Chronic kidney disease (CKD) impacts more than 25 million Americans and is associated with higher risk of all‐cause and cardiovascular mortality. Impaired kidney function leads to retention of metabolic waste products, termed uremic toxins, that negatively impact skeletal muscle resulting in increased fatigue, weakness, and muscle atrophy. Previous evidence has implicated mitochondria within the skeletal muscle as a primary mediator of muscle dysfunction in CKD, yet the underlying mechanisms are unknown. Therefore, the purpose of this study was to define the impact of uremic toxins on mitochondrial energetics. Skeletal muscle mitochondria were isolated from healthy C57BL/6N mice and exposed to vehicle (DMSO) or varying doses of the following uremic toxins: indoxyl sulfate, indole‐3‐acetic‐acid, L‐kynurenine, kynurenic acid, and methylguanidine. We employed a comprehensive mitochondrial phenotyping platform that included assessments of mitochondrial OXPHOS conductance across several levels of energy demand, hydrogen peroxide production (JH2O2), and dehydrogenase flux (using NADH autofluorescence). Exposure to uremic toxins resulted in a dose‐dependent decrease in OXPHOS conductance for all toxins, with 100nM exposure resulting in an average decrease of ~22% supported by pyruvate/malate (all P<0.05, n= 5–6/group). Uremic toxins did not decrease pyruvate dehydrogenase activity even at millimolar concentrations (all P>0.64), suggesting the decreased OXPHOS conductance occurs downstream of matrix dehydrogenases. Consistent with decreased OXPHOS conductance, uremic toxins dose‐dependently increased JH2O2 by 2–5‐fold (all P<0.01, n=4/group). These findings provide direct evidence that uremic toxins negatively impact skeletal muscle mitochondrial energetics, resulting in decreased energy transfer. Impaired mitochondrial energetics appears to be mediated downstream of matrix dehydrogenases, through either direct interaction within the electron transport system or ATP synthase. Support or Funding Information Partially supported by AHA Grant 18CDA34110044 to TER This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Preclinical models of chronic kidney disease (CKD) are critical to investigate the mechanisms of disease and to evaluate novel therapeutics aimed to treat CKD-associated pathologies. The objective of the present study was to compare the adenine diet and 5/6 nephrectomy (5/6 Nx) models in mice. Male and female 10-week-old C57BL/6J mice (N=5-9/sex/group) were randomly allocated to CKD groups (0.2-0.15% adenine-supplemented diet or 5/6 Nx surgery) or corresponding control groups (casein diet or sham surgery). Glomerular filtration rate was reduced to a similar level in adenine and 5/6 Nx mice (adenine male: 81.1 ± 41.9 µL/min vs. 5/6 Nx male: 160 ± 80.9 µL/min, P=0.5875; adenine female: 112.9 ± 32.4 µL/min vs. 5/6 Nx female: 107.0 ± 45.7 µL/min, P=0.9995). Serum metabolomics analysis indicated that established uremic toxins were robustly elevated in both CKD models, although some differences were observed between CKD models (i.e. p-Cresol sulfate). Dysregulated phosphate homeostasis was observed in the adenine model only, whereas calcium homeostasis was disturbed in male mice with both models. Muscle mass and myofiber cross-sectional area of extensor digitorum longus and soleus muscles were ~18-24% smaller in male CKD mice regardless of model, but were not different in female CKD mice (P>0.05). Skeletal muscle mitochondrial respiratory function was significantly decreased (19-24%) in CKD mice in both models and sexes. These findings demonstrate that adenine diet and 5/6 Nx models of CKD have similar levels of renal dysfunction and skeletal myopathy, but had less mortality (P<0.05 for both sexes) compared with the 5/6 Nx model.

Chronic kidney disease (CKD) substantially increases the severity of peripheral arterial disease (PAD) symptomology, however, the biological mechanisms remain unclear. The objective herein was to determine the impact of CKD on PAD pathology in mice. C57BL6/J mice were subjected to a diet-induced model of CKD by delivery of adenine for six weeks. CKD was confirmed by measurements of glomerular filtration rate, blood urea nitrogen, and kidney histopathology. Mice with CKD displayed lower muscle force production and greater ischemic lesions in the tibialis anterior muscle (78.1 ± 14.5% vs. 2.5 ± 0.5% in control mice, P < 0.0001, N = 5–10/group) and decreased myofiber size (1661 ± 134 μm2 vs. 2221 ± 100 μm2 in control mice, P < 0.01, N = 5–10/group). This skeletal myopathy occurred despite normal capillary density (516 ± 59 vs. 466 ± 45 capillaries/20x field of view) and limb perfusion. CKD mice displayed a ~50–65% reduction in muscle mitochondrial respiratory capacity in ischemic muscle, whereas control mice had normal mitochondrial function. Hydrogen peroxide emission was modestly higher in the ischemic muscle of CKD mice, which coincided with decreased oxidant buffering. Exposure of cultured myotubes to CKD serum resulted in myotube atrophy and elevated oxidative stress, which were attenuated by mitochondrial-targeted therapies. Taken together, these findings suggest that mitochondrial impairments caused by CKD contribute to the exacerbation of ischemic pathology.

Chronic limb threatening ischemia (CLTI) is the most severe manifestation of peripheral atherosclerosis. Patients with CLTI have poor muscle quality and function and are at high risk for limb amputation and death. The objective of this study was to interrogate the metabolome of limb muscle from CLTI patients. To accomplish this, a prospective cohort of CLTI patients undergoing either a surgical intervention (CLTI Pre-surgery) or limb amputation (CLTI Amputation), as well as non-peripheral arterial disease (non-PAD) controls were enrolled. Gastrocnemius muscle biopsy specimens were obtained and processed for nuclear magnetic resonance (NMR)-based metabolomics analyses using solution state NMR on extracted aqueous and organic phases and 1H high-resolution magic angle spinning (HR-MAS) on intact muscle specimens. CLTI Amputation specimens displayed classical features of ischemic/hypoxic metabolism including accumulation of succinate, fumarate, lactate, alanine, and a significant decrease in the pyruvate/lactate ratio. CLTI Amputation muscle also featured aberrant amino acid metabolism marked by elevated branched chain amino acids. Finally, both Pre-surgery and Amputation CLTI muscles exhibited pronounced accumulation of lipids, suggesting the presence of myosteatosis, including cholesterol, triglycerides, and saturated fatty acids. Taken together, these metabolite differences add to a growing body of literature that have characterized profound metabolic disturbance’s in the failing ischemic limb of CLTI patients.