Influence of sodium intake on circulating levels of neuropeptide Y.

Neuropeptide Y (NPY) is present in the adrenal medulla, in sympathetic neurons as well as in the circulation. This peptide not only exerts a direct vasoconstrictor effect, but also potentiates the vasoconstriction evoked by norepinephrine and sympathetic nerve stimulation. The vasoconstrictor effect of norepinephrine is also enhanced by salt loading and reduced by salt depletion. The purpose of this study was therefore to assess whether there exists a relationship between dietary sodium intake and the levels of circulating NPY. Uninephrectomized normotensive rats were maintained for 3 weeks either on a low, a regular or a high sodium intake. On the day of the experiment, plasma levels of NPY and catecholamines were measured in the unanesthetized animals. There was no significant difference in plasma norepinephrine and epinephrine levels between the 3 groups of rats. Plasma NPY levels were the lowest (65.4 +/- 8.8 fmol/ml, n-10, Mean +/- SEM) in salt-restricted and the highest (151.2 +/- 25 fmol/ml, n-14, p less than 0.02) in salt-loaded animals. Intermediate values were obtained in rats kept on a regular sodium intake (117.6 +/- 20.1 fmol/ml). These findings are therefore compatible with the hypothesis that sodium balance might to some extent influence blood pressure regulation via changes in circulating NPY levels which in turn modify blood pressure responsiveness.

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter.

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.

Sodium channels and mammalian sensory mechanotransduction.

BACKGROUND: Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear. RESULTS: Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro. CONCLUSION: Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.

A fast-track program reduces complications and length of hospital stay after open colonic surgery.

BACKGROUND & AIMS: A fast-track program is a multimodal approach for patients undergoing colonic surgery that combines stringent regimens of perioperative care (fluid restriction, optimized analgesia, forced mobilization, and early oral feeding) to reduce perioperative morbidity, hospital stay, and cost. We investigated the impact of a fast-track protocol on postoperative morbidity in patients after open colonic surgery. METHODS: A randomized trial of patients in 4 teaching hospitals in Switzerland included 156 patients undergoing elective open colonic surgery who were assigned to either a fast-track program or standard care. The primary end point was the 30-day complication rate. Secondary end points were severity of complications, hospital stay, and compliance with the fast-track protocol. RESULTS: The fast-track protocol significantly decreased the number of complications (16 of 76 in the fast-track group vs 37 of 75 in the standard care group; P = .0014), resulting in shorter hospital stays (median, 5 days; range, 2-30 vs 9 days, respectively; range, 6-30; P < .0001). There was a trend toward less severe complications in the fast-track group. A multiple logistic regression analysis revealed fluid administration greater than the restriction limits (odds ratio, 4.198; 95% confidence interval, 1.7-10.366; P = .002) and a nonfunctioning epidural analgesia (odds ratio, 3.365; 95% confidence interval, 1.367-8.283; P = .008) as independent predictors of postoperative complications. CONCLUSIONS: The fast-track program reduces the rate of postoperative complications and length of hospital stay and should be considered as standard care. Fluid restriction and an effective epidural analgesia are the key factors that determine outcome of the fast-track program.

Genetic dissection of sodium and potassium transport along the aldosterone-sensitive distal nephron: Importance in the control of blood pressure and hypertension.

In this review, we discuss genetic evidence supporting Guyton's hypothesis stating that blood pressure control is critically depending on fluid handling by the kidney. The review is focused on the genetic dissection of sodium and potassium transport in the distal nephron and the collecting duct that are the most important sites for the control of sodium and potassium balance by aldosterone and angiotensin II. Thanks to the study of Mendelian forms of hypertension and their corresponding transgenic mouse models, three main classes of diuretic receptors (furosemide, thiazide, amiloride) and the main components of the aldosterone- and angiotensin-dependent signaling pathways were molecularly identified over the past 20years. This will allow to design rational strategies for the treatment of hypertension and for the development of the next generation of diuretics.

The circadian clock modulates renal sodium handling.

The circadian clock contributes to the control of BP, but the underlying mechanisms remain unclear. We analyzed circadian rhythms in kidneys of wild-type mice and mice lacking the circadian transcriptional activator clock gene. Mice deficient in clock exhibited dramatic changes in the circadian rhythm of renal sodium excretion. In parallel, these mice lost the normal circadian rhythm of plasma aldosterone levels. Analysis of renal circadian transcriptomes demonstrated changes in multiple mechanisms involved in maintaining sodium balance. Pathway analysis revealed the strongest effect on the enzymatic system involved in the formation of 20-HETE, a powerful regulator of renal sodium excretion, renal vascular tone, and BP. This correlated with a significant decrease in the renal and urinary content of 20-HETE in clock-deficient mice. In summary, this study demonstrates that the circadian clock modulates renal function and identifies the 20-HETE synthesis pathway as one of its principal renal targets. It also suggests that the circadian clock affects BP, at least in part, by exerting dynamic control over renal sodium handling.

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast.

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans.

Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation.

Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

Photosensitizing effects of m-tetrahydroxyphenylchlorin on human tumor xenografts: correlation with sensitizer uptake, tumor doubling time and tumor histology.

