Tokunari Okayama

Shojiro Kikuchi
email: skikuchi@koto.kpu-m.ac.jp

Toshiya Ochiai

Hisashi Ikoma

Takeshi Kubota

Daisuke Ichikawa

Hitoshi Fujiwara

Kazuma Okamoto

Chouhei Sakakura

Teruhisa Sonoyama

Yukihito Kokuba

Yoshinori Doi

Sachiko Tsukita

D Montgomery Bissell

Eigo Otsuji

Attenuated Response to Liver Injury in Moesin-Deficient Mice: Impaired Stellate Cell Migration and Decreased Fibrosis

Keywords: ERM, thermal denaturation, wound healing, anti-fibrotic, collagen synthesis BDL, bile duct ligation, ERM, ezrin-radixin-moesin, Msn, moesin, HSC, hepatic stellate cell, GUS, β-glucuronidase, TGFβ-1, transforming growth factor

Please cite this article as: Tokunari Okayama, Shojiro Kikuchi, Toshiya Ochiai, Hisashi Ikoma, Takeshi Kubota, Daisuke Ichikawa, Hitoshi Fujiwara, Kazuma Okamoto, Chouhei Sakakura, Teruhisa Sonoyama, Yukihito Kokuba, Yoshinori Doi, Sachiko Tsukita, D. Montgomery Bissell, Eigo Otsuji, Attenuated Response to Liver Injury in Moesin-Deficient Mice: Impaired Stellate Cell Migration and Decreased Fibrosis, BBA -Molecular Basis of Disease (2008), doi: 10.1016/j.bbadis.2008.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Introduction

The response to liver injury is multi-faceted, involving several cell types and elaboration of a collagen-rich extracellular neomatrix, which serves both to strengthen the tissue and as a scaffolding upon which repair events proceed. Over time, the neomatrix undergoes chemical changes that render it resistant to degradation. Hence, the interest in strategies to modulate fibrogenesis. Hepatic stellate cells (HSCs) are prominent in the repair response as the principal source of extracellular matrix. They are also motile and migrate to the injury site. Migration appears to be among the initial responses to injury. The chemoattractant for HSC migration and intracellular signaling pathways has been defined [START_REF] Tangkijvanich | Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through rho-kinase-mediated alterations in the acto-myosin cytoskeleton[END_REF]. The cells appear to utilize a pathway that consists of

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Moesin and stellate cell migration -4hyaluronic acid and CD44, a cognate receptor for this matrix constituent [START_REF] Kikuchi | Role of CD44 in epithelial wound repair: migration of rat hepatic stellate cells utilizes hyaluronic acid and CD44v6[END_REF]. Migration also requires mechanical force generation, which is mediated by the actin cytoskeleton.

The latter structure comprises actin linked to the plasma membrane by a protein of the ezrin-radixin-moesin (ERM) family, which forms a complex with a variety of other membrane-associated proteins [START_REF] Charrin | Role of ERM (ezrin-radixin-moesin) proteins in T lymphocyte polarization, immune synapse formation and in T cell receptor-mediated signaling[END_REF][START_REF] Morales | Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia[END_REF][START_REF] Morales | NHERF1/EBP50 head-to-tail intramolecular interaction masks association with PDZ domain ligands[END_REF][START_REF] Yonemura | Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2[END_REF].

The ERM components in HSCs have not been defined, nor have their role in cell migration after acute injury. Many fibroblastic cells contain more than one ERM protein.

We showed that HSCs, in contrast, contain only moesin. We studied migration and activation of HSCs with or without moesin, after an injury challenge. HSCs from mice lacking moesin displayed significantly impaired migration and activation relative to those in wild-type cells. Fibrogenesis was decreased in parallel.

Materials and Methods

Animals

We have previously generated moesin-deficient (Msn -/-) mice on a mixed genetic background (129/Sv × C57BL/6J) [START_REF] Doi | Normal development of mice and unimpaired cell adhesion/cell motility/actin-based cytoskeleton without compensatory up-regulation of ezrin or radixin in moesin gene knockout[END_REF]. The mice were back-crossed through more than eight generations to create congenic C57BL/6J Msn -/-mice. All experiments were carried out in accordance with guidelines established by the Guidelines for Animal

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Moesin and stellate cell migration

-5 -Experiments of Kyoto Prefectural University of Medicine, Kyoto, Japan.

Antibodies and reagents

Rabbit polyclonal antibodies (TK89, anti-mouse ERM) and monoclonal antibodies (M11, anti-mouse ezrin; R21, anti-mouse radixin; M22, anti-mouse moesin) were provided by the Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan [START_REF] Kitajiri | Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia[END_REF].

Anti-α-smooth muscle actin (SMA)/FITC-conjugated monoclonal antibody was

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Moesin and stellate cell migration -6 -

Surgical procedures

Biliary obstruction model by common bile duct ligation: For studies of liver fibrosis and HSC activation in vivo, the common bile duct was ligated. Seven days later, the mice were sacrificed to isolate HSCs for liver perfusion, as described previously [START_REF] Kikuchi | Role of CD44 in epithelial wound repair: migration of rat hepatic stellate cells utilizes hyaluronic acid and CD44v6[END_REF]. For histological examination, the mice were sacrificed on 7 and 14 days after operation.

Hepatic injury model by thermal denaturation:

The injury device was an electrically heated probe with a beveled tip of 2 mm diameter, which was set at 150ºC (Taiyo Electric, Hiroshima, Japan). A 1-s application of the tip to the surface of the liver reproducibly resulted in a hemispheric injury of 3 mm diameter. Two lesions were created in the left lobe and one in the right lobe. Mice were sacrificed after 1 or 2 weeks, with removal of the whole liver, including the adherent peritoneal wall (including parietal peritoneum and muscle). The reason for this procedure was to remove completely and keep the fragile injured liver surface. Each experimental group consisted of 4-6 mice.

