Showing papers on "Calcineurin published in 2010"

Effect of CYP3A and ABCB1 Single Nucleotide Polymorphisms on the Pharmacokinetics and Pharmacodynamics of Calcineurin Inhibitors: Part II

Abstract: The calcineurin inhibitors ciclosporin (cyclosporine) and tacrolimus are immunosuppressant drugs used for the prevention of organ rejection following transplantation Both agents are metabolic substrates for cytochrome P450 (CYP) 3A enzymes — in particular, CYP3A4 and CYP3A5 — and are transported out of cells via P-glycoprotein (ABCB1) Several single nucleotide polymorphisms (SNPs) have been identified in the genes encoding for CYP3A4, CYP3A5 and P-glycoprotein, including CYP3A4 —392A>G (rs2740574), CYP3A5 6986A>G (rs776746), ABCB1 3435C>T (rs1045642), ABCB1 1236C>T (rs1 128503) and ABCB1 2677G>T/A (rs2032582) The aim of this review is to provide the clinician with an extensive overview of the recent literature on the known effects of these SNPs on the pharmacodynamics of ciclosporin and tacrolimus in solid-organ transplant recipients Literature searches were performed and all relevant primary research articles were critiqued and summarized There is no evidence that the CYP3A4 —392A>G SNP has an effect on the pharmacodynamics of either ciclosporin or tacrolimus; however, studies have been limited For patients prescribed ciclosporin, the CYP3A5 6986A>G SNP may influence long-term survival, possibly because of a different metabolite pattern over time This SNP has no clear association with acute rejection during ciclosporin therapy Despite a strong association between the CYP3A5 6986A>G SNP and tacrolimus pharmacokinetics, there is no consistent evidence of organ rejection as a result of genotype-related under-immunosuppression This is likely to be explained by the practice of performing tacrolimus dose adjustments in the early phase after transplantation The effect of the CYP3A5 6986A>G SNP on ciclosporin-and tacrolimus-related nephrotoxicity and development of hypertension is unclear Similarly, the ABCB1 SNPs exert no clear influence on either ciclosporin or tacrolimus pharmacodynamics, with studies showing conflicting results in regard to the main parameters of acute rejection and nephrotoxicity In kidney transplant patients, consideration of the donor kidney genotype rather than the recipient genotype may be more important when assessing development of nephrotoxicity Studies with low patient numbers may account for many inconsistent results to date The majority of studies have only evaluated the effects of individual SNPs; however, multiple polymorphisms may interact to produce a combined effect Further haplotype analyses are likely to be useful, particularly ones that consider both donor and recipient genotype The effects of polymorphisms associated with the pregnane X receptor, organic anion transporting polypeptides, calcineurin inhibitor target sites and immune response pathways need to be further investigated A large standardized clinical trial is now required to evaluate the relationship between the pharmacokinetics and pharmacodynamics of CYP3A5-mediated tacrolimus metabolism, particularly in regard to the outcomes of acute rejection and nephrotoxicity It is not yet clear whether pharmacogenetic profiling of calcineurin inhibitors will be a useful clinical tool for personalizing immunosuppressant therapy

MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling

Abstract: MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRs in myocardial disease processes. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure.

PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90

Abstract: Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Opposing roles for calcineurin and ATF3 in squamous skin cancer

Abstract: Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Inducing CTLA-4–Dependent Immune Regulation by Selective CD28 Blockade Promotes Regulatory T Cells in Organ Transplantation

Abstract: Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the eventual rejection of the organ. Blocking the CD28-mediated T cell costimulation pathway is a nontoxic alternative immunosuppression strategy that is now achieved by blockade of CD80/86, the receptor for CD28 on antigen-presenting cells. However, interaction of CD80/86 with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is required for immune regulation. Therefore, CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and preserve immune regulation. By using monovalent antibodies, we identified true CD28 antagonists that induced CTLA-4-dependent decreased T cell function compatible with regulatory T (Treg) cell suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood Treg cells, induced molecular signatures of immune regulation, and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 preserves CTLA-4-dependent immune regulation and promotes allograft survival.

