Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact

Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin–proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions.     

Strategies for suppressing muscle atrophy in chronic kidney disease: mechanisms activating distinct proteolytic systems.

Loss of protein and lean body mass occurs commonly in patients with chronic kidney disease (CKD). CKD or conditions associated with CKD will stimulate muscle loss, but the cellular mechanisms by which these conditions cause muscle atrophy are largely undefined. In animal models of uremia and other catabolic conditions or in peritoneal dialysis patients, there is evidence that the ubiquitin-proteasome proteolytic system is activated to degrade actomyosin and myofibrillar proteins in muscle. Before the ubiquitin system can degrade muscle proteins, however, an initial cleavage of actomyosin and myofibrils must occur. Caspase-3 performs this initial cleavage of actomyosin and leaves a footprint of its activity, accumulation of a 14-kDa actin fragment in muscle. A critical step in stimulating the ubiquitin-proteasome system in muscle was recently discovered, the activation of a specific E3 ubiquitin-conjugating enzyme, atrogin-1. Both caspase-3 and the ubiquitin system, including atrogin-1, are activated when insulin signaling is impaired, and specifically when phosphatidylinositol 3 kinase activity is suppressed. Strategies that prevent a decrease in phosphatidylinositol 3 kinase activity or inhibit caspase-3 activity could lead to treatments that prevent muscle wasting in CKD patients.     

Cachexia/Protein energy wasting syndrome in CKD: Causation and treatment

Cachexia is a multifactorial syndrome defined by significant body weight loss, fat and muscle mass reduction, and increased protein catabolism. Protein energy wasting (PEW) is characterized as a syndrome of adverse changes in nutrition and body composition being highly prevalent in patients with CKD, especially in those undergoing dialysis, and it is associated with high morbidity and mortality in this population. Multiple mechanisms are involved in the genesis of these adverse nutritional changes in CKD patients. There is no obvious distinction between PEW and cachexia from a pathophysiologic standpoint and should be considered as part of the spectrum of the same nutritional disorder in CKD with similar management approaches for prevention and treatment based on current understanding. A plethora of factors can affect the nutritional status of CKD patients requiring a combination of therapeutic approaches to prevent or reverse protein and energy depletion. At present, there is no effective pharmacologic intervention that prevents or attenuates muscle atrophy in catabolic conditions like CKD. Prevention and treatment of uremic muscle wasting involve optimal nutritional support, correction of acidosis, and physical exercise. There has been emerging consistent evidence that active treatment, perhaps by combining nutritional interventions and resistance exercise, may be able to improve but not totally reverse or prevent the supervening muscle wasting and weakness. Active research into more direct pharmacological treatment based on basic mechanistic research is much needed for this unmet medical need in patients with CKD.     

Cellular mechanisms causing loss of muscle mass in kidney disease

In stable adults or patients with kidney disease, the daily turnover of cellular proteins is very large, amounting to the quantity of protein in 1 to 1.5 kg of muscle. Consequently, even a small but persistent increase in protein degradation or decrease in protein synthesis leads to a substantial loss of muscle mass. In chronic kidney disease, the pathway that degrades muscle protein is the ubiquitin-proteasome system. We tested whether either of two complications of chronic kidney disease, metabolic acidosis or insulin resistance accelerates the loss of muscle protein. Metabolic acidosis activates the ubiquitin-proteasome system and this can explain an large number of clinical conditions in which metabolic acidosis also causes loss of muscle protein. Insulin deficiency as a model of insulin resistance also activates the ubiquitin-proteasome system. Both complications also activate caspase-3 and we found that this protease performs a critical initial step in breaking down the complex structure of muscle to provide actin, myosin and fragments of these proteins as substrates for the ubiquitin-proteasome system. Defects in insulin signalling processes can activate both caspase-3 and the ubiquitin-proteasome system to degrade muscle protein. Understanding mechanisms that activate protein breakdown will lead to therapies that successfully prevent the loss of muscle mass in patients with kidney disease.     

