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

Abstract 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.


| INTRODUC TI ON
Progressive decline in renal function leads to chronic kidney disease (CKD) and, ultimately, end-stage renal disease (ESRD). 1 Patients at this stage require renal replacement therapy, which is usually hemodialysis, to survive. Hemodialysis patients usually suffer from high cardiovascular morbidity and mortality due to chronic systemic inflammation. 2 Nevertheless, in many cases, plasma inflammatory cytokines increase with disease progression independent of antigenic stimulation, such as bacterrial DNA fragments. 3 In fact, inflammation not only occurs in response to pathogens but can also be induced without active infection. This so-called "sterile inflammation" may be triggered by damage-associated molecular patterns (DAMPs) derived from tissue injury and cell breakage.
Mitochondria play a central role in metabolism and are unique organelles that carry their own genome (mitochondrial DNA, MtDNA). 4 According to the endosymbiotic theory, mitochondria may have originated from energy-producing bacteria. Thus, most of MtDNA contains inflammatogenic unmethylated CpG motifs similar to those in bacterial DNA. 5 In this regard, if MtDNA is released out of the cell and becomes extracellular MtDNA, it may trigger an inflammatory response by binding to pattern recognition receptors (PRRs) that typically recognize DNA from bacterial pathogen. 6 Recent research has implicated MtDNA as a DAMP and marked increase in extracellular MtDNA was already found in different pathological disorders, such as trauma, sepsis, aging, caner, and immune-mediated disease, which are characterized by a chronic inflammatory status. [7][8][9][10][11] Likewise, an increased free-circulating MtDNA has also been reported in patients with kidney disease. 12,13 However, the role of circulating cell-free MtDNA (ccf-MtDNA) in the process of chronic inflammation in MHD patients and the precise mechanism of its emergence remain to be defined.
Abnormal mitochondrial structure has been demonstrated in muscle, heart, liver, lung, endothelial cells, and monocytes under uremic conditions. [14][15][16][17][18][19] According to previous research, it is not surprising that upon mitochondrial damage, its DAMP content can be easily released into the extracellular space, which has been proven to be a trigger for inflammatory response and oxidative injury. 20 Peripheral blood mononuclear cells (PBMCs) are abundantly rich in mitochondria. Recently, tests of PBMCs have been proposed to offer valid information about "general" mitochondrial health. [21][22][23] Hence, we chose PBMCs in lieu of tissue biopsy collection to assess the integrity of mitochondria and determine the relationship between ccf-MtDNA and mitochondria.
In the current study, we first sought to determine the association between ccf-MtDNA and inflammatory cytokines in MHD patients. Furthermore, we attempted to decipher the potential mechanisms affecting its levels. These findings provide novel mechanistic insights into the linkage between released MtDNA and inflammation, and enable the identification of new therapeutic targets for this disease.

| Participants
A cross-sectional study design was conducted in this research. All subjects were recruited from First Affiliated Hospital of Chengdu Medical College between January 2021 and May 2021. Inclusion criteria for the MHD group were as follows: (1) age above 40 and (2) patients undergoing regular hemodialysis prescription, three times a week, at least 6 months. The age-and sex-matched healthy control (HC) group included donors who attended routine health examinations at the same hospital; these patients had no history or clinical evidence of any renal diseases. The exclusion criteria for all subjects were as follows: (1)

| Sampling strategy
Sample size was determined prior to data collection by a power analysis (G*Power, Version 3.0). According to the previous research, 13 group sample sizes of 29 (MHD) and 29 (HC) achieved 95% power to reject the null hypothesis when the ccf-MtDNA mean difference was 86.4 with standard deviations of 101.3 for the MHD group and 8.5 for the HC group, with a significance level (alpha) of 0.01 using a two-sided two-sample unequal-variance t test. Power analyses indicated that the sample size in this study was appropriate

| Blood and urine sampling
Blood samples from hemodialysis patients were obtained immediately prior to dialysis. Simultaneously, urine samples were collected.
Samples were separated into cellular and cell-free fractions within 2 h of the samples being drawn.  termined by calculation from this standard curve, as previously described. 24 Results were presented as MtDNA×10 5 copies per μl.

| Measurement of reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) by flow cytometry
PBMCs were isolated from whole blood using the Ficoll-Hypaque density gradient separation technique, and then, the PBMCs were suspended in PBS at a final concentration of ~105 cells/ml for flow cytometry. Cellular ROS production was determined with

| Statistical analyses
Data are expressed as means ± standard deviation (SD) or medium (25th and 75th percentiles) for continuous variables. The distribution of the data was tested using the Kolmogorov-Smirnov test. Normally distributed data were analyzed using an independent t test. Nonnormally distributed data were analyzed using the Mann-Whitney test. Spearman's tests were applied to determine the associations between continuous variables. Categorical data between two groups were compared using the chi-square test (χ 2 test) with Fisher's exact test. Logistic regression was used to describe and explain the relationship between dependent binary variables and independent variables. Values of p < 0.05 were considered statistically significant. All statistical analyses were conducted using SPSS 18.0.

