Article Skeletal Muscle Mitochondrial Dysfunction Is Present in Patients

CJASN ePress. Published on June 26, 2020 as doi: 10.2215/CJN.10320819 Article

Skeletal Muscle Mitochondrial Dysfunction Is Present in Patients with CKD before Initiation of Maintenance Hemodialysis

Jorge L. Gamboa ,1 Baback Roshanravan,2 Theodore Towse,3 Chad A. Keller,1 Aaron M. Falck,1 Chang Yu,4 Walter R. Frontera,5,6 Nancy J. Brown ,1 and T. Alp Ikizler 7,8

Due to the number of Abstract contributing authors, Background and objectives Patients with CKD suffer from frailty and sarcopenia, which is associated with higher the affiliations are morbidity and mortality. Skeletal muscle mitochondria are important for physical function and could be a target to listed at the end of prevent frailty and sarcopenia. In this study, we tested the hypothesis that mitochondrial dysfunction is associated this article. with the severity of CKD. We also evaluated the interaction between mitochondrial function and coexisting comorbidities, such as impaired physical performance, intermuscular adipose tissue infiltration, inflammation, Correspondence: Dr. Jorge L. Gamboa, and oxidative stress. Vanderbilt University Medical Center, 2222 Design, setting, participants, & measurements Sixty-three participants were studied, including controls (n521), Pierce Avenue, 561B- patients with CKD not on maintenance hemodialysis (CKD 3–5; n520), and patients on maintenance hemodialysis PRB, Nashville, TN (n522). We evaluated in vivo knee extensors mitochondrial function using 31P magnetic resonance spectroscopy to 37232. Email: jorge. gamboa@ obtain the phosphocreatine recovery time constant, a measure of mitochondrial function. We measured physical vanderbilt.edu performance using the 6-minute walk test, intermuscular adipose tissue infiltration with magnetic resonance imaging, and markers of inflammation and oxidative stress in plasma. In skeletal muscle biopsies from a select number of patients on maintenance hemodialysis, we also measured markers of mitochondrial dynamics (fusion and fission).

Results We found a prolonged phosphocreatine recovery constant in patients on maintenance hemodialysis (53.3 [43.4–70.1] seconds, median [interquartile range]) and patients with CKD not on maintenance hemodialysis (41.5 [35.4–49.1] seconds) compared with controls (38.9 [32.5–46.0] seconds; P50.001 among groups). Mitochondrial dysfunction was associated with poor physical performance (r50.62; P50.001), greater intermuscular adipose tissue(r50.44; P50.001), andincreased markers of inﬂammation andoxidative stress (r50.60;P50.001).We found mitochondrial fragmentation and increased content of dynamin-related protein 1, a marker of mitochondrial ﬁssion, in skeletal muscles from patients on maintenance hemodialysis (0.86 [0.48–1.35] arbitrary units (A.U.), median [interquartile range]) compared with controls (0.60 [0.24–0.75] A.U.).

Conclusions Mitochondrial dysfunction is due to multifactorial etiologies and presents prior to the initiation of maintenance hemodialysis, including in patients with CKD stages 3–5. CJASN 15: ccc–ccc, 2020. doi: https://doi.org/10.2215/CJN.10320819

Introduction dysfunction is associated with slow walking speed, low Patients with CKD frequently present with skeletal physical function, and fatigue in older adults without muscle atrophy and weakness, both components of CKD (6). We and others have described muscle mito- sarcopenia (1,2). These factors contribute to physical chondrial abnormalities in patients with ESKD on MHD, frailty, a phenotype associated with fatigue, muscle such as decreased mitochondrial content, increased weakness, and low physical activity (3). It was mitophagy, and improper mitochondrial biogenesis reported that 73% of patients with advanced CKD (729). Other investigators have reported mitochondrial are frail at the time of initiation of maintenance dysfunction in patients on MHD compared with healthy hemodialysis (MHD) (4). Frailty and the coexisting controls using 31P magnetic resonance spectroscopy sarcopenia are associated with greater morbidity and (31P-MRS) (10212). In contrast, there is a paucity of mortality in these patients (2,4). data in patients with CKD not on MHD, with one study Mitochondria are important for proper skeletal muscle showing no difference in mitochondrial function between function, and there is evidence suggesting that mito- patients with CKD not on MHD and controls (13). It is chondrial dysfunction contributes to sarcopenia and still unclear if mitochondrial dysfunction is present in frailty (5). Recent studies have shown that mitochondrial patients with CKD prior to the initiation of MHD. www.cjasn.org Vol 15 July, 2020 Copyright © 2020 by the American Society of Nephrology 1 2 CJASN

Table 1. Participant characteristics

Parameter Controls, n521 CKD Stages 3–5, n520 Maintenance Hemodialysis, n522

Age, yr 47610 616948612 Sex, men 10 (48%) 10 (50%) 14 (64%) Race, black 12 (57%) 8 (40%) 18 (82%) Body mass index, kg/m2 306631653068 History of diabetes 2 (10%) 2 (10%) 4 (18%) History of cardiovascular disease 1 (5%) 2 (10%) 5 (23%) Systolic BP, mm Hg 128610 132610 137628 Hemoglobin, g/dl 13.861.3 13.461.2 11.561.3 Creatinine, mg/dl 0.960.1 1.861.0 10.163.2 Glucose, mg/dl 85612 84612.5 75612 Total cholesterol, mg/dl 199631 187646 176639 HDL cholesterol 55618 48610 46617 Triglycerides, mg/dl 94642 118653 156696

Data are presented as mean 6 SD.

