Clinically relevant mitochondrial-targeted therapy improves chronic outcomes after traumatic brain injury.

Pioglitazone, an FDA-approved compound, has been shown to target the novel mitochondrial protein mitoNEET and produce short-term neuroprotection and functional benefits following traumatic brain injury. To expand on these findings, we now investigate the dose- and time-dependent effects of pioglitazone administration on mitochondrial function after experimental traumatic brain injury. We then hypothesize that optimal pioglitazone dosing will lead to ongoing neuroprotection and cognitive benefits that are dependent on pioglitazone-mitoNEET signalling pathways. We show that delayed intervention is significantly more effective than early intervention at improving acute mitochondrial bioenergetics in the brain after traumatic brain injury. In corroboration, we demonstrate that mitoNEET is more heavily expressed, especially near the cortical contusion, in the 18 h following traumatic brain injury. To explore whether these findings relate to ongoing pathological and behavioural outcomes, mice received controlled cortical impact followed by initiation of pioglitazone treatment at either 3 or 18 h post-injury. Mice with treatment initiation at 18 h post-injury exhibited significantly improved behaviour and tissue sparing compared to mice with pioglitazone initiated at 3 h post-injury. Further using mitoNEET knockout mice, we show that this therapeutic effect is dependent on mitoNEET. Finally, we demonstrate that delayed pioglitazone treatment improves serial motor and cognitive performance in conjunction with attenuated brain atrophy after traumatic brain injury. This study illustrates that mitoNEET is the critical target for delayed pioglitazone intervention after traumatic brain injury, mitochondrial-targeting is highly time-dependent after injury and there is an extended therapeutic window to effectively treat mitochondrial dysfunction after brain injury.

Calcineurin A-α suppression drives nuclear factor-κB-mediated NADPH oxidase-2 upregulation.

Calcineurin inhibitors (CNIs) are vital immunosuppressive therapies in the management of inflammatory conditions. A long-term consequence is nephrotoxicity. In the kidneys, the primary, catalytic calcineurin (CnA) isoforms are CnAα and CnAß. Although the renal phenotype of CnAα-/- mice substantially mirrors CNI-induced nephrotoxicity, the mechanisms downstream of CnAα are poorly understood. Since NADPH oxidase-2 (Nox2)-derived oxidative damage has been implicated in CNI-induced nephrotoxicity, we hypothesized that CnAα inhibition drives Nox2 upregulation and promotes oxidative stress. To test the hypothesis, Nox2 regulation was investigated in kidneys from CnAα-/-, CnAß-/-, and wild-type (WT) littermate mice. To identify the downstream mediator of CnAα, nuclear factor of activated T cells (NFAT) and NF-κB regulation was examined. To test if Nox2 is transcriptionally regulated via a NF-κB pathway, CnAα-/- and WT renal fibroblasts were treated with the NF-κB inhibitor caffeic acid phenethyl ester. Our findings showed that cyclosporine A treatment induced Nox2 upregulation and oxidative stress. Furthermore, Nox2 upregulation and elevated ROS generation occurred only in CnAα-/- mice. In these mice, NF-κB but not NFAT activity was increased. In CnAα-/- renal fibroblasts, NF-κB inhibition prevented Nox2 upregulation and reactive oxygen species (ROS) generation. In conclusion, these findings indicate that 1) CnAα loss stimulates Nox2 upregulation, 2) NF-κB is a novel CnAα-regulated transcription factor, and 3) NF-κB mediates CnAα-induced Nox2 and ROS regulation. Our results demonstrate that CnAα plays a key role in Nox2 and ROS generation. Furthermore, these novel findings provide evidence of divergent CnA isoform signaling pathways. Finally, this study advocates for CnAα-sparing CNIs, ultimately circumventing the CNI nephrotoxicity.NEW & NOTEWORTHY A long-term consequence of calcineurin inhibitors (CNIs) is oxidative damage and nephrotoxicity. This study indicates that NF-κB is a novel calcineurin-regulated transcription factor that is activated with calcineurin inhibition, thereby driving oxidative damage in CNI nephropathy. These findings provide additional evidence of divergent calcineurin signaling pathways and suggest that selective CNIs could improve the long-term outcomes of patients by mitigating renal side effects.

Formoterol, a ß2-adrenoreceptor agonist, induces mitochondrial biogenesis and promotes cognitive recovery after traumatic brain injury.

Traumatic brain injury (TBI) leads to acute necrosis at the site of injury followed by a sequence of secondary events lasting from hours to weeks and often years. Targeting mitochondrial impairment following TBI has shown improvements in brain mitochondrial bioenergetics and neuronal function. Recently formoterol, a highly selective ß2-adrenoreceptor agonist, was found to induce mitochondrial biogenesis (MB) via Gßγ-Akt-eNOS-sGC pathway. Activation of MB is a novel approach that has been shown to restore mitochondrial function in several disease and injury models. We hypothesized that activation of MB as a target of formoterol after TBI would mitigate mitochondrial dysfunction, enhance neuronal function and improve behavioral outcomes. TBI-injured C57BL/6 male mice were injected (i.p.) with vehicle (normal saline) or formoterol (0.3 mg/kg) at 15 min, 8 h, 16 h, 24 h and then daily after controlled cortical impact (CCI) until euthanasia. After CCI, mitochondrial copy number and bioenergetic function were decreased in the ipsilateral cortex of the CCI-vehicle group. Compared to CCI-vehicle, cortical and hippocampal mitochondrial respiration rates as well as cortical mitochondrial DNA copy number were increased in the CCI-formoterol group. Mitochondrial Ca2+ buffering capacity in the hippocampus was higher in the CCI-formoterol group compared to CCI-vehicle group. Both assessments of cognitive performance, novel object recognition (NOR) and Morris water maze (MWM), decreased following CCI and were restored in the CCI-formoterol group. Although no changes were seen in the amount of cortical tissue spared between CCI-formoterol and CCI-vehicle groups, elevated levels of hippocampal neurons and improved white matter sparing in the corpus callosum were observed in CCI-formoterol group. Collectively, these results indicate that formoterol-mediated MB activation may be a potential therapeutic target to restore mitochondrial bioenergetics and promote functional recovery after TBI.

Zinc deficiency induces hypertension by promoting renal Na+ reabsorption.

Zn2+ deficiency (ZnD) is a common comorbidity of many chronic diseases. In these settings, ZnD exacerbates hypertension. Whether ZnD alone is sufficient to alter blood pressure (BP) is unknown. To explore the role of Zn2+ in BP regulation, adult mice were fed a Zn2+-adequate (ZnA) or a Zn2+-deficient (ZnD) diet. A subset of ZnD mice were either returned to the ZnA diet or treated with hydrochlorothiazide (HCTZ), a Na+-Cl- cotransporter (NCC) inhibitor. To reduce intracellular Zn2+ in vitro, mouse distal convoluted tubule cells were cultured in N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, a Zn2+ chelator)- or vehicle (DMSO)-containing medium. To replete intracellular Zn2+, TPEN-exposed cells were then cultured in Zn2+-supplemented medium. ZnD promoted a biphasic BP response, characterized by episodes of high BP. BP increases were accompanied by reduced renal Na+ excretion and NCC upregulation. These effects were reversed in Zn2+-replete mice. Likewise, HCTZ stimulated natriuresis and reversed BP increases. In vitro, Zn2+ depletion increased NCC expression. Furthermore, TPEN promoted NCC surface localization and Na+ uptake activity. Zn2+ repletion reversed TPEN effects on NCC. These data indicate that 1) Zn2+ contributes to BP regulation via modulation of renal Na+ transport, 2) renal NCC mediates ZnD-induced hypertension, and 3) NCC is a Zn2+-regulated transporter that is upregulated with ZnD. This study links dysregulated renal Na+ handling to ZnD-induced hypertension. Furthermore, NCC is identified as a novel mechanism by which Zn2+ regulates BP. Understanding the mechanisms of ZnD-induced BP dysregulation may have an important therapeutic impact on hypertension.

Chronic Calcineurin Inhibition via Cyclosporine A Impairs Visuospatial Learning After Isoflurane Anesthesia.

BACKGROUND: Clinical studies implicate the perioperative period in cognitive complications, and increasing experimental evidence shows that the anesthetic agents can affect neuronal processes that underpin learning and memory. Calcineurin, a Ca-dependent phosphatase critically involved in synaptic plasticity, is activated after isoflurane exposure, but its role in the neurological response to anesthesia is unclear. METHODS: We investigated the effect of chronic calcineurin inhibition on postanesthetic cognitive function. Mice were treated with 30 minutes of isoflurane anesthesia during a chronic cyclosporine A regimen. Behavioral end points during the perianesthesia period were quantified. Visuospatial learning was assessed with the water radial arm maze. Total and biotinylated surface protein expression of the α5ß3γ2 γ-aminobutyric acid (GABA) type A receptors was measured. Expression of the GABA synthesis enzyme glutamate decarboxylase (GAD)-67 was also measured. RESULTS: Mice treated with cyclosporine A before anesthesia showed significant deficits in visuospatial learning compared to sham and cyclosporine A-treated mice (n = 10 per group, P = .0152, Tukey post hoc test). Induction and emergence were unaltered by cyclosporine A. Analysis of hippocampal protein expression revealed an increased surface expression of the α5 GABA type A receptor subunit after isoflurane treatment (P = .019, Dunnett post hoc testing), as well as a decrease in GAD-67 expression. Cyclosporine A did not rescue either effect. CONCLUSIONS: Our results confirm the work of others that isoflurane induces changes to inhibitory network function and exclude calcineurin inhibition via cyclosporine A as an intervention. Further, our studies suggest that calcineurin mediates a protective role in the neurological response to anesthesia, and patients receiving cyclosporine A may be an at-risk group for memory problems related to anesthesia.

NADPH oxidase-2 mediates zinc deficiency-induced oxidative stress and kidney damage.

Zn2+ deficiency (ZnD) is comorbid with chronic kidney disease and worsens kidney complications. Oxidative stress is implicated in the detrimental effects of ZnD. However, the sources of oxidative stress continue to be identified. Since NADPH oxidases (Nox) are the primary enzymes that contribute to renal reactive oxygen species generation, this study's objective was to determine the role of these enzymes in ZnD-induced oxidative stress. We hypothesized that ZnD promotes NADPH oxidase upregulation, resulting in oxidative stress and kidney damage. To test this hypothesis, wild-type mice were pair-fed a ZnD or Zn2+-adequate diet. To further investigate the effects of Zn2+ bioavailability on NADPH oxidase regulation, mouse tubular epithelial cells were exposed to the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or vehicle followed by Zn2+ supplementation. We found that ZnD diet-fed mice develop microalbuminuria, electrolyte imbalance, and whole kidney hypertrophy. These markers of kidney damage are accompanied by elevated Nox2 expression and H2O2 levels. In mouse tubular epithelial cells, TPEN-induced ZnD stimulates H2O2 generation. In this in vitro model of ZnD, enhanced H2O2 generation is prevented by NADPH oxidase inhibition with diphenyleneiodonium. Specifically, TPEN promotes Nox2 expression and activation, which are reversed when intracellular Zn2+ levels are restored following Zn2+ supplementation. Finally, Nox2 knockdown by siRNA prevents TPEN-induced H2O2 generation and cellular hypertrophy in vitro. Together, these findings reveal that Nox2 is a Zn2+-regulated enzyme that mediates ZnD-induced oxidative stress and kidney hypertrophy. Understanding the specific mechanisms by which ZnD contributes to kidney damage may have an important impact on the treatment of chronic kidney disease.

Trends in hospital admissions due to antidepressant-related adverse drug events from 2001 to 2011 in the U.S.