Apoptosis and myostatin mRNA are upregulated in the skeletal muscle of patients with chronic kidney disease

Apoptosis and myostatin are major mediators of muscle atrophy and might therefore be involved in the wasting of uremia. To examine whether they are expressed in the skeletal muscle of patients with chronic kidney disease (CKD), we measured muscle apoptosis and myostatin mRNA and their related intracellular signal pathways in rectus abdominis biopsies obtained from 22 consecutive patients with stage 5 CKD scheduled for peritoneal dialysis. Apoptotic loss of myonuclei, determined by anti-single-stranded DNA antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, was significantly increased three to fivefold, respectively. Additionally, myostatin and interleukin (IL)-6 gene expressions were significantly upregulated, whereas insulin-like growth factor-I mRNA was significantly lower than in controls. Phosphorylated JNK (c-Jun amino-terminal kinase) and its downstream effector, phospho-c-Jun, were significantly upregulated, whereas phospho-Akt was markedly downregulated. Multivariate analysis models showed that phospho-Akt and IL-6 contributed individually and significantly to the prediction of apoptosis and myostatin gene expression, respectively. Thus, our study found activation of multiple pathways that promote muscle atrophy in the skeletal muscle of patients with CKD. These pathways appear to be associated with different intracellular signals, and are likely differently regulated in patients with CKD.     

Chronic kidney disease causes defects in signaling through the insulin receptor substrate/phosphatidylinositol 3-kinase/Akt pathway: implications for muscle atrophy

Complications of chronic kidney disease (CKD) include depressed responses to insulin/IGF-1 and accelerated muscle proteolysis as a result of activation of caspase-3 and the ubiquitin-proteasome system. Experimentally, proteolysis in muscle cells occurs when there is suppression of phosphatidylinositol 3-kinase (PI3-K) activity. Postreceptor signaling through the insulin receptor substrate (IRS)/PI3-K/Akt pathway was evaluated in muscles of acidotic, CKD and pair-fed control rats under physiologic conditions and in response to a dose of insulin that quickly stimulated the pathway. Basal IRS-1-associated PI3-K activity was suppressed by CKD; IRS-2-associated PI3-K activity was increased. The basal level of activated Akt in CKD muscles also was low, indicating that the higher IRS-2-associated PI3-K activity did not compensate for the reduced IRS-1-associated PI3-K activity. Insulin treatment overcame this abnormality. The low IRS-1-associated PI3-K activity in muscle was not due to a decrease in IRS-1 protein, but there was a higher amount of the PI3-K p85 subunit protein without a concomitant increase in the p110 catalytic subunit, offering a potential explanation for the lower IRS-1-associated PI3-K activity. Eliminating the acidosis of CKD partially corrected the decrease in basal IRS-1-associated PI3-K activity and protein degradation in muscle. It is concluded that in CKD, acidosis and an increase in the PI3-K p85 subunit are mechanisms that contribute to suppression of PI3-K activity in muscle, and this leads to accelerated muscle proteolysis.     

Catabolic Response to Stress and Injury: Implications for Regulation

Abstract Muscle catabolism is an important component of the metabolic response to stress and injury, including sepsis and burn injury. Muscle wasting and weakness in catabolic patients may adversely affect the outcome in these patients owing to delayed ambulation and involvement of respiratory muscles. An understanding of the regulation of muscle protein breakdown during sepsis and following injury therefore is of great importance from a clinical standpoint and is essential for the development of new therapeutic modalities to prevent protein loss from muscle tissue. Studies in experimental animals and in patients have provided evidence that the myofibrillar proteins actin and myosin are particularly sensitive to the effects of sepsis and injury. Glucocorticoids, interleukin-1, and tumor necrosis factor participate in the regulation of muscle protein breakdown. Most muscle proteins are degraded by the ubiquitin—proteasome-dependent proteolytic pathway. Because the proteasome does not degrade intact myofibrils, a calcium-dependent Z-band disintegration and release of myofilaments from the myofibrils may be an important initial step of muscle breakdown during sepsis and other catabolic conditions. Continued studies to define mechanisms of the catabolic response to stress and injury are important for improving the metabolic care of patients with muscle catabolism. E-pub: 31 October 2000     