Fat accumulation outside of the muscle fibers, called We recruited 63 participants divided into three groups: (1) intermuscular adipose tissue, correlates with low muscle control, participants with no history of CKD; (2) CKD 3–5, quality, and it is associated with reduced physical perfor- patients with eGFR,60 ml/min per 1.73 m2 (range, mance and frailty in older adults (14216). A previous study 13.9–54.4 ml/min per 1.73 m2) not on MHD; and (3) found increased intramuscular adipose tissue accumula- MHD, patients with ESKD on hemodialysis three times tion in patients on MHD compared with controls (17). per week for at least 6 months who were clinically stable, Other studies have found that intramuscular adipose tissue defined as not requiring hospitalization within the last month may correlate with physical performance in patients with before enrollment, and adequately dialyzed (single-pool CKD (18,19). Fat accumulation in the muscle may induce Kt/V .1.2). GFR was estimated from creatinine using the mitochondrial stress and affect mitochondrial function (20). Chronic Kidney Disease Epidemiology Collaboration for- Thus, intramuscular adipose tissue could be an important mula (30). The study was approved by the Vanderbilt mediator of mitochondrial dysfunction and the resultant University Human Research Protection Program Committee. decreased physical performance, sarcopenia, and frailty. Inflammation and oxidative stress are common in patients with CKD (21224). Dysfunctional mitochondria are Study Protocol An informed consent form was given and explained to all one of the sources of oxidative stress and inflammation of the participants. After the consent form was signed, (25). Conversely, inflammation and oxidative stress can participants were asked to come to the Vanderbilt General lead to mitochondrial dysfunction by damaging the mito- Clinical Research Center to evaluate the mitochondrial chondria. Removal of damaged mitochondria relies on function and physical performance. We then performed mitophagy and mitochondrial fission, a process of mito- muscle biopsies and blood sampling. For patients on MHD, chondrial dynamics that segregates damaged mitochondria all of these tests were performed on a nondialysis day. (26–28). Increased mitochondrial fission results in frag- Quadriceps mitochondrial function was measured using a mented mitochondria and may induce muscle wasting (29). 31P-MRS protocol (31). Intramuscular adipose tissue was In this study, we aimed to understand the complex inter- calculated in cross-sectional images of the midthigh region relationship between mitochondrial function and coexis- between the patella and ischial spine (32). We measured tent metabolic derangements in the setting of progressive physical performance with the 6-minute walk test (6MWT) CKD. We tested the hypothesis that these adverse meta- (33). Maximal static voluntary contraction of the quadri- bolic derangements, especially mitochondrial dysfunction, ceps was measured using with the leg suspended by a are present in patients with moderate to severe CKD even Velcro strap and attached to an ergometer by rigid metallic prior to the initiation of MHD. We also tested the hypoth- rings. Muscle biopsies were obtained from the vastus esis that mitochondrial function is associated with physical lateralis by a percutaneous needle biopsy in a subgroup performance, intramuscular adipose tissue, and systemic of patients (15 controls and 9 patients on MHD) (34). inflammation. The understanding of mitochondrial biology Samples were then prepared for electron microscopy or in CKD could help to understand the contribution of western blot (35). Cytokines and coenzyme Q (CoQ ) mitochondrial dysfunction to muscle wasting and will 10 10 levels were measured in plasma (36). allow the development of therapeutic targets for frailty and sarcopenia in patients with CKD. Statistical Analyses All of the participants completed 31P-MRS measurements Materials and Methods of mitochondrial function; ,5% of those (n53) had missing Participants 6MWT data. We used the Kruskal–Wallis test to compare All participants were recruited from Vanderbilt University the difference among the groups and implemented con- Medical Center Clinics from January 2015 to October 2017. trast for specific two-group comparisons. Differences in CJASN 15: ccc–ccc, July, 2020 Muscle Mitochondrial and CKD, Gamboa et al. 3

A phosphocreatine (PCr) recovery time (t) among the groups 100 were assessed using analysis of covariance with tertile of eGFR as ﬁxed effects and covariates, including sex, body 90 63.2% of PCr mass index (BMI), age, and 6MWT distance, as covariates. recovery We tested the association of mitochondrial function (PCr 80 recovery time) with physical performance, intramuscular Control adipose tissue, and markers of inﬂammation and oxidative Time constant (τ) MHD

PCr recovery 70 is the time to stress using linear regression and adjusting for potential

(% of resting value) 10 reach ~63.2% of confounders that may affect mitochondrial function. An 0 the plateau value additional comparison between control and MHD groups for mitochondrial fragmentation and markers of mitochon- 0 40 80 120 160 200 240 drial dynamics was performed using the Wilcoxon rank a5 Time (seconds) sum test. Hypotheses were tested at the level of 0.05. Statistical analysis was performed using SPSS version 25 (IBM). B p<0.001 More details of methods are available in Supplemen- p=0.007 tal Material. 100

80 Results Demographics 60 Sixty-three participants were enrolled in one of the following groups: controls (n521), CKD 3–5 not on MHD (n520) with an eGFR between 14 and 60 ml/min 40 per 1.73 m2,andMHD(n522). Participant characteristics

Time constant τ (seconds) are depicted in Table 1. The causes of CKD were hyper- 20 tension (52%), diabetes mellitus (10%), glomerular disease (17%), vasculitis (5%), and other (16%). Groups were matched Control CKD 3-5 MHD by sex, race, BMI, history of diabetes, and hypertension. Patients with CKD stages 3–5 were older and a smaller C p<0.001 proportion of patients was black compared with patients p=0.002 on MHD. Patients on MHD had a higher rate of stable 100 cardiovascular disease (Table 1).

80 p=0.03 Mitochondrial Function Measured by 31P-MRS Faster PCr recovery kinetics (reflected by a shorter time t vice 60 constant ) indicate better mitochondrial function and versa (Figure 1A) (37). We found that PCr recovery time constant (t) was longer in patients on MHD compared with 40 controls and patients with CKD 3–5 (Figure 1B). There was a statistically significant correlation between the PCr re- Time constant τ (seconds) 20 covery time constant and eGFR (Rho50.32; P50.04) in the non-MHD group of patients. This association was also Highest Middle Lowest MHD demonstrated when control and CKD 3–5 groups were (>90) (90-57) (56-14) divided into tertiles by the eGFR (Figure 1C), showing that Tertiles by eGFR (ml/min/1.73 m2) patients within the lowest eGFR tertile have a longer PCr recovery time constant than individuals within the highest eGFR tertile. These results suggest that mitochondrial Figure 1. | Mitochondrial function in patients with CKD. During dysfunction is associated with the severity of CKD. exercise, phosphocreatine (PCr) is broken down to synthesize ATP for the When grouped by stages of kidney disease, there was no workingmuscle.Duringrecovery,PCrisresynthesizedfromATPproduced difference in mitochondrial function between controls and by oxidative phosphorylation, and the rate of recovery of PCr is a measure patients with CKD 3–5(P50.40). Hemoglobin levels may of mitochondrial oxidative capacity. The recovery of PCr after light- and affect mitochondrial function. The difference in mitochon- moderate-intensity exercise follows a monoexponential pattern, and the t drial function among the groups persisted after adjusting time constant of PCr recovery is frequently used as an index of mito- P5 chondrial function. (A) Representative graph showing the PCr recovery for hemoglobin levels ( 0.01). kinetics after exercise in one healthy control and a patient on maintenance hemodialysis (MHD). (B) PCr recovery time constant t was prolonged in Mitochondrial Function, Physical Performance, and patientsonMHD(n522)comparedwithcontrols(n521)andpatientswith CKD stages 3–5(n520). (C) Patientswith CKD 3–5andcontrol participants Muscle Strength were divided into tertiles according to the eGFR. Patients within the lowest We measured physical performance using the 6MWT. fi eGFR tertile (n514) have a prolonged PCr recovery time compared with We found that patients on MHD walked a signi cantly individuals within the highest eGFR tertile (n514). shorter distance compared with both controls and patients 4 CJASN