Histology and immunofluorescent microscopy

For in situ histology, the liver was perfused sequentially with PBS (pH 7.4) and 4%

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Moesin and stellate cell migration -7phosphate-buffered formaldehyde. Tissue sections (5 µm) were treated with xylene, and rehydrated with decreasing graded ethanol. Hematoxylin stain and Sirius Red stain were performed for histological analysis and collagen visualization, respectively. For immunofluorescent staining of normal or bile duct ligation (BDL) mouse livers, the liver was removed from Msn +/+ and Msn -/-mice, cut into small pieces, and frozen in liquid nitrogen. Frozen sections were cut on a cryostat, mounted onto cover glasses, air dried, and fixed with 95% ethanol for 15 min at 4°C, followed by 100% acetone for 1 min at room temperature. A pair of frozen sections from the Msn +/+ and Msn -/-liver were placed on the same cover glass and processed for double staining with the appropriate antibodies.

The images were obtained with a fluorescence microscopy (model IX71; Olympus, Tokyo, Japan) and DP Manager (Olympus). In vivo-activated HSCs were cultured for 2 days, and then treated with rat anti-ERM monoclonal antibody (M11, anti-mouse ezrin;

R21, anti-mouse radixin; M22, anti-mouse moesin). Secondary antibodies were conjugates of Alexa Fluor 546 (Molecular Probes). FITC-conjugated anti-α-SMA was used for double staining. HSCs were fixed in 100% methanol and permeabilized by 0.2%

TritonX-100 avoiding autofluorescence for plenty of retinoids.

One-step real-time RT-PCR

Total RNA was extracted from whole liver with RNeasy kits (QIAGEN, Hilden, Germany). One-step real-time RT-PCR was performed using Quantitect™ SYBR Green

Immunoblotting

The liver was taken from Msn +/+ and Msn -/-mice and homogenized in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 2% 2-mercaptoethanol and 0.01%

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Moesin and stellate cell migration -9bromophenol blue). Total lysate (20 µg) was separated by SDS-PAGE, and electrophoretically transferred from gels to PVDF membranes for immunoblotting.

Immune complexes were detected using horseradish-conjugated goat anti-rabbit IgG fragments (dilution, 1:1000; DakoJapan, Tokyo, Japan) and the ECL Western blotting detection system (Amersham, Bucks, UK). We quantified the individual immunoblotted bands by comparing their intensities with those of serially-diluted reference standards for five separate combinations of Msn +/+ and Msn -/-mice .

Image analysis

Image analysis for fibrosis was performed with Image J (National Institutes of Health, Bethesda, MD, USA) by three people blinded to the experimental protocol. The same background threshold was used in all specimens to distinguish black and white. Then, the amount of black and white was analyzed, and the percentage of white was recorded as the fibrosis volume (V Fib ), which was represented by Sirius red-positive tissue.

HSC isolation

In vivo-activated HSCs were isolated from common-bile-duct-ligated Msn +/+ and Msn -/- mice 7 days after operation. The liver was perfused with PBS that contained 100 IU/ml heparin (30 ml/h, 5 min), 0.025% pronase (80 ml/h, 30 min), and 10.7 IU/ml collagenase

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Moesin and stellate cell migration -10 -(140 ml/h, 30 min). At the end of the perfusion period, the liver was removed and stirred with 0.001% DNase (37ºC, 30 min). HSCs were isolated on an 11.5% OptiPrep® density gradient. The purity of isolated HSCs was assessed as the percentage of cells that exhibited transient blue fluorescence under UV excitation. The average yield per liver was 1.0×10 6 cells with purity of 70-90%. The cells were plated in 20% FBS/DMEM.

HSC migration assay

Culture inserts were pre-coated with hyaluronic acid (1 mg/ml), then rinsed with PBS before use. Isolated HSCs (1×10 3 cells/each cell culture insert) were plated in DMEM with 20% FBS. After 12 h, cell culture inserts were transferred to 24-well cluster plates in serum-free ASF 104 medium for the migration assay, as described previously. At the indicated times of migration, membranes were removed from the culture inserts, fixed in 4% phosphate-buffered formaldehyde and stained with hematoxylin. Migration was scored as the number of HSCs on the bottom of the membrane (Lw) and the top surface of membrane (Up), in the same fields at 400× magnification; each field contained about 10 cells, and 20 fields were assessed for each study. The migration index was (Lw/Up + Lw = %). Each experimental group consisted of five mice.

Statistical analysis

Results

Wound healing and liver fibrosis

Common bile duct ligation raises cholestasis and acute liver injury. In Msn +/+ mouse liver collagen deposition and bile duct proliferation were significant around the intra-hepatic bile duct in zone 1 (red stain, Figure 1A andB). Collagen synthesis and proliferation were reduced in Msn -/-mice (Figure 1C andD). Focal thermal denaturation produced an injury with well-defined margins and a reproducible fibrotic response (Figure 2A andD). In Msn +/+ liver, the injury focus showed an inflammatory infiltrate and angiogenesis, followed by massive collagen production and fibrosis at the periphery (Figure 2B andC). Generally, the injury resolved within 3 weeks. In Msn -/-mice, the inflammatory infiltrate and fibrosis at the injury margin within the liver were clearly reduced (Figure 2E andF). Collagen deposition at the periphery of the lesion (Figure 2B and E, black arrow) was quantified and found to be 39.3±5.2% of the tissue section in Msn +/+ but only 11.4±3.4% in Msn -/-(Figure 2G).

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Moesin and stellate cell migration -12 -BDL and acute liver injuries increased markers of HSC activation, including expression of α1(І) and α2(І) procollagen, α-SMA and TGFβ-1 mRNAs (Figure 3). HSC activation and procollagen synthesis were significantly reduced in Msn -/-mice.

