The G protein-coupled receptor ligand apelin-13 ameliorates skeletal muscle atrophy induced by chronic kidney disease

Abstract

Background: Targeting of the apelin-apelin receptor (Apj) system may serve as a useful therapeutic intervention for the management of chronic kidney disease (CKD)-induced skeletal muscle atrophy. We investigated the roles and efficacy of the apelin-Apj system in CKD-induced skeletal muscle atrophy.

Methods: The 5/6-nephrectomized mice were used as CKD models. AST-120, a charcoal adsorbent of uraemic toxins (8 w/w% in diet), or apelin (1 μmol/kg) was administered to CKD mice to investigate the mechanism and therapeutic potential of apelin on CKD-induced skeletal muscle atrophy. The effect of indoxyl sulfate, a uraemic toxin, or apelin on skeletal muscle atrophy was evaluated using mouse myoblast cells (C2C12 cells) in vitro.

Results: Skeletal muscle atrophy developed over time following nephrectomy at 12 weeks, as confirmed by a significant increase of atrogin-1 and myostatin mRNA expression in the gastrocnemius (GA) muscle and a decrease of lower limb skeletal muscle weight (P < 0.05, 0.01 and 0.05, respectively). Apelin expression in GA muscle was significantly decreased (P < 0.05) and elabela, another Apj endogenous ligand, tended to show a non-significant decrease at 12 weeks after nephrectomy. Administration of AST-120 inhibited the decline of muscle weight and increase of atrogin-1 and myostatin expression. Apelin and elabela expression was slightly improved by AST-120 administration but Apj expression was not, suggesting the involvement of uraemic toxins in endogenous Apj ligand expression. The administration of apelin at 1.0 μmol/kg for 4 weeks to CKD mice suppressed the increase of atrogin-1 and myostatin, increased apelin and Apj mRNA expression at 30 min after apelin administration and significantly ameliorated weight loss and a decrease of the cross-sectional area of hindlimb skeletal muscle.

Conclusions: This study demonstrated for the first time the association of the Apj endogenous ligand-uraemic toxin axis with skeletal muscle atrophy in CKD and the utility of therapeutic targeting of the apelin-Apj system.

Keywords: Apj; apelin; chronic kidney disease; elabela; skeletal muscle atrophy; uraemic toxin.

Figure 1

Experimental scheme. (A) Evaluation of skeletal muscle atrophy progression and alteration of myokine expression in chronic kidney disease (CKD) mice. 5/6‐Nephrectomy was performed by two‐step surgery, and mice were maintained with standard food and water. Mice were sacrificed and evaluation was performed at 4, 8 and 12 weeks after second‐step surgery. (B) Evaluation of relationship between uraemic toxins and myokine expression in skeletal muscles of CKD mice. At 4 weeks after 5/6‐nephrectomy, mice were randomized and AST‐120 containing powder diet (8 w/w%) was administered for 8 weeks and mice were then sacrificed and evaluation was performed. (C) Evaluation of effect of apelin on CKD‐induced skeletal muscle atrophy. At 8 weeks after 5/6‐nephrectomy, mice were administered apelin once a day (1 μmol/kg, intraperitoneally) for 4 weeks and then mice were sacrificed and evaluation was performed.

Figure 2

Progression of skeletal muscle atrophy and alteration of myokine expression in chronic kidney disease (CKD) mice. (A–D, F) mRNA expression in the gastrocnemius muscle of CKD mice was determined by real‐time RT‐PCR. mRNA expression of Atrogin‐1, Apj, Myod, Myogenin and Pax7 was determined relative to the sham group that was used as a control. mRNA expression of Myostatin, Il‐6, Mif, Apelin, Irisin, Sparc and Elabela was determined relative to the sham‐operated group at 4 weeks. (E) Protein expression of APELIN and APJ or ELABELA was determined by western blot or enzyme‐linked immunosorbent assay (ELISA), respectively. Data are expressed as the means ± SEM (n = 4). *, **P < 0.05 or 0.01 compared with the respective sham‐operated group.

Figure 3

Relationship between uraemic toxins and myokine expression in skeletal muscles of chronic kidney disease (CKD) mice. (A) Cryosections of the tibial anterior muscle were immunostained with an anti‐laminin antibody to measure myofiber diameters. (B–E) mRNA expression of atrogin‐1, other myokines and Apj in the gastrocnemius muscle. (F) Protein expression of APELIN and APJ or ELABELA was determined by western blot or enzyme‐linked immunosorbent assay (ELISA), respectively. Data are expressed as the means ± SEM (n = 6–8). *P < 0.05 compared with each sham or indoxyl sulfate potassium (IS) (0 mM)‐treated group.^# P < 0.05 compared with CKD.

Figure 4

The effect of indoxyl sulfate potassium (IS) on apelin and elabela expression and efficacy of apelin in C2C12 cells. (A and B) The effect of IS (0, 0.25 and 1.0 mM) on apelin and elabela expression in C2C12 cells was determined by real‐time RT‐PCR. (C) The effect of apelin (1.0 or 10 nM) on IS (0.25 mM)‐induced skeletal muscle atrophy‐related genes expression in C2C12 cells was determined by real‐time RT‐PCR. Data are expressed as the means ± SEM (n = 3). *P < 0.05 compared with IS 0 mM.^{#, ##} P < 0.05 or 0.01.

Figure 5

Effect of apelin on chronic kidney disease (CKD)‐induced skeletal muscle atrophy. (A) The serum concentrations of apelin after apelin administration (1 μmol/kg, intraperitoneally) in CKD mice were determined by enzyme‐linked immunosorbent assay (ELISA). (B and C) The change in mRNA expression in the gastrocnemius muscle after apelin administration (1 μmol/kg, intraperitoneally) in CKD mice. (D) Cryosections of the tibial anterior muscle were immunostained with an anti‐laminin antibody to measure myofiber diameters. (E) mRNA expression of Atrogin‐1, other myokines and Apj in the gastrocnemius muscle at 4 weeks after apelin administration. Data are expressed as the means ± SEM (n = 5–6). *P < 0.05 compared with the 0 min group after apelin administration or the CKD group.^{#, ##} P < 0.05 or 0.01 compared with indoxyl sulfate potassium (IS) alone, respectively.

Figure 6

Proposed role of apelin in skeletal muscle atrophy in chronic kidney disease (CKD) and its therapeutic potential. In the early stage (approximately 8 weeks after 5/6‐nephrectomy) of CKD, myokine expressions such as myostatin, inflammation‐related, apelin, elabela, irisin and sparc were increased, and apj expression was also increased. In the late stage (>8 weeks after 5/6‐nephrectomy) of CKD, expression of apelin, elabela, irisin, sparc and apj decreased because of disruption of the compensatory mechanism, which may be the cause of the acceleration of skeletal muscle atrophy in CKD. Apelin administration transiently inhibits the increase of atrogin‐1 and myostatin expression and increases expression of apelin and Apj, which contributes to the amelioration of CKD‐induced skeletal muscle atrophy.