ﬁ A p<0.001 the knee extensors was also signi cantly lower in patients on MHD compared with controls and patients with CKD 800 p=0.04 p=0.02 3–5, but there was no difference in maximal static voluntary contraction between controls and patients with CKD 3–5 600 (Supplemental Figure 1). In unadjusted analysis, maximal static voluntary contraction is also inversely associated with 400 the PCr recovery time constant (Table 2). Mulitvariable

(meters) regression analysis adjusted by age, BMI, and sex showed 200 that only the 6MWT distance remained signiﬁcantly associ-

Six-minute walk test walk Six-minute ated with PCr recovery time constant (Table 2). We also 0 observed a statistically signiﬁcant association between mitochondrial function and the cross-sectional area (CSA) of the Control CKD 3-5 MHD quadriceps muscle in unadjusted (r520.34; P50.02) and adjusted analysis (Supplemental Table 1). B Mitochondrial dysfunction in CKD may be affected by Control different factors, such as physical performance, age, and 100 CKD 3-5 sex. Using analysis of covariance, we showed that the MHD difference in mitochondrial function among the tertile of eGFR groups remains signiﬁcant after adjustment for 6MWT distance, sex, BMI, and age (P50.05). Also, in a 50 subgroup of participants with similar physical performance (i.e., those who were within the 25th and 75th r=0.62 percentiles for the 6MWT), we found that mitochondrial P<0.001 function was impaired in patients on MHD compared with 0 Time constant (seconds) controls (Supplemental Figure 2). These results suggest that the worsening of kidney function has a considerable effect 0 200 400 600 800 on mitochondrial function independent of physical Six-minute walk (meters) performance.

Figure 2. | Physical performance and mitochondrial function. (A) Physical performance, measured by the 6-minute walk test, was im- Mitochondrial Function and Intramuscular Adipose Tissue paired in patients with CKD 3–5 (n520) and patients on MHD (n522) Lipid accumulation in the muscle usually coexists with compared with control individuals (n518). (B) Physical performance mitochondrial dysfunction, suggesting that ectopic fat may was significantly associated with mitochondrial function (n557). damage the mitochondria. In order to examine this association, we measure intramuscular adipose tissue on 51 participants (16 controls, 20 CKD 3–5, and 15 MHD) in the with CKD 3–5, whereas patients with CKD 3–5walkeda quadriceps muscle, the same muscle in which mitochon- shorter distance compared than controls (controls: 557.5 m drial function was measured. Intramuscular adipose tissue [489.9–586.7]; CKD 3–5: 493.0 m [447.4–544.1]; MHD: 419 m accumulation was significantly increased in the MHD [376.3–497.2]; median [interquartile range]; P50.001) group compared with the control and CKD 3–5groups (Figure 2A). There was a statistically significant inverse (Figure 3, A and B). Intramuscular adipose tissue accumu- correlation between PCr recovery time constant and phys- lation was also greater in the CKD 3–5 group compared ical performance (Figure 2B), suggesting that mitochon- with controls (Figure 3B). In addition, the PCr recovery drial dysfunction is associated with poor physical time constant also correlated with intramuscular adipose performance. The maximal static voluntary contraction of tissue, suggesting an association between mitochondrial

Table 2. Association of physical performance with mitochondrial function

b (95% Conﬁdence Interval) Variables Adjusted P Value Unadjusted, n557 Adjusted, n557

6MWT, m 20.08 (20.11 to 20.05) 20.06 (20.10 to 20.02) 0.004 Maximal Static Voluntary Contraction, N 20.10 (20.15 to 20.05) 20.05 (20.13 to 0.03) 0.22 Age, yr 0.13 (20.13 to 0.39) 0.32 BMI, kg/m2 20.51 (21.20 to 0.18 0.15 Sex, women 2.92 (25.37 to 11.20) 0.48 Race, white 24.76 (211.81 to 2.30) 0.18

Mitochondrial function (dependent variable) was measured using the t time constant (in seconds). Each 1-m increase in the 6-minute walk test (6MWT) was negatively associated with 0.08 s less in the t time constant. BMI, body mass index. CJASN 15: ccc–ccc, July, 2020 Muscle Mitochondrial and CKD, Gamboa et al. 5

A form of CoQ10 (or CoQ10 redox ratio), a validated marker of oxidative stress (38). We found that the total and reduced CoQ10 values were lower in patients on MHD compared with controls (Supplemental Figure 4). The CoQ10 redox ratio was diminished in patients with CKD and in patients on MHD compared with controls and correlated significantly with PCr recovery constant (Figure 5). The associ- a ation of TNF and CoQ10 redox ratio with mitochondrial function remains significant after adjusting for BMI, age, Control MHD sex, and race (Table 4). Also, after adjusting for physical performance, the correlation between mitochondrial func- B p<0.001 tion and TNF remain significant, whereas the association p=0.03 between mitochondrial function and CoQ10 redox ratio was p=0.03 not significant (Supplemental Table 2).