Innovation in the Treatment of Uremia: Proceedings from the Cleveland Clinic Workshop: Inflammation and Insulin Resistance as Novel Mechanisms of Wasting in Chronic Dialysis Patients

Over the last decade, there have been no proven therapies to lower the mortality and morbidity risk for chronic dialysis patients. One of the most important determinants of this poor clinical outcome is protein energy wasting (PEW), a unique and highly prevalent nutritional and metabolic abnormality primarily characterized by increased protein breakdown in the skeletal muscle compartment. Although the etiology and mechanisms leading to increased protein breakdown in chronic dialysis patients are complex and mostly ill‐defined, two well‐recognized and presumably interrelated metabolic abnormalities, insulin resistance and chronic inflammation, are likely to play a critical role in the pathogenesis of this condition. Multiple studies demonstrate the anabolic effects of insulin that extend beyond simple carbohydrate metabolism. Insulin is a mediator of accelerated protein breakdown in the catabolic condition such as advanced kidney disease. Chronic inflammation, a condition known to cause muscle catabolism in experimental conditions, has a strong association with advanced kidney disease in epidemiologic studies. Chronic inflammation is also known to induce insulin resistance, primarily by the induction of proinflammatory cytokines. The protein catabolic effects of inflammation and insulin resistance involve common cellular pathways. Thus, it is reasonable to speculate that chronic inflammation of advanced kidney disease mediates its protein catabolic effects by inducing insulin resistance of protein metabolism at both the physiologic and cellular levels. Modulating inflammatory response or insulin signaling by pharmacologic interventions could allow us to clarify the mechanisms contributing to the development of PEW in the setting of these particular metabolic derangements.     

Work-induced changes in skeletal muscle IGF-1 and myostatin gene expression in uremia

Resistance to growth hormone (GH)-induced insulin-like growth factor-1 (IGF-1) gene expression contributes to uremic muscle wasting. Since exercise stimulates muscle IGF-1 expression independent of GH, we tested whether work overload (WO) could increase skeletal muscle IGF-1 expression in uremia and thus bypass the defective GH action. Furthermore, to provide insight into the mechanism of uremic wasting and the response to exercise we examined myostatin expression. Unilateral plantaris muscle WO was initiated in uremic and pairfed (PF) normal rats by ablation of a gastrocnemius tendon and adjoining part of this muscle with the contralateral plantaris as a control. Some rats were GH treated for 7 days. WO led to similar gains in plantaris weight in both groups and corrected the uremic muscle atrophy. GH increased plantaris IGF-1 mRNA >twofold in PF rats but the response in uremia was severely attenuated. WO increased the IGF-1 mRNA levels significantly in both uremic and PF groups, albeit less brisk in uremia; however, after 7 days IGF-1 mRNA levels were elevated similarly, >2-fold, in both groups. In the atrophied uremic plantaris muscle basal myostatin mRNA levels were increased significantly and normalized after an increase in WO suggesting a myostatin role in the wasting process. In the hypertrophied uremic left ventricle the basal myostatin mRNA levels were reduced and likely favor the cardiac hypertrophy. Together the findings provide insight into the mechanisms of skeletal muscle wasting in uremia and the hypertrophic response to exercise, and suggest that alterations in the balance between IGF-1 and myostatin play an important role in these processes.     

Effects of glucose homeostasis on protein metabolism in patients with advanced chronic kidney disease.

Insulin deficiency is known to be associated with a state of increased muscle protein breakdown; this process is mediated by the ubiquitin-proteasome pathway. Convincing in vitro data are further supported by extensive studies in humans with insulin deprivation and are further substantiated by reversal of muscle protein breakdown with insulin treatment. In patients with end-stage renal disease (ESRD) and diabetes mellitus (DM), muscle protein breakdown is enhanced in both acute and chronic conditions. Recent data also point to the potential protein catabolic effects of insulin resistance combined with insulin deficiency. Because ESRD is associated with a state of insulin resistance, uremic muscle wasting may also be mediated by this pathway.     

