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Published in final edited form as: Kidney Int. 2009 Jul 29;76(7):10.1038/ki.2009.260. doi: 10.1038/ki.2009.260

Exercise ameliorates chronic kidney disease–induced defects in muscle protein metabolism and progenitor cell function

Xiaonan H Wang 1, Jie Du 2, Janet D Klein 1, James L Bailey 1, William E Mitch 2
PMCID: PMC3835682  NIHMSID: NIHMS512718  PMID: 19641484

Abstract

Chronic kidney disease (CKD) impairs muscle protein metabolism leading to muscle atrophy, and exercise can counteract this muscle wasting. Here we evaluated how resistance exercise (muscle overload) and endurance training (treadmill running) affect CKD-induced abnormalities in muscle protein metabolism and progenitor cell function using mouse plantaris muscle. Both exercise models blunted the increase in disease-induced muscle proteolysis and improved phosphorylation of Akt and the forkhead transcription factor FoxO1. Muscle overloading, but not treadmill running, corrected protein synthesis and levels of mediators of protein synthesis such as phosphorylated mTOR and p70S6K in the muscles of mice with CKD. In these mice, muscle overload, but not treadmill, running, increased muscle progenitor cell number and activity as measured by the amounts of MyoD, myogenin, and eMyHC mRNAs. Muscle overload not only increased plantaris weight and reduced muscle proteolysis but also corrected intracellular signals regulating protein and progenitor cell function in mice with CKD. Treadmill running corrects muscle proteolysis but not protein synthesis or progenitor cell function. Our results provide a basis for evaluating different types of exercise on muscle atrophy in patients with chronic kidney disease.

Keywords: chronic kidney disease, exercise, metabolism, nutrition, signaling

In patients with chronic kidney disease (CKD), a decline in protein stores along with decreased muscle mass is associated with increased morbidity and mortality.1,2 Mechanisms that could cause loss of muscle protein include an increase in protein degradation, a decrease in protein synthesis, and/or impairment in muscle progenitor cell function. We have found that CKD induces an increase in muscle protein degradation that is mediated by the activation of caspase-3 and the ubiquitin–proteasome proteolytic system (UPS).3,4 Not only are these proteolytic pathways stimulated in rodent models of CKD but there is also evidence that they are activated in the muscles of dialysis patients.5,6 Besides stimulating muscle protein degradation, CKD suppresses protein synthesis, but the mechanisms underlying this defect have not been systematically studied.4

Even less is known about the influence of CKD on the activation of muscle progenitor cells (sometimes referred to as satellite cells); these cells are necessary for repairing the loss of muscle mass due to atrophic stimuli or muscle damage.7,8 Specialized functions of these cells include their ability to fuse to produce myofibers that are crucial to the repair of injured muscle or to the prevention of muscle atrophy that occurs in catabolic conditions.7,8 The activation of muscle progenitor cells can be identified by their increased expression of regulatory proteins including MyoD and myogenin; MyoD mediates myoblast activation and proliferation, whereas myogenin mediates myoblast differentiation and the production of structural proteins in muscle such as embryonic myosin heavy chain (eMyHC).9

An important trigger of muscle wasting in CKD is abnormal insulin/insulin-like growth factor 1 (IGF-1) signaling.10 For example, the increase in muscle protein degradation induced by CKD or disorders associated with CKD, such as inflammation, excess angiotensin II, diabetes, and insulin resistance, arise because of defects in insulin/IGF-1 signaling.10–17 The impairment in insulin/IGF-1 signaling in muscle leads to the activation of caspase-3 and the UPS, resulting in accelerated protein degradation. Specifically, the muscle atrophy that occurs with CKD decreases the phosphorylation of Akt (p-Akt), which reduces the phosphorylation of the forkhead transcription factors (FoxO), including FoxO1, allowing it to increase the expression of the ubiquitin E3 ligase, atrogin-1/muscle atrophy F-box (MAFbx).10 The increase in atrogin-1/MAFbx is associated with accelerated muscle protein degradation by the UPS.10 As p-Akt also leads to phosphorylation of mediators of protein synthesis, a 70-kDa ribosomal protein S6 kinase (p70S6K) and the mammalian target of rapamycin (mTOR), impaired insulin/IGF-1 signaling could also suppress protein synthesis.10,18 Finally, there is evidence that p-Akt has a critical role in the activation of muscle progenitor cells.19

Exercise has the potential of blunting CKD-induced abnormalities in IGF-1 cellular signaling. In fact, Rabkin and colleagues14,20 showed that resistance exercise (muscle overloading) in rats with CKD increased the expression of IGF-1 in muscle as well as the levels of downstream mediators of the IGF-1 signaling pathway, IRS-1/P13K/p-Akt (insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt). It has been shown that a single session of resistance exercise can augment protein anabolism in the muscles of hemodialysis patients.21 Moreover, repeated exercise of patients with advanced CKD can improve protein metabolism in muscle: Kopple et al.22 studied hemodialysis patients during ~18 weeks of resistance or endurance exercise and found an increase in IGF-1 protein plus a decrease in myostatin mRNA in muscle. In the same study of hemodialysis patients who participated in a program of endurance (bicycle pedaling) exercise, there was reduction in the 14-kDa actin fragment in muscle biopsies of these patients. This indicates a suppression of the accelerated proteolysis observed in the muscles of dialysis patients.3,6 These reports stimulated us to examine how exercise could influence CKD-induced impairment of muscle metabolism.

We evaluated whether different types of exercise could counteract CKD-induced muscle wasting by studying two models of exercise in mice, muscle overloading and treadmill running. We confirmed that CKD increases protein degradation and decreases protein synthesis in muscle.4 However, CKD-induced defects in muscle protein metabolism responded to the two types of exercise differently. We found that the differences in responses of muscle protein metabolism were related to changes in cellular signaling pathways. We also found evidence that progenitor cell abnormalities may contribute to muscle atrophy in CKD.

RESULTS

The response of muscle weights to muscle overloading and treadmill running

In CKD mice, blood urea nitrogen (BUN) values were twoto three-fold higher than in control mice (Table 1). In normal mice with muscle overloading or treadmill running, plantaris muscle weights increased significantly compared with muscle weights in unexercised normal mice. CKD-induced muscle wasting in the two models of exercise differed; in CKD mice with muscle overloading, plantaris weights were 67% greater than in unexercised CKD mice (P<0.05), whereas plantaris weights of CKD mice trained in treadmill running did not differ from weights of pair-fed, unexercised, CKD mice. A statistical comparison among groups of mice with muscle overload and those with treadmill running is not appropriate as the mice were not paired. However, it appears that muscle overloading induced a more robust increase in plantaris weights compared with treadmill running.

Table 1.

Body weight and muscle weight

Body weight (g) Plantaris weight (mg) Muscle/body (×100, ratio) BUN (mg %)
Resistance
 Control (n=6) 30.7±2.1 16.8±1.0 0.54±1.5 31.6±1.0
 OL (n=6) 29.1±2.5 31.8±3.6* 1.09±1.4* 30.7±0.7
 CKD (n=9) 22.7±1.25* 12.9±0.8* 0.56±0.6 101.8±13.9*
 CKD+OL (n=9) 25.1±0.8* 21.6±1.6#,§ 0.86±1.2*,#,§ 93.3±10.6*
Endurance
 Control (n=6) 29.4±3.1 17.5±2.6 0.59±0.1 30.2±0.9
 Tm (n=6) 27.7±2.7# 23.4±1.7* 0.84±0.2* 29.9±0.3
 CKD (n=9) 22.9±1.8* 11.6±1.7* 0.51±0.1 75.8±9.9*
 CKD+Tm (n=9) 22.8±3.1* 13.1±1.8 0.57±0.1 69.3±9.6*
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BUN, blood urea nitrogen; CKD, chronic kidney disease; OL, overload; Tm, treadmill.

All data are presented as mean±s.e.

