Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases

Regulation of ClC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases.BackgroundClC-Ka and ClC-Kb, chloride channels participating in renal tubular Cl− transport, require the coexpression of barttin to become functional. Mutations of the barttin gene lead to the Bartter's syndrome variant BSND, characterized by congenital deafness and severe renal salt wasting. Barttin bears a proline-tyrosine motif, a target structure for the ubiquitin ligase Nedd4-2, which mediates the clearance of channel proteins from the cell membrane. Nedd4-2 is, in turn, a target of the serum- and glucocorticoid-inducible kinase SGK1, which phosphorylates and, thus, inactivates the ubiquitin ligase. ClC-Ka also possesses a SGK1 consensus site in its sequence. We hypothesized that ClC-Ka/barttin is stimulated by SGK1, and down-regulated by Nedd4-2, an effect that may be reversed by SGK1 and its isoforms, SGK2 or SGK3.MethodsTo test this hypothesis, ClC-Ka/barttin was heterologously expressed in Xenopus oocytes with or without the additional expression of Nedd4-2, SGK1, SGK2, SGK3, constitutively active S422DSGK1, or inactive K127NSGK1.ResultsExpression of ClC-Ka/barttin induced a slightly inwardly rectifying current that was significantly decreased upon coexpression of Nedd4-2, but not the catalytically inactive mutant C938SNedd4-2. The coexpression of S422DSGK1, SGK1, or SGK3, but not SGK2 or K127NSGK1 significantly stimulated the current. Moreover, S422DSGK1, SGK1, and SGK3 also phosphorylated Nedd4-2 and thereby inhibited Nedd4-2 binding to its target. The down-regulation of ClC-Ka/barttin by Nedd4-2 was abolished by elimination of the PY motif in barttin.ConclusionClC-Ka/barttin channels are regulated by SGK1 and SGK3, which may thus participate in the regulation of transport in kidney and inner ear

Systematic assessment of the quality of osteoporosis guidelines

BACKGROUND: Numerous agencies have developed clinical practice guidelines for the management of postmenopausal osteoporosis. The study objective was to conduct a systematic assessment of the quality of osteoporosis guidelines produced since 1998. METHODS: Guidelines were identified by searching MEDLINE (1998+), the world wide web, known guideline developer websites, bibliographies of retrieved guidelines, and through consultation with content experts. Each guideline was then assessed by three independent appraisers using the 'Appraisal Instrument for Clinical Guidelines' (version 1) by Cluzeau. RESULTS: We identified 26 unique guidelines from 1998–2001 and 21 met our inclusion criteria. Of the 21 guidelines reviewed, 8 were developed by medical societies, 6 by national groups, 6 by government agencies, and 1 by an international group. Twelve of the guidelines were published, 7 were organizational reports, and 2 were accessible only from the web. Half or more of the 20 items assessing the rigor of guideline development were met by 15% (median quality score 23%, range 5–80%, (95% CI 16.5, 34.7)), 81% met at least half of the 12 items assessing guideline content and context (median score 58%, range 17–83%, (95% CI 50.8, 65.5)), and none met half or more of the items assessing guideline application (median score 0%, range 0–47%, (95% CI -0.5 to 12.6)). Eight guidelines described the method used to assess the strength of evidence, and in 6 there was an explicit link between recommendations and the supporting evidence. Ten guidelines were judged not suitable for use in practice, 10 were acceptable with modification, and one was acceptable for use without modification. CONCLUSION: The methodological quality of current osteoporosis guidelines is low, although their scores for clinical content were higher. Virtually no guidelines covered dissemination issues. Few guidelines were judged as acceptable for use in their current format

TRPM6 and TRPM7—Gatekeepers of human magnesium metabolism

AbstractHuman magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems – next to passive paracellular magnesium flux – involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism

Electrophysiological effects of progesterone on hepatocytes

AbstractThe addition of progesterone (1–100 μmol/l) to the extracellular fluid bathing rat hepatocytes led to a rapid and fully reversible depolarization of the cell membrane. The progesterone-induced depolarization was paralleled by a decrease of potassium selectivity and an increase of cell membrane resistance and was abolished in the presence of the potassium channel blocker barium. Accordingly, in whole cell recordings, progesterone led to a decrease of the cell membrane conductance. 17 α-Hydroxyprogesterone and β-estradiol were less effective by a factor of 10, whereas cholesterol, corticosterone and hydrocortisone did not significantly alter the potential difference across the cell membrane. In conclusion, acute administration of progesterone depolarized rat hepatocytes by decreasing the potassium conductance of the cell membrane

A nongenomic mechanism for progesterone-mediated immunosuppression: Inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression

High glucose up-regulates ENaC and SGK1 expression in HCD-cells

Background/Aim: Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised. Methods: Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the a conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na+ concentration were assessed using single-cell microfluorimetry. Results: Incubation with glucose, the Ca2+-ionophore ionomycin and the cytokine TGF-beta 1 were all found to evoke significant and time-dependent increases in both SGK1 and alpha ENaC protein expression. These molecular changes were correlated to an increase in Na+-uptake at the single-cell level. Conclusion: Together these data offer a potential explanation for glucose-evoked Na+-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy

Down-Regulation of Serum/Glucocorticoid Regulated Kinase 1 in Colorectal Tumours Is Largely Independent of Promoter Hypermethylation

Background: We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples. Methodology/Principal Findings: We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG. Conclusions/Significance: Our results show that even though partial methylation of the promoter region of SGK1 is present

A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression

Cost-effectiveness of recommended nurse staffing levels for short-stay skilled nursing facility patients

BACKGROUND: Among patients in skilled nursing facilities for post-acute care, increased registered nurse, total licensed staff, and nurse assistant staffing is associated with a decreased rate of hospital transfer for selected diagnoses. However, the cost-effectiveness of increasing staffing to recommended levels is unknown. METHODS: Using a Markov cohort simulation, we estimated the incremental cost-effectiveness of recommended staffing versus median staffing in patients admitted to skilled nursing facilities for post-acute care. The outcomes of interest were life expectancy, quality-adjusted life expectancy, and incremental cost-effectiveness. RESULTS: The incremental cost-effectiveness of recommended staffing versus median staffing was $321,000 per discounted quality-adjusted life year gained. One-way sensitivity analyses demonstrated that the cost-effectiveness ratio was most sensitive to the likelihood of acute hospitalization from the nursing home. The cost-effectiveness ratio was also sensitive to the rapidity with which patients in the recommended staffing scenario recovered health-related quality of life as compared to the median staffing scenario. The cost-effectiveness ratio was not sensitive to other parameters. CONCLUSION: Adopting recommended nurse staffing for short-stay nursing home patients cannot be justified on the basis of decreased hospital transfer rates alone, except in facilities with high baseline hospital transfer rates. Increasing nurse staffing would be justified if health-related quality of life of nursing home patients improved substantially from greater nurse and nurse assistant presence

Membrane Cholesterol Content Modulates Activation of Volume-Regulated Anion Current in Bovine Endothelial Cells

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-β-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-β-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening