of urine samples from 5 FSGS patients and 5 healthy controls,respectively,and found the former higher than the latter;meanwhile the MV content in the urine samples of the FSGS patients was also found higher than that of healthy controls,4)we compared the mtDNA content in the microvesicles from human immortalized podocytes treated with or without PAN and found that the mtDNA content in the MV of PAN-treated podocytes was lower than that of untreated cells;in addition,the intracellular mtDNA content in PAN-treated podocytes was also lower than that of untreated cells.Conclusions:1)Mitochondrial DAMPs in circulation alone can have only subtle injurious effect on kidney,hence the elevated circulating mitochondrial DAMPs in the patients with trauma or burn may not the major factors responsible for the observed kidney injury or they require additional factors to induce kidney injury.2)MtDNA content in the biofluids or renal cells of the patients with a kidney disease can change,therefore it could be a promising biomarker for the diagnosis of kidney disease.