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.

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.

Background and objectivesFerric citrate is an oral medication approved for treatment of iron deficiency anemia in patients with CKD not requiring dialysis. The relative efficacy of ferric citrate versus ferrous sulfate in treating iron deficiency in patients with CKD is unclear.Design, setting, participants, & measurementsWe randomized 60 adults with moderate to severe CKD (eGFR 15–45 ml/min per 1.73 m2) and iron deficiency (transferrin saturation [TSAT] ≤30% and ferritin ≤300 ng/ml) to ferric citrate (2 g three times a day with meals, n=30) or ferrous sulfate (325 mg three times a day, n=30) for 12 weeks. Primary outcomes were change in TSAT and ferritin from baseline to 12 weeks. Secondary outcomes were change in hemoglobin, fibroblast growth factor 23 (FGF23), and hepcidin.ResultsBaseline characteristics were well balanced between study arms. There was a greater increase in TSAT (between-group difference in mean change, 8%; 95% confidence interval [95% CI], 1 to 15; P=0.02) and ferritin (between-group difference in mean change, 37 ng/ml; 95% CI, 10 to 64; P=0.009) from baseline to 12 weeks in participants randomized to ferric citrate as compared with ferrous sulfate. Similarly, as compared with ferrous sulfate, treatment with ferric citrate resulted in a greater increase in hepcidin from baseline to 12 weeks (between-group difference, 69 pg/ml; 95% CI, 8 to 130). There were no between-group differences in mean change for hemoglobin (0.3 g/dl; 95% CI, −0.2 to 0.8), intact FGF23 (−29 pg/ml; 95% CI, −59 to 0.1), or C-terminal FGF23 (61 RU/ml; 95% CI, −181 to 58). The incidence of adverse events did not differ between treatment arms.ConclusionsAs compared with ferrous sulfate, treatment with ferric citrate for 12 weeks resulted in a greater mean increase in TSAT and ferritin concentrations in individuals with moderate to severe CKD and iron deficiency.Clinical Trial registry name and registration numberImpact of Ferric Citrate vs Ferrous Sulfate on Iron Parameters and Hemoglobin in Individuals With Moderate to Severe Chronic Kidney Disease (CKD) With Iron Deficiency, NCT02888171.

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 InformationPartially supported by AHA Grant 18CDA34110044 to TERThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Patients with critical limb ischemia (CLI) have multiple concomitant comorbidities which are associated with poorer overall health outcomes. CLI patients with impaired kidney function and/or diabetes are at greater risk for limb amputation and death. While the negative impacts of renal dysfunction and diabetes on CLI outcomes are known clinically, the underlying biologic mechanism(s) responsible are poorly understood. Thus, we sought to evaluate the impact of impaired renal function and diabetes on the skeletal muscle pathology in mice following unilateral hindlimb ischemia (HLI). Renal dysfunction (RD) was induced by dietary administration of adenine which causes renal tubular injury, while diabetes was induced by streptozocin injection. HLI was induced by ligation and excision of the femoral artery, while the contralateral hindlimb was utilized as a control (no surgery). Three groups (n = 15; 5 per group) of C57BL/6J mice were analyzed: control, RD, and RD+ diabetes. Skeletal muscle ischemic injury was assessed using histological measurements of myofiber centralized nuclei, myofiber cross‐sectional area, ischemic lesion areas, and vascular density. High‐resolution O2 consumption was used to examine mitochondrial function in permeablized myofibers. Mice with RD, regardless of diabetes, exhibited greater ischemic lesions within the tibialis anterior muscle (Con = 2.53 ± 0.51% vs. RD = 60.554 ± 24.15% vs. RD+STZ = 79.99 ± 19.99%; P < 0.0001) despite similar capillary density (Con = 466.36 ± 44.98 vs. RD = 455.20 ± 79.55 vs. RD+STZ = 578.20 ± 87.16 capillaries/field; P = 0.9011). Mice with RD exhibited a ~45% decrease in mitochondrial respiratory capacity across numerous substrates, whereas control mice had normal respiration in the ischemic limb muscle seven days post‐HLI. These observations demonstrate that impaired kidney function exacerbates ischemic muscle injury in mice. Future studies are needed to uncover the molecular mechanisms by which RD negatively impacts skeletal muscle.Support or Funding InformationSupported 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.