10 Mitochondrial Fragmentation and Markers of Mitochondrial Dynamics in Skeletal Muscle 5 Damaged mitochondria by factors such as inﬂammation and/or oxidative stress are usually segregated and frag-

Intermuscular fat (%) mented by mitochondrial fission prior to being removed via 0 mitophagy. To determine changes in mitochondrial frag- Control CKD 3-5 MHD mentation, we evaluated mitochondria morphology using electron micrographs. We only evaluated a group of C patients with no history of CKD (controls) and patients on MHD. We found that mitochondria are smaller in 100 patients on MHD compared with controls (Figure 6, A and 80 B). The frequency distribution of individual mitochondrial areas showed a shift to the left in patients on MHD, 60 reflecting an increased proportion of smaller mitochondria (Figure 6C). We also evaluated markers of mitochondrial 40 fi

(seconds) fusion and ssion in skeletal muscle samples. We found r=0.44

Time constant τ 20 that levels of dynamin-related protein 1 (DRP1), a marker p=0.001 of mitochondrial ﬁssion, are increased in patients on MHD (Figure 7). There was no difference in Fis1 or on OPA-1 0 5 10 15 levels, other markers of mitochondrial dynamics. Intermuscular fat (%)

Figure 3. | Intermuscular adipose tissue and mitochondrial function. Discussion (A) Representative magnetic resonance imaging images showing in- In this study, we showed that mitochondrial dysfunction creasedintermuscularadipose tissue(IMAT)inapatient onMHDanda is present in patients with moderate CKD and occurs prior control individual. (B) IMATinfiltration (ratio of fat to muscle volume) to the initiation of MHD. CKD affects mitochondrial in the quadriceps muscle was increased in patients with CKD 3–5 dysfunction independent of age and physical performance, (n520) and patients on MHD (n515) comparedwith the control group despite the known association between CKD and lower (n516). (C) Linear regression showing the association between mi- levels of physical performance. Importantly, deficits in in t 5 tochondrial function (time constant ) and IMAT infiltration (n 49). vivo muscle mitochondrial function are associated with clinically important limitations in physical performance function and intramuscular adipose tissue accumulation measured by the 6MWT. Mitochondrial dysfunction in (Figure 3C). The association remained significant after patients with CKD may occur as a result of mitochondrial adjusting for BMI, age, sex, and race (Table 3). damage caused by inflammation, oxidative stress, and intramuscular adipose tissue infiltration. We also found increased mitochondrial fragmentation and fission in skel- Mitochondrial Function and Markers of Inflammation and etal muscle in patients on MHD, probably as an attempt to Oxidative Stress segregate and eliminate damaged mitochondria. Overall, As expected, we found that serum concentrations of these data re-emphasize the complex metabolic interplay TNFa, IL-6, and IL-1b were elevated in patients with CKD among mitochondrial dysfunction, inflammation, and ox- compared with controls (Figure 4, A and B, Supplemental idative stress that coexist in moderate to advanced CKD, Figure 3). There was no difference in levels of IL-8 or -10 which collectively may culminate in poor physical perfor- (Supplemental Figure 3). We also found a significant mance and poor quality of life. correlation between prolonged PCr recovery time constant Mitochondrial function has been previously evaluated in (worse mitochondrial function) and higher levels of both patients with CKD using 31P-MRS. As in other studies, we TNFa and IL-6 levels (Figure 4, C and D). We also found that mitochondrial function is impaired in patients 2 in vivo measured CoQ10 levels and the ratio of reduced to oxidized on MHD (10 12). We now found that mitochondrial 6 CJASN

Table 3. Association of intramuscular adipose tissue accumulation with mitochondrial function

b (95% Conﬁdence Interval) Variables Adjusted P Value Unadjusted, n551 Adjusted, n551

Intramuscular adipose tissue % 2.29 (0.86 to 3.72) 2.88 (1.35 to 4.40) ,0.001 Age, yr 0.12 (20.28 to 0.51) 0.56 BMI, kg/m2 20.95 (21.67 to 20.24) 0.01 Sex, women 0.42 (28.19 to 9.03) 0.92 Race, white 25.89 (214.57 to 2.79) 0.17

Mitochondrial function (dependent variable) was measured using the t time constant (in seconds). Each percentage increase in intramuscular adipose tissue was positively associated with 2.3 s more in the t time constant. BMI, body mass index.

dysfunction (i.e., prolonged PCr recovery constant) corre- inflammatory state in CKD by different mechanisms, lates with lower eGFR and occurs before the initiation of including the mitochondrial–reactive oxidative species- MHD. This finding suggests that the uremic environment dependent activation of the NLRP3 inflammasome (42). gradually damages the mitochondria. In fact, in vitro and Consistent with this, we observed a correlation between preclinical studies have shown that uremic toxins damage mitochondrial function and markers of inflammation. the mitochondria (39241). Thus, worsening kidney func- Another potential explanation is that inflammation induces tion, which leads to a gradual accumulation of uremic mitochondrial dysfunction. In fact, TNFa and IL-1b may toxins, may result in a progressive decline in mitochondrial damage mitochondria and impair mitochondrial function function. Indeed, uremic toxins, such as indoxyl sulfate and in different cell types. The use of anti-inflammatory agents, hippurate, have been shown to damage the mitochondria such as recombinant IL receptor antagonists, or CoQ10 and alter mitochondrial function (39,40), although we did supplementation to increase the CoQ10 redox ratio will be not explore this issue in detail. necessary to evaluate causality among oxidative stress, Mitochondrial dysfunction may result in increased pro- inflammation, and mitochondrial function in pa- duction of reactive oxygen species in CKD. Conversely, tients with CKD. systemic oxidative stress may damage the mitochondria. Mitochondrial quality control mechanisms (i.e.,mito- Thus, we found decreased CoQ10 redox ratio (a marker of chondrial dynamics, the continuous process of mitochon- oxidative stress) in patients with CKD, which correlates drial fission or fusion) are required to eliminate damaged with worsened mitochondrial function. Mitochondrial mitochondria through mitophagy. We have previously dysfunction may also contribute to the increased shown that markers of mitophagy are increased in patients

A B p<0.001 15 6 p<0.001 p=0.005

10 4 p=0.06 p=0.02

5 2 alpha (TNF α , pg/ml) Tumor necrosis factor 0 Interleukin 6 (IL6) pg/ml 0

Control CKD 3-5 MHD Control CKD 3-5 MHD C D 100 r=0.53 100 r=0.41 p<0.001 p=0.001 80 80

60 60

40 40

20 20 Time constant τ (seconds) Time constant τ (seconds)

0 5 10 15 0246 TNFα (pg/ml) IL6 (pg/ml)