The photodynamic effects of m-tetrahydroxyphenylchlorin (mTHPC) were assessed on human malignant mesothelioma, squamous cell carcinoma and adenocarcinoma xenografts grown in nude mice and were correlated with mTHPC uptake, histology and doubling time of the tumors. Non-thermal laser light was delivered to the tumor as surface radiation 4 days after intraperitoneal administration of 0.1 and 0.3 mg mTHPC/kg body weight, respectively. The extent of tumor necrosis was measured by histomorphometry. The mTHPC concentration in non-irradiated tumors was assessed by high-performance liquid chromatography (HPLC). The tumors were graded according to their doubling time and their vascular architecture as assessed by histology. The 0.1 mg/kg dose of mTHPC resulted in an equal uptake for all 3 tumor types but revealed a larger extent of photosensitized necrosis for adenocarcinoma, which displayed a delicate tumor stroma with numerous small capillary vessels, than for mesothelioma and squamous cell carcinoma, which were both poor in stroma and vessels. The 0.3 mg/kg dose of mTHPC resulted in a 2-fold higher tumor uptake for all 3 tumor types and in a larger extent of necrosis for mesothelioma and squamous cell carcinoma, but not for adenocarcinoma xenografts, compared with the lower drug dose. Our results demonstrate that different tumor xenografts respond differently to mTHPC-PDT for a given drug-light condition. In this setting, the photosensitizing effect was more closely related to the vascular architecture of the tumors than to the sensitizer uptake and doubling time of the different tumors

Modified Corneal Collagen Crosslinking (CXL) Reduces Corneal Edema and Diurnal Visual Fluctuations in Fuchs' Dystrophy

Purpose: Crosslinking of corneal collagen with riboflavin and ultraviolet-A irradiation (CXL) induces crosslinks within and between collagen fibers. CXL increases corneal biomechanical and biochemical stability and is currently used clinically to treat keratectasia. CXL also significantly reduces the stromal swelling capacity. We investigated whether a modified CXL treatment protocol would be beneficial in early Fuchs' dystrophy with various degrees of corneal edema and diurnal variations in visual acuity. Methods: CXL was performed as published previously with the following modification: in cases where the stroma was thicker than 450 µm after abrasion and 30 minutes of instillation of isoosmolar riboflavin solution, glycerol 70% solution was applied every 5 seconds for two minutes, and central corneal thickness (CCT) was measured using ultrasound pachymetry. Glycerol 70% solution was administered repeatedly until the target corneal thickness of 370-430 µm was reached. During irradiation, CCT was monitored by ultrasound pachymetry every five minutes and glycerol 70% solution was applied, if necessary. Results: Three eyes in two patients were treated using the modified CXL protocol. Representative case: a 50-year-old woman with Fuchs' dystrophy and a history of 3 years of diurnal visual fluctuations was referred to us in March 2008. Preoperative best spectacle-corrected visual acuity (BSCVA) was 20/50. We performed modified CXL in the left eye. At one month after CXL, Scheimpflug analysis of CCT showed a reduction of more than 100 µm, and the Corneal Thickness Spatial Profile (CTSP) and Percentage of Increase in Thickness (PIT) showed a regularization of the "flattening" typical for Fuchs' dystrophy. Accordingly, diurnal analysis of corneal thickness showed a distinct postoperative reduction in CCT at all time points measured. At one month after CXL, the patient reported a reduction of diurnal visual fluctuations and we measured an increase in BSCVA to 20/32. The patient showed stable topographical and visual acuity at the three months follow-up. Conclusions: We saw a distinct reduction in CCT, an improvement of the corneal thickness spatial profile (CTSP) and an increase in BSCVA at one month after treatment, which remained stable at the three months follow-up. Patients with early Fuchs' dystrophy and disturbing diurnal visual fluctuations represent a novel application for CXL. Although CXL may not prevent the outcome of the dystrophy, it may increase the patients' visual comfort until keratoplasty becomes necessary.

Dysfunction of epithelial sodium transport: from human to mouse.

The highly amiloride-sensitive epithelial sodium channel (ENaC) is an apical membrane constituent of cells of many salt-absorbing epithelia. In the kidney, the functional relevance of ENaC expression has been well established. ENaC mediates the aldosterone-dependent sodium reabsorption in the distal nephron and is involved in the regulation of blood pressure. Mutations in genes encoding ENaC subunits are causative for two human inherited diseases: Liddle's syndrome, a severe form of hypertension associated with ENaC hyperfunction, and pseudohypoaldosteronism (PHA-1), a salt-wasting syndrome caused by decreased ENaC function. Transgenic mouse technologies provide a useful tool to study the role of ENaC in vivo. Different mouse lines have been established in which each of the ENaC subunits was affected. The phenotypes observed in these mice demonstrated that each subunit is essential for survival and for regulation of sodium transport in kidney and colon. Moreover, the alpha subunit plays a specific role in the control of fluid absorption in the airways at birth. Such mice can now be used to study the role of ENaC in various organs and can serve as models to understand the pathophysiology of these human diseases.

Protective and Aggravating Effects of Nlrp3 Inflammasome Activation in IBD Models: Influence of Genetic and Environmental Factors.

Background: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1β and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3(-/-) mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3(-/-) mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. Methods: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we investigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3(-/-) mice, as well as by antibiotic treatment. Results: Nlrp3(-/-) mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3(-/-) mice in steady state conditions. Interestingly, after cohousing, Nlrp3(-/-) mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3(-/-) mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. Conclusions: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3(-/-) mice. This observation may - at least in part - explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD.