1. Volume 85, Issue 6, Pages 1330-1339 (June 2014)
Chronic kidney disease reduces muscle mitochondria and exercise endurance and its exacerbation by dietary protein through inactivation of pyruvate dehydrogenase 
Masanori Tamaki, Kazutoshi Miyashita, Shu Wakino, Masanori Mitsuishi, Koichi Hayashi, Hiroshi Itoh 
Kidney International 
Volume 85, Issue 6, Pages (June 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Muscle mitochondria and running distance are decreased in young 5/6 nephrectomized mice, despite that muscle volume and power are preserved. Mice were assigned to sham or 5/6Nx group. (a, b) Grip power (a) and running distance (b) of 5/6Nx mice at 8–16 weeks (8W–16W). (c) Macroscopic view of the gastrocnemius and soleus muscle at 20 weeks (20W). (d) SDH staining of the EDL muscle. Scale bar=100μm. (e) Weight of the gastrocnemius muscle. (f) Fiber size of the SDH-stained EDL muscle. (g, h) Western blot analysis of molecules involved in muscle degradation and hypertrophy: representative blots of actin fr., full length, Casp.3, β-actin, P-S6K1, and total S6K1 (g). Densitometry analysis of the blots of molecules related to muscle volume: ratio of actin fragment (actin fr.) to full-length actin (full length), Casp.3 to β-actin, and P-S6K1 to total S6K1 of the quadriceps muscle (h). (i) Muscle fiber type of the SDH-stained EDL muscle. (j, k) Mitochondrial amount (j) and ETC activity (k) of the quadriceps muscle. (l, m) Western blot analysis of molecules related to mitochondria: representative blots of P-AMPK, AMPK, porin, ATPsyn, and β-actin (l). Densitometry analysis of the blots of molecules related to muscle mitochondria: ratio of P-AMPK to total AMPK, porin to β-actin, and ATPsyn to β-actin of the quadriceps muscle (m). (n) Electron microscopic observation of the gastrocnemius muscle. Arrows: mitochondria. Scale bar=1μm. (o–r) Real-time PCR analysis of molecules related to muscle mitochondria: PGC-1α, Tfam (o), COX IV, ATPsyn (p), BNIP3L, PINK1 (q), PPAR-δ, and troponin I (r) of the quadriceps muscle; n=8 in each group. *P<0.05, **P<0.01 versus sham group. I, type I fiber (slow oxidative); IIa, type IIa fiber (fast oxidative glycolytic); IIb, type IIb fiber (fast glycolytic); actin fr., 14KDa actin fragment; AMPK, adenosine monophosphate–activated protein kinase; ATPsyn, adenosine triphosphate synthase; BNIP3L, BCL2/adenovirus E1B 19KDa protein-interacting protein 3-like; Casp.3, 17KDa cleaved caspase-3; COX IV, cytochrome c oxidase subunit IV; EDL, extensor digitorum longus; ETC, electron transport complex; full length, 42KDa full length actin; 5/6Nx, 5/6 nephrectomized mice; P-S6K1, phosphorylated S6K1; P-AMPK, phosphorylated AMPK; PGC-1α, peroxisome proliferator–activated receptor-γ coactivator 1α; PINK1, PTEN-induced putative kinase 1; PPAR-δ, peroxisome proliferator-activated receptor-δ; SDH, succinate dehydrogenase; S6K1, S6 kinase 1; Tfam, mitochondrial transcription factor A.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Muscle volume and grip power are decreased in aged 5/6 nephrectomized mice, in addition to muscle mitochondria and running distance. Mice were assigned to sham or 5/6Nx group. (a, b) Grip power (a) and running distance (b) at 48 weeks. (c) Macroscopic view of the gastrocnemius and soleus muscle at 52 weeks. (d, e) Body weight at 48 weeks (d) and weight of the gastrocnemius muscle (e). (f) Densitometry analysis of the blots of molecules related to muscle volume: ratio of actin fragment to full length actin, Casp.3 to β-actin, and P-S6K1 to total S6K1 of the quadriceps muscle. (g, h) Mitochondrial amount (g) and ETC activity (h). (i) Densitometry analysis of the blots of molecules related to muscle mitochondria: ratio of P-AMPK to total AMPK, porin to β-actin, and ATPsyn to β-actin of the quadriceps muscle. (j, k) Real-time PCR analysis of molecules related to muscle mitochondria: PGC-1α, ATPsyn (j), BNIP3L, and PINK1 (k) of the quadriceps muscle; n=8 in each group. *P<0.05, **P<0.01 versus P10% in each group. actin fr., 14KDa actin fragment; AMPK, adenosine monophosphate–activated protein kinase; ATPsyn, adenosine triphosphate synthase; BNIP3L, BCL2/adenovirus E1B 19KDa protein-interacting protein 3-like; Casp.3, 17KDa cleaved caspase-3; ETC, electron transport complex; full length, 42KDa full length actin; 5/6Nx, 5/6 nephrectomized mice; P-S6K1, phosphorylated S6K1; P-AMPK, phosphorylated AMPK; PGC-1α, peroxisome proliferator–activated receptor-γ coactivator 1α; PINK1, PTEN-induced putative kinase 1; S6K1, S6 kinase 1.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