Figure 4. | Inﬂammation and mitochondrial function. TNFa (A) and IL-6 (B) levels in control individuals (n521), patients with CKD 3–5 (n520), and patients on MHD (n521). Association of TNFa (C) and IL-6 (D) with mitochondrial function (n559). CJASN 15: ccc–ccc, July, 2020 Muscle Mitochondrial and CKD, Gamboa et al. 7

fi A p<0.001 mitochondrial ssion is increased in patients on MHD. Recent preclinical studies suggest that uremic toxins in- p=0.05 fi p=0.04 crease mitochondrial ssion through downregulation of fi 5 fusion proteins and upregulation of ssion proteins, including DRP1 (40,41). Although there is growing evidence 4 showing that altered mitochondrial dynamics affect muscle function, further mechanistic research is necessary to un- 3 derstand the relationship between mitochondrial dynamics and frailty in patients with CKD. 2 The presence of frailty and the level of habitual physical activity may influence mitochondrial function. In this 1 study, we did not measure physical activity, a major

Reduced/oxidized CoQ10 ratio limitation. Instead, we used physical performance as a proxy for physical activity. In our study, patients on MHD Control CKD 3-5 MHD have the lowest physical performance measured by the 6MWT. It is possible that the lack of physical activity B contributes to the impaired mitochondrial function. Ac- r=-0.42 cordingly, we found that low physical performance is p<0.001 associated with a worse mitochondrial function. Muscle 100 CSA has been used as a proxy of physical activity. Thus, we found an association between quadriceps CSA and mito- 75 chondrial function. This association is not as strong as the one between physical performance and mitochondrial 50 function. This is probably because CSA is a better predictor of muscle strength than of physical function (43). To 25 determine if the association between CKD and mitochondrial is independent of the physical performance, we Time constant τ (seconds) evaluated mitochondrial function in a subgroup of individuals with similar physical performance. We found that, 12345 despite a similar level of physical performance, patients on CoQ10 reduced/oxidized ratio MHD have impaired mitochondrial function. These results suggest that CKD affects mitochondrial function, to some Figure 5. | Oxidative stress and mitochondrial function. (A) Co- extent, independently of physical performance. Further enzyme Q10 (CoQ10) redox ratio (ratio of reduced to oxidized CoQ10) studies should measure physical activity and its relation to in control individuals (n521), patients with CKD 3–5 (n520), and mitochondrial function in patients with CKD. 5 patients on MHD (n 21). (B) Association between CoQ10 redox ratio Intramuscular adipose tissue may affect muscle quality 5 and mitochondrial function (n 59). and function, and it is associated with poor physical activity and performance (44,45). Few studies have eval- on MHD (7). We now found that mitochondria are small uated intramuscular adipose tissue in patients with CKD. A and fragmented in skeletal muscles from patients on MHD. previous study found increased intramuscular adipose We also found increased content of DRP1, a marker of tissue accumulation in patients on MHD (17). We found mitochondrial ﬁssion. These ﬁndings suggest that a progressive intramuscular adipose tissue accumulation

Table 4. Association of markers of inﬂammation and oxidative stress with mitochondrial function

b (95% Conﬁdence Interval) Variables Adjusted P Value Unadjusted, n559 Adjusted, n559

TNFa, pg/ml 3.03 (1.75 to 4.31) 3.30 (2.03 to 4.57) ,0.001 Age, yr 0.36 (20.08 to 0.64) 0.01 BMI, kg/m2 20.52 (21.07 to 20.03) 0.06 Sex, women 24.18 (210.92 to 2.55) 0.22 Race, white 23.772 (210.52 to 2.97) 0.27 2 2 2 2 2 2 CoQ10 redox ratio 7.55 ( 11.82 to 3.28) 6.81 ( 11.25 to 2.37) 0.003 Age, yr 0.18 (20.14 to 0.50) 0.25 BMI, kg/m2 20.41 (21.03 to 20.22) 0.20 Sex, women 0.74 (26.81 to 8.28) 0.85 Race, white 24.79 (212.42 to 2.85) 0.21

Mitochondrial function (dependent variable) was measured using the t time constant (in seconds). Every 1-pg/ml increase in TNFa t levels was positivelyassociated with 3.0s moreinthe timeconstant.EachunitincreaseinthecoenzymeQ10 (CoQ10) ratiowas negatively associated with 7.6 s less in the t time constant. BMI, body mass index. 8 CJASN

A A Cont MHD Cont MHD DRP-1

Coomassie

B 2.5 p=0.03 Control MHD 2.0

1.5 B p=0.02

0.08 DRP-1 1.0 ) 2 0.5

0.06 Optical density (A.U) 0.0 0.04 Control MHD 0.02

Mitochondrion area ( μ m Figure 7. | Western blot analysis of dynamin-related protein 1 0.00 (DRP1). (A) Representative western blot of DRP1, a marker of mitochondrial ﬁssion. (B) DRP1 is increased in patients on MHD when Control MHD compared with control subjects with no history of CKD. Skeletal muscle biopsies were obtained from the vastus lateralis (n59ineach C group). A.U., arbitrary units. 100

80 % of small potential mechanisms to prevent frailty and sarcopenia in mitochondria patients with CKD. 60 Control 38.4% Lower hemoglobin levels may affect mitochondrial func- 56.1% tion. A previous study evaluated mitochondrial function in 40 MHD patients with CKD before and after erythropoietin treat- distribution (%) 20 ment (46). This study found that there was no difference in