ERM expression in mouse liver

In normal mouse livers, ezrin is expressed in cholangiocytes, radixin in bile canaliculi in hepatocytes, and moesin in endothelium or HSCs (Figure 4 A, D and G).

BDL did not increase the expression of ezrin and radixin, but moesin-positive cells increased in zone 1 of Msn +/+ mouse livers (Figure 4 B, E and H). Otherwise, ezrin and radixin did not compensate for moesin deficiencies in Msn -/-BDL liver (Figure 4 C, F and I). Expression of ERM proteins obtained by immunoblotting was almost consistent with the results of immunofluorescence microscopy. Moesin increased in Msn +/+ BDL liver, but ezrin/radixin expression did not change in Msn -/-BDL livers (Figure 4J).

ERM expression in HSCs

In vivo-activated HSCs expressed moesin but not ezrin or radixin (Figure 5 A-C). As expected of these linker proteins, moesin co-localized with α-SMA, notably at the ruffling membrane of HSCs (Figure 5C, F and I; white arrow). Expression was

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Moesin and stellate cell migration -13particularly strong in tall and compact HSCs, which are the cells with the greatest mobility (Fig. 5I; white arrow head). The data suggest that linkage of moesin to α-SMA is essential for the reorganization of the cytoskeleton that occurs with activation of HSCs.

We did not examine the ERM phenotype in culture-activated HSCs, because our previous study found that these cells lack migratory capability [START_REF] Kikuchi | Role of CD44 in epithelial wound repair: migration of rat hepatic stellate cells utilizes hyaluronic acid and CD44v6[END_REF].

Migratory activity of HSCs

To confirm that activated HSCs from the Msn -/-liver were migration-impaired, we isolated cells from mouse liver that had been subjected to total ligation of the biliary duct, an injury that induces activation of HSCs within 4 days. The proportion of Msn +/+ HSCs that migrated through the filter averaged 12.5±0.1% at 24 h, and 22.1±1.5% at 36 h. In sharp contrast, Msn -/-HSCs were almost completely quiescent, with a migration index of only 1.3±1.2% at 36 h (Figure 6). These data provide direct evidence for impaired migration of moesin-deficient HSCs.

Discussion

To the best of our knowledge, this is the first report of moesin being the ERM protein present in HSCs. Previous studies of this family of proteins in the liver have found

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Moesin and stellate cell migration -14that radixin is abundant in hepatocytes, ezrin in cholangiocytes and moesin in sinusoidal endothelial cells [START_REF] Fouassier | Ezrin-radixin-moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia[END_REF]. Interestingly, brain astrocytes, which share several properties with HSCs, express ezrin (and, in culture, radixin), but not moesin [START_REF] Derouiche | Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins[END_REF]. The counterpart of the HSC in the kidney is the mesangial cell, which reacts to injury by acquiring myofibroblast-like properties and producing collagens. It expresses both radixin and moesin [START_REF] Hugo | The cytoskeletal linking proteins, moesin and radixin, are upregulated by platelet-derived growth factor, but not basic fibroblast growth factor in experimental mesangial proliferative glomerulonephritis[END_REF]. The predominant linker protein in various types of fibroblasts in culture is ezrin [START_REF] Lamb | Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts[END_REF]. We found that repair of the liver capsule (visceral peritoneum), in contrast to the parenchyma, was unaffected by moesin deficiency. Presumably, the capsule fibroblasts, which migrate from the abdominal cavity or ascites, utilize ezrin and/or radixin. We examined the ERM phenotype of mouse embryo fibroblasts isolated from C57BL/6J mice (E15.5), and found that all three proteins were expressed (data not shown). Thus, it appears that expression of moesin alone may be unique to HSCs, just as individual ERM proteins are unique to other cell types in the liver (see above). The implications of exclusive expression of a single ERM family member by a specific cell type in the liver are speculative. The function of moesin in HSCs is likely to be similar to that of ERM proteins generally. These are linker proteins, which when phosphorylated are responsible for anchoring the actin cytoskeleton to the plasma membrane, and are particularly prominent in filopodia and microvilli of the plasma membrane [START_REF] Tsukita | ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons[END_REF]. An early

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Moesin and stellate cell migration -15facet of the injury response is migration of HSCs to the affected area. We have previously shown that HSCs use CD44 for migration on a substratum of hyaluronic acid [START_REF] Kikuchi | Role of CD44 in epithelial wound repair: migration of rat hepatic stellate cells utilizes hyaluronic acid and CD44v6[END_REF]. ERM linker proteins are known to form a complex with CD44 and other migration-related proteins such as osteopontin [START_REF] Zohar | Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration[END_REF]. They also likely participate in the formation of focal contacts between cell and substratum, which provide the traction for migratory movements. We now report that HSCs without moesin demonstrated minimal migratory capability.

Fibrogenesis by HSCs accompanies migration, although it is not clear whether these respective responses to injury are functionally linked. It is of interest in this regard that fibrogenesis was sharply attenuated in Msn -/-mice. In our study, moesin deficiencies reduced collagen deposition and bile duct proliferation in zone 1 and HSC activation.

These findings indicated that moesin specifically interacts with membrane-associated proteins, some of which play important roles in HSC activation and fibrogenesis. It forms a complex with the beta receptor for platelet-derived growth factor (PDGF-Rβ). The ligand for this receptor, PDGF, is known to stimulate proliferation, migration and collagen production by HSCs [START_REF] Borkham-Kamphorst | Dominant-negative soluble PDGF-beta receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis[END_REF][START_REF] Borkham-Kamphorst | Pro-fibrogenic potential of PDGF-D in liver fibrosis[END_REF][START_REF] Friedman | Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell[END_REF][START_REF] Kinnman | PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury[END_REF]. Another member of the moesin-PDGF-R complex is sodium-hydrogen exchanger factor-1, which associates with G-protein-coupled receptors and receptor tyrosine kinase, which mediate intracellular signaling [START_REF] James | A NHERF binding site links the betaPDGFR to the cytoskeleton and regulates cell spreading and migration[END_REF][START_REF] Wu | The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival[END_REF].

