Reduced synthesis of muscle proteins in chronic renal failure

Deborah Adey1, Rajiv Kumar1, James T. McCarthy1, and K. Sreekumaran Nair2

Divisions of 1 Nephrology and 2 Endocrinology, Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle wasting and weakness occur frequently in patients with chronic renal failure. The mechanism(s) by which these abnormalities occur is unclear. We hypothesized that such findings were due to defective muscle protein synthesis. We measured synthetic rates of mixed muscle proteins, myosin heavy chain, and mitochondrial proteins in serial muscle biopsy samples during a continuous infusion of L[1-13C]leucine from 12 patients with chronic renal failure and 10 healthy control subjects under identical study conditions. Patients with chronic renal failure have significantly lower synthetic rates of mixed muscle proteins and myosin heavy chain (27 and 37% reductions, respectively, P < 0.05 and P < 0.02). Significant declines in the synthetic rates of muscle mitochondrial protein (27%) (P < 0.05), muscle cytochrome c-oxidase activity (42%) (P < 0.007), and citrate synthase (27%) (P < 0.007) were also observed in patients with chronic renal failure. The synthetic rates of muscle proteins and activity of mitochondrial enzymes were negatively correlated to the severity of renal failure. These results indicate that in chronic renal failure there is a decrease in the synthesis of muscle contractile and mitochondrial proteins and a decrease in muscle mitochondrial oxidative enzymes. Reduced synthetic rate of several muscle proteins is the likely biochemical basis of muscle loss and muscle weakness in people with chronic renal failure.

mitochondria; myosin heavy chain


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MUSCLE WASTING AND WEAKNESS are common clinical features in people with chronic renal failure (17). This cachexic state contributes to increased morbidity in chronic renal failure patients. Little is known about the mechanism of muscle wasting in these patients with chronic renal failure (20). Most of the studies investigating the mechanism of muscle wasting in chronic renal failure have been performed in rodent models (3, 14, 16, 20, 28). In rats with acute and chronic renal failure, increased protein breakdown has been reported to occur (3, 14, 20, 28). It has been proposed on the basis of animal studies that the mechanism of muscle wasting in people with chronic renal failure is related to increased muscle protein breakdown (30). There is, however, no direct proof of this phenomenon in humans, and the effect of renal failure on muscle protein synthesis has not been investigated.

Muscle loss occurs either because of increased muscle protein breakdown or decreased muscle protein synthesis or a combination of both. Decreased muscle protein synthesis occurs in many chronic muscle-wasting conditions, such as Duchenne muscular dystrophy and myotonic dystrophy (18, 38). Muscle wasting and weakness also occur in aging as a result of decreased synthesis rates of muscle proteins (7, 39). We hypothesized that muscle wasting in chronic renal failure is related to decreased synthesis rates of several muscle proteins and muscle contractile proteins, such as myosin heavy chain (2, 5). Because protein synthesis is dependent on the availability of energy, we also investigated whether synthesis of mitochondrial proteins (39) is altered in this condition. We show that in patients with chronic renal failure, mixed muscle, myosin heavy chain, and mitochondrial protein synthetic rates are diminished.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

L[1-13C]leucine (99 atom percent excess) and 13C-labeled sodium bicarbonate were purchased from Cambridge Isotopes Laboratories (Andover, MA). Chemical and isotopic purities were confirmed before use. Various isotopically labeled leucine and sodium bicarbonate solutions were prepared in sterile normal saline. The absence of pyrogens and bacteria was confirmed before human use. All studies were approved by the Mayo Foundation Institutional Review Board, and written informed consent was obtained from participants.

Subjects

Twelve patients with chronic renal failure of varying degrees and ten healthy control subjects were studied. All of the renal failure patients were in a predialysis phase and had never undergone dialysis therapy. Exclusion criteria included diabetes mellitus, proteinuria >2.0 g/24 h, use of glucocorticoids, anticoagulants, or cytotoxic agents within the last year, chronic antibiotic therapy, dialysis therapy, or renal transplantation. The subjects studied had polycystic kidney disease (n = 6), hypertension with nephrosclerosis (n = 1), or parenchymal renal disease (n = 5). Glomerular filtration rate (GFR) was determined by the clearance of iothalamate. In patients with chronic renal failure, iothalamate clearance ranged between 8 and 60 ml/min, and in healthy control subjects, it ranged between 75 and 117 ml/min. All control subjects were determined to be healthy after review of their medical records, physical examination, and review of laboratory values.

