Skeletal Muscle Phenotype in Patients Undergoing Long-Term Hemodialysis Awaiting Kidney Transplantation

Abstract

Background and objectives: Age and comorbidity-related sarcopenia represent a main cause of muscle dysfunction in patients on long-term hemodialysis. However, recent findings suggest muscle abnormalities that are not associated with sarcopenia. The aim of this study was to isolate functional and cellular muscle abnormalities independently of other major confounding factors, including malnutrition, age, comorbidity, or sedentary lifestyle, which are common in patients on maintenance hemodialysis. To overcome these confounding factors, alterations in skeletal muscle were analyzed in highly selected patients on long-term hemodialysis undergoing kidney transplantation.

Design, setting, participants, & measurements: In total, 22 patients on long-term hemodialysis scheduled for kidney transplantation with few comorbidities, but with a long-term uremic milieu exposure, and 22 age, sex, and physical activity level frequency-matched control participants were recruited. We compared biochemical, functional, and molecular characteristics of the skeletal muscle using maximal voluntary force and endurance of the quadriceps, 6-minute walking test, and muscle biopsy of vastus lateralis. For statistical analysis, mean comparison and multiple regression tests were used.

Results: In patients on long-term hemodialysis, muscle endurance was lower, whereas maximal voluntary force was not significantly different. We observed a transition from type I (oxidative) to type II (glycolytic) muscle fibers, and an alteration of mitochondrial structure (swelling) without changes in DNA content, genome replication (peroxisome proliferator activator receptor γ coactivator-1α and mitochondrial transcription factor A), regulation of fusion (mitofusin and optic atrophy 1), or fission (dynamin-related protein 1). Notably, there were autophagosome structures containing glycogen along with mitochondrial debris, with a higher expression of light chain 3 (LC3) protein, indicating phagophore formation. This was associated with a greater conversion of LC3-I to LC3-II and the expression of Gabaralp1 and Bnip3l genes involved in mitophagy.

Conclusions: In this highly selected long-term hemodialysis population, a low oxidative phenotype could be defined by a poor endurance, a fiber-type switch, and an alteration of mitochondria structure, without evidence of sarcopenia. This phenotype could be related to uremia through the activation of autophagy/mitophagy.

Clinical trial registration numbers: NCT02794142 and NCT02040363.

Keywords: cell signaling; chronic hemodialysis; chronic kidney disease; mitochondria; phenotype; signal transduction; transplantation.

Graphical abstract

Figure 1.

Endurance and 6-minute walking parameters are changed in patients associated with a transition from type I (oxidative) to type II (glycolytic) fibers. (A) (Left panel) Maximal voluntary force of the quadriceps (C, n=10), (middle) endurance test (C, n=10), (right) 6-minute walk test (C, n=21). (C, control; P, patient; n=22). Data are presented as mean±SD ($, P<0.05). (B) Representative pictures of transverse cryosections of vastus lateralis from a healthy participant (C) or a patient (P), showing the superposition of the bright field picture with the myosin heavy chain 1 (red) and Hoechst 33258 (blue) staining pictures. Scale bar: 100 µm. (C) Myosin percentage and cross-section area (CSA) from control participants (C) or patients (P): type I graph shows the percentage of type I myofibers stained with an anti-myosin I (MHC I). Type N-I graph shows the myofibers not stained by the anti-myosin I antibody (indicated as MHC N-1). (n=21). Data are presented as mean±SD ($, P<0.05). (D) Electron microscopy of vastus lateralis. Scale bar: 1 µm, arrows show the zones of proteolysis and zones of glycogen or lipid accumulation. MVF, maximal voluntary force; N-I, no fiber type 1.

Figure 2.

The ubiquitin-proteasome is not involved in muscle wasting in patients who are hemodialyis selected. Real-time quantitative PCR analysis of IGF-1 (A, left), Murf 1, Atrogin 1, Foxo1, Foxo3 expression. (B) Western blot analysis of myostatin (A, right) and Murf 1 protein expression (C) on muscle biopsies. (C, control, n=10; P, patient, n=22). Data are presented as mean±SD ($, P<0.05).

Figure 3.

The ultrastructure of the mitochondria is altered in patients. (A) Zoom of muscle electron microscopic views, which show sarcomeric disorder and degradation of patients vastus lateralis muscles (Mit, mitochondries; Z, Z line; scale bar: 500 nm). (B) Quantification of mitochondrial density and size. (C, control, n=10; P, patient, n=22). Data are presented as mean±SD ($, P<0.01). (C) Example of mitochondria analysis on control (C) or patient (P); electron microscopic views of large swollen or damaged mitochondria are shown.

Figure 4.

Selected patients who are on long-term hemodialysis present autophagy system activation. (A) Representative example of electron microscopy image detected on muscle biopsies in patients (the images on the right were an enlargement of black square). Arrowhead shows autophagosome structure, (B) western blot analysis of LC3 protein activation. The ratio LC3-II/LC3-I, which reflects LC3 activation, was quantified ($, P<0.05). (C) Real-time quantitative PCR analysis of Gabaralp 1, Bnip 3l, and Bnip 3. (C, control, n=10; P, patient, n=22). Data are presented on the histograms as mean±SD ($, P<0.05).