Protein and Energy Intake in Advanced Chronic Kidney Disease: How Much is Too Much?

Uremic wasting is strongly associated with increased risk of death and hospitalization events in patients with advanced chronic kidney disease (CKD). Recent evidence indicates that patients with advanced chronic kidney disease are prone to uremic wasting due to several factors, which include the dialysis procedure and certain comorbid conditions, especially chronic inflammation and insulin resistance or deficiency. While the catabolic effects of dialysis can be readily avoided with intradialytic nutritional supplementation, there are no established alternative strategies to avoid the catabolic consequences of comorbid conditions other than treatment of their primary etiology. To this end, there is no indication that simply increasing dietary protein and energy intake above the required levels based on level of kidney disease is beneficial in patients with advanced chronic kidney disease. However, aside from the potential adverse effects such as uremic toxin production, dietary protein and energy intake in excess of actual needs might be beneficial in maintenance dialysis patients as it may lead to weight gain over time. Clearly, the role of obesity in advanced uremia needs to be examined in detail prior to making any clinically applicable recommendations, both in terms of 'low' and 'high' dietary protein and energy intake.     

MicroRNAs: a new therapeutic frontier for muscle wasting in chronic kidney disease

Multiple microRNAs (miRs) are implicated in muscle cell differentiation and muscle mass regulation. Pharmacological agents targeting miR-486 and other miRs, involved in muscle mass regulation, could potentially be developed into therapeutic agents for muscle wasting. Muscle wasting is prevalent among patients with chronic kidney disease (CKD). Xu et al. showed that miR-486 mimetic ameliorated muscle wasting in mice with CKD. miR mimetics may represent a new therapeutic frontier for muscle wasting in CKD.     

Malnutrition is an unusual cause of decreased muscle mass in chronic kidney disease.

Patients with chronic kidney disease (CKD), including those who are treated with hemodialysis, frequently develop hypoalbuminemia and a decrease in body weight. These abnormalities are usually attributed to malnutrition, but true malnutrition (ie, a disorder due to an abnormal diet) is rarely the mechanism causing decreased protein stores. Hypoalbuminemia is closely related to evidence of inflammation, and a decrease in muscle mass is caused by activation of muscle protein breakdown. In uremic rodents and patients, the initial step in the loss of muscle protein is activation of caspase-3, which cleaves the complex structure of muscle to provide substrates for the ubiquitin-proteasome pathway (UPP). The activity of caspase-3 can be detected by the presence of a characteristic 14-kDa actin fragment in the insoluble fraction of a muscle biopsy specimen. Abnormalities in cell signaling activate caspase-3 and the UPP; a key abnormality is decreased activity in the phosphatidylinositol-3-kinase/Akt pathway, leading to activation of caspase-3 and a specific E3 ubiquitin conjugating enzyme, atrogin-1/MAFbx. Inflammatory cytokines also represent a potential cell signaling abnormality that activates muscle protein breakdown, possibly because cytokines activate the E3 ubiquigin conjugating enzyme, MuRF1. An understanding of these pathways could help the clinician to identify therapeutic targets for preventing loss of muscle protein.     