A high-protein diet decreases exercise endurance in young 5/6 nephrectomized mice without drastic change in muscle mitochondria. Mice were assigned to P3%, P10%, P30%, and P50% group. (a, b) Grip power (a) and running distance (b) of 5/6Nx mice at 16 weeks fed P3% to P50%. (c) Macroscopic view of the gastrocnemius and soleus muscle at 20 weeks. (d) SDH staining of the EDL muscle. Scale bar=100μm. (e) Weight of the gastrocnemius muscle. (f) Fiber size of the SDH-stained EDL muscle. (g, h) Mitochondrial amount (g) and ETC activity (h). (i) Densitometry analysis of the blots of molecules related to muscle volume or mitochondria: ratio of P-S6K1 to total S6K1, P-AMPK to total AMPK, porin to β-actin, and ATPsyn to β-actin. (j–m) Real-time PCR analysis of molecules related to muscle mitochondria: PGC-1α, Tfam (j), COX IV, ATPsyn (k), BNIP3L, PINK1 (l), PPAR-δ, and troponin I (m) of the quadriceps muscle; n=8 in each group. *P<0.05, **P<0.01 versus P10% in each group. AMPK, adenosine monophosphate–activated protein kinase; ATPsyn, adenosine triphosphate synthase; BNIP3L, BCL2/adenovirus E1B 19KDa protein-interacting protein 3-like; 5/6Nx, 5/6 nephrectomized mice; P3%, 3% Kcal protein in diet; P10%, 10% Kcal protein in diet; P30%, 30% Kcal protein in diet; P50%, 50% Kcal protein in diet; P-S6K1, phosphorylated S6K1; P-AMPK, phosphorylated AMPK; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator 1α; PINK1, PTEN-induced putative kinase 1; PPAR-δ, peroxisome proliferator-activated receptor-δ; SDH, succinate dehydrogenase; S6K1, S6 kinase 1; Tfam, mitochondrial transcription factor A.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Pyruvate dehydrogenase activity is a key factor to regulate exercise endurance in 5/6 nephrectomized mice fed a high-protein diet. Mice were assigned to P10% or P30% group. (a–e) Analysis of the effects of dietary protein on young 5/6Nx mice. (a) Blood lactate levels before and after exercise. (b) PDH activity of the quadriceps muscle. (c, d) Analysis of the effect of DCA (PDH activator) in 5/6Nx mice fed P10% or P30%. (c) Running distance and (d) PDH activity of the quadriceps muscle. (e) Serum total amino acids; n=8 in each group for in vivo experiments. **P<0.01 versus DCA-. #P<0.05, ##P<0.01 versus P10%. The cultured myocytes were treated with or without a commercially available amino acid cocktail for 24h. (f–i) Mitochondrial energy metabolism in C2C12 myocytes. (f) O2 utilization and (g) ATP content of myocytes. (h) Lactate levels in cultured medium. (i) PDH activity of myocytes. Inflammatory cytokines and mediators of oxidative stress reduce mitochondria in C2C12 myocytes. The cultured myocytes were treated with inflammatory cytokines (TNF-α or IL-6; 0.01, 0.1, or 1ng/ml each) or mediators of oxidative stress (acrolein or 4-HNE; 1, 10, or 100μmol/l each), with or without inhibitors of each substance (SPD-304 (100μmol/l) for TNF-α; D7715A7 (100μg/ml) for IL-6; N-acetylcystein (100mmol/l) for acrolein and 4-HNE) for 24h. (j–l) Regulation of mitochondrial amount in C2C12 myocytes. Real-time PCR analysis of molecules related to mitochondrial biogenesis or degradation: (j) PGC-1α and (k) BNIP3L. (l) Quantification of mitochondrial amount in the myocytes by use of fluorescent probe; n=4 independent experiments for in vitro. *P<0.05, **P<0.01 versus control in each group. #P<0.05, ##P<0.01 versus the treated group without inhibitors. AA, amino acid cocktail; ATP, adenosine triphosphate; BNIP3L, BCL2/adenovirus E1B 19KDa protein-interacting protein 3-like; DCA, dichloroacetate; 4-HNE, 4-hydroxynonenal; IL-6, interleukin-6; 5/6Nx, 5/6 nephrectomized mice; O2 utilization, oxygen utilization; P10%, 10% Kcal protein in diet; P30%, 30% Kcal protein in diet; PDH, pyruvate dehydrogenase; PGC-1α, peroxisome proliferator–activated receptor-γ coactivator 1-α; TNF-α, tumor necrosis factor-α.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Schematic representation of the findings in this study. Muscle insufficiency in renal failure and harmful effects of dietary protein, which were identified in this study, are demonstrated. AMPK, adenosine monophosphate–activated protein kinase; PDH, pyruvate dehydrogenase; S6K1, S6 kinase 1.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions