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Phosphatidylinositol 3-kinase (PI3K) is a pivotal enzyme involved in the control of a variety of diverse metabolic functions. Glucocorticoids have been shown to attenuate PI3K signaling in some nonrenal cell types, raising the possibility that some physiological effects of glucocorticoids in renal cells may be achieved by a similar mechanism. Therefore, we tested whether glucocorticoids affect signaling through the insulin receptor substrate (IRS)-1/PI3K/Akt signaling cascade in LLC-PK1-GR101 renal epithelial cells. Treatment of cells with dexamethasone for 24 h: 1) suppressed IRS-1-associated PI3K activity and Akt phosphorylation, 2) increased the level of the PI3K p85 regulatory subunit but not the p110 catalytic subunit, and 3) induced the phosphorylation of IRS-1 on inhibitory [Ser.sup.307]. We have previously reported that glucocorticoids increase branched-chain ketoacid dehydrogenase (BCKD) activity in LLC-PK1-GR101 cells. This response was achieved, in part, by alterations in the transcription of BCKD subunits and BCKD kinase, which inactivates the enzyme complex by phosphorylation. Therefore, we tested whether inhibition of PI3K signaling would mimick glucocorticoids by increasing branched-chain amino acid degradation. Expression of a dominant negative PI3K p85 regulatory subunit (Adp85[DELTA]iSH2) increased BCKD activity, and dexamethasone did not further stimulate enzyme activity. Inhibition of PI3K using LY-294002 increased the transcription of the BCKD E2 subunit but not the E1[alpha] subunit or BCKD kinase. Thus, glucocorticoids inhibit signaling through the IRS-1/PI3K/Akt pathway with a consequence of increased branched-chain amino acid catabolism. phosphatidylinositol 3-kinase; kidney doi:10.1152/ajpcell.00617.2006

Acidosis impairs insulin receptor substrate-1-associated phosphoinositide 3-kinase signaling in muscle cells: consequences on proteolysis. Am J Physiol Renal Physiol 287: F700-F706, 2004. First published May 25, 2004; 10.1152/ajprenal.00440.2003.--Chronic acidosis is a stimulus for proteolysis in muscle in vivo, but the mechanism of this response is unknown. We tested the hypothesis that acidosis or TNF-[alpha], a cytokine whose production increases in acidosis, regulates proteolysis by inhibiting insulin signaling through phosphoinositide 3-kinase (PI3K). In cultured L6 myotubes, acidified (pH 7.1) media did not accelerate the basal protein degradation rate, but it inhibited insulin's ability to suppress proteolysis. Insulin receptor substrate-1 (IRS-1)-associated PI3K activity was not altered in ceils acidified for 10 min but was strongly inhibited in cells incubated at pH 7.1 for 24 h. Phosphorylation of Akt was also suppressed by acidification for 24 h. Acidification did not induce changes in IRS-1 abundance, insulin-stimulated IRS-1 tyrosine phosphorylation, or the amount of PI3K p85 regulatory subunit. In contrast to acidification, TNF-[alpha] suppressed proteolysis in the presence or absence of insulin but had no effect on IRS-1associated PI3K activity. To establish that the PI3K pathway can regulate protein degradation in muscle, we measured proteolysis in cells after inhibition of PI3K activity with LY-294002 or infection with an adenovirus encoding a dominant negative PI3K p85[alpha]-subunit. Both approaches inhibited insulin-induced suppression of proteolysis to a degree similar to that seen with acidification. We conclude that acidosis accelerates protein degradation by impairing insulin signaling through PI3K in muscle cells. protein degradation; tumor necrosis factor; metabolic acidosis; skeletal muscle

Acidosis and glucocorticoids (GC) are two catabolic signals associated with chronic renal disease. Previously, we reported that these signals stimulate branched-chain amino acid (BCAA) oxidation in renal tubule cells by increasing both the amount and activation state of branched-chain [alpha]-ketoacid dehydrogenase (BCKD). Activation of the BCKD complex could result from decreased expression of BCKD kinase, which inhibits BCKD by phosphorylating its El[alpha] subunit. To investigate this possibility, we examined how dexamethasone and acidification (pH 7.0) influence BCKD kinase expression in LLC-P[K.sub.1]-GR101 cells. Dexamethasone, a synthetic GC, decreased BCKD kinase protein by 65% (P < 0.05 vs. control), whereas a low pH (i.e., pH 7.0) decreased the amount of kinase by 71% (P < 0.05 vs. control). Either GC or acidification reduced BCKD kinase mRNA by 46% (P < 0.05 vs. control), but the two signals together did not reduce kinase mRNA more than either signal alone. To examine the mechanism(s) leading to lower kinase mRNA, kinase transcription was evaluated by transiently transfecting LLC-PKt-GR101 cells with BCKD kinase promoter-luciferase minigenes containing ~3.5 kb of proximal rat kinase promoter. GC, but not acidification, decreased luciferase activity 42% (P < 0.05 vs. control). Nuclear run-on assays confirmed that GC decrease kinase mRNA by attentuating its transcription. Thus two catabolic signals associated with renal failure, GC and acidification, reduce BCKD kinase expression by different mechanisms. These responses lead to an increase in the activation state of BCKD and a resulting acceleration of BCAA degradation. branched-chain amino acids; acidosis; gene transcription

A study was conducted to analyze signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting. rats underwent bilateral adrenalectomy to determine the role of glucocorticoids in the proteolytic response in muscle. Nuclei were then isolated from hindquarter muscles to carry out nuclear run-off assays. Experimental results indicated that glucocorticoids and a low insulin level in tandem activate the ubiquitin-proteasome proteolytic system.

The role of extracellular acidification and glucocorticoids in the regulation of BCAA oxidation was analyzed via BCKAD flux in transfected cells that express stable glucocorticoid receptors. Analysis of branched-chain alpha-ketoacid dehydrogenase flux indicated the stimulatory effects of intracellular hydrogen-ion concentration in LLC-PK1 cells without glucocorticoid receptors. Furthermore, BCKAD flux was greates when extracellular pH was low and glucocorticoids were present.

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and mycBK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway. [ABSTRACT FROM AUTHOR]

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-β, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-β, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson’s trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO. [ABSTRACT FROM AUTHOR]

Acupuncture with low-frequency electrical stimulation (Acu/LFES) can prevent muscle atrophy by increasing muscle protein anabolism in mouse models of chronic kidney disease. During the treatment of muscle wasting, we found that Acu/LFES on the gastrocnemius muscle of the leg enhances renal blood flow. We also found that Acu/LFES increases exosome abundance and alters exosome-associated microRNA expression in the circulation. When exosome secretion was blocked using GW4869, the Acu/LFES-induced increase in renal blood flow was limited. This provided evidence that the increased renal blood flow is exosome mediated. To identify how exosomes regulate renal blood flow, we performed microRNA deep sequencing in exosomes isolated from treated and untreated mouse serum and found that the 34 microRNAs are altered by Acu/LFES. In particular, miR-181d-5p is increased in the serum exosome of Acu/ LFES-treated mice. In silico searching suggested that miR-181d-5p could target angiotensinogen. Using a luciferase reporter assay, we demonstrated that miR-181 directly inhibits angiotensinogen. When Acu/LFES-treated muscle was excised and incubated in culture medium, we found that the amount of exosomes and miR-181d-5p was increased in the medium providing evidence that Acu/LFES can increase miR-181 secretion. We conclude that Acu/LFES on leg hindlimb increases miR-181 in serum exosome leading to increased renal blood flow. This study provides important new insights about the mechanism(s) by which acupuncture may regulation of muscle-organ cross talk through exosome-derived microRNA. [ABSTRACT FROM AUTHOR]