XIAP Reduces Muscle Proteolysis Induced by CKD

X-chromosome-linked inhibitor of apoptosis protein (XIAP) is an endogenous caspase inhibitor. Caspase-3 contributes to the muscle wasting associated with chronic kidney disease (CKD) and other systemic illnesses, but whether XIAP modulates muscle wasting in CKD is unknown. Here, overexpression of XIAP in cultured skeletal muscle cells decreased protein degradation induced by serum deprivation, suggesting that caspase-mediated proteolysis contributes to muscle atrophy. We generated transgenic mice that overexpress human XIAP specifically in skeletal muscle (mXIAP) and evaluated muscle protein degradation induced by CKD. mXIAP mice with normal kidney function exhibited mild skeletal muscle hypertrophy. Muscle weights of mXIAP mice with CKD (mXIAP-CKD) were indistinguishable from wild-type mice, suggesting that overexpression of XIAP in skeletal muscle protects from CKD-induced muscle atrophy. The rate of total protein degradation, proteasome chymotrypsin–like activity, and caspase-3–mediated actin cleavage all were lower in muscle isolated from mXIAP-CKD mice compared with wild-type CKD mice. Concomitant with the reduction in overall proteolysis, mRNA levels of ubiquitin, muscle-specific ring finger 1, and atrogin-1/muscle atrophy F-box were lower in mXIAP-CKD mice, suggesting that decreased expression of the ubiquitin–proteasome pathway components may contribute to the protein-sparing effects of XIAP. In summary, these results demonstrate that XIAP inhibits multiple aspects of protein degradation in skeletal muscle during CKD.The loss of muscle mass as a result of disease¹ and inactivity² is commonly known as muscle wasting or atrophy. It occurs in chronic kidney disease (CKD),^3,4 sepsis,⁵ cancer,⁶ diabetes,^7,8 trauma,⁹ burn injury,¹⁰ AIDS,¹¹ and heart failure.^12,13 Regardless of the triggering event, common mechanisms contribute to the muscle-wasting process. Here we provide information about muscle wasting in CKD.Malnutrition is a serious complication of CKD, and virtually every survey of dialysis patients has revealed that weight loss with decreased muscle mass is common. These abnormalities are associated with excess morbidity and mortality and often occur before dialysis begins.^14–17 Because population screens indicate that the prevalence of CKD will increase sharply over the next decades,^18,19 it is important to understand fully the mechanisms that cause muscle atrophy in CKD.Muscle atrophy in CKD has been attributed to activation of the ATP-dependent ubiquitin–proteasome system that degrades the bulk of protein in cells. In this system, the multisubunit proteasome complex degrades proteins that have been modified by the covalent addition of polyubiquitin chains. A variety of genes involved in this pathway are increased in CKD, including ubiquitin and two muscle-specific E3 ubiquitin ligases, muscle-specific ring finger 1 (MuRF-1) and atrogin-1 (also called muscle atrophy F-box [MAFbx]).^4,20 An increase in these ligases has been correlated with muscle atrophy in CKD, diabetes, insulin resistance, fasting, cancer, denervation, muscle immobilization, and hind-limb suspension.^8,20,21 Consistent with this, mice with a genetic knockout of MuRF-1 are resistant to atrophy-inducing effects such as denervation.²¹Other degradation systems are also increased in muscle during CKD, including lysosomal and caspase-3–mediated proteolysis.^1,4,22–24 Caspase-3 facilitates the degradation of several proteins, including actin present in actomyosin. The actin cleavage process yields peptides that include a 14-kD actin fragment that is rapidly degraded by the ubiquitin–proteasome system.¹ This fragment serves as a biomarker of muscle wasting in CKD and other conditions.²⁵Inhibitors of apoptosis proteins are endogenous inhibitors of caspases, and X-chromosome linked inhibitor of apoptosis protein (XIAP) is the most potent member of the group.²⁶ XIAP directly inhibits both the initiation (e.g., caspase-9) and execution (e.g., caspase-3) phases of the caspase cascade, which is crucial for cell growth, differentiation, and apoptosis.²⁶ XIAP, however, has other roles in cells. For example, it contains a RING finger domain that functions as an E3 ubiquitin ligase.²⁷ Previously, we showed that XIAP can alter overall protein degradation, including myofibrillar protein breakdown, in the muscles of diabetic mice by attenuating caspase-3 and reducing proteasome-dependent proteolysis.²⁸ Others have shown that XIAP can influence cellular functions unrelated to caspase inhibition,^29,30 such as enhancing Akt signaling³¹ that might inhibit protein turnover.In this study, we tested whether overexpression of XIAP could prevent or diminish muscle wasting in CKD. We developed transgenic mice that overexpress XIAP specifically in skeletal muscle (mXIAP mice). Protein degradation rates were examined in wild-type (WT) and mXIAP mice with CKD to investigate the impact of XIAP on muscle protein loss. We also studied the effect of XIAP on several components of the ubiquitin–proteasome pathway. Our results provide evidence that XIAP regulates muscle mass via a multifaceted mechanism.     

