Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

Proteolytic Cleavage of the ENaC γ Subunit - Impact Upon Na + and K + Handling.

The ENaC gamma subunit is essential for homeostasis of Na + , K + , and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro . Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 ( Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo .

The role of dietary magnesium deficiency in inflammatory hypertension.

Nearly 30% of adults consume less than the estimated average daily requirement of magnesium (Mg2+), and commonly used medications, such as diuretics, promote Mg2+ deficiency. Higher serum Mg2+ levels, increased dietary Mg2+ in-take, and Mg2+ supplementation are each associated with lower blood pressure, suggesting that Mg2+-deficiency contributes to the pathogenesis of hypertension. Antigen-presenting cells, such as monocytes and dendritic cells, are well-known to be involved in the pathogenesis of hypertension. In these cells, processes implicated as necessary for increased blood pressure include activation of the NLRP3 inflammasome, IL-1ß production, and oxidative modification of fatty acids such as arachidonic acid, forming isolevuglandins (IsoLGs). We hypothesized that increased blood pressure in response to dietary Mg2+-depletion leads to increased NLRP3, IL-1ß, and IsoLG production in antigen presenting cells. We found that a Mg2+-depleted diet (0.01% Mg2+ diet) increased blood pressure in mice compared to mice fed a 0.08% Mg2+ diet. Mg2+-depleted mice did not exhibit an increase in total body fluid, as measured by quantitative magnetic resonance. Plasma IL-1ß concentrations were increased (0.13 ± 0.02 pg/mL vs. 0.04 ± 0.02 pg/mL). Using flow cytometry, we observed increased NLRP3 and IL-1ß expression in antigen-presenting cells from spleen, kidney, and aorta. We also observed increased IsoLG production in antigen-presenting cells from these organs. Primary culture of CD11c+ dendritic cells confirmed that low extracellular Mg2+ exerts a direct effect on these cells, stimulating IL-1ß and IL-18 production. The present findings show that NLRP3 inflammasome activation and IsoLG-adduct formation are stimulated when dietary Mg2+ is depleted. Interventions and increased dietary Mg2+ consumption may prove beneficial in decreasing the prevalence of hypertension and cardiovascular disease.

Influence of glycoprotein MUC1 on trafficking of the Ca2+-selective ion channels, TRPV5 and TRPV6, and on in vivo calcium homeostasis.

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.

Salt sensitivity of volume and blood pressure in a mouse with globally reduced ENaC γ-subunit expression.

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γmt) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γmt/mt mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γmt/mt mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γmt/mt mice and controls. γmt/mt mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na+ diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γmt/mt mice relative to the controls. Lower blood pressures were variably observed in γmt/mt mice on a high-salt diet compared with the controls. γmt/mt also exhibited reduced diurnal blood pressure variation, a "nondipping" phenotype, on a high-Na+ diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure.NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na+ channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na+ channel in regulating body fluid and blood pressure beyond classical transepithelial Na+ transport mechanisms.

L-WNK1 is required for BK channel activation in intercalated cells.

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet.

How does thyroidectomy for benign thyroid disease impact upon quality of life? A prospective study.

BACKGROUND: Choosing which patients to recommend surgery for benign thyroid conditions can be difficult due to the subjective nature of compressive thyroid and hormonal symptoms. The aim of this prospective study was to analyse changes in quality of life (QOL) following thyroid surgery using a validated disease-specific assessment tool, the thyroid-related patient-reported outcome (ThyPRO) questionnaire. METHODS: Participants undergoing elective thyroid surgery for benign conditions were recruited. Patient demographics and clinical data were collected. ThyPRO consists of 85 questions grouped into 13 physical, mental and social symptom domains. Patients completed a ThyPRO questionnaire pre-operatively and at 6 weeks and 6 months post-operatively. ThyPRO items were scored according to protocol to produce 13 subscales. Repeated measures linear models with no random effects were performed using data for each outcome. RESULTS: Results were available for a total of 72 patients. The sample was predominately female (n = 63, 88%) with average age 49.8 years. The majority of patients underwent surgery for multi-nodular goitre. At 6 weeks post-operatively, significant improvement was demonstrated in the goitre, hypothyroid, hyperthyroid and anxiety symptom domains. At 6 months post-operatively, significant improvement was demonstrated in all but four domains. No domains demonstrated significant increase in impairment post-operatively. CONCLUSION: Patients had significant improvement in nine of 13 symptom domains following surgery. Patients did not experience a negative impact on QOL following surgery. Further studies with larger patient cohorts may be able to identify potential pre-operative predictive factors for a post-operative improvement in QOL for benign thyroid disease.

Environmental enrichment and amantadine confer individual but nonadditive enhancements in motor and spatial learning after controlled cortical impact injury.