In catabolic conditions such as aging and diabetes, IGF signaling is impaired and fibrosis develops in skeletal muscles. To examine whether impaired IGF signaling initiates muscle fibrosis, we generated IGF-IR+/- heterozygous mice by crossing loxP-floxed IGF-IR (exon 3) mice with MyoD-cre mice. IGF-IR+/- mice were studied because we were unable to obtain homozygous IGF-IR-KO mice. In IGF-IR+/- mice, both growth and expression of myogenic genes (MyoD and myogenin; markers of satellite cell proliferation and differentiation, respectively) were depressed. Likewise, in injured muscles of IGF-IR+/- mice, there was impaired regeneration, depressed expression of MyoD and myogenin, and increased expression of TGF-β1, α-SMA, collagen I, and fibrosis. To uncover mechanisms stimulating fibrosis, we isolated satellite cells from muscles of IGFIR +/- mice and found reduced proliferation and differentiation plus increased TGF-β1 production. In C2C12 myoblasts (a model of satellite cells), IGF-I treatment inhibited TGF-β1-stimulated Smad3 phosphorylation, its nuclear translocation, and expression of fibronectin. Using immunoprecipitation assay, we found an interaction between p-Akt or Akt with Smad3 in wild-type mouse muscles and in C2C12 myoblasts; importantly, IGF-I increased p-Akt and Smad3 interaction, whereas TGF-β1 decreased it. Therefore, in muscles of IGF-IR+/- mice, the reduction in IGF-IR reduces p-Akt, allowing for dissociation and nuclear translocation of Smad3 to enhance the TGF-β1 signaling pathway, leading to fibrosis. Thus, strategies to improve IGF signaling could prevent fibrosis in catabolic conditions with impaired IGF signaling. [ABSTRACT FROM AUTHOR]

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca 2+ -activated K + (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K + challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K + excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/CTGF/Nov). CCN1 mRNA was upregulated 113-fold in muscle of aged vs. young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1-treated MPCs did express a senescence marker (SA-βgal). Interestingly, we found CCN1 increased p53, p16(Ink4A), and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by sevenfold and protein increased by threefold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time- and dose-dependent manner. We conclude that in aging muscle, the circulating factor wnt-3a acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins.

In catabolic conditions such as aging and diabetes, IGF signaling is impaired and fibrosis develops in skeletal muscles. To examine whether impaired IGF signaling initiates muscle fibrosis, we generated IGF-IR(+/-) heterozygous mice by crossing loxP-floxed IGF-IR (exon 3) mice with MyoD-cre mice. IGF-IR(+/-) mice were studied because we were unable to obtain homozygous IGF-IR-KO mice. In IGF-IR(+/-) mice, both growth and expression of myogenic genes (MyoD and myogenin; markers of satellite cell proliferation and differentiation, respectively) were depressed. Likewise, in injured muscles of IGF-IR(+/-) mice, there was impaired regeneration, depressed expression of MyoD and myogenin, and increased expression of TGF-β1, α-SMA, collagen I, and fibrosis. To uncover mechanisms stimulating fibrosis, we isolated satellite cells from muscles of IGF-IR(+/-) mice and found reduced proliferation and differentiation plus increased TGF-β1 production. In C2C12 myoblasts (a model of satellite cells), IGF-I treatment inhibited TGF-β1-stimulated Smad3 phosphorylation, its nuclear translocation, and expression of fibronectin. Using immunoprecipitation assay, we found an interaction between p-Akt or Akt with Smad3 in wild-type mouse muscles and in C2C12 myoblasts; importantly, IGF-I increased p-Akt and Smad3 interaction, whereas TGF-β1 decreased it. Therefore, in muscles of IGF-IR(+/-) mice, the reduction in IGF-IR reduces p-Akt, allowing for dissociation and nuclear translocation of Smad3 to enhance the TGF-β1 signaling pathway, leading to fibrosis. Thus, strategies to improve IGF signaling could prevent fibrosis in catabolic conditions with impaired IGF signaling.

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.