BACKGROUND: Depression is a prevalent mental health disorder and the fourth leading cause of disability in the world as per the World Health Organization. Use of antidepressants can lead to adverse drug events (ADEs), defined as any injury resulting from medication use. This study aimed to examine changes in hospital admissions due to antidepressant-related ADEs (ArADEs) among different socio-demographic groups and changes in lengths of stay (LOS) and hospital charges in ArADE admissions from 2001 to 2011. METHODS: The Healthcare Cost and Utilization Project database was used. ArADE admissions in different socio-demographic groups were examined including characteristics such as age, gender, rural/urban, and income. LOS and hospital charges for ArADE cases were compared between 2001 and 2011. Chi-square test and t test were used for statistical analyses. RESULTS: There were 17,375 and 20,588 ArADE related admissions in 2001 and 2011, respectively. There was a 17.6% increase among the group of 18 to 64 years old and a 64.8% increase among the group of 65 years or older while the other age groups experienced decreased admission rates. Males and females had similar increases. Patients from the lower income areas experienced a two-fold increase while those from the higher income areas experienced a decrease. The mean LOS for all ArADE related admissions increased from 2.18 to 2.81 days and mean hospital charges increased from $8,456.2 to $21,572.5. CONCLUSIONS: There was an increase in ArADE hospital admissions. The greater increase in ArADE admissions among elderly, urban or low-income patients should be noted and addressed by practitioners and policy makers. The large increase in hospital charges needs further research.

Calcineurin Aß regulates NADPH oxidase (Nox) expression and activity via nuclear factor of activated T cells (NFAT) in response to high glucose.

Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144-F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin Aß is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414-422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnAα(-/-) or CnAß(-/-)), we found that high glucose selectively activates CnAß, whereas CnAα is constitutively active. Furthermore, CnAß but not CnAα mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnAß(-/-) cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnAß reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnAß-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnAß/NFAT pathway modulates Nox. These data reveal that the CnAß/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney.

miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export.

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or ß-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAß in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

Compensatory renal hypertrophy following uninephrectomy is calcineurin-independent.

Calcineurin is a calcium-dependent phosphatase that is involved in many cellular processes including hypertrophy. Inhibition or genetic loss of calcineurin blocks pathological cardiac hypertrophy and diabetic renal hypertrophy. However, calcineurin does not appear to be involved in physiological cardiac hypertrophy induced by exercise. The role of calcineurin in a compensatory, non-pathological model of renal hypertrophy has not been tested. Therefore, in this study, we examined activation of calcineurin and the effect of calcineurin inhibition or knockout on compensatory hypertrophy following uninephrectomy (UNX). UNX induces ~15% increase in the size of the remaining kidney; the data show no change in the generation of reactive oxygen species (ROS), Nox4 or transforming growth factor-ß expression confirming the model as one of compensatory hypertrophy. Next, analyses of the remaining kidney reveal that total calcineurin activity is increased, and, to a lesser extent, transcriptional activity of the calcineurin substrate nuclear factor of activated T cell is up-regulated following UNX. However, inhibition of calcineurin with cyclosporine failed to prevent compensatory renal hypertrophy. Likewise, hypertrophy was comparable to WT in mice lacking either isoform of the catalytic subunit of calcineurin (CnAα-/- or CnAß-/-). In conclusion, similar to its role in the heart, calcineurin is required for pathological but not compensatory renal hypertrophy. This separation of signalling pathways could therefore help further define key factors necessary for pathological hypertrophy including diabetic nephropathy.

Targeting the immune system to treat hypertension: where are we?

PURPOSE OF REVIEW: Research over the past decade has significantly deepened our understanding of mechanisms that drive the development of hypertension. In particular, a novel paradigm of inflammation as a common mediator of cardiovascular and kidney disease has emerged. This review will summarize the role of the immune system in cardiovascular disease, explore some of the most promising new therapeutic directions and consider their potential as new treatments for hypertension. RECENT FINDINGS: Recent data continue to demonstrate that targeting the immune system can prevent hypertension in a variety of experimental models. Tempering the enthusiasm for a long-awaited new approach to treating hypertension is decades of clinical data, showing that classic immunosuppression regimens are associated with significant side-effects - including cardiovascular disease - that effectively preclude their use in the setting of chronic hypertension. New, more specific therapies are being developed that target cytokines including IL-17, IL-6 and TNFα. SUMMARY: Preclinical data convincingly demonstrate a key role for the immune system and specific cytokine mediators. Several biotherapeutics targeting these pathways are on the market and more are in development. Side-effects, however, continue to resemble those of classic immunosuppressants, highlighting the challenge of translating these research advances into new therapies for hypertension. VIDEO ABSTRACT: http://links.lww.com/CONH/A9.

