Regulation of 11β-hydroxysteroid dehydrogenase type 2 by microRNA.

The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is selectively expressed in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor. A diminished activity causes salt-sensitive hypertension. The mechanism of the variable and distinct 11β-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) expression in the cortical collecting duct is poorly understood. Here, we analyzed for the first time whether the 11β-HSD2 expression is modulated by microRNAs (miRNAs). In silico analysis revealed 53 and 27 miRNAs with potential binding sites on human or rat HSD11B2 3'-untranslated region. A reporter assay demonstrated 3'-untranslated region-dependent regulation of human and rodent HSD11B2. miRNAs were profiled from cortical collecting ducts and proximal convoluted tubules. Bioinformatic analyses showed a distinct clustering for cortical collecting ducts and proximal convoluted tubules with 53 of 375 miRNAs, where 13 were predicted to bind to the rat HSD11B2 3'-untranslated region. To gain insight into potentially relevant miRNAs in vivo, we investigated 2 models with differential 11β-HSD2 activity linked with salt-sensitive hypertension. (1) Comparing Sprague-Dawley with low and Wistar rats with high 11β-HSD2 activity revealed rno-miR-20a-5p, rno-miR-19b-3p, and rno-miR-190a-5p to be differentially expressed. (2) Uninephrectomy lowered 11β-HSD2 activity in the residual kidney with differentially expressed rno-miR-19b-3p, rno-miR-29b-3p, and rno-miR-26-5p. In conclusion, miRNA-dependent mechanisms seem to modulate 11β-HSD2 dosage in health and disease states.

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. Experimental approach: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg·kg−1) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. Key results: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone Cmax was correlated with SPT assessment (ρS= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. Conclusions and implications: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism.

Calcium reabsorption in the distal tubule: regulation by sodium, pH, and flow.

We developed a mathematical model of Ca transport along the late distal convoluted tubule (DCT2) and the connecting tubule (CNT) to investigate the mechanisms that regulate Ca reabsorption in the DCT2-CNT. The model accounts for apical Ca influx across transient receptor potential vanilloid 5 (TRPV5) channels and basolateral Ca efflux via plasma membrane Ca-ATPase pumps and type 1 Na/Ca exchangers (NCX1). Model simulations reproduce experimentally observed variations in Ca uptake as a function of extracellular pH, Na, and Mg concentration. Our results indicate that amiloride enhances Ca reabsorption in the DCT2-CNT predominantly by increasing the driving force across NCX1, thereby stimulating Ca efflux. They also suggest that because aldosterone upregulates both apical and basolateral Na transport pathways, it has a lesser impact on Ca reabsorption than amiloride. Conversely, the model predicts that full NCX1 inhibition and parathyroidectomy each augment the Ca load delivered to the collecting duct severalfold. In addition, our results suggest that regulation of TRPV5 activity by luminal pH has a small impact, per se, on transepithelial Ca fluxes; the reduction in Ca reabsorption induced by metabolic acidosis likely stems from decreases in TRPV5 expression. In contrast, elevations in luminal Ca are predicted to significantly decrease TRPV5 activity via the Ca-sensing receptor. Nevertheless, following the administration of furosemide, the calcium-sensing receptor-mediated increase in Ca reabsorption in the DCT2-CNT is calculated to be insufficient to prevent hypercalciuria. Altogether, our model predicts complex interactions between calcium and sodium reabsorption in the DCT2-CNT.

Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation.

Sequentially along B cell differentiation, the different classes of membrane Ig heavy chains associate with the Ig alpha/Ig beta heterodimer within the B cell receptor (BCR). Whether each Ig class conveys specific signals adapted to the corresponding differentiation stage remains debated. We investigated the impact of the forced expression of an IgA-class receptor throughout murine B cell differentiation by knocking in the human C alpha Ig gene in place of the S mu region. Despite expression of a functional BCR, homozygous mutant mice showed a partial developmental blockade at the pro-B/pre-BI and large pre-BII cell stages, with decreased numbers of small pre-BII cells. Beyond this stage, peripheral B cell compartments of reduced size developed and allowed specific antibody responses, whereas mature cells showed constitutive activation and a strong commitment to plasma cell differentiation. Secreted IgA correctly assembled into polymers, associated with the murine J chain, and was transported into secretions. In heterozygous mutants, cells expressing the IgA allele competed poorly with those expressing IgM from the wild-type allele and were almost undetectable among peripheral B lymphocytes, notably in gut-associated lymphoid tissues. Our data indicate that the IgM BCR is more efficient in driving early B cell education and in mucosal site targeting, whereas the IgA BCR appears particularly suited to promoting activation and differentiation of effector plasma cells.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

A homozygous missense mutation in SCNN1A is responsible for a transient neonatal form of pseudohypoaldosteronism type 1.

Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disorder of mineralocorticoid resistance characterized by salt wasting, hyperkalemia, high aldosterone levels, and failure to thrive. An autosomal recessive form (AR-PHA1) is caused by mutations in the epithelial sodium channel ENaC with usually severe and persisting multiorgan symptoms. The autosomal dominant form of PHA1 (AD-PHA1) is due to mutations in the mineralocorticoid receptor causing milder and transient symptoms restricted to the kidney. We identified a homozygous missense mutation in the SCNN1A gene (c.727T>C/p.Ser(243)Pro), encoding α-subunit of ENaC (α-ENaC) in a prematurely born boy with a severe salt-losing syndrome. The patient improved rapidly under treatment, and dietary salt supplementation could be stopped after 6 mo. Interestingly, the patient's sibling born at term and harboring the same homozygous Ser(243)Pro mutation showed no symptom of salt-losing nephropathy. In vitro expression of the αSer(243)Pro ENaC mutant revealed a slight but significant decrease in ENaC activity that is exacerbated in the presence of high Na(+) load. Our study provides the first evidence that ENaC activity is critical for the maintenance of salt balance in the immature kidney of preterm babies. Together with previous studies, it shows that, when the kidney is fully mature, the severity of the symptoms of AR-PHA1 is related to the degree of the ENaC loss of function. Finally, this study identifies a novel functional domain in the extracellular loop of ENaC.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.

Blocking Junctional Adhesion Molecule C Enhances Dendritic Cell Migration and Boosts the Immune Responses against Leishmania major.

The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.

Neoadjuvant treatment with docetaxel plus lapatinib, trastuzumab, or both followed by an anthracycline-based chemotherapy in HER2-positive breast cancer: results of the randomised phase II EORTC 10054 study.

BACKGROUND: Neoadjuvant trials conducted using a double HER2 blockade with lapatinib and trastuzumab, combined with different paclitaxel-containing chemotherapy regimens, have shown high pathological complete response (pCR) rates, but at the cost of important toxicity. We hypothesised that this toxicity might be due to a specific interaction between paclitaxel and lapatinib. This trial assesses the toxicity and activity of the combination of docetaxel with lapatinib and trastuzumab. PATIENTS AND METHODS: Patients with stage IIA to IIIC HER2-positive breast cancer received six cycles of chemotherapy (three cycles of docetaxel followed by three cycles of fluorouracil, epirubicin, cyclophosphamide). They were randomised 1 : 1 : 1 to receive during the first three cycles either lapatinib (1000 mg orally daily), trastuzumab (4 mg/kg loading dose followed by 2 mg/kg weekly), or trastuzumab + lapatinib at the same dose. The primary end point was pCR rate defined as ypT0/is. Secondary end points included safety and toxicity. pCR rate defined as ypT0/is ypN0 was assessed as an exploratory analysis. In June 2012, arm A was closed for futility based on the results from other studies. RESULTS: From October 2010 to January 2013, 128 patients were included in 14 centres. The percentage of the 122 assessable patients with pCR in the breast, and pCR in the breast and nodes, was numerically highest in the lapatinib + trastuzumab group (60% and 56%, respectively), intermediate in the trastuzumab group (52% and 52%), and lowest in the lapatinib group (46% and 36%). Frequency (%) of the most common grade 3-4 toxicities in the lapatinib /trastuzumab/lapatinib + trastuzumab arms were: febrile neutropenia 23/15/10, diarrhoea 9/2/18, infection (other) 9/4/8, and hepatic toxicity 0/2/8. CONCLUSIONS: This study demonstrates a numerically modest pCR rate increase with double anti-HER2 blockade plus chemotherapy, but suggests that the use of docetaxel rather than paclitaxel may not reduce toxicity. CLINICALTRIALSGOV: NCT00450892.

Cardiorenal end points in a trial of aliskiren for type 2 diabetes.