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Moesin and stellate cell migration -16 -Moesin appears to be required for the topographically and spatially correct formation of this complex. In its absence, the component proteins may drift within the plasma membrane and fail to generate the appropriate signals in response to the ligand, which leads to loss of the activation response. The expression of TGFβ-1 in Msn -/-control (sham operation) and Msn -/-BDL mice was significantly low. These results suggest that there is less fibrosis in Msn -/-mouse liver. The mechanism responsible for low levels of TGFβ-1 is still unclear. In future, we would like to investigate actin dynamics and ERM function in HSCs when exposed to mitogens.

Progression in many chronic liver diseases can be characterized as inappropriately active or dysregulated repair, and the goal of many antifibrotic strategies is suppressing the repair response. The present results suggest that interventions that target moesin merit consideration. Despite their altered repair response, Msn -/-mice are phenotypically normal and fertile, which suggests that moesin is not critical for homeostasis in the adult [START_REF] Doi | Normal development of mice and unimpaired cell adhesion/cell motility/actin-based cytoskeleton without compensatory up-regulation of ezrin or radixin in moesin gene knockout[END_REF]. In principle, this implies a margin of safety for agents that compromise moesin function in reactive HSCs, towards slowing disease progression in chronic liver injury. In Msn -/-mice, collagen synthesis and proliferation were significantly reduced (C and D).

Original magnification, ×200. Asterisk, bile duct. Moesin co-localized with α-SMA at the ruffling membrane of HSCs (C, F and I; white arrow). The expression level of moesin was greatest in compact HSCs, which are migration capable (I; white arrow head). Original magnification, ×400.

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  as means±SD. Significance was established using Student's t test and analysis of variance. Differences were considered significant at P<0.05. The data were analyzed by Graph Pad Prism5 (GraphPad Software, San Diego, CA, USA).

Figure 1 .

 1 Figure 1. Periductal fibrosis by common bile duct ligation

Moesin and stellate cell migration - 22 -Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 222345 Figure 2. Moesin deficiency reduced collagen synthesis in liver injury induced by

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Acknowledgements 285 This study was supported in part by a Grant-in-Aid for Scientific Research (C) from Ministry of Education, Science, and Culture of Japan and by the UCSF Liver Center (P30 DK26743).


Vik Rajan

M.D William E Mitch
email: mitch@bcm.edu

Nephrology

Ubiquitin, Proteasomes and Proteolytic Mechanisms Activated by Kidney Disease

Keywords: ubiquitin-proteasome system (UPS), muscle wasting, protein degradation, chronic kidney disease (CKD), 14kD actin fragment, caspase-3

The ubiquitin-proteasome system (UPS) includes 3 enzymes that conjugate ubiquitin to intracellular proteins that are then recognized and degraded in the proteasome. The process participates in the regulation of cell metabolism. In the kidney, the UPS regulates the turnover of transporters and signaling proteins and its activity is down regulated in acidosis-induced proximal tubular cell hypertrophy. In chronic kidney disease (CKD), muscle wasting occurs because complications of CKD including acidosis, insulin resistance, inflammation, and increased angiotensin II levels stimulate the UPS to degrade muscle proteins. This response also includes caspase-3 and calpains which act to cleave muscle proteins to provide substrates for the UPS. For example, caspase-3 degrades actomyosin, leaving a 14kD fragment of actin in muscle.

The 14 kD actin fragment is increased in muscle of patient with kidney disease, burn injury and surgery. In addition, acidosis, insulin resistance, inflammation and angiotensin II stimulate glucocorticoid production. Glucocorticoids are also required for the muscle wasting that occurs in CKD. Thus, the UPS is involved in regulating kidney function and participates in highly organized responses that degrade muscle protein in response to loss of kidney function.

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All intracellular proteins and many extracellular proteins are continually "turning over", being degraded to their constituent amino acids and replaced by synthesis of new proteins. Specific nuclear or cytosolic proteins, including proteins in the endoplasmic reticulum and mitochondria, are degraded at widely different rates; these rates vary from minutes for some regulatory enzymes, to days or weeks for proteins like actin and myosin in skeletal muscle, to months for hemoglobin in the red cell. Complex regulatory mechanisms ensure that proteolytic and synthetic processes are highly selective and precisely balanced because even a small decrease in synthesis or a small acceleration of degradation, if sustained, will result in marked loss of protein stores [START_REF] Mitch | Mechanisms of muscle wasting: The role of the ubiquitin-proteasome system[END_REF].

There are different proteolytic processes but in all tissues, the majority of intracellular proteins are degraded by the ubiquitin-proteasome system (UPS) [START_REF] Rock | Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class 1 molecules[END_REF]. However, extracellular proteins and some cell surface proteins are engulfed by endocytosis and degraded within lysosomes containing several acid-optimal proteases (e.g., cathepsins). Degradation of some cytosolic proteins in muscle are degraded in in lysosomes [START_REF] Zhao | FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells[END_REF]. In mammalian cells, there also are calciumactivated, ATP-independent, cysteine proteases (calpains) and caspases. The latter cytosolic proteases cleave proteins at aspartic acid residues and are critical for the apoptotic process [START_REF] Salvesen | Caspases: intracellular signaling by proteolysis[END_REF].