Postoperative cognitive dysfunction, Alzheimer's disease, and anesthesia.

Although aging itself is not a disease, there are many comorbidities that become more common with aging. Heart disease, cancer, and other chronic illnesses are either more common or more severe in aging patients. Approximately 5.5 million people in the United States have Alzheimer's disease (AD), with the principal risk factor being age. It is estimated that the incidence of AD diagnosis doubles every 5 years after the age of 65. Therefore, as the population ages, the impact of AD on the healthcare landscape will increase. Understanding how to manage patients with AD is critical as we begin to care for more elderly patients in the perioperative period. In addition to their other health considerations, aging surgical patients are increasingly more likely to have pre-existing AD or be at risk for developing AD. There is growing interest to determine how anesthesia affects the development or progression of AD. Similarly, a best practice for the anesthetic management of patients with AD is not yet defined. Finally, the relationship between AD and susceptibility to or exacerbation of postoperative cognitive dysfunction (POCD) is not well understood. In this review, we will discuss both the clinical and the preclinical data related to anesthesia and AD, describe the overlapping pathophysiology of neurodegeneration and provide some insight into the anesthetic care of patients with AD.

Calcineurin inhibitors and immunosuppression - a tale of two isoforms.

Organ transplantation is the state of the art for treating end-stage organ failure. Over 25000 organ transplants are performed in the USA each year. Survival rates following transplantation are now approaching 90% for 1 year and 75% for 5 years. Central to this success was the introduction of drugs that suppress the immune system and prevent rejection. The most commonly used class of immunosuppressing drugs are calcineurin inhibitors (CNIs). Calcineurin is a ubiquitous enzyme that is important for T-cell function. With more people taking CNIs for longer and longer periods of time the consequences of calcineurin inhibition on other organ systems - particularly the kidney - have become a growing concern. Virtually all people who take a CNI will develop some degree of kidney toxicity and up to 10% will progress to kidney failure. In the past 15 years, research into calcineurin action has identified distinct actions of the two main isoforms of the catalytic subunit of the enzyme. The α-isoform is required for kidney function whereas the ß-isoform has a predominant role in the immune system. This review will discuss the current state of knowledge about calcineurin isoforms and how these new insights may reshape post-transplant immunosuppression.

Rescue of calcineurin Aα(-/-) mice reveals a novel role for the α isoform in the salivary gland.

Calcineurin is an important signal transduction mediator in T cells, neurons, the heart, and kidneys. Recent evidence points to unique actions of the two main isoforms of the catalytic subunit. Although the ß isoform is required for T-cell development, α is important in the brain and kidney. In addition, mice lacking α but not ß suffer from failure to thrive and early mortality. The purpose of this study was to identify the cause of postnatal death of calcineurin α null (CnAα(-/-)) mice and to determine the mechanism of α activity that contributes to the phenotype. CnAα(-/-) mice and wild-type littermate controls were fed a modified diet and then salivary gland function and histology were examined. In vitro studies were performed to identify the mechanism of α action. Data show that calcineurin is required for normal submandibular gland function and secretion of digestive enzymes. Loss of α does not impair nuclear factor of activated T-cell activity or expression but results in impaired protein trafficking downstream of the inositol trisphosphate receptor. These findings show a novel function of calcineurin in digestion and protein trafficking. Significantly, these data also provide a mechanism to rescue to adulthood a valuable animal model of calcineurin inhibitor-mediated neuronal and renal toxicities.

Calcineurin A-ß is required for hypertrophy but not matrix expansion in the diabetic kidney.

