American Journal of Physiology-Cell Physiology, 1997
The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta... more The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta 1) causes hypertrophy in renal epithelial cells. One mechanism contributing to hypertrophy is that EGF induces activation of the cell cycle and increases protein synthesis, whereas TGF-beta 1 prevents cell division, thereby converting hyperplasia to hypertrophy. To assess whether suppression of proteolysis is another mechanism causing hypertrophy induced by these growth factors, we measured protein degradation in primary cultures of proximal tubule cells and in cultured NRK-52E kidney cells. A concentration of 10(-8) M EGF alone or EGF plus 10(-10) M TGF-beta 1 decreased proteolysis by approximately 30%. TGF-beta 1 alone did not change protein degradation. Using inhibitors, we examined which proteolytic pathway is suppressed. Neither proteasome nor calpain inhibitors prevented the antiproteolytic response to EGF + TGF-beta 1. Inhibitors of lysosomal proteases eliminated the antiproteoly...
Muscle wasting increases the morbidity and mortality associated with chronic kidney disease (CKD)... more Muscle wasting increases the morbidity and mortality associated with chronic kidney disease (CKD) and has been attributed to malnutrition. In most patients, this is an incorrect diagnosis because simply feeding more protein aggravates uremia. Instead, there are complex mechanisms that stimulate loss of skeletal muscle, involving activation of mediators that stimulate the ATP-dependent ubiquitinproteasome system (UPS). Identified mediators of muscle protein breakdown include inflammation, metabolic acidosis, angiotensin II, and neural and hormonal factors that cause defects in insulin/ insulin-like growth factor I (IFG-I) intracellular signaling processes. Abnormalities in insulin/IGF-I signaling activate muscle protein degradation in the UPS and caspase-3, a protease that disrupts the complex structure of muscle proteins to provide substrates for the UPS. During the cleavage of muscle proteins, caspase-3 leaves behind a characteristic 14-kD actin fragment in the insoluble fraction of muscle, and characterization of this fragment identifies the presence of muscle catabolism. Thus, it could become a marker of excessive muscle wasting, providing a method for early detection of muscle wasting. Another consequence of activation of caspase-3 in muscle is stimulation of the activity of the proteasome, which increases the degradation of muscle proteins. Treatment strategies for blocking muscle wasting include correction of metabolic acidosis, which can suppress muscle protein losses in patients with CKD who are or are not being treated by dialysis. Correcting acidosis also improves bone metabolism in CKD and hence should be a goal of therapy. Exercise training is a potentially beneficial approach, but more information is needed to optimize exercise regimens. Replacing testosterone deficits can improve muscle mass in men, but dosing and side effects in women have not been adequately tested. Although insulin resistance occurs early in the course of CKD, there are no effective means of correcting it. Consequently, new therapies that can safely suppress muscle wasting are needed.
Chronic uremia and metabolic acidosis impair vascular responses to norepinephrine (NE) and also c... more Chronic uremia and metabolic acidosis impair vascular responses to norepinephrine (NE) and also cause multiple metabolic defects in skeletal muscle. These studies were conducted to determine whether decreased vascular responsiveness resulted from putative second messenger metabolism. Tail arteries were used from rats with metabolic acidosis or nonacidotic uremia and from normal controls. In normal arteries, the maximal responses were the same for arginine vasopressin (AVP), NE, and mixtures of the two agonists, suggesting that the two receptor types use the same transduction mechanism. Nonetheless, qualitative differences exist between AVP- and NE-induced responses: (a) Concentration-response curves are steeper for AVP than for NE, (b) EC50 values are similar for AVP between inositol phosphates (IP assays) and contraction, but not for NE, and (c) arteries from rats with metabolic acidosis or uremia show selective blunting of biochemical and contractile responses to NE but not to AVP. We conclude that these metabolic derangements selectively affect alpha-adrenergic receptors, but not AVP receptors or the transduction mechanism leading to contraction.
With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically... more With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically to increase the degradation of muscle proteins. Whether proteasome activity is also elevated in response to catabolic conditions is unknown. We find that caspase-3 increases proteasome activity in myotubes but not in myoblasts. This difference is related to the cleavage of specific 19 S proteasome subunits. In mouse muscle or myotubes, caspase-3 cleaves Rpt2 and Rpt6 increasing proteasome activity. In myoblasts, caspase-3 cleaves Rpt5 to decrease proteasome activity. To confirm the caspase-3 dependence, caspase-3 cleavage sites in Rpt2, Rpt6, or Rpt5 were mutated. This prevented the cleavage of these subunits by caspase-3 as well as the changes in proteasome activity. During differentiation of myoblasts to myotubes, there is an obligatory, transient increase in caspase-3 activity, accompanied by a corresponding increase in proteasome activity and cleavage of Rpt2 and Rpt6. Therefore, differentiation changes the proteasome type from sensitivity of Rpt5 to caspase-3 in myoblasts to sensitivity of Rpt2 and Rpt6 in myotubes. This novel mechanism identifies a feed-forward amplification that augments muscle proteolysis in catabolic conditions. Indeed, we found that in mice with a muscle wasting condition, chronic kidney disease, there was cleavage of subunits Rpt2 and Rpt6 and stimulation of proteasome activity.