*

P<0.05 vs control

#

P<0.05 vs CKD

§

P<0.05 vs CKD+Tm.

Muscle overloading and treadmill running blunt CKD-induced muscle protein degradation

With CKD, the rate of protein degradation was 28% higher in the plantaris muscle of unexercised mice compared with the results in control mice (Figure 1a). Muscle overloading lowered the CKD-induced proteolysis by 57% (P<0.01) to a level only 12% above the value in normal, unexercised mice. A similar pattern occurred in treadmill running mice; the actual rates of protein breakdown are shown in Supplementary Figure S1. Thus, both types of exercise blunted CKD-induced acceleration of protein degradation.

Figure 1. Both plantaris overloading and treadmill running suppress CKD-induced muscle protein degradation.

Figure 1

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Rates of protein degradation were measured in isolated plantaris muscles from the following groups of mice: unexercised, control; CKD; overloading (OL); CKD with overloading (CKD+OL); treadmill running (TM); and CKD with treadmill running (CKD + TM). The results are expressed as the percentage change from values in the muscles of unexercised, control mice (baseline). Values are presented as the mean±s.e. (n=9 per group). Differences detected were *P<0.05 vs values in the muscle of unexercised, control mice; and #P<0.05 vs values in unexercised mice with CKD. CKD, chronic kidney disease.

To investigate why exercise suppressed CKD-induced muscle protein degradation, we examined cell signaling pathways that influence muscle proteolysis.11,15,23,24 In the muscle of CKD mice, the level of Akt serine 473 phosphorylation (p-Akt) was depressed and both models of exercise reversed this defect to control levels (Figure 2a and b). Similarly, the forkhead transcription factor-1 (p-FoxO1) phosphorylation levels at threonine 24/32 were decreased by CKD, and these lower levels were reversed in mice by either muscle overloading or treadmill running. These measurements are relevant because a decrease in p-Akt results in dephosphorylated FoxO1, which migrates into the nucleus to stimulate expression of the E3 ubiquitin ligase, atrogin-1/MAFbx, resulting in increased protein degradation.15,23,24 In agreement with this signaling pathway, we found a significant increase in the mRNAs of ubiquitin E3 ligases, atrogin-1/MAFbx and MuRF1, in the muscle of CKD mice with muscle overload or in those exercised by treadmill running compared with values in the muscle of unexercised, CKD mice (Supplementary Figure S3). We also evaluated the activation of another proteolytic system in muscle, caspase-3, by measuring the level of the 14-kDa actin fragment.3,6,10,14 Both muscle overloading and treadmill running significantly suppressed CKD-induced actin cleavage (Figure 2c).

Figure 2. Both plantaris overloading and treadmill running attenuate the CKD-induced decline in phosphorylation of Akt and FoxO1 and suppression of actin cleavage.

Figure 2

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(a) The phosphorylation of Akt serine 473 and FoxO1 threonine 24/32 in plantaris muscles was assessed by western blot analysis in the following groups of mice: control (C); CKD; overloading (OL); and CKD + overloading (CKD+OL) (top). The bars represent the ratio of the phosphorylated mediator to the total amount of Akt (p-Akt; left panel) and FoxO1 (p-FoxO1; right panel). Values are expressed as a percentage change from values in unexercised, control mice. The data are expressed as a mean±s.e. of six mice in each group. Differences detected were *P<0.05 vs control muscle, and #P<0.05 vs CKD muscle. (b) Values of p-Akt and p-FoxO1 were assessed in six mice in each group of treadmill-running (TM) mice as described for panel 2a. (c) Western blots of the 14-kDa actin fragment in plantaris muscle representing caspase-3-mediated proteolysis. The quantity of this actin fragment was calculated as the density of the 14-kDa band divided by the density of the 42-kDa actin band. Values are the mean±s.e. and are expressed as a percentage of the values found in the muscle of unexercised, control mice. Four mice in each group were examined and differences detected were *P<0.05 vs value in unexercised control mice or #P<0.05 vs values in unexercised mice with CKD. Left bars: overloading; right bars: treadmill running. CKD, chronic kidney disease.