Calcineurin is an important signalling protein that regulates a number of molecular and cellular processes. Previously, we found that inhibition of calcineurin with cyclosporine reduced renal hypertrophy and blocked glomerular matrix expansion in the diabetic kidney. Isoforms of the catalytic subunit of calcineurin are reported to have tissue specific expression and functions. In particular, the ß isoform has been implicated in cardiac and skeletal muscle hypertrophy. Therefore, we examined the role of calcineurin ß in diabetic renal hypertrophy and glomerular matrix expansion. Type I diabetes was induced in wild-type and ß(-/-) mice and then renal function, extracellular matrix expansion and hypertrophy were evaluated. The absence of ß produced a significant decrease in total calcineurin activity in the inner medulla (IM) and reduced nuclear factor of activated T-cells (NFATc) activity. Loss of ß did not alter diabetic renal dysfunction assessed by glomerular filtration rate, urine albumin excretion and blood urea nitrogen. Similarly, matrix expansion in the whole kidney and glomerulus was not different between diabetic wild-type and ß(-/-) mice. In contrast, whole kidney and glomerular hypertrophy were significantly reduced in diabetic ß(-/-) mice. Moreover, ß(-/-) renal fibroblasts demonstrated impaired phosphorylation of Erk1/Erk2, c-Jun N-terminal kinases (JNK) and mammalian target of rapamycin (mTOR) following stimulation with transforming growth factor-ß and did not undergo hypertrophy with 48 hrs culture in high glucose. In conclusion, loss of the ß isoform of calcineurin is sufficient to reproduce beneficial aspects of cyclosporine on diabetic renal hypertrophy but not matrix expansion. Therefore, while multiple signals appear to regulate matrix, calcineurin ß appears to be a central mechanism involved in organ hypertrophy.

Calcineurin signaling and PGC-1alpha expression are suppressed during muscle atrophy due to diabetes.

PGC-1alpha is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1alpha expression and oxidative capacity are decreased in skeletal muscle of patients and animals undergoing atrophy, suggesting that PGC-1alpha participates in the regulation of muscle mass. PGC-1alpha gene expression is controlled by calcium- and cAMP-sensitive pathways. However, the mechanism regulating PGC-1alpha in skeletal muscle during atrophy remains unclear. Therefore, we examined the mechanism responsible for decreased PGC-1alpha expression using a rodent streptozotocin (STZ) model of chronic diabetes and atrophy. After 21days, the levels of PGC-1alpha protein and mRNA were decreased. We examined the activation state of CREB, a potent activator of PGC-1alpha transcription, and found that phospho-CREB was paradoxically high in muscle of STZ-rats, suggesting that the cAMP pathway was not involved in PGC-1alpha regulation. In contrast, expression of calcineurin (Cn), a calcium-dependent phosphatase, was suppressed in the same muscles. PGC-1alpha expression is regulated by two Cn substrates, MEF2 and NFATc. Therefore, we examined MEF2 and NFATc activity in muscles from STZ-rats. Target genes MRF4 and MCIP1.4 mRNAs were both significantly reduced, consistent with reduced Cn signaling. Moreover, levels of MRF4, MCIP1.4, and PGC-1alpha were also decreased in muscles of CnAalpha-/- and CnAbeta-/- mice without diabetes indicating that decreased Cn signaling, rather than changes in other calcium- or cAMP-sensitive pathways, were responsible for decreased PGC-1alpha expression. These findings demonstrate that Cn activity is a major determinant of PGC-1alpha expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass.

Inhibition of calcineurin phosphatase promotes exocytosis of renin from juxtaglomerular cells.