Diet

All study volunteers followed a similar diet with respect to protein intake (1 g · kg body wt-1 · day-1) for the month before study. Food records were kept by the subjects and were reviewed by dietitians on every 10-14 days during this 1-mo period before the study. Adjustments in protein intake were made by the subjects as needed. During the 3 days before the study, all meals were prepared by the metabolic kitchen of the Mayo Clinic General Clinical Research Center. They were comparable in composition with respect to carbohydrate, protein, and fat (55:15:30, respectively), with a protein intake of 1 g/kg body wt. Such standard diets 3 days before the protein turnover measurements in six subjects resulted in <4% coefficient variation in leucine flux, leucine oxidation, and nonoxidative leucine flux measurements in five repeated studies at >2-wk intervals between each study (10).

Study Design

All subjects were studied after an overnight fast. Priming doses of L-[1-13C]leucine (1.0 mg/kg) and [13C]sodium bicarbonate (0.2 mg/kg) were given intravenously to prime the respective pools and bring about early isotope plateaus (26). Immediately after the priming dose, a continuous infusion of L-[1-13C]leucine (1.0 mg · kg-1 · h-1) was given for 10 h. Percutaneous needle muscle biopsies were performed under sterile conditions at 5 and 10 h after the beginning of the infusion (31, 34). Blood and breath samples were drawn at baseline and every hour from 5 to 10 h. Rest CO2 production was measured for 60 min by indirect calorimetry at 6-7 h.

Body Composition

All study subjects underwent evaluation of body composition by dual-energy X-ray absorptiometry (DEXA), which provided an assessment of fat mass (21).

Analytical Techniques

Breath and plasma sample measurement. Measurement of 13CO2 was performed with a gas chromatograph-isotope ratio mass spectrometer, as previously described (4). Isotopic enrichment of plasma [13C]ketoisocaproate (KIC) was measured with a gas chromatograph-mass spectrometer as the quinoxalinol-trimethylsilyl derivative (27) by use of electron impact ionization conditions. Selective mass-to-charge (m/z) fragment ions 233/232 were monitored. Plasma KIC concentration was measured using ketovaleric acid as an internal standard. Muscle tissue fluid was separated as previously described (9), and the amino acids were derivatized as their trifluroacetyl isopropyl esters (1). Enrichment of [13C]leucine was measured in a gas chromatograph-mass spectrometer under chemical ionization conditions with ammonia as a carrier gas.

Muscle proteins. Mixed muscle proteins in the biopsy sample were separated and hydrolyzed as previously described (9, 35). The constituent amino acids in the hydrolysate were purified by cation exchange chromatography (Dowex 50, H+ form, Bio-Rad Laboratories, Hercules, CA), and amino acids were eluted with the use of 4 M ammonium hydroxide, as previously described (9). The purification of muscle mitochondrial proteins (39, 40) and myosin heavy chains (5, 6) was performed as previously described. These proteins were also hydrolyzed, and the constituent amino acids were purified as noted above. Leucine from the purified amino acids of the hydrolysate (both mixed muscle protein and myosin heavy chain) was purified by high-performance liquid chromatography, without derivatization, by use of a reversed-phase C18 column, as previously described (8). Carbon dioxide from the carboxyl group of purified leucine was liberated by the ninhydrin reaction, and isotopic enrichment was measured with a gas chromatograph-isotope ratio mass spectrometer (Delta S, Finnigam MAT, Bremen, Germany) as described (4). The isotopic enrichment of leucine in the mitochondrial hydrolysate was also measured with the same instrument, by use of a combustion system as previously described (4).