Muscle overloading also prevented the CKD-induced decrease in MGF (the mechano-growth factor, muscle isoform of IGF-1; Supplementary Figure S4). Treadmill running also significantly increased MGF mRNA, but did not prevent the decrease in MGF induced by CKD.

Muscle overloading counteracts the CKD-induced decrease in protein synthesis

In normal mice, muscle overloading increased the rate of protein synthesis to a level 18% higher than that in the muscle of unexercised, control animals (Figure 3; P<0.01). In unexercised CKD mice, the rate of protein synthesis was 12% lower than that in unexercised, control mice; muscle overloading reversed the inhibition of protein synthesis. In contrast, treadmill running did not reverse CKD-induced suppression of muscle protein synthesis. This was unexpected because results from normal mice indicate that treadmill running significantly stimulated muscle protein synthesis compared with the level found in the muscle of normal, unexercised mice. The rates of muscle protein synthesis are showed in Supplementary Figure S2. To determine whether other muscles show the same responses, we measured protein synthesis in the soleus (predominately oxidative, red fibers) and the extensor digitorum longus (predominately glycolytic, white fibers) muscles of mice trained in treadmill running. There was no prevention of the effects of CKD on protein synthesis in both muscles (data not shown).

Figure 3. Plantaris overloading counteracts the CKD-induced suppression of muscle protein synthesis.

Figure 3

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The rates of protein synthesis were measured in plantaris muscles from the same groups of mice as reported in Figure 1. The results are expressed as the percentage change from values in unexercised, control mice (baseline). Values are presented as the mean±s.e. (n=9 per group). Differences detected were *P<0.05 vs values in the muscle of unexercised, control mice; and #P<0.05 vs values in unexercised mice with CKD.

To examine potential reasons why the responses to the two models of exercise differed, we assessed the activation of mediators of protein synthesis by measuring the phosphorylation of threonine 389 of p70S6K (p-p70S6K) and of serine 2448 of mTOR (p-mTOR) (Figure 4a and b). Muscle overloading increased the phosphorylation of p70S6K by five-fold (P<0.01) compared to control mice. In CKD mice, muscle overloading reversed the lower values of p-p70S6K (P<0.01). Changes in p-mTOR levels showed a similar pattern: p-mTOR was significantly decreased in the muscle of CKD mice (P<0.05) and muscle overloading reversed the suppression of p-mTOR (P<0.01). Thus, muscle overloading not only reversed the increase in muscle protein degradation but also reversed the decrease in muscle protein synthesis (reversed the suppression of mediators of protein synthesis) caused by CKD. These beneficial responses were not found in the muscle of mice after treadmill running: CKD was associated with a 60% decrease in p-p70S6K compared with values in the muscle of control mice and this was not reversed by treadmill running. Similarly, in the muscle of normal mice, p-mTOR was increased 1.5-fold by treadmill running, but this response was not found in the muscle of CKD mice.

Figure 4. Plantaris overloading counteracts CKD-induced suppression of the phosphorylation of p70S6K and mTOR in muscle.

Figure 4

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(a) Phosphorylated mediators, p70S6K threonine 389 and mTOR serine 2448, were assessed by western blotting of plantaris muscle from the following groups of mice: control (C); CKD; overloading (OL); and CKD overloading (CKD+OL) mice. The bars represent the ratio of the phosphorylated p70S6K (p-p70S6K; left panel) or mTOR (p-mTOR; right panel) to GAPDH. The values are expressed as a percentage change from values in unexercised, control mice. The data are expressed as a mean±s.e. of six mice in each group. Differences detected were *P<0.05 vs control muscle; and #P<0.05 vs CKD muscle. (b) The same evaluations were made as in panel 4a except that the mice were subjected to treadmill running (TM) rather than muscle overloading. CKD, chronic kidney disease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Muscle overloading increases progenitor cell nuclei and prevents CKD-induced suppression of muscle progenitor cell markers

Muscle progenitor cells are located between the sarcolemma and the basal lamina outside myofibers.7,8 We counted the number of DAPI-positive nuclei outside dystrophin-stained sarcolemma to estimate the number of muscle progenitor cells. In the muscle of CKD mice, there was a significant decrease (18%) in the number of nuclei outside myofibers. However, in CKD mice with muscle overloading, the number of these nuclei increased 1.8-fold compared with values in the muscle of unexercised, CKD mice (Figure 5a). In CKD mice trained in treadmill running, there was no increase in the number of nuclei compared with unexercised CKD mice (Figure 5b).