Cumulative frequency mitochondrial function after treatment with erythropoietin, 0 despite an increase in hemoglobin by 50%. The authors also 0.00 0.05 0.10 0.15 0.20 0.25 showed that maximal oxygen flow from the microcircula- tion to the mitochondria did not increase after erythropoi- Mitochondrial area (μm2) etin treatment. This is consistent with the observation that the difference in mitochondrial function among the groups Figure 6. | Mitochondrial fragmentation in patients on MHD in persisted after adjusting by hemoglobin levels. skeletal muscle biopsies from the vastus lateralis. (A) Electron mi- The frailty phenotype among patients with CKD may be croscopies showing that mitochondria (white arrows) are smaller in a consequence of uremic solutes retention, which initiates a patients on MHD. (B) Quantification of mitochondrial areas in control vicious cycle of impaired mitochondrial function and 5 5 individuals (n 15) and patients on MHD (n 9). Each dot represents decreased physical functioning. Worsening of kidney the median area of hundreds of mitochondria in each subject. (C) disease may affect both physical and mitochondrial func- Frequency distribution of individual mitochondria areas showing a greater proportion of smaller mitochondria in patients on MHD. tion. In turn, mitochondrial dysfunction may negatively affect physical functioning and vice versa, contributing to the frailty phenotype. Also, uremia may have a direct with increased severity of kidney disease. Intramuscular deleterious effect on frailty and physical function or in- adipose tissue may damage the mitochondria, resulting in directly by affecting muscle quality (i.e., increasing in- mitochondrial dysfunction. Conversely, mitochondria are tramuscular adipose tissue infiltration). Other factors that the primary site for lipid metabolism, and mitochondrial may play a role in the frail phenotype include inflamma- dysfunction may play a role in intramuscular adipose tion, oxidative stress, and intramuscular adipose tissue tissue accumulation. The cross-sectional study design infiltration, either directly or by inducing mitochondrial precludes the assessment of causality. Nevertheless, we dysfunction. Thus, the pathophysiologic link among frailty, found a strong association between mitochondrial function impaired physical activity, and mitochondrial dysfunction and intramuscular adipose tissue. Further prospective in patients with CKD is very complex (Figure 8) and studies should evaluate changes in intramuscular adipose deserved further investigation. Therapeutic approaches to tissue, physical activity, and mitochondrial function as prevent frailty in CKD may require interventions that CJASN 15: ccc–ccc, July, 2020 Muscle Mitochondrial and CKD, Gamboa et al. 9

Acknowledgments Oxidative stress We thank all of the study coordinators and participants for their dedication to the study. Uremia Inflammation The sponsors had no inﬂuence on the design, execution, and analysis of the results of the study.

Mitochondrial Physical Disclosures dysfunction inactivity T.A. Ikizler reports personal fees from Fresenius Kabi and Abbott Nutrition during the conduct of the study and personal fees from IMAT Reata, ISN, Elsevier, and ABIM, outside the submitted work. B. Roshanravan reports grants from Dialysis Clinics Incorporated Physical during the conduct of the study. All remaining authors have dysfunction nothing to disclose.

Frailty Funding The following funding was received for this study: National Center for Research Resources grant 1UL-1RR024975; National Figure 8. | The possible association among factors that could be Institute of Diabetes and Digestive and Kidney Diseases grants implicated in the pathogenesis of frailty in patients with CKD. K23DK0099442, K23DK100533, and R03DK114502; National Institute of General Medical Sciences grant T32GM07569; and US reverse uremia, such as kidney transplantation. Exercise is Department of Veterans Affairs grant CX001755. another potential intervention that may prevent or reverse frailty by improving physical and mitochondrial function. fi Supplemental Material Whereas many studies show some bene tofexerciseon This article contains the following supplemental material online at physical function in CKD, the effect of exercise on mito- http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/CJN. chondrial function in patients with CKD remains largely 10320819/-/DCSupplemental. unexplored. Likewise, mitochondrial-targeted therapies, Supplemental Figure 1. PCr recovery time constant t was pro- which could be used in combination with exercise, may longed in patients on MHD compared with controls and patients prevent frailty by improving mitochondrial bioenergetics with CKD with similar physical performance measured by the fi and exercise ef ciency. 6-minute walk. This study has several strengths. Importantly, it exam- Supplemental Figure 2. Maximal voluntary contraction of the ines and compares mitochondrial function in patients at quadriceps muscle was diminished in the MHD group (n518) different stages of CKD not yet on MHD and in patients on compared with control (n516) and CKD 3–5(n520) groups. MHD. We evaluated mitochondrial function in the quad- Supplemental Figure 3. IL-1b, IL-8, and IL-10 levels in control riceps muscle whose proper function is important for daily individuals (n521), patients with CKD 3–5(n520), and patients on activities, such as rising from a chair, but also for prevent- MHD (n522). 31 ing falls in elderly frail individuals (47). We used P-MRS, Supplemental Figure 4. Levels of total coenzyme Q and reduced in vivo 10 a state-of-the-art technique, to measure mitochon- coenzyme Q in control individuals (n521), patients with CKD 3–5 in vivo 10 drial function. The combination of studying mito- (n520), and patients on MHD (n521). chondrial function and muscle biopsies was important to Supplemental Table 1. Association of total quadriceps cross- in vivo provide mechanistic insight for the studies. There sectional area with mitochondrial function. are also some limitations. This is a cross-sectional study not Supplemental Table 2. Association of physical performance with – suitable for the evaluation of causality. Also, the CKD 3 5 mitochondrial function after adjusting by inflammation and oxi- group was not properly matched to the other groups; the dative stress markers. patients were older, and there was a lower proportion of black patients. Another limitation of this study is that we did not assess physical activity, which has more clinical References relevance to patients with CKD. We only evaluated mitochon- 1. Johansen KL, Chertow GM, Jin C, Kutner NG: Significance of drial fragmentation and makers of mitochondrial dynamics in frailty among dialysis patients. J Am Soc Nephrol 18: 2960–2967, muscles from patients on MHD and control individuals. 2007 2. Isoyama N, Qureshi AR, Avesani CM, Lindholm B, Ba`ra`ny P, Further analysis of muscle tissue from patients with CKD Heimbu¨rger O, Cederholm T, Stenvinkel P, Carrero JJ: Compar- not yet on MHD would be important for a better understand- ative associations of muscle mass and muscle strength with ing of the effect of uremia on mitochondrial dynamics. mortality in dialysis patients. Clin J Am Soc Nephrol 9: In conclusion, we found that mitochondrial dysfunction 1720–1728, 2014 occurs prior to the initiation of MHD. We found that 3. Fried LP, Tangen CM, Walston J, Newman AB, Hirsch C, Gottdiener J, Seeman T, Tracy R, Kop WJ, Burke G, McBurnie MA; mitochondrial dysfunction correlates with physical perfor- Cardiovascular Health Study Collaborative Research Group: mance, intramuscular adipose tissue, oxidative stress, and Frailty in older adults: Evidence for a phenotype. J Gerontol A Biol inflammation. Our findings suggest that strategies aimed at Sci Med Sci 56: M146–M156, 2001 improving mitochondrial function should be investigated 4. Bao Y, Dalrymple L, Chertow GM, Kaysen GA, Johansen KL: Frailty,dialysis initiation, and mortality in end-stage renal disease. in patients with CKD with the ultimate goal of preventing Arch Intern Med 172: 1071–1077, 2012 or treating frailty and sarcopenia and improving overall 5. Marzetti E, Calvani R, Cesari M, Buford TW,Lorenzi M, Behnke BJ, physical function in this high-risk patient population. Leeuwenburgh C: Mitochondrial dysfunction and sarcopenia of 10 CJASN

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AFFILIATIONS

1Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 2Division of Nephrology, Department of Medicine, University of California, Davis, California 3Department of Biomedical Sciences, Grand Valley State University, Allendale, Michigan 4Department of Biostatics, Vanderbilt University Medical Center, Nashville, Tennessee 5Department of Physical Medicine, Rehabilitation, and Sports Medicine, University of Puerto Rico, San Juan, Puerto Rico 6Department of Physiology and Biophysics, University of Puerto Rico, San Juan, Puerto Rico 7Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 8Veterans Administration Tennessee Valley Healthcare System, Nashville, Tennessee Supplemental Material Table of Contents

Methods

Supplemental Table 1. Association of total quadriceps cross-sectional area with mitochondrial function.

Supplemental Table 2. Association of physical performance with mitochondrial function after adjusting by inflammation and oxidative stress markers.

Supplemental Figure 1. A. PCr recovery time constant tau (τ) was prolonged in patients on MHD compared to controls and patients with CKD with similar physical performance measured by the 6-minute walk (B).

Supplemental Figure 2. Maximal voluntary contraction (MVC) of the quadriceps muscle was diminished in the MHD group (n=18) compared to control (n=16) and CKD 3-5 (n=20) groups.

Supplemental Figure 3. Interleukin 1 beta (A), interleukin-8 (B), and interleukin-10 (C) levels in control individuals (n=21), patients with CKD 3-5 (n=20), and patients on MHD (n=22).

Supplemental Figure 4. Levels of total coenzyme Q10 (CoQ10, A), reduced CoQ10 (B), in control individuals (n=21), patients with CKD 3-5 (n=20), and patients on MHD (n=21).

References

METHODS

Participants

We recruited 63 participants divided into three different groups; a) Control, participants with no history of CKD, b) CKD 3-5, patients with eGFR <60 ml/min/1.73m2 not on maintenance hemodialysis, and c) MHD, patients with ESRD on maintenance hemodialysis, three times per week for at least six months, who were clinically stable and adequately dialyzed (single-pool

Kt/V > 1.2). GFR was estimated from creatinine using the CKD-EPI formula. Exclusion criteria were a history of functional kidney transplant less than six months prior to the study, use of immunosuppressive drugs within one month prior to the study, active connective tissue disease, acute infectious disease, history of myocardial infarction or cerebrovascular event within three months prior to the study, advanced liver disease, gastrointestinal dysfunction requiring parenteral nutrition, active malignancy, left ventricular ejection fraction less than 40%, history of poor adherence to hemodialysis or medical regimen, use of vitamin E>60 IU/day or vitamin C

>500 mg/day, and inability to undergo MRI evaluation. Patients with a history of stable cardiovascular disease were included in the study (Table 1). The study was approved by the

Vanderbilt University Human Research Protection Program Committee.

31-Phosphorus magnetic resonance spectroscopy (31P-MRS)

Quadriceps muscle mitochondrial bioenergetics were measured using 31P-MRS protocol that has been previously published.[1] Briefly, each participant lay prone with a coil positioned over the belly of the rectus femoris muscle. The knee was flexed and suspended with elastic bands secured to a non-magnetic ergometer at one end and ankle Velcro® strap at the other end. The level of resistance of the elastic tubing was selected to an intensity that decreased PCr to approximately 30% of the baseline during the exercise protocol. After basal measurements, participants were asked to perform two knee extensions every three seconds against the

2 resistance of approximately 30- 40% of the MVC. The exercise protocol lasted 90 seconds (a total of 60 knee extensions) followed by four minutes of rest. The exercise/rest cycle was repeated three times. The intensity of the exercise decreases phosphocreatine (PCr) levels with minimal change in muscle pH. Spectra analysis was performed with AMARES from the jMRUI software package.[2] Spectra were used to calculate the relative concentrations of inorganic phosphate (Pi), PCr, and ATP. The recovery of PCr after the exercise was fit with a monoexponential model that calculated the time constant tau (τ), which is the time to restore approximately 63% of the recovery response.

Magnetic resonance imaging and intermuscular fat (IMAT) measurements

IMAT was calculated using nine consecutive cross-sectional images of the mid-thigh region between the patella and ischial spine. Each section was 3 mm thickness and at a 14 mm interval. The analysis was performed in all the quadriceps muscle heads using a custom-written

Matlab (Mathwworks, Natick, MA) program, as previously described.[3] IMAT was defined as the fat beneath the deep fascia of the muscle. IMAT infiltration was quantified as the ratio between IMAT and muscle volumes.

Six-minute walk and maximal voluntary contraction

We measured physical performance with the six-minute walk test. Briefly, this test consists of instructing the patients to walk back and forth on an indoor 30-meter measured corridor.[4]

Maximal static voluntary contraction (MVC) of the quadriceps was measured with the participant lying prone, the leg suspended by a Velcro strap, and with a knee angle of approximately 20°.

The strap was attached to an ergometer by two rigid metallic rings. MVC was repeated until we obtained at least three consistent and reproducible MVCs.

Skeletal muscle biopsies

We performed muscle biopsies in a subgroup of patients (15 controls and 9 patients on MHD).

Biopsies were obtained from the vastus lateralis, one of the heads of the quadriceps femoris, by a percutaneous needle biopsy with the modified Bergström technique.[5] Briefly, after proper aseptic technique and administration of local anesthesia, the needle was then inserted through a skin incision to the skeletal muscle. The inner trocar of the needle was retracted, and suction applied to pull muscle into the outer trocar. The inner trocar was then closed to cut the muscle.

The procedure was repeated two more times. Muscle biopsies were immediately placed into the fixative solution, or instantly frozen in liquid nitrogen and stored at -80ºC.

Transmission electron microscopy

Samples were prepared for electron microscopy as previously described.[6] Briefly, samples were post-fixed in osmium tetraoxide (1%), dehydrated and embedded for further sectioning.

Thin (80 nm) fiber transverse sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope.