The ubiquitin-proteasome system

Initial steps in protein degradation by the UPS involve a series of 3 enzymes that link the cofactor, ubiquitin (Ub), onto proteins [START_REF] Lecker | Protein degradation by the ubiquitinproteasome pathway in normal and disease states[END_REF][START_REF] Mitch | Mechanisms of muscle wasting: The role of the ubiquitin-proteasome system[END_REF]. The enzymatic components that link chains of Ub onto proteins include the E1 (Ub-

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activating enzyme) and E2 proteins (Ub-carrier or conjugating proteins), which prepare Ub for conjugation. The third enzyme, E3 Ub-protein ligase, is a key factor in terms of the specificity of proteolysis because specific E3 enzymes will recognize a specific protein substrate. E3 enzymes catalyze the transfer of activated Ub to the substrate until a chain of 4-5 Ub's are attached. The conjugation reactions form Ub-conjugated proteins which can be recognized by the 26S proteasome. The proteasome removes Ub and degrades the substrate protein into small peptides [START_REF] Baumeister | The proteasome: paradigm of a self-comparmentalizing protease[END_REF]. In some cases, only a single ubiquitin is conjugated to a substrate protein (i.e. monoubiqutination). These substrate proteins are not degraded; instead, they are sorted into endocytic compartments and the Golgi network for routing to the plasma membrane or lysosomes [START_REF] Schnell | Non-traditional functions of ubiquitin and ubiquitinbinding proteins[END_REF].

The discovery of Ub and the biochemistry of its conjugation to substrate proteins led to the 2004 Nobel Prize in Chemistry awarded to Avram Hershko, Aaron

Ciechanover and Irwin Rose (http://nobelprize.org/chemistry/laureates/2004/).

The initial step in the conjugation of Ub onto proteins is the activation of Ub at its carboxy-terminus by the E1, Ub-activating enzyme. A single E1 enzyme uses ATP to generate a Ub thiolester which is subsequently transferred to a sulfhydryl group of one of 30-40 E2 carrier proteins [START_REF] Jentsch | The ubiquitin-conjugation system[END_REF]. The major determinant of the specificity of the Ub conjugating process, however, is the presence of more than a thousand E3's which can recognize specific proteins and transfer Ub to it.

For example, in muscle wasting conditions, two E3 Ub-conjugating enzymes, Atrogin-1 (also known as MAFbx) and MuRF-1, are critical for the breakdown of muscle proteins [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF]. In cultured muscle cells, the content of atrogin-1 mRNA

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5 correlates closely with rates of protein breakdown [START_REF] Sacheck | IGF-1 stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1[END_REF][START_REF] Sandri | Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy[END_REF][START_REF] Stitt | The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors[END_REF]. In models of muscle wasting conditions, their expression increases dramatically (8-20 fold) and this increased expression of Atrogin- [START_REF] Jensen | Multiple proteolytic systems, including the proteasome contribute CFTR processing[END_REF]. 4) The UPS functions to present antigen on the major histocompatibility complex class I molecules [START_REF] Rock | Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class 1 molecules[END_REF]. 5) Finally, the UPS degrades cellular proteins (including muscle proteins) when calories are inadequate or in response to catabolic illnesses. These reactions provide amino acids that are used for gluconeogenesis and the synthesis of new proteins.

Protein metabolism and the UPS in the kidney

In the kidney, the UPS plays a critical role in the turnover of proteins that affect cellular function, including the regulation of ion channels. Malik et al.,

found that the UPS played a key role in regulating the turnover of the amiloridesensitive epithelial sodium channel (ENac) in renal cells [START_REF] Malik | ENaC degradation in A6 cells by the ubiquitin-proteasome proteolytic pathway[END_REF][START_REF] Malik | Role of Nedd4-2 and polyubiquitination in epithelial sodium channel degradation in untransfected renal A6 cells expressing endogenous ENaC subunits[END_REF]. The physiologic relevance of changing sodium channel activity was demonstrated by inhibiting the UPS which led to greater activity of ENac, a condition reminiscent of Liddle's syndrome, characterized by hypertension. Evidence that the UPS is involved in functions of the kidney by regulating transcriptional responses was found in studies of hypoxia-Inducible Factor 1 or HIF-1 [START_REF] Haase | Vascular tumors in livers with targeted inactivation of the von Hippel-Lindau tumor suppressor[END_REF]. It was determined that blocking the UPS changes expression of HIF-1 and hence affects the kidney response to low oxygen supply. Another example of how the UPS affects the regulation of kidney cell function was found in studies of the responses to TGF-β.

It was shown that Smurf-2 (Smad ubiquitination regulartory factor-2), an E3 ubiquitin ligase was upregulated in the kidney tubule cells of patients with obstructive kidney disease [START_REF] Tan | Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication[END_REF]. Smurf-2 can suppress Smad transcriptional corepressors, SnoN and Ski thereby altering TGF-β signaling transmission to promote fibrosis in the kidney.

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The UPS is also critically involved in regulating the bulk of proteins in kidney cells in conditions associated with an increase in kidney cell mass. In response to accumulation of excess acid, proximal tubule epithelial cells hypertrophy and increase glutamine/glutamate metabolism to promote ammonia formation and the excretion of acid. The hypertrophic response involves a decrease in protein degradation along with a minor increase in protein synthesis [START_REF] Jurkovitz | Influence of ammonia and pH on protein and amino acid metabolism in LLC-PK1 cells[END_REF]. Franch et al., examined mechanisms underlying the increase in proximal tubule cell mass by studying the response to EGF as a signal for cell hypertrophy [START_REF] Franch | Mechanisms of renal tubular cell hypertrophy: Mitogen-induced suppression of proteolysis[END_REF]. EGF stimulated two hypertrophic responses, an increase in protein synthesis and suppression of protein breakdown. The mechanism involved both the UPS and lysosomes. The mechanisms underlying the hypertrophy of kidney cells in response to diabetes or unilateral nephrectomy has not been established.