Figure 5. Overloading attenuates the CKD-induced decline in progenitor cell nuclei.

Figure 5

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(a) A representative section from plantaris muscles is shown after staining for dystrophin (red) and counterstaining with DAPI (blue). The white arrows point to nuclei outside of myofibers. These nuclei were used to estimate the number of progenitor cells. The following groups of mice were assessed: control (C); CKD; overloading (OL); and CKD overloading (CKD+OL). The bars indicate the number of nuclei of cells present outside myofibers in each group. Results are the mean±s.e. (n=4 per group); *P<0.05 vs values from the muscle of unexercised, control mice; and #P<0.05 vs values from the muscle of CKD mice. (b) The same evaluations were made as in panel a except that the mice were subjected to treadmill running (TM) rather than muscle overloading.

The expression of MyoD and myogenin was assessed as a measure of the muscle progenitor cell activation.7,8 We isolated muscle progenitor cells from exercised or unexercised mice with or without CKD. Muscle overloading led to increased levels of the mRNAs of MyoD (a marker of muscle progenitor cell activation and proliferation), myogenin (a marker of proliferation and differentiation), and eMyHC in exercised mice (a marker of differentiation and fusion) compared with those of muscle progenitor cells isolated from the muscle of unexercised, normal mice. As shown in Figure 6a, the differences included a 2.5-fold higher level of myoD, a 1.8-fold increase in myogenin, and a 2.7-fold increase in eMyHC expression (each, P<0.01 vs control). In muscle progenitor cells from the muscle of CKD, the mRNAs of MyoD, myogenin, and eMyHC were decreased by 38, 49, and 55%, respectively, compared with the results obtained in progenitor cells from control mice. In CKD mice with muscle overload, these suppressed levels were reversed, surpassing the levels present in overloaded muscles of normal mice.

Figure 6. Overloading attenuates the CKD-induced decline in mRNAs of myoD, myogenin, and eMyHC.

Figure 6

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(a) mRNA of myoD, myogenin, and eMyHC in plantaris muscles of the following groups of mice: control (C); CKD; overloading (OL); and CKD overloading (CKD+OL). The bars represent the ratio of myoD (left panel), myogenin (middle panel), or eMyHC (right panel) to 18S rRNA. The values are expressed as a percentage change from values in unexercised, control mice. The bars represent the mean±s.e (n=6 per group). Differences detected are *P<0.05 vs values in unexercised, control mice; and #P<0.05 vs values in the muscle of CKD mice. (b) Expression of mRNA of myoD, myogenin, and eMyHC in plantaris muscles of the following groups of mice (n=6 per group): control (C); CKD; treadmill (TM); and CKD treadmill (CKD+TM) mice. Results (means±s.e.) are presented as described in the legend for panel a. CKD, chronic kidney disease; eMyHC, embryonic myosin heavy chain.

In normal mice trained in treadmill running, the expression of MyoD and myogenin was significantly (P<0.05) increased to 2- and 1.6-fold higher, respectively, compared with values from the muscle of unexercised, normal mice (Figure 6b). CKD depressed these values. Although treadmill running of CKD mice raised their MyoD mRNA level, there was no increase in the mRNA levels of myogenin and eMyHC compared with the values in unexercised CKD mice.