Measurement of glucose and amino acids. Serum total CO2 content was measured using the bicarbonate kit based on phosphoenolpyruvate conversion of bicarbonate to oxaloacetate and ultaviolet detection of the decline of NADH in the solution (Boehringer Mannheim, Indianapolis, IN). Plasma glucose concentrations were measured enzymatically with an auto analyzer (Beckman Instruments, Fullerton, CA). Plasma concentrations of amino acids were measured as previously reported (19, 32). Hormone levels were measured using established assays. Insulin was measured by chemiluminescent sandwich assay (Sanofi Diagnostics, Chaska, MN) (46). Growth hormone was measured using a locally developed two-site chemiluminescent sandwich assay using antibodies from Sanofi Diagnostics. Insulin-like growth factor (IGF)-I, IGF-II, and IGF-binding proteins were measured using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX). The latter analyses included a simple extraction step in which IGF-I is separated from its binding proteins (22). Plasma concentrations of epinephrine and norepinephrine (11) and parathyroid hormone (23) were measured by RIA, and cortisol was measured by a competitive binding immunoenzymatic assay based on a commercial kit (Sanofi Diagnostics, Chaska, MN).

Measurement of glomerular filtration. GFR was measured using a short iothalamate clearance method (47) and standard 24-h urine creatinine clearance.

Calculations

Leucine kinetics. Leucine flux (Q) is calculated on the basis of a stochastic model, and the underlying assumptions are discussed in detail elsewhere (31). Q in an isotopic steady state represents both leucine appearance rate (Ra) or protein breakdown (B) and leucine disappearance rate (Rd). Q is calculated as follows
<IT>Q</IT> = (E<SUB>i</SUB>/E<SUB>p</SUB> − 1) × I<SUB>i</SUB>
where Ei is the isotopic enrichment of the infusate (99%), Ep is the mean isotope enrichment of [13C]KIC (transamination product of leucine) in plasma at plateau, and Ii is the rate of infusion isotope. Plasma KIC enrichment is used as a surrogate measure of intracellular leucine enrichment (27, 42).

The rate of 13CO2 release from isotopically labeled leucine, with the assumption of 81% recovery of 13CO2 released from leucine oxidation, is calculated as
F<SUP>13</SUP>CO<SUB>2</SUB> = (F<SC>co</SC><SUB>2</SUB> × E<SC>co</SC><SUB>2</SUB>/FFM) × 33.037
where FCO2 is the production rate of CO2, ECO2 is the enrichment of 13CO2 in expired air, FFM represents fat-free mass, and 33.037 is a constant to account for standard conditions and CO2 retention (48).

The rate of leucine oxidation (OL) is
O<SUB>L</SUB> = F<SUP>13</SUP>CO<SUB>2</SUB>[1/E<SUB>p</SUB> − 1/E<SUB>i</SUB>] × 100

Fractional synthesis rate of muscle proteins. Fractional synthesis rate (FSR) of a muscle protein (29) is determined as
FSR = (E<SUB>10 h</SUB> − E<SUB>5 h</SUB>/TFE<SUB>L</SUB> × 5 h) × 100
where E10h and E5h are the respective enrichment values in the muscle protein (e.g., mixed muscle protein, myosin heavy chain, and mitochondrial protein obtained at biopsy at 10 and 5 h, respectively; 5 h refers to the amount of time elapsed between the first and second biopsies), and TFEL represents the mean isotopic enrichment of leucine in muscle tissue fluid at 5 and 10 h. It has been demonstrated that muscle tissue fluid leucine enrichment best represents the isotopic enrichment of leucine acylated to tRNA, the obligate precursor of protein synthesis (24), and it therefore was used in the calculations.

As we previously reported, the coefficient of variation of repeated isotopic enrichment measurements in plasma and tissue proteins is <5% (24).

Statistics

All values are given as means ± SE. First, we performed unpaired analysis to compare the outcome measurements of chronic renal failure patients with healthy control subjects. We performed a one-way ANOVA and tested whether the chronic renal failure group differed from the control group by use of the ANOVA mean square error term. When a significant difference was found, we also performed linear regression of the outcome measures against GFR to determine whether the differences between the groups were related to the severity of renal failure.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subject Data

Table 1 gives the subject data. There were no significant differences between the two groups of subjects in their body weight or body composition, although the renal failure patients tended to have higher fat mass. Patients with chronic renal failure had lower GFR than the control group (chronic renal failure: range 8-60 ml/min, normal: range 75-117 ml/min, P < 0.01).

                              
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Table 1.   Subject data

Plasma concentrations of hormones are given in Table 2 and show that concentrations of insulin, IGF-binding protein (IGFBP)-2, IGFBP-3, and parathyroid hormone (PTH) were significantly higher in patients with chronic renal failure than in the control subjects.