To examine the role of the calcium/calmodulin-dependent phosphatase calcineurin in regulation of renin release, we assayed exocytosis using whole-cell patch clamp of single juxtaglomerular cells in culture. The calcineurin inhibitor, cyclosporine A (CsA), significantly increased juxtaglomerular cell membrane capacitance, an index of cell surface area and an established measure of exocytosis in single-cell assays. This effect was mimicked by intracellular delivery of a calcineurin inhibitory peptide, the calcium chelator ethylene glycol tetraacetic acid (EGTA), or the calmodulin inhibitor W-13. Simultaneous exposure to EGTA and CsA had no additive effect. The protein kinase A (PKA) blocker RpcAMPs had no effect on the CsA-induced increase in membrane capacitance. Intra- and extracellular application of tacrolimus did not alter membrane capacitance. A calmodulin antagonist (calmidazolium) and CsA, but not tacrolimus, significantly stimulated renin release from cultured juxtaglomerular cells. Juxtaglomerular cells expressed the calcineurin isoforms A-beta and A-gamma but not A-alpha. Plasma renin concentrations (PRCs) were not different in wild-type, calcineurin A-alpha, or A-beta knockout mice but increased after CsA treatment of the A-alpha knockout, while renin mRNA was suppressed. We conclude that calcineurin and calcium/calmodulin suppress exocytosis of renin from juxtaglomerular cells independent of PKA.

Muscle atrophy in chronic kidney disease results from abnormalities in insulin signaling.

Muscle atrophy is a significant consequence of chronic kidney disease that increases a patient's risk of mortality and decreases their quality of life. The loss of lean body mass results, in part, from an increase in the rate of muscle protein degradation. In this review, the proteolytic systems that are activated during chronic kidney disease and the key insulin signaling pathways that regulate the protein degradative processes are described.

T-cell receptor-stimulated calcineurin activity is inhibited in isolated T cells from transplant patients.

The addition of calcineurin inhibitors, including cyclosporine A (CsA) and FK-506 (tacrolimus), to transplant protocols has markedly reduced acute allograft rejection and prolonged patient survival. Although monitoring of serum drug levels has been shown to be a poor indicator of efficacy, there is little data on calcineurin enzymatic activity in humans. Therefore, we measured calcineurin in isolated CD3(+)/4(+) T cells from 81 non-transplant controls and 39 renal allograft patients by using a (32)PO(4)-labeled calcineurin-specific substrate. A gender difference was observed in the control cohort, with activity in males significantly higher than that in females (1073 +/- 134 versus 758 +/- 75 fmol/microg/min, respectively). Activity of both groups was comparably inhibited by 5 ng/ml tacrolimus (27 +/- 4 versus 30 +/- 4%). Calcineurin is a downstream target of the T-cell receptor (TCR). Therefore, activity was measured in isolated T cells after incubation with anti-CD3/CD28 antibodies to stimulate the TCR. Calcineurin activity increased significantly from 1214 +/- 111 to 1652 +/- 138 fmol/microg/min; addition of either tacrolimus or CsA (500 ng/ml) blocked CD3/CD28 stimulation. Despite therapeutic levels of tacrolimus and CsA (mean 11.4 and 172 ng/ml), basal calcineurin activity was significantly higher among renal transplant recipients than controls (1776 +/- 175 versus 914 +/- 78 fmol/microg/min). In contrast, anti-CD3/CD28 antibodies failed to stimulate calcineurin activity in transplant subjects. Finally, we found that basal and stimulated calcineurin activities are inversely related. Consistent with this finding, basal activity in resting T cells rose over time after transplant but stimulation fell (r(2) = 0.785, p < 0.05). These data suggest that examination of TCR-stimulated calcineurin activity after renal transplantation may be useful for monitoring immunosuppression of individual patients.

A fluorimetric method for determination of calcineurin activity.

Calcineurin is a calcium-dependent, serine/threonine phosphatase that is involved in a variety of signaling pathways. Calcineurin is distinct among phosphatases because its activity requires calcium and is not sensitive to inhibition by compounds that block the related phosphatases PP1A and PP2A. Therefore, the most common methods to measure calcineurin activity rely on calcium-dependent dephosphorylation of a substrate derived from the RII subunit of protein kinase A in the presence of PP1A/PP2A inhibitors. However, current techniques quantify activity by measurement of released radioactive phosphate or detection of free phosphate with malachite green. Both methods involve technical challenges and have undesirable features. We report a new calcineurin fluorimetric assay that utilizes a fluorescently labeled phosphopeptide substrate and separation of dephosphorylated peptide product by titanium-oxide. The method is rapid, quantitative, involves no radioactivity and is suitable for high throughput assays. Furthermore, with the use of a standard curve, precise measurements of calcineurin activity can be obtained.