DISCUSSION

In addition to nutritional approaches in overcoming muscle atrophy in CKD patients, there is evidence that exercise could improve muscle metabolism in CKD.6,21,22,25,26 To evaluate the influence of exercise on CKD-induced abnormalities in muscle protein turnover, we studied muscle overloading and treadmill running as models of resistance and endurance exercise. In CKD mice, we found that the two models elicited different responses in terms of muscle growth, the components of protein turnover and cellular signaling pathways that can affect protein metabolism, and progenitor cells function in muscle. Specifically, we found that both exercise models suppressed CKD-induced muscle protein degradation. This response was linked to the increased phosphorylation of Akt and FoxO1 and suppressed activation of caspase-3 and the UPS.3,15,23,25 However, the two exercise models exhibited different responses in terms of correcting the CKD-induced decrease in muscle protein synthesis. In mice with muscle overloading, the decrease in muscle protein synthesis was reversed, but with treadmill running there was only a slight improvement in protein synthesis. Interestingly, differences in the phosphorylation of p70S6K and mTOR reflected these responses, linking these mediators to the improvement of protein synthesis. In particular, muscle overloading corrected the low levels of phosphorylated mediators of protein synthesis, p-p70S6K and p-mTOR (Figure 4a), but treadmill running did not significantly improve the levels of these mediators nor the rate of protein synthesis (Figure 4b).

The beneficial influence of muscle overloading was also present when we evaluated CKD-induced abnormalities in muscle progenitor cells. In the muscle of unexercised, CKD mice, there were decreased numbers of progenitor cells compared with findings in control mice. This abnormality was reflected in lower levels of the mRNAs of MyoD, myogenin, and eMyHC, mediators of progenitor cell function. The beneficial influence of muscle overloading on protein synthesis in the muscle of CKD mice was reflected in the increased expression of MyoD, myogenin, and eMyHC, indicating increased production of muscle progenitor cells (Figure 6a). In contrast, treadmill running of CKD mice did not increase the number of progenitor cells nor did it significantly improve their expression of progenitor cell markers (Figure 6b).

These results indicate that muscle overloading can exert at least three changes to blunt the development of muscle atrophy, even when CKD is present: protein degradation is suppressed, protein synthesis is stimulated, and progenitor cells are activated. It can be noted that these functions can be linked to increased phosphorylation of Akt.10 This conclusion is supported by reports indicating that an increase in p-Akt will suppress proteolysis by caspase-3 and the UPS, will increase phosphorylation of p70S6K and mTOR to stimulate protein synthesis, and will activate muscle progenitor cell differentiation and function.15,16,23 The increase in p-Akt related to muscle overloading presumably relates to improved insulin/IGF-1 signaling plus the increase in MGF expression shown in Supplementary Figure S4.10,14

As noted in the Results section, differences in protein turnover and progenitor cell function between muscle overloading and treadmill running cannot be strictly compared, as the mice were not appropriately paired. The differences in responses of these metabolic parameters could be influenced by a constant influence of muscle overloading compared with the intermittent nature of treadmill running. We attempted to examine this possibility by increasing the intensity of treadmill running by CKD mice. However, raising the intensity and duration of exercise increased the mortality of CKD mice; hence, we were unable to assess how muscle metabolism in CKD mice would respond to more prolonged treadmill running exercise. We also did not evaluate whether exercise-induced transitions of myofiber types contributed to the changes we observed. It is relevant to consider the changes in fiber types because the fiber type pattern of a given muscle is specific for that muscle, but it can be modified by changes in muscle activity.27 Unfortunately, assigning outcomes to changes in fiber types is difficult because exercise also induces intermediate fibers expressing several MyHC isoforms.27