| Participant characteristics
The study comprised 52 subjects with ESRD undergoing hemodialysis therapy thrice a week and 32 normal healthy controls. Groups were matched for age and sex. Baseline clinical characteristics and laboratory data of the study population are summarized in Table 1.
Of the included MHD patients, 55.8% were male, and the mean age was 51.59 ± 11.15 years. Glomerulonephritis (54.1%) was the most common primary cause of ESRD, followed by diabetic nephropathy (21.1%), hypertensive nephropathy (11.5%), and nephrosclerosis (5.7%). The mean dialysis vintage was 3.95 ± 1.74 years, and a total of 38 (73.0%) patients presented with anuria. There was a statistically significant difference between the two groups in terms of Cr, SBP, FBS, Ab, Hb, and VitD (all p < 0.05).

| Level of ccf-MtDNA and its relationship with cytokines in MHD patients
Free-circulating MtDNA was extracted from the plasma of all subjects. Cytb was applied to evaluate the quantity of MtDNA. As shown in Table 1, the content of ccf-MtDNA in MHD patients was significantly higher than that in healthy controls (p < 0.05). Then, to evaluate systemic inflammation, the levels of circulating proinflammatory cytokines TNFα, IL-1β, and IL-6 in the plasma were measured ( Table 2). Among these cytokines, TNFα (p = 0.002) and IL-6 (p < 0.001) levels were significantly higher in MHD groups than controls, whereas no significant difference was observed in IL-1β.

| Urinary MtDNA and association with ccf-MtDNA in MHD patients
In the present study, we confirmed that patients with anuria were more likely to have higher levels of ccf-MtDNA. This prompted us to investigate whether the excessive ccf-MtDNA might be partly due to the lack of urinary MtDNA excretion. We measured urinary MtDNA content in patients with residual (n = 14) and found that MtDNA was readily detectable in urinary supernatant (median, 0.21 × 10 5 copies/μl; range, 0.01-1.16 × 10 5 copies/μl). Meanwhile, although not statistically significant, an inverse correlative trend was observed between urinary MtDNA and ccf-MtDNA (Figure 2; r = −0.398, p = 0.158). As previously reported, kidney is responsible for scavenging circulatory MtDNA via glomerular hyperfiltration. 25 Based on this, it is tempting to speculate that preserving more renal function might be an effective way to eliminate MtDNA in the blood.

| Mitochondria appear to be impaired more severely in patients with higher ccf-MtDNA
MtDNA is packaged into nucleoids in mitochondria. In order to define the relationship between mitochondrial impairment and MtDNA Abbreviations: Ab, albumin (g/L); ccf-MtDNA, circulating cell-free mitochondrial DNA (10 5 × 10 5 copies/μl); Cr, creatinine (μmol/L); DBP, diastolic blood pressure (mmHg); FBS, fasting blood sugar (mmol/L); Hb, hemoglobin (g/L); LDL-C, low-density lipoprotein cholesterol (mmol/L); NR, not recorded; SBP, systolic blood pressure (mmHg); TC, total cholesterol (mmol/L); TG, triglyceride (mmol/L); VitD, vitamin D (ng/ml).  Many chronic diseases such as cardiovascular disease, cancer, chronic kidney disease, diabetes, and neurodegenerative diseases, among others, are initiated or worsened by systemic inflammation. 27,28 Nevertheless, evidence from recent works suggest that in many case, plasma inflammatory cytokines increase with disease progresses independently of antigenic stimulation, such as infections. 29 Mitochondria are derived from bacteria that were engulfed by the ancestors of today's eukaryotic cells more than a billion years ago and now produce virtually all of the cellular energy. 30 Due to their bacterial ancestry, mitochondrial-derived moleculars, such as

TA B L E 2 Plasma cytokine levels among the groups
MtDNA, cardiolipin, and formyl peptides, can act as DAMP agents, triggering the same pathways that respond to pathogen-associated molecular patterns (PAMPs). 10 That is, mitochondria not only participate in danger signaling inside the cell but are also a major source of molecule able to activate an innate immune response.
Recently, it has been shown that ccf-MtDNA can be detected in many pathological conditions which are characterized by a chronic inflammatory status. [7][8][9][10][11] Similarly, the clinical significance of ccf-MtDNA in kidney disease has also been reported. 12,13 Despite that, the possible contribution of this mitochondrial DAMP to the inflammatory milieu that characterizes MHD has not been clarified clearly.
In our study, we found that ccf-MtDNA was significantly elevated in MHD patients, and that IL-6 and TNFα had a positive correla-

| CON CLUS IONS
In conclusion, we found that ccf-MtDNA is highly implicated in the immune response, and its level may be causally associated with urinary excretion and mitochondrial damage in MHD patients. A schematic diagram of the proposed pathway is shown in Figure 4.
From a future perspective, identification of the role of ccf-MtDNA in diseases is importance for designing new therapeutic strategies against MtDNA or its receptors to reduce a harmful immune activation.

ACK N OWLED G EM ENT
The study was founded by Sichuan Provincial Cadre Health Research

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data used to support the findings of this study are included within the article.