Maintenance of protein stores in chronic kidney disease

Based on epidemiologic studies, chronic kidney disease afflicts millions of adults in the U.S. and the world and sharply increases their risk of cardiovascular disease [START_REF] Coresh | Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey[END_REF]. In addition, CKD is associated with muscle wasting and loss fo protein stores, conditions associated with excessive morbidity [START_REF] Kopple | National Kidney Foundation K/DOQI clinical practice guidelines for nutrition in chronic renal failure[END_REF]. The loss of protein stores in CKD has been attributed to "malnutrition" (i.e., abnormalities caused by an insufficient or imbalanced diet). This is incorrect because changing the diet generally does not correct these abnormalities because they are due to complex metabolic adaptation [START_REF] Mitch | Malnutrition: a frequent misdiagnosis for hemodialysis patients[END_REF]. With progressive loss of kidney function, the overall rates of muscle protein synthesis may decrease, but the more prominent response is an increase in rates of protein degradation [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATPdependent, ubiquitin-proteasome pathway[END_REF][START_REF] Goodship | Adaptation to low-protein diets in renal failure: Leucine turnover and nitrogen balance[END_REF][START_REF] Graham | Correction of acidosis in CAPD decreases whole body protein degradation[END_REF][START_REF] Graham | Correction of acidosis in hemodialysis decreases whole-body protein degradation[END_REF][START_REF] May | Mechanisms for defects in muscle protein metabolism in rats with chronic uremia: The influence of metabolic acidosis[END_REF][START_REF] Reaich | Correction of acidosis in humans with CRF decreases protein degradation and amino acid oxidation[END_REF]. Because

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protein turnover in humans is very high (3.5-4.5 g protein/kg/day) [START_REF] Mitch | Mechanisms of muscle wasting: The role of the ubiquitin-proteasome system[END_REF], even a small, persistent increase in proteolysis will cause marked protein depletion.

Programmed activation of the UPS accounts for most of the accelerated muscle protein degradation CKD.

Mechanisms causing loss of muscle protein in CKD

Recent studies in rodent models of CKD have established that the accelerated muscle wasting induced by uremia involves cellular mechanisms that are similar to the mechanisms causing muscle wasting in other catabolic conditions, such as cancer cachexia, starvation, insulin deficiency/resistance, or sepsis [START_REF] Lecker | Protein degradation by the ubiquitinproteasome pathway in normal and disease states[END_REF][START_REF] Mitch | Mechanisms of muscle wasting: The role of the ubiquitin-proteasome system[END_REF]. The mechanism of muscle wasting in these catabolic states involves accelerated proteolysis via the UPS plus higher levels of mRNAs encoding certain components of the UPS. There are also changes (both increases or decreases) in the expression of about 100 atrophy-related genes called atrogenes [START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF]. The increase in muscle mRNA levels of atrophy-related genes in muscle wasting states occurs because of increased gene transcription [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATPdependent, ubiquitin-proteasome pathway[END_REF][START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF][START_REF] Mitch | Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting[END_REF][START_REF] Price | Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome pathway by a mechanism including gene transcription[END_REF]. In fact, there is a common transcriptional program that includes decreased expression of various growth-related genes in atrophying muscle leading to the conclusion that multiple transcriptional factors change coordinately, resulting in loss of muscle mass [START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF]. The strongest evidence for activation of the UPS in muscles of animals undergoing atrophy due to uremia (or other catabolic diseases) is that inhibitors of the proteasome provided in vitro will block the increase in protein degradation present in muscles isolated from rodents with various models of catabolic human diseases [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATPdependent, ubiquitin-proteasome pathway[END_REF][START_REF] Price | Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome pathway by a mechanism including gene transcription[END_REF][START_REF] Tawa | Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles[END_REF]. In humans as well,

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catabolic conditions stimulate activation of the UPS in muscle (i.e., an increase in mRNAs encoding Ub and proteasome subunits) [START_REF] Mansoor | Increased mRNA levels for components of the lysosomal, Ca++-activated and ATP-ubiquitindependent proteolytic pathways in skeletal muscle from head trauma patients[END_REF][START_REF] Pickering | Nutrition in CAPD: Serum bicarbonate and the ubiquitin-proteasome system in muscle[END_REF][START_REF] Tiao | Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle[END_REF][START_REF] Williams | The expression of genes in the ubiquitin-proteasome proteolytic pathway is increased in skeletal muscle from patients with cancer[END_REF].

Loss of kidney function is associated with many abnormalities, some of which have been identified as signals stimulating protein degradation in muscle.

For example, the accumulation of acid from the reduced capacity to excrete acid stimulates protein degradation in muscle [START_REF] May | Mechanisms for defects in muscle protein metabolism in rats with chronic uremia: The influence of metabolic acidosis[END_REF]. Secondly, insulin resistance can be present in CKD patients with only moderate renal insufficiency and it stimulates protein degradation in muscle [START_REF] Kobayashi | Insulin resistance in patients with chronic kidney disease[END_REF][START_REF] Lee | Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitinproteasome systems by phosphatidylinositol 3 kinase[END_REF][START_REF] Mitch | Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting[END_REF][START_REF] Price | Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome pathway by a mechanism including gene transcription[END_REF][START_REF] Wang | Insulin resistance accelerates muscle protein degradation: activation of the ubiquitin-proteasome pathway by defects in muscle cell signaling[END_REF].

Another common problem for CKD patients is hypertension and cardiovascular disease linked to an increase in angiotensin II levels. This response, like those related to acidosis or insulin resistance, causes muscle atrophy in rodents [START_REF] Brink | Angiotensin II induces skeletal muscle wasting through enhanced protein degradation and down regulation of autocrine insulinlike growth factor-1[END_REF][START_REF] Song | Musclespecific expression of insulin-like growth factor-1 blocks angiotensin IIinduced skeletal muscle wasting[END_REF]. Finally, CKD patients frequently have high levels of inflammatory cytokines, raising the possibility that muscle protein degradation is accelerated because of inflammation [START_REF] Cai | IKKbeta/NF-kappaB activation causes severe muscle wasting in mice[END_REF][START_REF] Kimmel | Immunologic function and survival in hemodialysis patients[END_REF].

