A pegylated leptin antagonist ameliorates CKD-associated cachexia in mice

Abstract

Elevated serum leptin levels correlate with inflammation and predict changes in lean body mass in patients with CKD, and activation of the melanocortin system by leptin signaling mediates the pathophysiology of CKD-associated cachexia. We tested whether treatment with a pegylated leptin receptor antagonist (PLA) attenuates cachexia in mice with CKD. CKD and Sham mice received vehicle or PLA (2 or 7 mg/kg per day). At these doses, PLA did not influence serum leptin levels in mice. Treatment with 7 mg/kg per day PLA stimulated appetite and weight gain, improved lean mass and muscle function, reduced energy expenditure, and normalized the levels of hepatic TNF-α and IL-6 mRNA in mice with CKD. Furthermore, treatment with 7 mg/kg per day PLA attenuated the CKD-associated increase in the transcriptional and protein abundance of uncoupling proteins that mediates thermogenesis, and it normalized the molecular signatures of processes associated with muscle wasting in CKD, including proteolysis, myogenesis and muscle regeneration, and expression of proinflammatory muscle cytokines, such as IL-1α, -1β, and -6 and TNF-α. Our results suggest that leptin antagonism may represent a viable therapeutic strategy for cachexia in CKD.

Figure 1.

Schematic representation of the study design.

Figure 2.

Average daily calorie intake in mice. For CKD/V, CKD/PLA (2 mg/kg per day), CKD/PLA (7 mg/kg per day), Sham/PLA, and Sham/V mice, calorie intake (kilocalories) was calculated by multiplication of daily food intake of Diet 5015 (grams) with physiologic fuel value of 3.8 kcal/g. For CKD/Supp mice, total calorie intake (kilocalories) is the sum of calorie intake derived from daily food intake and nutrient supplementation. Calorie intake from nutrient supplementation is the volume of supplemented nutrient (in milliliters)×3.0 kcal/ml. Calorie intakes of CKD/V, CKD/PLA (2 mg/kg per day), CKD/PLA (7 mg/kg per day), CKD/Supp, and Sham/PLA mice were compared with calorie intakes of Sham/V mice. Calorie intakes of CKD/PLA (2 mg/kg per day) and CKD/PLA (7 mg/kg per day) mice were also compared with calories intakes of CKD/V mice. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 3.

Weight change during the course of the study. Mice were weighted and normalized with initial weight. Weight changes in CKD/V, CKD/PLA (2 mg/kg per day), CKD/PLA (7 mg/kg per day), CKD/Supp, and Sham/PLA mice were compared with Sham/V mice. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

Change of (A) fat and (B) lean mass in mice. Animals were scanned before the initiation of the study followed 28 days later by a second quantitative magnetic resonance imaging scan. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

Muscle function in mice. (A) Forelimb grip strength and (B) motor coordination. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01; ***P<0.001.

Figure 6.

Basal metabolic rate in mice. (A) Light- and (B) dark-phase VO2. VO2 (milliliters per kilogram per hour) is calculated as the average readings obtained during the recording period. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01; ***P<0.001.

Figure 7.

Gene expression of UCPs. (A) Gene expression of UCP-1 in brown adipose tissue. (B) Gene expression of UCP-2 in white adipose tissue. (C) Gene expression of UCP-3 in gastrocnemius muscle. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01; ***P<0.001.

Figure 8.

Protein contents of UCPs. (A) Protein content of UCP-1 in brown adipose tissue. (B) Protein content of UCP-2 in white adipose tissue. (C) Protein content of UCP-3 in gastrocnemius muscle. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01; ***P<0.001.

Figure 9.

Hepatic inflammatory cytokine mRNA levels. (A) Hepatic TNF-α mRNA. (B) Hepatic IL-6 mRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Results were analyzed and expressed as in Figure 3. **P<0.01.

Figure 10.

Protein concentrations of cytokines in gastrocnemius muscle. (A) Muscle IL-1α content. (B) Muscle IL-1β content. (C) Muscle IL-6 content. (D) Muscle IL-10 content. (E) Muscle IFN-γ content. (F) Muscle TNF-α content. (G) Muscle CXCL-16 content. (H) Muscle MCP-1 content. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01.

Figure 11.

Protein concentration of IGF-I and myostatin in gastrocnemius muscle. (A) Muscle IGF-I protein content. (B) Muscle myostatin protein content. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01.

Figure 12.

Gene expression of key molecules implicated in the pathogenesis of CKD-associated muscle wasting. (A) Muscle Atrogin-1 mRNA expression. (B) Muscle MuRF-1 mRNA expression. (C) Muscle Sirt-1 mRNA expression. (D) Muscle PGC-1α mRNA expression. (E) Muscle Pax-3 mRNA expression. (F) Muscle Pax-7 mRNA expression. (G) Muscle myogenin mRNA expression. (H) Muscle MyoD mRNA expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Results were analyzed and expressed as in Figure 3. *P<0.05; **P<0.01.

Figure 13.

Summary of the beneficial effects of PLA treatment on food intake, energy expenditure, and muscle wasting in CKD mice.