                              
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Table 2.   Hormone levels

Protein Metabolism

Plasma amino acid concentrations and KIC are given in Table 3. Plasma concentrations of tyrosine and leucine and KIC were lower in people with chronic renal failure. Whole body leucine kinetics: leucine flux, leucine oxidation, and nonoxidative leucine disposal per unit FFM (DEPX), were not different between patients with chronic renal failure and normal control subjects (Table 4).

                              
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Table 3.   Plasma concentrations of amino acids and KIC


                              
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Table 4.   Whole body leucine kinetics

Muscle

Muscle protein synthesis and muscle mitochondrial enzyme data are summarized in Figs. 1 and 2. Fractional synthesis rates (FSR) of mixed muscle protein (P < 0.05), myosin heavy chain (P < 0.02) (Fig. 1), and mitochondrial protein (P <=  0.05) were significantly lower in subjects with chronic renal failure (Fig. 2). In addition, muscle mitochondrial enzyme activity (citrate synthase and cytochrome c-oxidase) were also significantly lower in patients with chronic renal failure than in the healthy control subjects (P <= 0.01).


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Fig. 1.   Fractional synthesis rate (FSR) of mixed muscle protein (A) and myosin heavy chain (B) in people with chronic renal failure and normal control subjects. People with renal failure have a lower FSR of mixed muscle protein (* P < 0.05) and myosin heavy chain (* P < 0.02) than healthy control subjects.



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Fig. 2.   FSR of mitochondrial protein (A) and muscle activity of citrate synthase (B) and cytochrome c-oxidase (C) in people with renal failure and normal control subjects. Those with renal failure have a lower FSR of mitochondrial protein (* P < 0.05), citrate synthase (* P < 0.01), and cytochrome c-oxidase (* P < 0.01).

Based on regression analysis, FSR of myosin heavy chain (r = 0.53, P = 0.01), cytochrome c-oxidase (r = 0.54, P = 0.009), citrate synthase (r = 0.48, P = 0.02) (Fig. 3), and mixed muscle proteins (r = 0.42, P = 0.054) were correlated with GFR, suggesting that as GFR decreased, the synthesis rates of muscle proteins and mitochondrial enzymes also decreased.


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Fig. 3.   FSR of myosin heavy chain (MHC, A), citrate synthase (B), and cytochrome c-oxidase (C) are significantly correlated to glomerular filtration rate (GFR).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The current study demonstrated that human subjects with chronic renal failure synthesize many muscle proteins at a slower rate than healthy humans with normal renal function. A substantial decline in the synthesis rates of myosin heavy chain and mitochondrial proteins was noted. In addition, muscle mitochondrial oxidative enzymes, such as citrate synthase and cytochrome-c oxidase, were substantially lower in human subjects with chronic renal failure than in the healthy control group. The studies were performed in both groups under identical study conditions. There were also significant correlations between many changes in muscle protein synthesis and mitochondrial enzymes and GFRs.

Muscle mass is determined by the balance between muscle protein breakdown and synthesis; the two processes are essential for the remodeling of muscle tissue. The lack of increase in leucine flux (a measure of whole body protein breakdown) argues against an increase of protein breakdown. It is, however, possible that muscle protein breakdown may alter without any change in whole body protein breakdown estimated from leucine flux. The observed lower synthesis rates of mixed muscle proteins (an average of all muscle proteins) suggest that decreased muscle protein synthesis is likely the biochemical basis of muscle wasting in our patients with chronic renal failure. Although we studied only 12 patients, all showed a decrease in muscle protein synthesis without any change in whole body protein breakdown. In animal models of renal failure, the enhanced muscle protein breakdown that occurs with metabolic acidosis is believed to play a pivotal role in mass loss (3, 17, 28). The subjects who participated in our study were not acidotic on the basis of serum bicarbonate concentrations. The chronic renal failure patients had slightly lower serum bicarbonate concentrations than the control group, but these bicarbonate levels were within normal range. Severe metabolic acidosis may cause increased muscle protein breakdown in humans, thus further accelerating muscle loss in decompensated renal failure. Because our subjects were relatively well compensated, we were unable to test this possibility. We cannot be certain that modest changes in muscle protein breakdown were not present, which can only be ascertained by direct measurement of muscle protein breakdown. A decrease in muscle protein synthesis alone is sufficient to explain the observed net catabolic state of muscle in humans with chronic renal failure. The decrease in muscle protein synthesis at the rate we observed during the postabsorptive state without any change in muscle protein breakdown could account for substantial muscle loss.