Muscle wasting in CKD occurs when there is metabolic acidosis, insulin resistance, excessive angiotensin II, or the presence of inflammation.4,11,28,29 Several of these complications cause abnormalities in IRS-1/P13K/Akt signaling resulting in decreased Akt phosphorylation.4,5,12,15,28,30 This is relevant because exercise stimulates the local release of growth factors, including IGF-1. We have confirmed the report of Chen et al.14 and others31 that muscle overloading led to an increase in p-Akt even when CKD is present. The increase in p-Akt resulted in phosphorylation of p70S6K and mTOR, and this in turn improved muscle protein synthesis (Figure 3). Our finding that treadmill running did not stimulate phosphorylation of p70S6K or mTOR was unexpected because this exercise did increase pAkt slightly. Perhaps the increase was insufficient to see a corresponding increase in protein synthesis. Another possibility is that mTOR could be phosphorylated by a p-Akt-independent pathway.18,32 Clearly, the major effect of treadmill exercise was through decreased protein degradation. We have no evidence that CKD influences other hormones or growth factors released by exercise that might affect changes in muscle protein metabolism or progenitor cell function.20,31

An increase in muscle progenitor cell function in the muscle overload model is especially interesting because these cells produce new myofibers or fuse with existing myofibers to increase muscle mass.33 The ability of muscle progenitor cells to accomplish these tasks depends on highly regulated expression of muscle regulatory factors. These include MyoD, which is responsible for the exit from the cell cycle to proliferate in order to form myotubes, and myogenin, which promotes myoblast differentiation into myotubes. These processes include eMyHC formation that provides a structural protein for myotube formation.9,33 We found that muscle overloading but not treadmill running reversed the suppression of MyoD, myogenin, and eMyHC mRNAs induced by CKD. How CKD and/or exercise affect signaling mechanisms to change the expression of these factors has not been reported, but others have provided information about the influence of exercise on muscle progenitor cells. Consistent with our results, Pislander et al.34 reported that myogenin and myoD mRNA levels in human muscle were 100–400% higher after a single bout of resistance training. Yang et al.35 found an increase in the mRNAs of MyoD (5.8-fold) and myogenin (3.5-fold) with resistance exercise; these factors did not change with a running exercise regimen.

In conclusion, impaired muscle growth due to CKD can be improved in a mouse model of resistance exercise. Mechanisms for this response include improvements in protein synthesis, protein degradation, and muscle progenitor cell function, and each is related to improved signaling through the insulin/IGF-1 pathway. In contrast, the model of treadmill running partially suppressed protein degradation but did not stimulate protein synthesis or improve muscle progenitor cell function. We recognize that the two types of exercise cannot be rigorously compared because of differences in experimental design. We also acknowledge that patients with CKD might respond differently to the two types of exercise. The results, however, do provide insights into the mechanisms that might correct CKD-induced muscle wasting, and they provide experimental information that could be useful in designing a clinical trial of exercise in CKD.

MATERIALS AND METHODS

Animals and CKD model

The experiments were approved by the Institutional Animal Care and Use Committee of Emory University (protocol 141-2008). Mice (C57BL/6J) were kept in a 12-h light/12-h dark cycle. CKD was induced by subtotal nephrectomy in anesthetized mice (12 mg/kg xylazine and 60 mg/kg ketamine) as described.30 A right kidney was removed through a right flank incision. The two poles of the left kidney were excised and hemostasis was achieved by cautery and pressure. Initially, mice were fed 14% Protein Rodent Diet Chow (Harlan Teklad, Madison, WI, USA) ad libitum; after 7 days, control, sham-surgery, and weight-matched mice were pair-fed (40% protein chow) for at least 2 weeks.30 BUN was measured using the Reflotron diagnostic device (Roche, Indianapolis, IN, USA).

Exercise training

Treadmill running

Running on a Model 1055MSD Exer-6M treadmill (Columbus Instruments, Columbus, OH, USA) was studied as a model of endurance exercise. Before subtotal nephrectomy, mice ran at 15 m/min. Exercise intensity was progressively increased over the next week until they ran at 30 m/min. In week 2, a subtotal nephrectomy was carried out, and after 1 week of recovery, mice were exercised by running at 30 m/min for 30 min each day for 2 weeks. Age- and weight-matched control and pair-fed mice were exercised at the same time rate and intensity.