Environmental enrichment (EE) and amantadine (AMT) enhance motor and cognitive outcome after experimental traumatic brain injury (TBI). However, there are no data on the effects of combining these two therapies. Hence, the aim of the current study was to combine EE and AMT after TBI to determine if their net effect further enhances motor and cognitive performance. Anesthetized adult male rats received either a cortical impact of moderate severity or sham injury and then were randomly assigned to EE or standard (STD) housing and once daily administration of AMT (20 mg/kg; i.p.) or saline vehicle (VEH, 1 mL/kg; i.p.) beginning 24 h after injury for 19 days. Motor and cognitive function were assessed on post-surgical days 1-5 and 14-19, respectively. Cortical lesion volume was quantified on day 21. There were no statistical differences among the sham groups regardless of therapy, so the data were pooled. EE, AMT, and their combination (EE + AMT) improved beam-balance, but only EE and EE + AMT enhanced beam-walking. All three treatment paradigms improved spatial learning and memory relative to the VEH-treated STD controls (p < 0.05). No differences were revealed between the EE groups, regardless of treatment, but both were better than the AMT-treated STD group on beam-walking and spatial learning (p < 0.05). Both EE groups equally reduced cortical lesion volume relative to the STD-housed AMT and VEH groups (p < 0.05). The results indicate that although beneficial on their own, EE + AMT do not provide additional benefits after TBI. It is important to note that the lack of additive effects using the current treatment and behavioral protocols does not detract from the benefits of each individual therapy. The findings provide insight for future combination studies.

Dose-dependent neurorestorative effects of amantadine after cortical impact injury.

Numerous pharmacotherapies have been evaluated after experimental traumatic brain injury (TBI). While amantadine (AMT) has shown potential for clinical efficacy, the few studies on its effectiveness have been mixed. It is possible that suboptimal dosing, due to the evaluation of only one dose, may be causing the discrepancies in outcomes. Hence, the goal of the current study was to conduct a dose response of AMT after TBI to determine an optimal behavioral benefit. Anesthetized adult male rats received either a cortical impact of moderate severity or sham injury and then were randomly assigned to receive once daily intraperitoneally injections of AMT (10, 20, or 40 mg/kg) or saline vehicle (VEH, 1 mL/kg) commencing 24 h after injury for 19 days. Motor and cognitive function were assessed on post-operative days 1-5 and 14-19, respectively. There were no statistical differences among the sham groups treated with AMT or VEH so the data were pooled. AMT (20 mg/kg) facilitated beam-balance recovery and spatial learning relative to VEH-treated controls (p < 0.05). No other doses of AMT were effective. These results indicate that dosing should be carefully considered when assessing the effects of pharmacotherapies after TBI so that potential benefits are not inadvertently missed.

Systematic differences between ultrasound and pathological evaluation of thyroid nodules: a method comparison study.

BACKGROUND: The size of thyroid nodules as measured by ultrasound (ultrasound size, USS) is routinely used in clinical decision-making. Reports of discrepancy between USS and pathological size (PS) evaluation have not analysed their systematic differences. The objective of this study was to uncover the lack of agreement (bias) between USS and PS measurements. METHODS: A retrospective study was performed on 121 patients who had a total or hemi-thyroidectomy for a solitary nodule. Ordinary least product regression was used to detect and distinguish constant and proportional bias in unidimensional size measurements between USS and PS evaluation. Three-dimensional volume measurements were compared in a subgroup of 31 patients. Pre-specified acceptable limits of interchange were defined as 20% difference. RESULTS: Ordinary least product regression demonstrated no constant or proportional bias between the two methods; regression equation: USS = (0.863) + (1.040) × PS. When nodules were grouped by size, discrepancies between the two methods were observed in nodules <10 mm (P = 0.004). However, potential overtreatment of patients with USS >10 mm but PS <10 mm only accounted for 4.1% of total patients. Subgroup analysis of volume measurements showed no bias between USS and PS evaluation. CONCLUSIONS: USS and PS measurements were interchangeable, as there was no evidence of constant or proportional bias between the two measurements. However, USS may misclassify the size for smaller nodules and potentially lead to unnecessary workup and treatment. Discrepancy in size measurements between USS and PS should be taken into account in clinical practice, particularly in smaller nodules.

Assessing the Methodological Quality of Glaucoma Clinical Practice Guidelines and Their Recommendations on Microinvasive Glaucoma Surgery: A Systematic Review.