Reciprocal Repression Between MicroRNA-133 and Calcineurin Regulates Cardiac Hypertrophy. A Novel Mechanism for Progressive Cardiac Hypertrophy

Abstract: Cardiac hypertrophy involves a remodeling process of the heart in response to diverse pathological stimuli. Both calcineurin/nuclear factor of activated T cells pathway and microRNA-133 ( miR-133 ) have been shown to play a critical role in cardiac hypertrophy. It has been recognized that the expression and activity of calcineurin increases and miR-133 expression decreases in the hypertrophic heart, and inhibition of calcineurin or increase of miR-133 expression protects against cardiac hypertrophy. Here we tested the interaction between miR-133 and calcineurin in cardiac hypertrophy. Cardiac hypertrophy in vivo and in vitro was induced by transverse aortic constriction and phenylephrine treatment. mRNA levels were measured by using real-time PCR methods. Luciferase assays showed that transfection of miR-133 in HEK293 cells downregulated calcineurin expression, which was reversed by cotransfection with the miR-133 –specific 2′- O -methyl antisense inhibitory oligoribonucleotides. These results were confirmed in cultured primary cardiomyocytes. miR-133 expression was downregulated, and calcineurin activity was enhanced in both in vivo and in vitro cardiac hypertrophy models. Treatment of cells and animals with cyclosporin A, an inhibitor of calcineurin, prevented miR-133 downregulation. Moreover, the antisense oligodeoxynucleotides against the catalytic subunits of calcineurin Aβ and the decoy oligodeoxynucleotides targeting nuclear factor of activated T cells transcription factor, a calcineurin downstream effector, increased miR-133 expression in cultured primary cardiomyocytes. Our data show that reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy.

Calcineurin regulates innate antifungal immunity in neutrophils

Abstract: Patients taking immunosuppressive drugs, like cyclosporine A (CsA), that inhibit calcineurin are highly susceptible to disseminated fungal infections, although it is unclear how these drugs suppress resistance to these opportunistic pathogens. We show that in a mouse model of disseminated Candida albicans infection, CsA-induced susceptibility to fungal infection maps to the innate immune system. To further define the cell types targeted by CsA, we generated mice with a conditional deletion of calcineurin B (CnB) in neutrophils. These mice displayed markedly decreased resistance to infection with C. albicans, and both CnB-deficient and CsA-treated neutrophils showed a defect in the ex vivo killing of C. albicans. In response to the fungal-derived pathogen-associated molecular pattern zymosan, neutrophils lacking CnB displayed impaired up-regulation of genes (IL-10, Cox2, Egr1, and Egr2) regulated by nuclear factor of activated T cells, the best characterized CnB substrate. This activity was Myd88 independent and was reproduced by stimulation with the β(1,3) glucan curdlan, indicating that dectin-1, rather than toll-like receptors, is the upstream activator of calcineurin. Our results suggest that disseminated fungal infections seen in CsA-treated patients are not just a general consequence of systemic suppression of adaptive immunity but are, rather, a result of the specific blockade of evolutionarily conserved innate pathways for fungal resistance.

Transcriptional Regulation of Chitin Synthases by Calcineurin Controls Paradoxical Growth of Aspergillus fumigatus in Response to Caspofungin

Abstract: Attenuated activity of echinocandin antifungals at high concentrations, known as the “paradoxical effect,” is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca2+/calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the ΔcnaA and ΔcrzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.

Islet Transplantation in Type 1 Diabetic Patients Using Calcineurin Inhibitor-Free Immunosuppressive Protocols Based on T-Cell Adhesion or Costimulation Blockade