Muscle cachexia: current concepts of intracellular mechanisms and molecular regulation

OBJECTIVE: To review present knowledge of intracellular mechanisms and molecular regulation of muscle cachexia. SUMMARY BACKGROUND DATA: Muscle cachexia, mainly reflecting degradation of myofibrillar proteins, is an important clinical feature in patients with severe injury, sepsis, and cancer. The catabolic response in skeletal muscle may result in muscle wasting and weakness, delaying or preventing ambulation and rehabilitation in these patients and increasing the risk for pulmonary complications. RESULTS: Muscle cachexia, induced by severe injury, sepsis, and cancer, is associated with increased gene expression and activity of the calcium/calpain- and ubiquitin/proteasome-proteolytic pathways. Calcium/calpain-regulated release of myofilaments from the sarcomere is an early, and perhaps rate-limiting, component of the catabolic response in muscle. Released myofilaments are ubiquitinated in the N-end rule pathway, regulated by the ubiquitin-conjugating enzyme E2(14k) and the ubiquitin ligase E3 alpha, and degraded by the 26S proteasome. CONCLUSIONS: An understanding of the mechanisms regulating muscle protein breakdown is important for the development of therapeutic strategies aimed at treating or preventing muscle cachexia in patients with severe injury, sepsis, cancer, and perhaps other catabolic conditions as well.     

Regulation of skeletal muscle proteolysis by amino acids.

Skeletal muscle is the major reservoir of body protein that can be mobilized in a number of muscle wasting conditions, that include kidney failure. Increased proteolysis in such conditions provides free amino acids that are used for acute-phase protein synthesis or that are degraded for energy purposes. Amino acids act as signals to regulate both protein synthesis and protein breakdown. We review the current but limited information available on the regulation of proteolytic systems in muscle cells. In particular, recent data have shown that amino acid deprivation in C2C12 myotubes stimulates autophagic sequestration by mechanisms that implicate the Apg system through a class III phosphoinositide-3'-kinase (PI3K III ) signaling cascade.     

Insulin Resistance and Protein Energy Metabolism in Patients with Advanced Chronic Kidney Disease

Insulin resistance (IR), the reciprocal of insulin sensitivity is a known complication of advanced chronic kidney disease (CKD) and is associated with a number of metabolic derangements. The complex metabolic abnormalities observed in CKD such as vitamin D deficiency, obesity, metabolic acidosis, inflammation, and accumulation of “uremic toxins” are believed to contribute to the etiology of IR and acquired defects in the insulin‐receptor signaling pathway in this patient population. Only a few investigations have explored the validity of commonly used assessment methods in comparison to gold standard hyperinsulinemic hyperglycemic clamp technique in CKD patients. An important consequence of insulin resistance is its role in the pathogenesis of protein energy wasting, a state of metabolic derangement characterized by loss of somatic and visceral protein stores not entirely accounted for by inadequate nutrient intake. In the general population, insulin resistance has been associated with accelerated protein catabolism. Among end‐stage renal disease (ESRD) patients, enhanced muscle protein breakdown has been observed in patients with Type II diabetes compared to ESRD patients without diabetes. In the absence of diabetes mellitus (DM) or severe obesity, insulin resistance is detectable in dialysis patients and strongly associated with increased muscle protein breakdown, primarily mediated by the ubiquitin‐proteasome pathway. Recent epidemiological data indicate a survival advantage and better nutritional status in insulin‐free Type II DM patients treated with insulin sensitizer thiazolidinediones. Given the high prevalence of protein energy wasting in ESRD and its unequivocal association with adverse clinical outcomes, insulin resistance may represent an important modifiable target for intervention in the ESRD population.