INTRODUCTION: Clinical practice guidelines (CPG) are regarded by many as critical communications providing guidance within specific medical fields. Over a decade ago, the first microinvasive glaucoma surgical (MIGS) procedures were introduced. Since then, a number of these novel intraocular pressure controlling surgical options have been approved worldwide. Governing bodies and health care administration often utilize CPGs when considering funding for newer technologies. This highlights the importance of well-written, accurate, and up-to-date CPGs in the rapidly evolving field of MIGS. If CPGs are unable to fill this role, their use in treatment decision-making is doing a disservice to patients, who will be denied currently available and potentially superior care. To determine the overall value of a CPG, the methodological quality with which it was developed, in addition to the current relevance and appropriateness of its recommendations, should be evaluated. The objective of the present study was to assess the methodological quality of currently available international glaucoma CPGs, as well as their coverage of MIGS as a surrogate marker of relevance and appropriateness to policy-makers and ophthalmologists alike. MATERIALS AND METHODS: To identify potentially relevant CPGs, a predefined search strategy was used to search the following databases: Medline, EMBASE, BIOSIS, and Web of Science. All CPGs related to adult glaucoma and published in English were included. CPG methodological quality was assessed by 3 individuals using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) tool. Studies were then assessed for coverage of MIGS devices and procedures. RESULTS: Search strategy and subsequent screening identified 11 CPGs for analysis. Eight were of high quality according to the AGREE II criteria. Three included basic information on MIGS, but none provided specific recommendations regarding their indications or which patient populations would benefit most. CONCLUSIONS: Many international glaucoma CPGs are of high methodological quality. However, coverage of MIGS is sparse, nonspecific and in many instances, absent. This causes CPGs to be a suboptimal source in guiding physicians and health policy-makers in areas characterized by novel and/or rapidly evolving technologies. Mechanisms to incorporate updated evidence in CPGs would have to be considered before they can be used as a source of contemporary clinical decision-making.

Antidepressants Are Effective in Decreasing Neuropathic Pain After SCI: A Meta-Analysis.

OBJECTIVE: To systematically review and assess the effectiveness and safety of antidepressants for neuropathic pain among individuals with spinal cord injury (SCI). METHODS: A systematic search was conducted using multiple databases for relevant articles published from 1980 to April 2014. Randomized controlled trials (RCTs) involving antidepressant treatment of neuropathic pain with ≥ 3 individuals and ≥ 50% of study population with SCI were included. Two independent reviewers selected studies based on inclusion criteria and then extracted data. Pooled analysis using Cohen's d to calculate standardized mean difference, standard error, and 95% confidence interval for primary (pain) and other secondary outcomes was conducted. RESULTS: Four RCTs met inclusion criteria. Of these, 2 studies assessed amitriptyline, 1 trazadone, and 1 duloxetine among individuals with neuropathic SCI pain. A small effect was seen in the effectiveness of antidepressants in decreasing pain among individuals with SCI (standardized mean difference = 0.34 ± 0.15; 95% CI, 0.05-0.62; P = .02). A number needed to treat of 3.4 for 30% or more pain relief was found by pooling 2 studies. Of these, significantly higher risk of experiencing constipation (risk ratio [RR] = 1.74; 95% CI, 1.09-2.78; P = .02) and dry mouth (RR = 1.39; 95% CI, 1.04-1.85; P = .02) was found amongst individuals receiving antidepressant treatment compared to those in the control group. CONCLUSIONS: The current meta-analysis demonstrates that antidepressants are effective in reducing neuropathic SCI pain. However, this should be interpreted with caution due to the limited number of studies. Further evaluation of long-term therapeutic options may be required.

Are we getting the critical view? A prospective study of photographic documentation during laparoscopic cholecystectomy.

BACKGROUND: At laparoscopic cholecystectomy, most surgeons have adopted the operative approach where the 'critical view of safety' (CVS) is achieved prior to dividing the cystic duct and artery. This prospective study evaluated whether an adequate critical view was achieved by scoring standardized intra-operative photographic views and whether there were other factors that might impact on the ability to obtain an adequate critical view. METHODS: One hundred consecutive patients undergoing a laparoscopic cholecystectomy were studied. At each operation, two photographs were taken. Two independent experienced hepatobiliary surgeons scored the photographs on whether a critical view of safety was achieved. Inter-observer agreement was calculated using the weighted kappa coefficient. The Cochran-Mantel-Haenszel test was used to analyse the scores with potential confounding clinical factors. RESULTS: The kappa coefficient for adequate display of the cystic duct and artery was 0.49; 95% confidence interval (CI) 0.33 to 0.64; P = 0.001. No bias was detected in the overall scorings between the two observers (χ(2) 1.33; P = 0.312). Other clinical factors including surgeon seniority did not alter the outcome [odds ratio (OR) 0.902; 95% confidence interval 0.622 to 1.264]. CONCLUSION: Heightened awareness of the CVS through mandatory documentation may improve both trainee and surgeon technique.