Recent technological advances in mass spectrometry (4) enabled us to measure the in vivo synthesis rates of individual muscle protein components by use of small needle biopsy samples. The decline in synthesis rates of mitochondrial proteins is of interest, as mitochondria are responsible for oxidative phosphorylation and ATP production. These mitochondrial functions depend on the integrity of electron transport, for which five protein complexes in the inner mitochondrial membrane are essential. The decline in mitochondrial protein synthesis and decreased levels of two crucial mitochondrial enzymes (cytochrome c-oxidase and citrate synthase) suggest a compromised ability to produce ATP. Durozard et al. (15), on the basis of their 31P NMR spectroscopic studies, reported a reduced ATP production, supporting impaired mitochondrial function in chronic renal failure patients. This reduced ATP production may not have much functional consequence in the resting muscle because of the abundance of muscle mitochondria but may be a limiting factor for continued muscle contraction, thus limiting the endurance, as reported in chronic renal failure patients (36). In addition, decreased synthetic rate of myosin heavy chain, the contractile protein responsible for hydrolyzing ATP to ADP, may also affect muscle strength (7). An excellent correlation between muscle strength and synthesis rate of muscle myosin heavy chain has been previously observed (7). Reduced oxidative capacity of mitochondria and reduced synthesis rate of myosin heavy chain may contribute to the reported muscle weakness in patients with renal failure (36). Insulin levels were increased in patients with chronic renal failure. This finding, noted by several other groups in the past, could be due to tissue resistance to insulin and may contribute to some of the impaired muscle protein synthesis. Reduced bioavailability of IGF-I due to increased binding proteins, as observed in the current study and by others (44), may also play a role in decreasing muscle protein synthesis. In addition, there is reported resistance of muscle protein synthesis to IGF-I in chronic renal failure (13, 29).

In conclusion, the current study clearly demonstrated that, under identical study conditions, the patients with chronic renal failure have reduced synthesis rates of myosin heavy chain and mitochondrial protein, indicating that chronic renal failure causes a defect in the remodeling process of muscle proteins. Accumulation of toxins and altered hormonal levels, chemical milieu, and tissue redox potential, lack of exercise, or a decreased oxygen supply to muscle due to accelerated atherosclerosis are among many possibilities that need to be considered as the cause of reduced chronic muscle protein synthesis in chronic renal failure patients. We measured several hormones that could potentially affect muscle protein synthesis. The current data do not show any correlation between the changes in muscle protein synthesis and any of the anabolic hormones or bicarbonate levels. The renal failure patients have elevated circulating insulin levels, which may reflect relative insulin resistance observed in these people (25). In conditions with insulin resistance to glucose metabolism, such as lipodystrophy (12), obesity, or type II diabetes (33, 37, 43, 45), there is no evidence of decreased protein synthesis. Increased insulin levels may inhibit protein breakdown in humans (31, 32), but the observed decline in muscle protein synthesis cannot be explained on the basis of insulin's effect on muscle protein synthesis. Other hormones, such as cortisol, catecholamines, IGF-I, and IGF-II, which can potentially affect protein synthesis (41), have not changed significantly. The PTH levels are elevated, but to our knowledge PTH has never been shown to have any effect on muscle protein synthesis. The current study demonstrated decreased muscle protein synthesis as an underlying mechanism of muscle wasting in chronic renal failure. This decrease in protein synthesis could be related to multiple factors, including the effects of many metabolites and toxins not disposed by the kidney.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the staff of the General Clinical Research Center (GCRC), and Maureen Bigelow, for help in the conduct of this study, and Jane Kahl, Dawn Morse, Rebecca Miller, Jill Schimke, G. C. Ford, and Larry Ward for skilled technical assistance.


    FOOTNOTES

This study was supported by National Institutes of Health Grants RO1 DK-41973 and AG-09531 and GCRC Grant RR-00585.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. S. Nair, Mayo Clinic, 200 First St. SW, 5-194 Joseph, Rochester, MN 55905 (E-mail: nair.sree{at}mayo.edu).

Received 22 March 1999; accepted in final form 15 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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