The muscle protein catabolism present in rats with insulin-dependent diabetes and other catabolic... more The muscle protein catabolism present in rats with insulin-dependent diabetes and other catabolic conditions is generally associated with increased glucocorticoid production and mRNAs encoding components of the ubiquitin-proteasome system. The mechanisms that increase ubiquitin (UbC) expression have not been identified. We studied the regulation of UbC expression in L6 muscle cells because dexamethasone stimulates the transcription of this gene and others encoding components of the ubiquitin-proteasome pathway. Results of in vivo genomic DNA footprinting experiments indicate that a protein(s) binds to Sp1 sites approximately 50 bp upstream from the UbC transcription start site; dexamethasone changes the methylation pattern at these sites. Sp1 binds to DNA probes corresponding to the rat or human UbC promoter, and treating cells with dexamethasone increases this binding. Deletion and mutation analyses of the rat and human UbC promoters are consistent with an important role of Sp1 in UbC induction by glucocorticoids. Dexamethasone-induced ubiquitin expression is blocked by mithramycin, an inhibitor of Sp1 binding. UO126, a pharmacologic inhibitor of MEK1, also blocks UbC transcriptional activation by dexamethasone; L6 cells transfected to express constitutively active MEK1 exhibit increased UbC promoter activity. Thus, glucocorticoids increase UbC expression in muscle cells by a novel transcriptional mechanism involving Sp1 and MEK1.
Angiotensin II (Ang II) is known to act as a growth factor and may be involved in cardiac remodel... more Angiotensin II (Ang II) is known to act as a growth factor and may be involved in cardiac remodeling. We have shown that insulin-like growth factor-I (IGF-I) is an autocrine mediator of growth responses to Ang II in vascular smooth muscle cells in vitro, and we hypothesized that IGF-I also serves as an important modulator of cardiovascular growth in vivo. To study the effect of Ang II on cardiac IGF-I, we infused rats for 3, 7, or 14 days with Ang II through osmotic minipumps. After 7 days, left ventricular mass normalized for body weight was increased by 20% (PϽ0.01) in Ang II rats compared with pair-fed control rats that were given a restricted amount of food identical to that eaten by the anorexic, Ang II-infused rats. Ang II increased left ventricular IGF-I mRNA levels by 1.5-to 1.8-fold compared with ad libitum-fed or pair-fed control rats (PϽ0.05). Cardiac IGF-I protein was increased correspondingly and was localized on the cardiomyocytes. Treatment with hydralazine abolished the induction of IGF-I mRNA, which indicates that Ang II induces cardiac IGF-I mRNA expression through a pressor-mediated mechanism. IGF-I receptor (IGF-IR) mRNA was induced 2.1-fold in Ang II rats compared with ad libitum-fed rats (PϽ0.01). However, this increase was also observed in pair-fed controls and is thus due to the anorexigenic effect of Ang II. We have recently shown that circulating IGF-I levels are reduced in response to Ang II infusion. Elevation of IGF-I levels by coinfusion of IGF-I and Ang II significantly increased left ventricular index by 16% compared with rats infused with Ang II alone (PϽ0.05). In conclusion, autocrine upregulation of cardiac IGF-I and IGF-IR mRNA by Ang II occurs through hemodynamic and nonhemodynamic mechanisms, respectively, and may modulate cardiac structural changes that occur in hypertension.
Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanism... more Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day(-1) prednisolone on protein synthesis and degradation in human skeletal muscle. Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6(-3)H(5)]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0.023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0.007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs' encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0.007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.
We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a... more We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a ghrelin receptor agonist. Currently, we examine a surgical model of CKD in rats, resulting in uremia and decreased accrual of lean body mass. Treatment with ghrelin and two ghrelin receptor agonists (BIM-28125 and BIM-28131) resulted in increased food intake and an improvement in lean body mass accrual that was related in part to a decrease in muscle protein degradation as assessed by muscle levels of the 14-kDa actin fragment resulting from cleaved actomyosin. Addition-ally, there was a decrease in circulating inflammatory cytokines in nephrectomized animals treated with ghrelin relative to saline treatment. Ghrelin-treated animals also had a decrease in the expression of IL-1 receptor in the brainstem and a decrease in expression of prohormone convertase-2, an enzyme involved in the processing of proopiomelanocortin to the anorexigenic peptide ␣-MSH. We conclude that ghrelin treatment in uremia results in improved lean mass accrual in part due to suppressed muscle proteolysis and possibly related to antiinflammatory effects. (Endocrinology 149: 827-835, 2008) P ATIENTS WITH CHRONIC kidney disease (CKD) often exhibit symptoms of anorexia, loss of lean body mass, and increased energy expenditure, matching the syndrome of cachexia seen in other chronic diseases such as cancer, chronic heart failure, and AIDS (1, 2). In addition, patients with CKD-induced uremia frequently exhibit a dangerous decrease in protein nutritional status, including decreased levels of albumin and prealbumin as well as catabolism of actinomycin in muscle tissue via caspase-3 and the ubiquitinproteasome pathway (3-8). As with other diseases associated with cachexia, these catabolic processes seen in CKD are linked to systemic inflammatory changes, including increases in acute-phase reactants such as C-reactive protein and inflammatory cytokines (9 -15). Indeed, this inflammation is felt to be in the causative pathway of the production of cachexia (16 -19).