Abstract: Background. The applicability of islet transplantation as treatment for type 1 diabetes is limited by long-term graft dysfunction, immunosuppressive drug toxicity, need for multiple donors, and increased risk of allosensitization. We describe two immunosuppressive regimens based on the costimulation blocker belatacept (BELA) or the antileukocyte functional antigen-1 antibody efalizumab (EFA), which permit long-term islet allograft survival and address some of these concerns. Methods. Ten patients with type I diabetes with hypoglycemic unawareness received intraportal allogeneic islet transplants. Immunosuppression consisted of antithymocyte globulin induction and maintenance with sirolimus or mycophenolate and BELA (n=5) or EFA (n=5). Results. All five BELA-treated patients achieved independence after single transplants; one resumed partial insulin use 305 days after transplant but is now independent after a second transplant. All five patients treated with EFA achieved independence after one (3/5) or two (2/5) islet transplants and remained independent while on EFA (392-804 days). After EFA was discontinued because of withdrawal of the drug from the market, two patients resumed intermittent insulin use; the others remain independent. No patient in either group developed significant side effects related to the study drugs, and none have been sensitized to alloantigens. All have stable renal function. Conclusions. These two novel immunosuppressive regimens are effective, well tolerated, and the first calcineurin inhibitor/steroid-sparing islet protocols resulting in long-term insulin independence. Although EFA is no longer available for clinical use, these early results demonstrate that a regimen using BELA may be an effective alternative to improve graft function and longevity while minimizing renal and β-cell toxicity.

Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help

Abstract: In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T-B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T-B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses.

Effect of different immunosuppressive schedules on recurrence-free survival after liver transplantation for hepatocellular carcinoma.

Abstract: Background Tumor recurrence represents the main limitation of liver transplantation in patients with hepatocellular carcinoma (HCC) and can be favored by exposure to calcineurin inhibitors. Methods We investigated the effect of an immunosuppressant schedule that minimizes the exposure to calcineurin inhibitors on patients transplanted for HCC to ascertain whether this can reduce the tumor recurrence rate. For this purpose, we conducted a matched-cohort study: 31 patients with HCC transplanted between 2004 and 2007 who received sirolimus as part of their immunosuppression (group A) were compared with a control group of 31 patients (group B) transplanted in the same period who had the same prognostic factors but were given standard immunosuppression based on tacrolimus. Results Three-year recurrence-free survival was 86% in group A and 56% in group B (P=0.04). Although the prevalence of microvascular invasion G3-G4 grading and alpha-fetoprotein more than 200 ng/mL was identical in the two groups, exposure to tacrolimus was significantly higher in patients of group B (median, 8.54; range, 5.5-13.5) in comparison with those of group A (median, 4.6; range, 1.8-9.1) (P=0.0001). Conclusions By using sirolimus, exposure to calcineurin inhibitors can be minimized, reducing the risk of HCC recurrence.

Tacrolimus-Associated Posterior Reversible Encephalopathy Syndrome after Solid Organ Transplantation

Abstract: Tacrolimus (TAC) is an immunosuppressant drug discovered in 1984 by Fujisawa Pharmaceutical Co., Ltd. This drug belongs to the group of calcineurin inhibitors, which has been proven highly effective in preventing acute rejection after transplantation of solid organs. However, neurotoxicity and nephrotoxicity are its major adverse effects. Posterior reversible encephalopathy syndrome (PRES) is the most severe and dramatic consequence of calcineurin inhibitor neurotoxicity. It was initially described by Hinchey et al. in 1996 [N Engl J Med 1996;334:494-450]. Patients typically present with altered mental status, headache, focal neurological deficits, visual disturbances, and seizures. Magnetic resonance imaging is the most sensitive imaging test to detect this. With the more deep-going studies done recently, we have learnt more about this entity. It was noted that this syndrome is frequently reversible, rarely limited to the posterior regions of the brain, and often located in gray matter and cortex as well as in white matter. Therefore, in this review, the focus is on the current understanding of clinical recognition, pathogenesis, neuroimaging and management of TAC-associated PRES after solid organ transplantation.

Inhibition of Drp1-dependent mitochondrial fragmentation and apoptosis by a polypeptide antagonist of calcineurin

Abstract: During apoptosis, mitochondria lose their membrane potential and undergo fragmentation around the time of release of cytochrome c. Apoptotic fission is at least in part sustained by the translocation of dynamin-related protein 1 (Drp1), normally located in the cytosol, to mitochondria. This process depends on dephosphorylation of Drp1 by the phosphatase calcineurin. Here, we report the identification of a novel inhibitor of this process. A polypeptide (PPD1) from the immunophilin FKBP52 inhibits calcineurin activation triggered by mitochondrial dysfunction. PPD1 blocks Drp1 translocation to mitochondria and fragmentation of the organelle. PPD1 delays apoptosis by intrinsic stimuli by preventing fragmentation and release of cytochrome c. Cells expressing PPD1 display enhanced clonogenic ability after exposure to staurosporine. A genetic analysis revealed that the activity of PPD1 is independent of the BH3-only protein BAD, another target of calcineurin during apoptosis, and is not additive to inhibition of Drp1. Thus, PPD1 is a novel inhibitor of apoptosis that elucidates the function of calcineurin-dependent mitochondrial fragmentation in the amplification of cell death.