BACKGROUND: This study was undertaken to determine whether use of the direct renin inhibitor aliskiren would reduce cardiovascular and renal events in patients with type 2 diabetes and chronic kidney disease, cardiovascular disease, or both. METHODS: In a double-blind fashion, we randomly assigned 8561 patients to aliskiren (300 mg daily) or placebo as an adjunct to an angiotensin-converting-enzyme inhibitor or an angiotensin-receptor blocker. The primary end point was a composite of the time to cardiovascular death or a first occurrence of cardiac arrest with resuscitation; nonfatal myocardial infarction; nonfatal stroke; unplanned hospitalization for heart failure; end-stage renal disease, death attributable to kidney failure, or the need for renal-replacement therapy with no dialysis or transplantation available or initiated; or doubling of the baseline serum creatinine level. RESULTS: The trial was stopped prematurely after the second interim efficacy analysis. After a median follow-up of 32.9 months, the primary end point had occurred in 783 patients (18.3%) assigned to aliskiren as compared with 732 (17.1%) assigned to placebo (hazard ratio, 1.08; 95% confidence interval [CI], 0.98 to 1.20; P=0.12). Effects on secondary renal end points were similar. Systolic and diastolic blood pressures were lower with aliskiren (between-group differences, 1.3 and 0.6 mm Hg, respectively) and the mean reduction in the urinary albumin-to-creatinine ratio was greater (between-group difference, 14 percentage points; 95% CI, 11 to 17). The proportion of patients with hyperkalemia (serum potassium level, ≥6 mmol per liter) was significantly higher in the aliskiren group than in the placebo group (11.2% vs. 7.2%), as was the proportion with reported hypotension (12.1% vs. 8.3%) (P<0.001 for both comparisons). CONCLUSIONS: The addition of aliskiren to standard therapy with renin-angiotensin system blockade in patients with type 2 diabetes who are at high risk for cardiovascular and renal events is not supported by these data and may even be harmful. (Funded by Novartis; ALTITUDE ClinicalTrials.gov number, NCT00549757.).

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Expériences cliniques avec le facteur natriurétique auriculaire [Clinical experiences with atrial natriuretic factor]

Myocardial cells of mammals release a peptide with diuretic, natriuretic and vasodilating properties into the circulation. This peptide, called atrial natriuretic factor, is also involved in the regulation of plasma volume and, in addition, is instrumental in suppressing the activity of the renin-angiotensin-aldosterone system. The renal effects of the atrial natriuretic factor become less pronounced when systemic blood pressure is lowered. The auricular natriuretic factor seems to play an important role in cardiovascular regulation due to both its renal and extrarenal actions.

Salt-dependent renal effects of an angiotensin II antagonist in healthy subjects.

This study was designed to evaluate in healthy volunteers the renal hemodynamic and tubular effects of the orally active angiotensin II receptor antagonist losartan (DuP 753 or MK 954). Losartan or a placebo was administered to 23 subjects maintained on a high-sodium (200 mmol/d) or a low-sodium (50 mmol/d) diet in a randomized, double-blind, crossover study. The two 6-day diet periods were separated by a 5-day washout period. On day 6, the subjects were water loaded, and blood pressure, renal hemodynamics, and urinary electrolyte excretion were measured for 6 hours after a single 100-mg oral dose of losartan (n = 16) or placebo (n = 7). Losartan induced no significant changes in blood pressure, glomerular filtration rate, or renal blood flow in these water-loaded subjects, whatever the sodium diet. In subjects on a low-salt diet, losartan markedly increased urinary sodium excretion from 115 +/- 9 to 207 +/- 21 mumol/min (P < .05). The fractional excretion of endogenous lithium was unchanged, suggesting no effect of losartan on the early proximal tubule in our experimental conditions. Losartan also increased urine flow rate (from 10.5 +/- 0.4 to 13.1 +/- 0.6 mL/min, P < .05); urinary potassium excretion (from 117 +/- 6.9 to 155 +/- 11 mumol/min); and the excretion of chloride, magnesium, calcium, and phosphate. In subjects on a high-salt diet, similar effects of losartan were observed, but the changes induced by the angiotensin II antagonist did not reach statistical significance. In addition, losartan demonstrated significant uricosuric properties with both sodium diets.(ABSTRACT TRUNCATED AT 250 WORDS)