We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decre... more We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decreases circulating insulin-like growth factor I (IGF-I). The weight loss derives from an anorexigenic response and a catabolic effect of ang II. In these experiments we assessed potential catabolic mechanisms and the involvement of the IGF-I system in these responses to ang II. Ang II infusion caused a significant decrease in body weight compared with that of pair-fed control rats. Kidney and left ventricular weights were significantly increased by ang II, whereas fat tissue was unchanged. Skeletal muscle mass was significantly decreased in the ang II-infused rats, and a reduction in lean muscle mass was a major reason for their overall loss of body weight. In skeletal muscles, ang II did not significantly decrease protein synthesis, but overall protein breakdown
Conditions such as acidosis, uremia, and sepsis are characterized by insulin resistance and muscl... more Conditions such as acidosis, uremia, and sepsis are characterized by insulin resistance and muscle wasting, but whether the insulin resistance associated with these disorders contributes to muscle atrophy is unclear. We examined this question in db/db mice with increased blood glucose despite high levels of plasma insulin. Compared with control littermate mice, the weights of different muscles in db/db mice and the cross-sectional areas of muscles were smaller. In muscle of db/db mice, protein degradation and activities of the major proteolytic systems, caspase-3 and the proteasome, were increased. We examined signals that could activate muscle proteolysis and found low values of both phosphatidylinositol 3 kinase (PI3K) activity and phosphorylated Akt that were related to phosphorylation of serine 307 of insulin receptor substrate-1. To assess how changes in circulating insulin and glucose affect muscle protein, we treated db/db mice with rosiglitazone. Rosiglitazone improved indices of insulin resistance and abnormalities in PI3K/Akt signaling and decreased activities of caspase-3 and the proteasome in muscle leading to suppression of proteolysis. Underlying mechanisms of proteolysis include increased glucocorticoid production, decreased circulating adiponectin, and phosphorylation of the forkhead transcription factor associated with increased expression of the E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1. These abnormalities were also corrected by rosiglitazone. Thus, insulin resistance causes muscle wasting by mechanisms that involve suppression of PI3K/ Akt signaling leading to activation of caspase-3 and the ubiquitin-proteasome proteolytic pathway causing muscle protein degradation.
OBJECTIVE-Mechanisms impairing wound healing in diabetes are poorly understood. To identify mecha... more OBJECTIVE-Mechanisms impairing wound healing in diabetes are poorly understood. To identify mechanisms, we induced insulin resistance by chronically feeding mice a high-fat diet (HFD). We also examined the regulation of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) during muscle regeneration because augmented IGF-1 signaling can improve muscle regeneration.
On December 13th and 14th a group of scientists and clinicians met in Washington, DC, for the cac... more On December 13th and 14th a group of scientists and clinicians met in Washington, DC, for the cachexia consensus conference. At the present time, there is no widely agreed upon operational definition of cachexia. The lack of a definition accepted by clinician and researchers has limited identification and treatment of cachectic patient as well as the development and approval of potential therapeutic agents. The definition that emerged is: ''cachexia, is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass. The prominent clinical feature of cachexia is weight loss in adults (corrected for fluid retention) or growth failure in children (excluding endocrine disorders). Anorexia, inflammation, insulin resistance and increased muscle protein breakdown are frequently associated with cachexia. Cachexia is distinct from starvation, age-related loss of muscle mass, primary depression, malabsorption and hyperthyroidism and is associated with increased morbidity. While this definition has not been tested in epidemiological or intervention studies, a consensus operational definition provides an opportunity for increased research. ª
Clinical Journal of the American Society of Nephrology, 2006
Diabetic nephropathy is the most important cause of ESRD. The aim of this study was to develop a ... more Diabetic nephropathy is the most important cause of ESRD. The aim of this study was to develop a risk score from risk predictors for ESRD, with and without death, in the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study and to compare ability of the ESRD risk score and its components to predict ESRD. The risk score was developed from coefficients of independent risk factors from multivariate analysis of baseline variables and equals (1.96 x log [urinary albumin:creatinine ratio]) - (0.78 serum albumin [g/dl]) + (1.28 x serum creatinine [mg/dl]) - (0.11 x hemoglobin [g/dl]). It was robust with respect to severity of nephropathy, gender, race, and treatment group. The risk score for ESRD or death was comparable. The four risk predictors for progression of kidney disease were independent of therapy. For combined treatment groups, the hazard ratio between the fourth and first quartiles of the ESRD risk score was 49.0, as compared with the corresponding hazard ratios for each component: 14.7 for urinary albumin:creatinine ratio, 9.2 for serum creatinine, 5.5 for hemoglobin, and 10.2 for serum albumin. The RENAAL risk scores for ESRD with or without death emphasize the importance of identification of level of albuminuria, serum albumin, serum creatinine, and hemoglobin to predict development of ESRD in patients with type 2 diabetes and nephropathy. Although albuminuria is a strong risk factor for ESRD, the contribution of serum albumin, serum creatinine, and hemoglobin level further enhances prediction of ESRD. Future trials with a similar patient population and outcomes measures should consider adjusting analyses for baseline risk factors.
Background-Albuminuria is an established risk marker for both cardiovascular and renal outcomes. ... more Background-Albuminuria is an established risk marker for both cardiovascular and renal outcomes. Albuminuria can be reduced with drugs that block the renin-angiotensin system (RAS). We questioned whether the short-term drug-induced change in albuminuria would predict the long-term cardioprotective efficacy of RAS intervention. Methods and Results-We analyzed data from Reduction in Endpoints in Non-insulin dependent diabetes mellitus with the Angiotensin II Antagonist Losartan (RENAAL), a double-blind, randomized trial in 1513 type 2 diabetic patients with nephropathy, focusing on the relationship between the prespecified cardiovascular end point (composite) or hospitalization for heart failure and baseline or reduction in albuminuria. Patients with high baseline albuminuria (Ն3 g/g creatinine) had a 1.92-fold (95% CI, 1.54 to 2.38) higher risk for the cardiovascular end point and a 2.70-fold (95% CI, 1.94 to 3.75) higher risk for heart failure compared with patients with low albuminuria (Ͻ1.5 g/g). Among all available baseline risk markers, albuminuria was the strongest predictor of cardiovascular outcome. The association between albuminuria and cardiovascular outcome was driven by those patients who also had a renal event. Modeling of the initial 6-month change in risk parameters showed that albuminuria reduction was the only predictor for cardiovascular outcome: 18% reduction in cardiovascular risk for every 50% reduction in albuminuria and a 27% reduction in heart failure risk for every 50% reduction in albuminuria. Conclusions-Albuminuria is an important factor predicting cardiovascular risk in patients with type 2 diabetic nephropathy. Reducing albuminuria in the first 6 months appears to afford cardiovascular protection in these patients. (Circulation. 2004;110:921-927.)
Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mecha... more Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models with CKD or acute diabetes. A small-molecule that inhibits Stat3 activation produced similar responses, suggesting a potential for translation strategies. Using CCAAT/enhancer-binding protein δ (C/EBPδ) KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ, and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting.
In otherwise healthy adults, high C-reactive protein (CRP) levels are associated with cardiovascu... more In otherwise healthy adults, high C-reactive protein (CRP) levels are associated with cardiovascular disease and have been linked to an inflammatory state. The presence of vascular disease is also associated with increased expression of adhesion molecules, including soluble intercellular adhesion molecule (sICAM), vascular endothelial growth factor (VEGF) and leukocyte-derived myeloperoxidase (MPO). These associations suggest potential mechanisms whereby inflammation may injure the vascular endothelium, but the recognition of how these mediators act in concert remain poorly characterized. That the prevalence of atherosclerosis and markers of inflammation are increased in renal failure patients suggests that inflammation causes accelerated vascular disease. In hemodialysis patients, we examined the relationships between plasma CRP and sICAM, VEGF and MPO longitudinally. We determined whether episodes of a high CRP value were paralleled by simultaneous increases in mediators of inflammatory injury or molecules associated with endothelial cell adhesion or growth and whether CRP levels correlated with those of VEGF and MPO. Episodic increases in CRP were accompanied by higher levels of VEGF, sICAM and MPO. However, there was no correlation between serum CRP levels or other acute phase proteins and either MPO or VEGF, nor was there a constant temporal relationship between MPO and CRP. By contrast, MPO and VEGF levels were closely correlated with one another during episodes of inflammation (p = 0.0001), and CRP and interleukin-6 levels were also correlated. Increases in MPO tended to be restricted to patients with grafts or catheters, and not those with AV fistulas. These results suggest that high plasma levels of CRP or other acute phase proteins in cross-sectional studies should be interpreted cautiously when defining mechanisms underlying cardiovascular disease in the hemodialysis patient population. One, or more than one inflammatory repertoire may be activated, one involving hepatic acute phase proteins and the other neutrophil activation and each may contribute separately to outcomes. Better prognostic information may be obtained by measurement of more markers than CRP alone, such as MPO and VEGF.
A fundamental clinical problem in treating patients with chronic kidney disease (CKD) is designin... more A fundamental clinical problem in treating patients with chronic kidney disease (CKD) is designing their diets: an excess of protein leads to the accumulation of uremic toxins, whereas a diet insufficient in protein could lead to loss of lean body mass. The benefits of dietary protein restriction include reducing the accumulation of metabolic waste products that can suppress appetite and stimulate muscle protein wasting. There also is a potential for slowing the loss of kidney function. Unfortunately, advanced CKD is strongly associated with a protein wasting syndrome that is directly correlated with morbidity and mortality. Fortunately, the mechanisms underlying negative responses to an excess of dietary protein, including the causes of the wasting syndrome, are beginning to be understood. We have examined how dietary protein influences the mechanisms causing protein wasting, and we propose a framework for approaching the variable dietary protein requirements in patients with CKD or end-stage kidney disease.
Only a few specific chemokines that mediate interactions between inflammatory and satellite cells... more Only a few specific chemokines that mediate interactions between inflammatory and satellite cells in muscle regeneration have been identified. The chemokine CXCL16 differs from other chemokines because it has both a transmembrane region and active , soluble chemokine forms. Indeed, we found increased expression of CXCL16 and its receptor, CXCR6, in regenerating myofibers. Muscle regeneration in CXCL16deficient (CXCL16KO) mice was severely impaired compared with regeneration in wild-type mice. In addition, there was decreased MyoD and myogenin expression in regenerating muscle in CXCL16KO mice, indicating impaired satellite cell proliferation and differentiation. After 1 month, new myofibers in CXCL16KO mice remained significantly smaller than those in muscle of wild-type mice. To understand how CXCL16 regulates muscle regeneration, we examined cells infiltrating injured muscle. There were more infiltrating neutrophils and fewer macrophages in injured muscle of CXCL16KO mice compared with events in wild-type mice. Moreover , absence of CXCL16 led to different expression of cytokines/ chemokines in injured muscles: mRNAs of macrophage-inflammatory protein (MIP)-1␣, MIP-1, and MIP-2 were increased, whereas regulated on activation normal T cell expressed and secreted, T-cell activation-3, and monocyte chemoattractant protein-1 mR-NAs were lower compared with results in muscles of wild-type mice. Impaired muscle regeneration in CXCL16KO mice also resulted in fibrosis, which was linked to transforming growth factor-1 expression. Thus, CXCL16 expression is a critical mediator of muscle regeneration , and it suppresses the develop-ment of fibrosis.