The Functional Role of Calcineurin in Hypertrophy, Regeneration, and Disorders of Skeletal Muscle

Abstract: Skeletal muscle uses calcium as a second messenger to respond and adapt to environmental stimuli. Elevations in intracellular calcium levels activate calcineurin, a serine/threonine phosphatase, resulting in the expression of a set of genes involved in the maintenance, growth, and remodeling of skeletal muscle. In this review, we discuss the effects of calcineurin activity on hypertrophy, regeneration, and disorders of skeletal muscle. Calcineurin is a potent regulator of muscle remodeling, enhancing the differentiation through upregulation of myogenin or MEF2A and downregulation of the Id1 family and myostatin. Foxo may also be a downstream candidate for a calcineurin signaling molecule during muscle regeneration. The strategy of controlling the amount of calcineurin may be effective for the treatment of muscular disorders such as DMD, UCMD, and LGMD. Activation of calcineurin produces muscular hypertrophy of the slow-twitch soleus muscle but not fast-twitch muscles.

Tacrolimus dose requirements and CYP3A5 genotype and the development of calcineurin inhibitor-associated nephrotoxicity in renal allograft recipients.

Abstract: Objectives:Prolonged calcineurin inhibitor maintenance therapy in kidney allograft recipients is complicated by the development of chronic irreversible drug-induced nephrotoxicity (CNIT).Methods:In 304 de novo renal graft recipients, the association among tacrolimus exposure indices (dose, C0, AUC0-

A systematic review on steroid withdrawal between 3 and 6 months after kidney transplantation

Abstract: Background Steroid withdrawal (SW) after the first posttransplant months in patients receiving a kidney transplant has been recently discouraged in clinical guidelines. Methods A systematic review and meta-analysis of randomized controlled trials assessing SW (beyond the second week after kidney transplantation) was performed. Only trials using a calcineurin inhibitor plus mycophenolic acid were included. Results The nine trials (1820 participants) randomly withdrew steroids between 3 and 6 months after transplantation. Death and graft loss were similar in SW and control patients. Including all trials, acute rejection was not more frequent after SW, but stratifying by the drug used, cyclosporine A (CsA) was associated with an increased incidence of overall acute rejection (risk ratio 1.42, 95% confidence interval 1.08-1.87) or biopsy-proven acute rejection (risk ratio 1.61 95% confidence interval 1.20-2.17). Contrarily, tacrolimus allowed SW without increased biopsy-proven acute rejection (P interaction=0.005). Serum cholesterol level was lower after SW than in controls using CsA or tacrolimus. Serum creatinine, blood pressure, serum triglycerides, new-onset diabetes mellitus, infections, or malignancies were similar in SW and control patients. Conclusions SW after 3 to 6 months of kidney transplantation is associated with increased rates of acute rejection only if CsA is used but not with tacrolimus. Graft function and survival remain stable up to 3 years after transplantation, the longest follow-up reported. The interest for late SW has decreased during the past years in the literature. More trials with carefully designed outcome measures are needed in patients treated with low-exposure tacrolimus and mycophenolic acid derivatives.

