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 × 105 copies/mL; p = 0.000). Subsequently, the MHD patients were classified into two groups based on the MtDNA median (4.34 × 105 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 × 105 copies/mL; p = 0.001) or hemodiafiltration (pre 4.85 vs. post 4.09 × 105 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.

Increased level of free-circulating MtDNA in maintenance hemodialysis patients: Possible role in systemic inflammation.

BACKGROUND: Mitochondrial DNA (MtDNA) exposed to the extracellular space due to cell death and stress has immunostimulatory properties. However, the clinical significance of circulating MtDNA in maintenance hemodialysis (MHD) patients and the precise mechanism of its emergence have yet to be investigated. METHODS: This cross-sectional study consisted of 52 MHD patients and 32 age- and sex-matched healthy controls. MHD patients were further categorized into high and low circulating cell-free MtDNA (ccf-MtDNA) groups based on the median value. Copy number of MtDNA was quantified using TaqMan-based qPCR. Plasma cytokines were measured using ELISA kits. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) in peripheral blood mononuclear cells (PBMCs) were detected using DCFH-DA or JC-1 staining. RESULTS: The copy numbers of ccf-MtDNA in patients with MHD were higher than those in healthy controls, and these alterations were correlated with changes of cytokines TNF-α and IL-6. Adjusted model in multivariate analysis showed that the presence of anuria and longer dialysis vintage were independently associated with higher levels of ccf-MtDNA. Meanwhile, although not statistically significant, an inverse correlative trend between urinary MtDNA and ccf-MtDNA was observed in patients with residual urine. Afterward, using PBMCs as surrogates for mitochondria-rich cells, we found that patients in the high ccf-MtDNA group exhibited a significantly higher ROS production and lower Δψm in cells. CONCLUSIONS: Our data suggested that changes in ccf-MtDNA correlate with the degree of inflammatory status in MHD patients, and that the excessive MtDNA may be caused by mitochondrial dysfunction and reduced urinary MtDNA excretion.

Altered levels of circulating mitochondrial DNA in elderly people with sarcopenia: Association with mitochondrial impairment.

BACKGROUND: Age-related chronic inflammatory process is often referred to as "inflammaging", which had been described in several human disorders, including sarcopenia. Recently, mitochondrial DNA (MtDNA) has moved into the spotlight as a "damage-associated molecular pattern" (DAMP) agent that can potentially elicit inflammation. Yet, the roles of this mitochondrial DAMP have never been investigated in sarcopenia. DESIGN: Cross-sectional study. PARTICIPANTS: From January 2021 to June 2021, elderly outpatients ≥65 years and able to finish a comprehensive geriatric assessment were recruited in our study. METHODS: Participants were divided into sarcopenia group and non-sarcopenia group according to the DXA scans and grip strength. Genomic DNA was extracted from plasma and peripheral blood mononuclear cells (PBMCs), and changes in MtDNA copies were quantified using qPCR. Plasma levels of inflammatory cytokines were measured using ELISA kits. Loss of mitochondrial membrane potential (Δψm) in PBMCs was analyzed using the fluorescent probe JC-1. RESULTS: Participants with sarcopenia were significantly older, more likely to be physically inactive, and had higher levels of circulating cell-free MtDNA (ccf-MtDNA) (all p < 0.05). After adjusting for potential confounders, ccf-MtDNA was independently associated with increased odds of sarcopenia (adjusted odds ratio (AOR), 1.576; p = 0.009). Furthermore, ROC curve analysis showed that ccf-MtDNA had an area under the curve (AUC) of 0.726 (95% CI: 0.607-0.844; p < 0.05) for distinguishing elderly subjects from sarcopenia. Compared with non-sarcopenia subjects, plasma interleukin (IL)-6 and IL-8 were significantly higher in sarcopenia subjects (both p < 0.05). By performing a correlation test, it was found that the level of IL-6 was positively correlated with ccf-MtDNA (r = 0.301; p < 0.05). Then, PBMCs were used as surrogates for mitochondria-rich cells, and the results showed that the relative amplification of MtDNA in PBMCs was significantly reduced (p < 0.05), whereas the depolarization of Δψm was significantly increased in sarcopenia subjects (p < 0.05). CONCLUSIONS: Taken together, our data suggested that circulating MtDNA might be a novel and important source of inflammatory stimuli potentially relevant for sarcopenia in elderly people, and this would provide an attractive therapeutic target to improve this disease.

Circulating Cell-Free Mitochondrial DNA: A Potential Blood-Based Biomarker for Sarcopenia in Patients Undergoing Maintenance Hemodialysis.

BACKGROUND Mitochondrial impairment and exaggerated inflammation are hallmarks of sarcopenia. Recently, cell-free mitochondrial DNA (cf-mtDNA) has been in the spotlight as an endogenous danger molecule that can potentially elicit inflammation. Yet, its actual impact on sarcopenia, especially in patients with maintenance hemodialysis (MHD), is still at an early stage of investigation. MATERIAL AND METHODS A total of 105 MHD patients were enrolled in this study. The subjects were classified into sarcopenia group (SP) and non-sarcopenia group (NSP) according to the DXA scan and grip strength. Plasma and peripheral blood mononuclear cells (PBMCs) were separated from whole blood. Circulating cf-mtDNA (ccf-mtDNA) was detected using Taq Man RT-qPCR. Cytosolic mtDNA and inflammation- and mitophagy-related genes in PBMCs were quantitated using SYBR Green RT-qPCR. ΔΨm was analyzed using the fluorescent probe JC-1. RESULTS ccf-mtDNA content was significantly higher in SP group than in NSP group. Multivariate regression analysis showed a significant correlation of ccf-mtDNA with sarcopenia after adjusting for potential confounders. A similar trend of increased mtDNA was also observed in the mitochondria-free cytoplasm of PBMCs from SP patients, together with higher expression of TLR9 and IL-6 in this group. Next, using PBMCs as surrogates for mitochondria-rich cells, we found that ΔΨm was dramatically decreased in the SP group. In parallel, the mRNA levels of mitophagy-related genes Parkin and LAMP2 were increased in the SP group. CONCLUSIONS The results obtained demonstrated that ccf-mtDNA, as a potential driver of inflammatory component, may be involved in the pathogenesis of the MHD-related sarcopenia.