American Journal of Physiology-Cell Physiology, 1997
The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta... more The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta 1) causes hypertrophy in renal epithelial cells. One mechanism contributing to hypertrophy is that EGF induces activation of the cell cycle and increases protein synthesis, whereas TGF-beta 1 prevents cell division, thereby converting hyperplasia to hypertrophy. To assess whether suppression of proteolysis is another mechanism causing hypertrophy induced by these growth factors, we measured protein degradation in primary cultures of proximal tubule cells and in cultured NRK-52E kidney cells. A concentration of 10(-8) M EGF alone or EGF plus 10(-10) M TGF-beta 1 decreased proteolysis by approximately 30%. TGF-beta 1 alone did not change protein degradation. Using inhibitors, we examined which proteolytic pathway is suppressed. Neither proteasome nor calpain inhibitors prevented the antiproteolytic response to EGF + TGF-beta 1. Inhibitors of lysosomal proteases eliminated the antiproteoly...
Muscle wasting increases the morbidity and mortality associated with chronic kidney disease (CKD)... more Muscle wasting increases the morbidity and mortality associated with chronic kidney disease (CKD) and has been attributed to malnutrition. In most patients, this is an incorrect diagnosis because simply feeding more protein aggravates uremia. Instead, there are complex mechanisms that stimulate loss of skeletal muscle, involving activation of mediators that stimulate the ATP-dependent ubiquitinproteasome system (UPS). Identified mediators of muscle protein breakdown include inflammation, metabolic acidosis, angiotensin II, and neural and hormonal factors that cause defects in insulin/ insulin-like growth factor I (IFG-I) intracellular signaling processes. Abnormalities in insulin/IGF-I signaling activate muscle protein degradation in the UPS and caspase-3, a protease that disrupts the complex structure of muscle proteins to provide substrates for the UPS. During the cleavage of muscle proteins, caspase-3 leaves behind a characteristic 14-kD actin fragment in the insoluble fraction of muscle, and characterization of this fragment identifies the presence of muscle catabolism. Thus, it could become a marker of excessive muscle wasting, providing a method for early detection of muscle wasting. Another consequence of activation of caspase-3 in muscle is stimulation of the activity of the proteasome, which increases the degradation of muscle proteins. Treatment strategies for blocking muscle wasting include correction of metabolic acidosis, which can suppress muscle protein losses in patients with CKD who are or are not being treated by dialysis. Correcting acidosis also improves bone metabolism in CKD and hence should be a goal of therapy. Exercise training is a potentially beneficial approach, but more information is needed to optimize exercise regimens. Replacing testosterone deficits can improve muscle mass in men, but dosing and side effects in women have not been adequately tested. Although insulin resistance occurs early in the course of CKD, there are no effective means of correcting it. Consequently, new therapies that can safely suppress muscle wasting are needed.
Chronic uremia and metabolic acidosis impair vascular responses to norepinephrine (NE) and also c... more Chronic uremia and metabolic acidosis impair vascular responses to norepinephrine (NE) and also cause multiple metabolic defects in skeletal muscle. These studies were conducted to determine whether decreased vascular responsiveness resulted from putative second messenger metabolism. Tail arteries were used from rats with metabolic acidosis or nonacidotic uremia and from normal controls. In normal arteries, the maximal responses were the same for arginine vasopressin (AVP), NE, and mixtures of the two agonists, suggesting that the two receptor types use the same transduction mechanism. Nonetheless, qualitative differences exist between AVP- and NE-induced responses: (a) Concentration-response curves are steeper for AVP than for NE, (b) EC50 values are similar for AVP between inositol phosphates (IP assays) and contraction, but not for NE, and (c) arteries from rats with metabolic acidosis or uremia show selective blunting of biochemical and contractile responses to NE but not to AVP. We conclude that these metabolic derangements selectively affect alpha-adrenergic receptors, but not AVP receptors or the transduction mechanism leading to contraction.
With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically... more With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically to increase the degradation of muscle proteins. Whether proteasome activity is also elevated in response to catabolic conditions is unknown. We find that caspase-3 increases proteasome activity in myotubes but not in myoblasts. This difference is related to the cleavage of specific 19 S proteasome subunits. In mouse muscle or myotubes, caspase-3 cleaves Rpt2 and Rpt6 increasing proteasome activity. In myoblasts, caspase-3 cleaves Rpt5 to decrease proteasome activity. To confirm the caspase-3 dependence, caspase-3 cleavage sites in Rpt2, Rpt6, or Rpt5 were mutated. This prevented the cleavage of these subunits by caspase-3 as well as the changes in proteasome activity. During differentiation of myoblasts to myotubes, there is an obligatory, transient increase in caspase-3 activity, accompanied by a corresponding increase in proteasome activity and cleavage of Rpt2 and Rpt6. Therefore, differentiation changes the proteasome type from sensitivity of Rpt5 to caspase-3 in myoblasts to sensitivity of Rpt2 and Rpt6 in myotubes. This novel mechanism identifies a feed-forward amplification that augments muscle proteolysis in catabolic conditions. Indeed, we found that in mice with a muscle wasting condition, chronic kidney disease, there was cleavage of subunits Rpt2 and Rpt6 and stimulation of proteasome activity.