Proteasome functional insufficiency activates the calcineurin–NFAT pathway in cardiomyocytes and promotes maladaptive remodelling of stressed mouse hearts

Abstract: Aims Proteasome functional insufficiency (PFI) may play an important role in the progression of congestive heart failure but the underlying molecular mechanism is poorly understood. Calcineurin and nuclear factor of activated T-cells (NFAT) are degraded by the proteasome, and the calcineurin–NFAT pathway mediates cardiac remodelling. The present study examined the hypothesis that PFI activates the calcineurin–NFAT pathway and promotes maladaptive remodelling of the heart. Methods and results Using a reporter gene assay, we found that pharmacological inhibition of 20S proteasomes stimulated NFAT transactivation in both mouse hearts and cultured adult mouse cardiomyocytes. Proteasome inhibition stimulated NFAT nuclear translocation in a calcineurin-dependent manner and led to a maladaptive cell shape change in cultured neonatal rat ventricular myocytes. Proteasome inhibition facilitated left ventricular dilatation and functional decompensation and increased fatality in mice with aortic constriction while causing cardiac hypertrophy in the sham surgery group. It was further revealed that both calcineurin protein levels and NFAT transactivation were markedly increased in the mouse hearts with desmin-related cardiomyopathy and severe PFI. Expression of an aggregation-prone mutant desmin also directly increased calcineurin protein levels in cultured cardiomyocytes. Conclusions The calcineurin–NFAT pathway in the heart can be activated by proteasome inhibition and is activated in the heart of a mouse model of desmin-related cardiomyopathy that is characterized by severe PFI. The interplay between PFI and the calcineurin–NFAT pathway may contribute to the pathological remodelling of cardiomyocytes characteristic of congestive heart failure.

The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses

Abstract: Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses—slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knock-out of calcineurin. These manipulations significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7- to 10-d-old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These results suggest that calcium influx during nerve firing activates calcium/calmodulin-dependent calcineurin, which controls the speed of both rapid and slow endocytosis at synapses by dephosphorylating endocytic proteins. The calcium/calmodulin/calcineurin signaling pathway may underlie regulation of endocytosis by nerve activity and calcium as reported at many synapses over the last several decades.

Mechanisms of the sensory effects of tacrolimus on the skin.

Abstract: Summary Background Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported. Objectives To understand the mechanisms underlying these burning sensations. Methods We examined the impact of tacrolimus on substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation. Because phosphorylation of TRPV1 (transient receptor potential subtype vanilloid 1) plays a role in the induction of pain, we investigated whether tacrolimus regulates the phosphorylation state of TRPV1. Finally, we used a macropatch to evaluate the impact of tacrolimus on voltage-gated calcium currents of sensory neurons. Results Tacrolimus was able to induce initial SP release by extracellular calcium influx and inhibited SP release induced by capsaicin after 1, 24 and 72 h of pretreatment. Analysis of TRPV1 phosphorylation by Western blot confirmed the capacity of tacrolimus to favour phosphorylation. An electrophysiological study showed inhibitory effects on calcium currents. Conclusions The efficacy of tacrolimus in pruritus, as well as the sensory side-effects, could be explained by a direct effect on neurons through an effect on calcineurin, possibly by a desensitization of TRPV1 and calcium currents through the PIP2 regulation pathway.

CIB1 is a regulator of pathological cardiac hypertrophy

Abstract: Increased levels of Ca2+ in cardiomyocytes promote cell growth that, under stressful conditions, such as those caused by hypertension, can contribute to heart remodeling and failure. Joerg Heineke et al. identify a new regulator of this type of maladaptive cardiac muscle growth in mice, the calcium-binding protein CIB1, which they show regulates the membrane-association of calcineurin and downstream signaling.

Renal function, efficacy, and safety of sirolimus and mycophenolate mofetil after short-term calcineurin inhibitor-based quadruple therapy in de novo renal transplant patients: one-year analysis of a randomized multicenter trial.

Abstract: Background De novo sirolimus in calcineurin inhibitor-free regimens, although potentially useful to improve early renal function, are complicated by various drug-related side effects. Methods We report a prospective open-label, multicenter, randomized trial to evaluate early conversion from a CsA-based to a sirolimus (SRL)-based regimen 10 to 24 days after renal transplantation. Of the 196 patients, 141 patients with a low-to-moderate immunological risk were eligible to be converted to SRL or to continue CsA. All patients received antithymocyte globulin-F single-bolus induction, mycophenolate mofetil, and steroids. Results The primary endpoint, renal function determined by S-creatinine and estimated glomerular filtration rate calculated by Nankivell formula at 12 months was significantly better in the SRL group (1.51+/-0.59 vs. 1.87+/-0.98 mg/dL or 64.5+/-25.2 vs. 53.4+/-18.0 mL/min/1.73 m). Patient survival, graft survival, and incidence of biopsy-proven acute rejection after conversion were not statistically different. Drug discontinuations were significantly higher in the SRL group (36.2% vs. 19.7%). Significantly, more patients in the SRL group reported acne, aphtous, and temporary hyperlipidemia, whereas cytomegalovirus viremia was significantly decreased (7.3% vs. 28.2%). Conclusions Early conversion to a calcineurin inhibitor-free regimen with SRL in combination with mycophenolate mofetil may be a useful strategy to improve renal function. The identification of appropriate candidates and safe management of SRL-related adverse events will be a key to avoid the high rate of dropouts, which currently limit the broad applicability of this protocol.