Caspase-3 and the initial cleavage of myofibrillar proteins in CKD

Muscle atrophy in catabolic conditions specifically affects contractile proteins [START_REF] Clarke | The E3 Ligase MuRF1 degrades myosin heavy chain protein in dexamethasonetreated skeletal muscle[END_REF]. Since myofibrillar proteins comprise about 2/3 of the protein in muscle, loss of these proteins is largely responsible for the disability of patients who experience muscle wasting. Notably, degradation of myofibrillar proteins requires more than one proteolytic step because the UPS can readily degrade major components of the myofibril (actin, myosin, troponin or tropomyosin) but when these same proteins are present in complexes or in intact myofibrils, they are degraded very slowly by the UPS [START_REF] Solomon | Importance of the ATP-ubiquitin-proteasome pathway in degradation of soluble and myofibrillar proteins in rabbit muscle extracts[END_REF]. Therefore, other proteases must

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10 initially cleave proteins to break down the complex structure of muscle to produce substrates for the UPS. In exploring proteases that could initiate cleavage of myofibrillar proteins, we examined caspases because several catabolic states are characterized by high circulating levels of TNFα and insulin resistance, conditions that activate the caspase cascade [START_REF] Defronzo | Glucose intolerance following chronic metabolic acidosis in man[END_REF][START_REF] Hotamisligil | IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-a-and obesity-induced insulin resistance[END_REF][START_REF] Kimmel | Immunologic function and survival in hemodialysis patients[END_REF]. We found that activated caspase-3 cleaves actomyosin in vitro and when stimulated in cultured muscle cells, there is cleavage of myofibrillar proteins that are rapidly degraded by the UPS.

Notably, caspase-3 activation produces a "footprint" of its activity, a 14kD

C-terminal fragment of actin that is found in the insoluble fraction of muscle [START_REF] Du | Activation of caspase 3 is an initial step triggering muscle proteolysis in catabolic conditions[END_REF].

Localization of the fragment to the insoluble fraction presumably occurs because it is less susceptible to degradation by the UPS. We find accumulation of the 14 kD actin fragment in muscles of animals with accelerated protein degradation due to acidosis, diabetes, and angiotensin II-induced hypertension, all complications of CKD [START_REF] Du | Activation of caspase 3 is an initial step triggering muscle proteolysis in catabolic conditions[END_REF][START_REF] Lee | Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitinproteasome systems by phosphatidylinositol 3 kinase[END_REF][START_REF] Wang | Insulin resistance accelerates muscle protein degradation: activation of the ubiquitin-proteasome pathway by defects in muscle cell signaling[END_REF][START_REF] Wang | Insulin resistance accelerates muscle protein degradation: activation of the ubiquitin-proteasome pathway by defects in muscle cell signaling[END_REF]. In patients with CKD or other causes of muscle wasting as well, we find that the level of the 14 kD actin fragment is increased in muscle. In addition, the level of the 14 kD actin fragment in muscle of CKD patients decreases in response to an exercise program directed at increasing the patient's endurance. In addition, we found that the level of the fragment was highly correlated (r = 0.78) with the measured rate of protein degradation in muscles of patients with muscle wasting from osteoarthritis.

Finally, the 14 kD actin fragment in unburned muscle of patients who had suffered a major burn injury was sharply increased [START_REF] Workeneh | Development of a diagnostic method for detecting increased muscle protein degradation in patients with catabolic conditions[END_REF]. These results indicate

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that at least in these conditions, the level of the 14 kD fragment in muscle is closely related to the rate of protein degradation and is present in specific disorders characterized by muscle wasting [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATPdependent, ubiquitin-proteasome pathway[END_REF][START_REF] Clark | Systemic response to thermal injury in rats: Increased protein degradation and altered glucose utilization in muscle[END_REF]. Additional testing will be needed to determine if this method could serve as a biomarker of accelerated muscle protein degradation in other conditions causing muscle wasting.

Another family of proteases, the calpains, have also been suggested as the protease that initially cleaves myofibrillar proteins in conditions associated with muscle wasting. Calpains are calcium-dependent, cysteine proteases that could play a role in disorders that include muscular dystrophy or sepsis-induced muscle wasting [START_REF] Tidball | Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse[END_REF][START_REF] Wei | Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3[END_REF]. At least in rodents with uremia or certain other types of atrophy the inhibition of calcium-activated proteases in muscle does not block the increase in protein degradation, the degradation of myofibrillar proteins, or the accumulation of the 14kD actin fragment in muscle cells [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATPdependent, ubiquitin-proteasome pathway[END_REF][START_REF] Du | Activation of caspase 3 is an initial step triggering muscle proteolysis in catabolic conditions[END_REF]. Additional studies are needed to (a) determine whether different catabolic disorders stimulate caspase-3 and/or calpains and (b) explain the specific roles of these proteases in the breakdown of different muscle proteins.

Signals triggering muscle wasting in CKD or other catabolic states

CKD is associated with several complications that can trigger the UPS to degrade muscle protein. These include metabolic acidosis, decreased insulin action and angiotensin II levels, and/or inflammation [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATPdependent, ubiquitin-proteasome pathway[END_REF][START_REF] Price | Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome pathway by a mechanism including gene transcription[END_REF][START_REF] Song | Musclespecific expression of insulin-like growth factor-1 blocks angiotensin IIinduced skeletal muscle wasting[END_REF][START_REF] Stenvinkel | Strong association between malnutrition, inflammation and atherosclerosis in chronic kidney failure[END_REF]. There is evidence that these complications can function in concert to cause muscle wasting. For example, metabolic acidosis stimulates protein breakdown by the UPS in muscle by integrating abnormalities, including an increase in

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glucocorticoid production and the development of insulin resistance in muscle [START_REF] Lee | Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitinproteasome systems by phosphatidylinositol 3 kinase[END_REF][START_REF] May | Metabolic acidosis stimulates protein degradation in rat muscle by a glucocorticoid-dependent mechanism[END_REF]. Notably, however, acidification alone does not stimulate muscle protein breakdown unless a physiological amount of glucocorticoids is also present [START_REF] May | Metabolic acidosis stimulates protein degradation in rat muscle by a glucocorticoid-dependent mechanism[END_REF][START_REF] Price | Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle[END_REF]. Likewise, glucocorticoids are required to for the accelerated protein degradation documented to occur in models of diabetes, high levels of angiotensin II, and sepsis [START_REF] Mitch | Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting[END_REF][START_REF] Song | Musclespecific expression of insulin-like growth factor-1 blocks angiotensin IIinduced skeletal muscle wasting[END_REF][START_REF] Tiao | Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids[END_REF].