The muscle protein catabolism present in rats with insulin-dependent diabetes and other catabolic... more The muscle protein catabolism present in rats with insulin-dependent diabetes and other catabolic conditions is generally associated with increased glucocorticoid production and mRNAs encoding components of the ubiquitin-proteasome system. The mechanisms that increase ubiquitin (UbC) expression have not been identified. We studied the regulation of UbC expression in L6 muscle cells because dexamethasone stimulates the transcription of this gene and others encoding components of the ubiquitin-proteasome pathway. Results of in vivo genomic DNA footprinting experiments indicate that a protein(s) binds to Sp1 sites approximately 50 bp upstream from the UbC transcription start site; dexamethasone changes the methylation pattern at these sites. Sp1 binds to DNA probes corresponding to the rat or human UbC promoter, and treating cells with dexamethasone increases this binding. Deletion and mutation analyses of the rat and human UbC promoters are consistent with an important role of Sp1 in UbC induction by glucocorticoids. Dexamethasone-induced ubiquitin expression is blocked by mithramycin, an inhibitor of Sp1 binding. UO126, a pharmacologic inhibitor of MEK1, also blocks UbC transcriptional activation by dexamethasone; L6 cells transfected to express constitutively active MEK1 exhibit increased UbC promoter activity. Thus, glucocorticoids increase UbC expression in muscle cells by a novel transcriptional mechanism involving Sp1 and MEK1.
Angiotensin II (Ang II) is known to act as a growth factor and may be involved in cardiac remodel... more Angiotensin II (Ang II) is known to act as a growth factor and may be involved in cardiac remodeling. We have shown that insulin-like growth factor-I (IGF-I) is an autocrine mediator of growth responses to Ang II in vascular smooth muscle cells in vitro, and we hypothesized that IGF-I also serves as an important modulator of cardiovascular growth in vivo. To study the effect of Ang II on cardiac IGF-I, we infused rats for 3, 7, or 14 days with Ang II through osmotic minipumps. After 7 days, left ventricular mass normalized for body weight was increased by 20% (PϽ0.01) in Ang II rats compared with pair-fed control rats that were given a restricted amount of food identical to that eaten by the anorexic, Ang II-infused rats. Ang II increased left ventricular IGF-I mRNA levels by 1.5-to 1.8-fold compared with ad libitum-fed or pair-fed control rats (PϽ0.05). Cardiac IGF-I protein was increased correspondingly and was localized on the cardiomyocytes. Treatment with hydralazine abolished the induction of IGF-I mRNA, which indicates that Ang II induces cardiac IGF-I mRNA expression through a pressor-mediated mechanism. IGF-I receptor (IGF-IR) mRNA was induced 2.1-fold in Ang II rats compared with ad libitum-fed rats (PϽ0.01). However, this increase was also observed in pair-fed controls and is thus due to the anorexigenic effect of Ang II. We have recently shown that circulating IGF-I levels are reduced in response to Ang II infusion. Elevation of IGF-I levels by coinfusion of IGF-I and Ang II significantly increased left ventricular index by 16% compared with rats infused with Ang II alone (PϽ0.05). In conclusion, autocrine upregulation of cardiac IGF-I and IGF-IR mRNA by Ang II occurs through hemodynamic and nonhemodynamic mechanisms, respectively, and may modulate cardiac structural changes that occur in hypertension.
Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanism... more Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day(-1) prednisolone on protein synthesis and degradation in human skeletal muscle. Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6(-3)H(5)]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0.023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0.007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs' encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0.007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.
We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a... more We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a ghrelin receptor agonist. Currently, we examine a surgical model of CKD in rats, resulting in uremia and decreased accrual of lean body mass. Treatment with ghrelin and two ghrelin receptor agonists (BIM-28125 and BIM-28131) resulted in increased food intake and an improvement in lean body mass accrual that was related in part to a decrease in muscle protein degradation as assessed by muscle levels of the 14-kDa actin fragment resulting from cleaved actomyosin. Addition-ally, there was a decrease in circulating inflammatory cytokines in nephrectomized animals treated with ghrelin relative to saline treatment. Ghrelin-treated animals also had a decrease in the expression of IL-1 receptor in the brainstem and a decrease in expression of prohormone convertase-2, an enzyme involved in the processing of proopiomelanocortin to the anorexigenic peptide ␣-MSH. We conclude that ghrelin treatment in uremia results in improved lean mass accrual in part due to suppressed muscle proteolysis and possibly related to antiinflammatory effects. (Endocrinology 149: 827-835, 2008) P ATIENTS WITH CHRONIC kidney disease (CKD) often exhibit symptoms of anorexia, loss of lean body mass, and increased energy expenditure, matching the syndrome of cachexia seen in other chronic diseases such as cancer, chronic heart failure, and AIDS (1, 2). In addition, patients with CKD-induced uremia frequently exhibit a dangerous decrease in protein nutritional status, including decreased levels of albumin and prealbumin as well as catabolism of actinomycin in muscle tissue via caspase-3 and the ubiquitinproteasome pathway (3-8). As with other diseases associated with cachexia, these catabolic processes seen in CKD are linked to systemic inflammatory changes, including increases in acute-phase reactants such as C-reactive protein and inflammatory cytokines (9 -15). Indeed, this inflammation is felt to be in the causative pathway of the production of cachexia (16 -19).