Prospects for mTOR Inhibitor Use in Patients with Polycystic Kidney Disease and Hamartomatous Diseases

Abstract: Mammalian target of rapamycin (mTOR) is the core component of two complexes, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin and analogues. mTORC2 is impeded only in some cell types by prolonged exposure to these compounds. mTOR activation is linked to tubular cell proliferation in animal models and human autosomal dominant polycystic kidney disease (ADPKD). mTOR inhibitors impede cell proliferation and cyst growth in polycystic kidney disease (PKD) models. After renal transplantation, two small retrospective studies suggested that mTOR was more effective than calcineurin inhibitor-based immunosuppression in limiting kidney and/or liver enlargement. By inhibiting vascular remodeling, angiogenesis, and fibrogenesis, mTOR inhibitors may attenuate nephroangiosclerosis, cyst growth, and interstitial fibrosis. Thus, they may benefit ADPKD at multiple levels. However, mTOR inhibition is not without risks and side effects, mostly dose-dependent. Under certain conditions, mTOR inhibition interferes with adaptive increases in renal proliferation necessary for recovery from injury. They restrict Akt activation, nitric oxide synthesis, and endothelial cell survival (downstream from mTORC2) and potentially increase the risk for glomerular and peritubular capillary loss, vasospasm, and hypertension. They impair podocyte integrity pathways and may predispose to glomerular injury. Administration of mTOR inhibitors is discontinued because of side effects in up to 40% of transplant recipients. Currently, treatment with mTOR inhibitors should not be recommended to treat ADPKD. Results of ongoing studies must be awaited and patients informed accordingly. If effective, lower dosages than those used to prevent rejection would minimize side effects. Combination therapy with other effective drugs could improve tolerability and results.

A calcineurin/AKAP complex is required for NMDA receptor-dependent long-term depression.

Abstract: AKAP79/150 is a protein scaffold that is thought to position specific kinases (protein kinase A and C) and phosphatases (calcineurin) in appropriate synaptic domains so that their activities can regulate excitatory synaptic strength. Using a viral-mediated molecular replacement strategy in rat hippocampal slices, we found that AKAP is required for NMDA receptor-dependent long-term depression solely because of its interaction with calcineurin.

Rituximab efficiency in children with steroid-dependent nephrotic syndrome

Abstract: Although most patients with idiopathic nephrotic syndrome (NS) respond to steroid treatment, development of steroid dependency may require a long-term multidrug therapy including steroid and calcineurin inhibitor. Rituximab was shown to allow a reduction of the doses of steroid and immunosuppressive drugs in those patients. In the present series, 22 patients with steroid-sensitive, but steroid-dependent nephrotic syndrome were treated with rituximab. Rituximab reduced B cell count down to an undetectable level in all patients. A second treatment was necessary in 18 patients in order to maintain B cell depletion for up to 18 months. B cell depletion lasted 4.9 to 26 months (mean 17.2 months). At last follow-up, 9 patients were in remission without oral steroid or calcineurin inhibitor, although B cell count had recovered for 2.9 to 17 months (mean 9.5 months). A remission under ongoing B cell depletion was observed in 10 other patients in the absence of oral steroid or calcineurin inhibitor. Rituximab failed in 2 patients and 1 refused any additional treatment, despite B cell recovery and relapse. Toxicity of rituximab was limited to reversible cytokine shock in 2 patients and reversible neutropenia in 1 patient. No severe infection was observed.