Inflammatory conditions are frequently present in CKD patients but it is not clear how they affect muscle protein degradation [START_REF] Kaysen | Inflammation and reduced albumin synthesis associated with stable decline in serum albumin in hemodialysis patients[END_REF]. One possibility is that Inflammation suppresses insulin signaling and increases glucocorticoid production. These responses could explain why there is a requirement for glucocorticoids in the accelerated muscle protein degradation that occurs in response to sepsis [START_REF] Tiao | Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids[END_REF].

Thirdly, decreased insulin/IGF-1 signaling could be the stimulus for accelerated muscle protein breakdown. One consequence of decreased insulin signaling would be a decrease in protein synthesis. The mechanism would involve decreased signaling through the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway [START_REF] Bodine | Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[END_REF][START_REF] Lee | Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitinproteasome systems by phosphatidylinositol 3 kinase[END_REF]. Specifically, when insulin or IGF-1 signaling is low, Besides stimulating the expression of specific E3 Ub ligases, the insulin/IGF-1-PI3K/Akt pathway regulates the activation of caspase-3 to initiate the breakdown of muscle proteins. For example, in insulin-deficient rats with accelerated muscle protein degradation, we found activation of the pro-apoptotic factor, Bax. This leads to the release of cytochrome C from mitochondria and activation of caspase-3 [START_REF] Lee | Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitinproteasome systems by phosphatidylinositol 3 kinase[END_REF]. The result is increased production of the 14 kD actin fragment. This pathway was confirmed in cultured muscle cells with inhibited PI3K activity using genetic or pharmacological techniques. Together, these results provide evidence that muscle wasting in response to the complications of CKD is due to a common signaling pathway that alters key enzymes modulating protein synthesis and degradation.

A key endocrine factor that participates in the regulation of muscle protein turnover is glucocorticoids. Pharmacologic doses of glucocorticoids have been used as a mechanism to activate the UPS to degrade muscle proteins and to study the activation of Atrogin-1 [START_REF] Dardevet | Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats: Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging[END_REF][START_REF] Sacheck | IGF-1 stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1[END_REF][START_REF] Tran | The many forks in FOXO's road[END_REF]. However, the response to In vivo, catabolic conditions do not stimulate muscle wasting unless a physiologic level of glucocorticoids is present. Evidence for this difference in the responses is present in studies of adrenalectomized rodents: when they were starved or treated with NH 4 Cl to induce metabolic acidosis or made insulin-deficient with streptozotocin, muscle protein degradation did not increase unless the animals are also given a physiological dose of glucocorticoids [START_REF] Mitch | Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting[END_REF][START_REF] Price | Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle[END_REF][START_REF] Wing | Glucocorticoids activate the ATP-ubiquitindependent proteolytic system in skeletal muscle during fasting[END_REF]. Similarly, the increase in muscle wasting induced by angiotensin II or sepsis can be blocked by inhibiting the glucocorticoid receptor [START_REF] Song | Musclespecific expression of insulin-like growth factor-1 blocks angiotensin IIinduced skeletal muscle wasting[END_REF]. Thus, glucocorticoids exert a permissive role because the same dose of glucocorticoids does not stimulate muscle wasting in adrenalectomized animals. These results as well as the analysis of the complex transcriptional responses that are present in catabolic conditions indicate that muscle wasting results from activation of a regulated pathway that is of protein degradation that is integrated with changes in protein synthesis.

In this brief review of the UPS in kidney disease, we have emphasized that activity of the UPS in kidney cells is important for controlling levels of

PI3K

  activity falls, reducing the production of phosphadidylinositol-3,4,5 phosphate, the active product of PI3K. This results in a decrease in the phosphorylation and activity of the serine/threonine kinase, Akt. With a decrease in Akt activation, there would be decreased phosphorylation of downstream kinases, GSK1 and mTOR/S6kinase, suppressing protein synthesis. Similarly, decreased PI3K/Akt signaling is the key step in stimulating protein degradation in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 13 muscle. Decreased PI3K/Akt signaling will induce the expression of the E3's Ub conjugating enzymes, Atrogin-1 and MuRF-1, enhancing muscle protein degradation [28,44,49]. Expression of these E3 enzymes occurs because there is decreased phosphorylation of the forkhead family of transcription factors (FoxO1, 3, 4). When FoxO1, 3 and 4 are not phosphorylated, they can migrate into the nucleus to stimulate transcription of Atrogin-1 [28,44,49]. Insulin or IGF-1 can block this process by stimulating activity of the PI3K/Akt pathway to suppress the expression of Atrogin-1.

  glucocorticoids differs from the physiologic responses.

22 Fig. 1

 221 Fig.1Complications of CKD including increased levels of angiotensin II, the presence of inflammation, insulin resistance and/or acidosis, impair insulin/IGF-1 signaling. The impairment decreases Akt phosphorylation, resulting in an increase in caspase-3 activity which disrupts the complex structure of muscle proteins and creates substrates for the UPS. The UPS activity is stimulated by Forkhead transcription factor (FoxO) which promotes expression of atrogenes including components of the UPS. The result is stimulation of muscle protein degradation.

Acknowledgements: Supported by NIH R01 grants DK37175 and P50 DK64233.