We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decre... more We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decreases circulating insulin-like growth factor I (IGF-I). The weight loss derives from an anorexigenic response and a catabolic effect of ang II. In these experiments we assessed potential catabolic mechanisms and the involvement of the IGF-I system in these responses to ang II. Ang II infusion caused a significant decrease in body weight compared with that of pair-fed control rats. Kidney and left ventricular weights were significantly increased by ang II, whereas fat tissue was unchanged. Skeletal muscle mass was significantly decreased in the ang II-infused rats, and a reduction in lean muscle mass was a major reason for their overall loss of body weight. In skeletal muscles, ang II did not significantly decrease protein synthesis, but overall protein breakdown
Conditions such as acidosis, uremia, and sepsis are characterized by insulin resistance and muscl... more Conditions such as acidosis, uremia, and sepsis are characterized by insulin resistance and muscle wasting, but whether the insulin resistance associated with these disorders contributes to muscle atrophy is unclear. We examined this question in db/db mice with increased blood glucose despite high levels of plasma insulin. Compared with control littermate mice, the weights of different muscles in db/db mice and the cross-sectional areas of muscles were smaller. In muscle of db/db mice, protein degradation and activities of the major proteolytic systems, caspase-3 and the proteasome, were increased. We examined signals that could activate muscle proteolysis and found low values of both phosphatidylinositol 3 kinase (PI3K) activity and phosphorylated Akt that were related to phosphorylation of serine 307 of insulin receptor substrate-1. To assess how changes in circulating insulin and glucose affect muscle protein, we treated db/db mice with rosiglitazone. Rosiglitazone improved indices of insulin resistance and abnormalities in PI3K/Akt signaling and decreased activities of caspase-3 and the proteasome in muscle leading to suppression of proteolysis. Underlying mechanisms of proteolysis include increased glucocorticoid production, decreased circulating adiponectin, and phosphorylation of the forkhead transcription factor associated with increased expression of the E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1. These abnormalities were also corrected by rosiglitazone. Thus, insulin resistance causes muscle wasting by mechanisms that involve suppression of PI3K/ Akt signaling leading to activation of caspase-3 and the ubiquitin-proteasome proteolytic pathway causing muscle protein degradation.
OBJECTIVE-Mechanisms impairing wound healing in diabetes are poorly understood. To identify mecha... more OBJECTIVE-Mechanisms impairing wound healing in diabetes are poorly understood. To identify mechanisms, we induced insulin resistance by chronically feeding mice a high-fat diet (HFD). We also examined the regulation of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) during muscle regeneration because augmented IGF-1 signaling can improve muscle regeneration.
On December 13th and 14th a group of scientists and clinicians met in Washington, DC, for the cac... more On December 13th and 14th a group of scientists and clinicians met in Washington, DC, for the cachexia consensus conference. At the present time, there is no widely agreed upon operational definition of cachexia. The lack of a definition accepted by clinician and researchers has limited identification and treatment of cachectic patient as well as the development and approval of potential therapeutic agents. The definition that emerged is: ''cachexia, is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass. The prominent clinical feature of cachexia is weight loss in adults (corrected for fluid retention) or growth failure in children (excluding endocrine disorders). Anorexia, inflammation, insulin resistance and increased muscle protein breakdown are frequently associated with cachexia. Cachexia is distinct from starvation, age-related loss of muscle mass, primary depression, malabsorption and hyperthyroidism and is associated with increased morbidity. While this definition has not been tested in epidemiological or intervention studies, a consensus operational definition provides an opportunity for increased research. ª
Clinical Journal of the American Society of Nephrology, 2006
Diabetic nephropathy is the most important cause of ESRD. The aim of this study was to develop a ... more Diabetic nephropathy is the most important cause of ESRD. The aim of this study was to develop a risk score from risk predictors for ESRD, with and without death, in the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study and to compare ability of the ESRD risk score and its components to predict ESRD. The risk score was developed from coefficients of independent risk factors from multivariate analysis of baseline variables and equals (1.96 x log [urinary albumin:creatinine ratio]) - (0.78 serum albumin [g/dl]) + (1.28 x serum creatinine [mg/dl]) - (0.11 x hemoglobin [g/dl]). It was robust with respect to severity of nephropathy, gender, race, and treatment group. The risk score for ESRD or death was comparable. The four risk predictors for progression of kidney disease were independent of therapy. For combined treatment groups, the hazard ratio between the fourth and first quartiles of the ESRD risk score was 49.0, as compared with the corresponding hazard ratios for each component: 14.7 for urinary albumin:creatinine ratio, 9.2 for serum creatinine, 5.5 for hemoglobin, and 10.2 for serum albumin. The RENAAL risk scores for ESRD with or without death emphasize the importance of identification of level of albuminuria, serum albumin, serum creatinine, and hemoglobin to predict development of ESRD in patients with type 2 diabetes and nephropathy. Although albuminuria is a strong risk factor for ESRD, the contribution of serum albumin, serum creatinine, and hemoglobin level further enhances prediction of ESRD. Future trials with a similar patient population and outcomes measures should consider adjusting analyses for baseline risk factors.
Background-Albuminuria is an established risk marker for both cardiovascular and renal outcomes. ... more Background-Albuminuria is an established risk marker for both cardiovascular and renal outcomes. Albuminuria can be reduced with drugs that block the renin-angiotensin system (RAS). We questioned whether the short-term drug-induced change in albuminuria would predict the long-term cardioprotective efficacy of RAS intervention. Methods and Results-We analyzed data from Reduction in Endpoints in Non-insulin dependent diabetes mellitus with the Angiotensin II Antagonist Losartan (RENAAL), a double-blind, randomized trial in 1513 type 2 diabetic patients with nephropathy, focusing on the relationship between the prespecified cardiovascular end point (composite) or hospitalization for heart failure and baseline or reduction in albuminuria. Patients with high baseline albuminuria (Ն3 g/g creatinine) had a 1.92-fold (95% CI, 1.54 to 2.38) higher risk for the cardiovascular end point and a 2.70-fold (95% CI, 1.94 to 3.75) higher risk for heart failure compared with patients with low albuminuria (Ͻ1.5 g/g). Among all available baseline risk markers, albuminuria was the strongest predictor of cardiovascular outcome. The association between albuminuria and cardiovascular outcome was driven by those patients who also had a renal event. Modeling of the initial 6-month change in risk parameters showed that albuminuria reduction was the only predictor for cardiovascular outcome: 18% reduction in cardiovascular risk for every 50% reduction in albuminuria and a 27% reduction in heart failure risk for every 50% reduction in albuminuria. Conclusions-Albuminuria is an important factor predicting cardiovascular risk in patients with type 2 diabetic nephropathy. Reducing albuminuria in the first 6 months appears to afford cardiovascular protection in these patients. (Circulation. 2004;110:921-927.)
Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mecha... more Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models with CKD or acute diabetes. A small-molecule that inhibits Stat3 activation produced similar responses, suggesting a potential for translation strategies. Using CCAAT/enhancer-binding protein δ (C/EBPδ) KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ, and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting.
In otherwise healthy adults, high C-reactive protein (CRP) levels are associated with cardiovascu... more In otherwise healthy adults, high C-reactive protein (CRP) levels are associated with cardiovascular disease and have been linked to an inflammatory state. The presence of vascular disease is also associated with increased expression of adhesion molecules, including soluble intercellular adhesion molecule (sICAM), vascular endothelial growth factor (VEGF) and leukocyte-derived myeloperoxidase (MPO). These associations suggest potential mechanisms whereby inflammation may injure the vascular endothelium, but the recognition of how these mediators act in concert remain poorly characterized. That the prevalence of atherosclerosis and markers of inflammation are increased in renal failure patients suggests that inflammation causes accelerated vascular disease. In hemodialysis patients, we examined the relationships between plasma CRP and sICAM, VEGF and MPO longitudinally. We determined whether episodes of a high CRP value were paralleled by simultaneous increases in mediators of inflammatory injury or molecules associated with endothelial cell adhesion or growth and whether CRP levels correlated with those of VEGF and MPO. Episodic increases in CRP were accompanied by higher levels of VEGF, sICAM and MPO. However, there was no correlation between serum CRP levels or other acute phase proteins and either MPO or VEGF, nor was there a constant temporal relationship between MPO and CRP. By contrast, MPO and VEGF levels were closely correlated with one another during episodes of inflammation (p = 0.0001), and CRP and interleukin-6 levels were also correlated. Increases in MPO tended to be restricted to patients with grafts or catheters, and not those with AV fistulas. These results suggest that high plasma levels of CRP or other acute phase proteins in cross-sectional studies should be interpreted cautiously when defining mechanisms underlying cardiovascular disease in the hemodialysis patient population. One, or more than one inflammatory repertoire may be activated, one involving hepatic acute phase proteins and the other neutrophil activation and each may contribute separately to outcomes. Better prognostic information may be obtained by measurement of more markers than CRP alone, such as MPO and VEGF.
A fundamental clinical problem in treating patients with chronic kidney disease (CKD) is designin... more A fundamental clinical problem in treating patients with chronic kidney disease (CKD) is designing their diets: an excess of protein leads to the accumulation of uremic toxins, whereas a diet insufficient in protein could lead to loss of lean body mass. The benefits of dietary protein restriction include reducing the accumulation of metabolic waste products that can suppress appetite and stimulate muscle protein wasting. There also is a potential for slowing the loss of kidney function. Unfortunately, advanced CKD is strongly associated with a protein wasting syndrome that is directly correlated with morbidity and mortality. Fortunately, the mechanisms underlying negative responses to an excess of dietary protein, including the causes of the wasting syndrome, are beginning to be understood. We have examined how dietary protein influences the mechanisms causing protein wasting, and we propose a framework for approaching the variable dietary protein requirements in patients with CKD or end-stage kidney disease.
Only a few specific chemokines that mediate interactions between inflammatory and satellite cells... more Only a few specific chemokines that mediate interactions between inflammatory and satellite cells in muscle regeneration have been identified. The chemokine CXCL16 differs from other chemokines because it has both a transmembrane region and active , soluble chemokine forms. Indeed, we found increased expression of CXCL16 and its receptor, CXCR6, in regenerating myofibers. Muscle regeneration in CXCL16deficient (CXCL16KO) mice was severely impaired compared with regeneration in wild-type mice. In addition, there was decreased MyoD and myogenin expression in regenerating muscle in CXCL16KO mice, indicating impaired satellite cell proliferation and differentiation. After 1 month, new myofibers in CXCL16KO mice remained significantly smaller than those in muscle of wild-type mice. To understand how CXCL16 regulates muscle regeneration, we examined cells infiltrating injured muscle. There were more infiltrating neutrophils and fewer macrophages in injured muscle of CXCL16KO mice compared with events in wild-type mice. Moreover , absence of CXCL16 led to different expression of cytokines/ chemokines in injured muscles: mRNAs of macrophage-inflammatory protein (MIP)-1␣, MIP-1, and MIP-2 were increased, whereas regulated on activation normal T cell expressed and secreted, T-cell activation-3, and monocyte chemoattractant protein-1 mR-NAs were lower compared with results in muscles of wild-type mice. Impaired muscle regeneration in CXCL16KO mice also resulted in fibrosis, which was linked to transforming growth factor-1 expression. Thus, CXCL16 expression is a critical mediator of muscle regeneration , and it suppresses the develop-ment of fibrosis.
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Papers by William Mitch