Plantaris muscle overloading

To evaluate a model of resistance exercise, we studied muscle overloading. At 1 week after subtotal nephrectomy, plantaris muscles of mice were overloaded by removing gastrocnemius and soleus muscles from both hindlimbs. Control mice were sham-operated and pair-fed to muscle overloaded CKD mice. After 2 weeks, plantaris muscles were harvested for study.

Skeletal muscle protein metabolism

Protein degradation in plantaris muscles was assessed as tyrosine released into the media, whereas protein synthesis was measured as phenylalanine incorporation into muscle as described.4,30 These amino acids were used because muscle neither synthesizes nor degrades them and tyrosine does not accumulate in the intracellular pool of muscle; hence, its release into the media is an estimate of protein degradation. Phenylalanine rapidly equilibrates with the intracellular amino acid pool in muscle and can be used to measure protein synthesis. Details are given in Supplementary materials.

Western blot and antibodies

Plantaris muscles were homogenized in RIPA buffer, and signaling proteins in the soluble fraction of tissue homogenates were assessed by western blotting.36 When we assessed the 14-kDa actin fragment, muscles were homogenized in hypotonic buffer.3 Details of the western blot procedure are given in the Supplementary materials.

Muscle progenitor cell isolation

To isolate muscle progenitor cells from plantaris muscles, muscles were minced to create a coarse slurry followed by gentle agitation in DMEM (Dulbecco’s modified Eagle’s medium) containing 25 mm HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4) plus 0.1% pronase (Calbiochem, San Diego, CA, USA) for 1 h at 37 °C. The digest was passed through a 100-μm filter before centrifugation through an isotonic Percoll gradient.8 The cells were collected from the interface of the Percoll gradient.

Muscle progenitor cell number estimates

Nuclei numbers outside muscle fibers were counted in three transverse cross-sections from frozen sections of plantaris muscles after staining using the MOM Immunodetection Kit (Vector Laboratories, Burlingame, CA, USA). The sections were incubated with anti-dystrophin antibody for 30 min, followed by Fluorescein Avidin DCS for 5 min and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 500 ng/ml) for 10 min. The myofiber sarcolemma was defined by dystrophin staining (red). The nuclei outside the sarcolemma were stained with DAPI (blue) and visualized using an Olympus 1×51 inverted fluorescence microscope; the images were captured by a SIS-CC12 CLR camera. At least 300 individual myofibers per muscle were measured using MicroSuite Five Biological Software (Olympus, Melville, NY, USA).

Reverse transcription-PCR and qPCR

mRNAs for MyoD, myogenin, and eMyHC were measured by RT-PCR, and atrogin-1/MAFbx, MuRF1and MGF mRNA were measured by quantitative PCR (qPCR) as described.36 The detail protocol and primer sequence are provided in Supplementary materials.

Statistical analysis

Results are presented as mean±s.e. Data were analyzed using t-test when comparing results from two experimental groups; ANOVA for results from more groups. After ANOVA, pair-wise comparisons were made using the Student–Newman–Keuls test.

Supplementary Material

supplement

ACKNOWLEDGMENTS

This work was supported in part by a Norman S. Coplon Extramural research grant from Satellite Health, and a grant from the University Research Committee of Emory University to XHW; NIH DK062081 and AHA GIA0655280B to JDK; NIH R37 DK37175 and P50 DK64233 to WEM; and R01HL70762 to JD. We thank Drs Karyn A. Esser and John J. McCarthy of the University of Kentucky for their advice and assistance with evaluating the influence of exercise in mouse models.

Footnotes

DISCLOSURE All the authors declared no competing interests.

SUPPLEMENTARY MATERIAL Figure S1. Both plantaris overloading and treadmill running suppress the increase in muscle protein degradation by CKD.

Figure S2. Plantaris overloading counteracts the suppression of muscle protein synthesis induced by CKD.

Figure S3. The mRNA levels of atrogin-1/MAFbx and MuRF1 in plantaris muscles were measured using qPCR.

Figure S4. The mRNA values of MGF in plantaris muscles were measured using qPCR.

Supplementary material is linked to the online version of the paper at http://www.nature.com/ki

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