Western blot analysis

One piece of the muscle biopsy was homogenized, denatured, and resolved electrophoretically in 4-20% precast acrylamide gels (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-FL, Millipore, Billerica, MA). Membranes were then incubating overnight with primary antibodies against dynamic-related protein 1 (DRP-1, Cell

Signaling, Danvers, MA, catalog number 8570), mitochondrial fission 1 protein (Fis-1, Novus

Biological, Littleton, CO, catalog number NB110-56646), and optic atrophy protein 1 (OPA-1,

Novus Biological, catalog number NB110-55290). After incubating the membranes with fluorescent secondary antibodies, we used the Odyssey® Infrared Imaging System (LI-COR

Biosciences, Lincoln, NE) to detect the antibody fluorescence intensity. Band densities were analyzed using NIH Image J software.

Measurements of inflammatory cytokines

Cytokines were measured in serum. Blood was collected in serum tubes and allowed to clot at room temperature followed by centrifugation at 2,000g for 10 minutes at 4°C. Serum was collected and stored at -80°C. Inflammatory cytokines (IL1β, IL6, IL8, IL10, and TNFα) were measured using the multiplex proinflammatory human panel 1 (Meso Scale Discovery,

Rockville, MD) according to the manufacturer’s instruction.

Coenzyme Q10 (CoQ10) measurements

CoQ10 was measured as previously published.[7] Briefly, 100 µl of plasma samples were mixed with 200 µl of ice-cold propanol containing coenzyme Q9 and reduced CoQ9 as internal standards. Precipitated proteins were removed by centrifugation and the supernatant was analyzed by liquid chromatography-tandem mass spectrophotometry.

Statistical Analysis

We performed standard graphing and screening to detect any outliers and verify the data accuracy. The distribution of endpoints was examined for normality. We used the Kruskal-Wallis test to compare the difference among the groups and implemented contrast for specific two- group comparisons. Differences in PCr recovery time (tau) among the groups were assessed using ANCOVA with tertile of eGFR as fixed effects, and covariates including gender, BMI, age, six-minute walk distance as covariates. We tested the association of mitochondrial function (PCr recovery time) with physical performance, IMAT, and markers of inflammation and oxidative stress using linear regression and adjusting for potential confounders. An additional comparison between Control and MHD groups for mitochondrial fragmentation and markers of mitochondrial dynamics was performed using the Wilcoxon rank-sum test. Hypotheses were tested at the level of α=0.05. Statistical analysis was performed using SPSS version 25 (IBM, North Carolina).

Supplemental Table 1. Association of total quadriceps cross-sectional area with mitochondrial function.

Unadjusted Adjusted Adjusted (n=57) (n=57) p value β (95% CI) β (95% CI) Quadriceps CSA (cm2) -0.137 (-0.246, -0.028) -0.211 (-0.372, -0.050) 0.011 Age (years) 0.164 (-0.256, 0.583) 0.436 BMI (kg/m2) -0.145 (-0.906, -0.615) 0.702 Gender (female) 8,559 (-4.110, 21.228) 0.180 Race (Caucasian) -9.289 (-19.036, 0.458) 0.061 CSA, cross-sectional area; BMI, body mass index. Mitochondrial function (dependent variable) was measured using the tau time constant (in seconds). Every cm2 increase in CSA was negatively associated with 0.137 seconds lesser in the tau time constant.

Supplemental Table 2. Association of physical performance with mitochondrial function after adjusting by inflammation and oxidative stress markers.

Unadjusted Adjusted Adjusted (n=59) (n=57) p value β (95% CI) β (95% CI) TNF (pg/ml) 3.032 (1.754, 4,310) 1.986 (0.731, 3.242) 0.003

CoQ10 redox ratio -7.550 (-11.817, -3.282) -3.034 (0.142, -7.121) 0.14 6MWT (meters) -0.047 (-0.078, -0.016) 0.004 Mitochondrial function (dependent variable) was measured using the tau time constant (in seconds). Each one pg/ml of TNF increase was positively associated with 0.078 seconds greater in the tau time constant.

Supplemental Figure 1

Supplemental Figure 1. A. PCr recovery time constant tau (τ) was prolonged in patients on

MHD compared to controls and patients with CKD with similar physical performance measured by the 6-minute walk (B).

Supplemental Figure 2

Supplemental Figure 2. A. Maximal voluntary contraction (MVC) of the quadriceps muscle was diminished in the MHD group (n=18) compared to control (n=16) and CKD 3-5 (n=20) groups.

Supplemental Figure 3

Supplemental Figure 3. Interleukin 1 beta (A), interleukin-8 (B), and interleukin-10 (C) levels in control individuals (n=21), patients with CKD 3-5 (n=20), and patients on MHD (n=22).

Supplemental Figure 4

Supplemental Figure 4. Levels of total coenzyme Q10 (CoQ10, A), reduced CoQ10 (B), in control individuals (n=21), patients with CKD 3-5 (n=20), and patients on MHD (n=21).

References

1 Forbes SC, Slade JM, Meyer RA: Short-term high-intensity interval training improves phosphocreatine recovery kinetics following moderate-intensity exercise in humans. Appl Physiol Nutr Metab 2008;33:1124-1131.

2 Vanhamme L, van den Boogaart A, Van Huffel S: Improved Method for Accurate and Efficient Quantification of MRS Data with Use of Prior Knowledge. Journal of Magnetic Resonance 1997;129:35-43.

3 Kumar D, Karampinos D, MacLeod T, Lin W, Nardo L, Li X, Link T, Majumdar S, Souza R: Quadriceps intramuscular fat fraction rather than muscle size is associated with knee osteoarthritis. Osteoarthritis and cartilage 2014;22:226-234.

4 ATS Statement: Guidelines for the Six-Minute Walk Test: Am J Respir Crit Care Med 2002;166:111-117.

5 Tarnopolsky MA, Pearce E, Smith K, Lach B: Suction-modified Bergstrom muscle biopsy technique: experience with 13,500 procedures. Muscle Nerve 2011;43:717-725.

6 Gamboa JL, Andrade FH: Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia. Am J Physiol Regul Integr Comp Physiol 2010;298:R575-R583.

7 Rivara MB, Yeung CK, Robinson-Cohen C, Phillips BR, Ruzinski J, Rock D, Linke L, Shen DD, Ikizler TA, Himmelfarb J: Effect of Coenzyme Q10 on Biomarkers of Oxidative Stress and Cardiac Function in Hemodialysis Patients: The CoQ10 Biomarker Trial. American Journal of Kidney Diseases 2017.