Association of circulating MtDNA with CVD in hemodialysis patients and in vitro effect of exogenous MtDNA on cardiac microvascular inflammation

BACKGROUND: Chronic kidney disease (CKD) patients sustain a fairly high prevalence of cardiovascular disease (CVD). Microvascular inflammation is an early manifestation of CVD, and the released mitochondrial DNA (MtDNA) has been proposed to be a crucial integrator of inflammatory signals. Herein, the aim of this study was to determine the relationship between CVD, microvessel, and circulating MtDNA in the settings of uremia. METHODS: Forty-two maintenance hemodialysis (MHD) patients and 36 health controls were enrolled in this study. Plasma cell-free MtDNA was detected by TaqMan-based qPCR assay. CVD risk markers including high-sensitive C-reactive protein (Hs-CRP), monocyte chemoattractant protein-1 (MCP-1), fibrinogen, and erythrocyte sedimentation rate (ESR) were measured by standard assays. Ten-year CVD risk was calculated from the Framingham risk score (FRS) model. In vitro study, human cardiac microvascular endothelial cells (HCMECs) were incubated with normal or uremic serum, with or without exogenous MtDNA. Intracellular toll-like receptor 9 (TLR9), adhesion molecule 1 (ICAM-1), MCP-1 and tumor necrosis factor-α (TNF-α) and cytosolic MtDNA were detected by qPCR. RESULTS: Plasma MtDNA in MHD patients was significantly higher than healthy controls (4.74 vs. 2.41 × 10(5) copies/mL; p = 0.000). Subsequently, the MHD patients were classified into two groups based on the MtDNA median (4.34 × 10(5) copies/mL). In stratified analyses, the levels of Hs-CRP (5.02 vs. 3.73 mg/L; p = 0.042) and MCP-l (99.97 vs. 64.72 pg/mL; p = 0.008) and FRS (21.80 vs. 16.52; p = 0.016) in the high plasma MtDNA group were higher than those in the low plasma MtDNA group. In vitro study, we found that exogenous MtDNA aggravated uremic serum-induced microvascular inflammation (ICAM-1 and TNF-α) in HCMECs (all p < 0.05). Besides, the addition of MtDNA to the medium resulted in a further increase in cytosolic MtDNA and TLR9 levels in uremic serum-treated cells (all p < 0.05). In patients with MHD, MtDNA levels in plasma were significantly reduced after a single routine hemodialysis (pre 4.47 vs. post 3.45 × 10(5) copies/mL; p = 0.001) or hemodiafiltration (pre 4.85 vs. post 4.09 × 10(5) copies/mL; p = 0.001). These two approaches seem similar in terms of MtDNA clearance rate (21.26% vs. 11.94%; p = 0.172). CONCLUSIONS: Overall, the present study suggests that MtDNA released into the circulation under the uremic toxin environment may adversely affect the cardiovascular system by exacerbating microvascular inflammation, and that reducing circulating MtDNA might be a future therapeutic strategy for the prevention of MHD-related CVD.