Active Sodium Transport Research Articles

Enteric Nervous System: Identification of a Novel Neuronal Sensory Network in the Duodenal Epithelium.

The communication between the intestinal epithelium and the enteric nervous system has been considered indirect. Mechanical or chemical stimuli activate enteroendocrine cells inducing hormone secretion, which act on sub-epithelial nerve ends, activating the enteric nervous system. However, we identified an epithelial cell that expresses NKAIN4, a neuronal protein associated with the β-subunit of Na+/K+-ATPase. This cell overexpresses Na+/K+-ATPase and ouabain-insensitive Na+-ATPase, enzymes involved in active sodium transport. NKAIN4-positive cells also express neuronal markers as NeuN, acetylcholine-esterase, acetylcholine-transferase, α3- and α7-subunits of ACh receptors, glutamic-decarboxylase, and serotonin-receptor-7, suggesting they are neurons. NKAIN4-positive cells show a polarized shape with an oval body, an apical process finished in a knob-like terminal in contact with the lumen, a basal cilia body at the base of the apical extension, and basal axon-like soma projections connecting sub-epithelial nerve terminals, lymphoid nodules, glial cells, and enterochromaffin cells, forming a network that reaches the epithelial surface. We also showed, using retrograde labeling and immunofluorescence, that these cells receive afferent signals from the enteric nervous system. Finally, we demonstrated that acetylcholine activates NKAIN4-positive cells inducing Ca2+ mobilization and probably serotonin secretion in enterochromaffin cells. NKAIN4-positive cells are neurons that would form a part of a duodenal sensory network for physiological or noxious luminal stimuli.

#3293 ESTRADIOL REPLACEMENT MITIGATED BLOOD PRESSURE ELEVATION VIA SUPPRESSION OF NCC IN ANGIOTENSIN II-INFUSED OVARIECTOMIZED FEMALE RATS

Abstract Background and Aims Female rats excrete more urine sodium than male rats with similar blood pressure (BP). However, it is not clear whether the sex difference in pressure natriuresis is conserved in menopausal rats. We assessed whether the natriuretic response and renal sodium transporter activity in ovariectomized female rats infused with angiotensin II (ANGII) could be affected by estrogen supplementation. Method Six-week-old female Sprague-Dawley rats (n=30) were ovariectomized (OVX, n =20) or sham-operated (n=10) (−4 weeks, −4W). After 2 weeks, subcutaneous estradiol was administered to half of the OVX rats (n=10) for 4 weeks (-2W∼2W). ANGII was infused via the osmotic minipump in 60% of the rats for 2 weeks (0W∼2W). Six groups (female saline: FS, female ANGII: FA, OVX saline: OS, OVX ANGII: OA, OVX estradiol saline: OES, OVX estradiol ANGII: OEA) were sacrificed after 2 weeks of the ANGII infusion (2W). We also analyzed human data from the Korean Genome and Epidemiology Study (KoGES). Results ANGII-induced BP elevation was highest in the OA group, followed by the FA and OEA groups. Urine sodium tended to increase after ANGII infusion in FA and OEA but decreased in OA. Western blot results showed that cortical phosphorylated sodium chloride cotransporter (pNCC) tended to be augmented by ANGII treatment in FA and OA, but mitigated in OEA. Cortical ANGII type 2 receptor (AT2R) expression was significantly higher in estradiol-treated OVX rats than in OVX rats, irrespective of ANGII infusion. Similarly, the enhanced pNCC in ANGII-treated distal convoluted tubular cells was reversed by estradiol treatment. In KoGES data, urine sodium was significantly lower in postmenopausal women than in premenopausal women, despite the higher BP. Conclusion Estradiol reversed BP elevation and augmented the natriuretic response in ANGII-infused OVX rats and distal tubular cells via suppression of pNCC and increase of AT2R.

Impact of KvLQT1 potassium channel modulation on alveolar fluid homeostasis in an animal model of thiourea-induced lung edema.

Alveolar ion and fluid absorption is essential for lung homeostasis in healthy conditions as well as for the resorption of lung edema, a key feature of acute respiratory distress syndrome. Liquid absorption is driven by active transepithelial sodium transport, through apical ENaC Na+ channels and basolateral Na+/K+-ATPase. Our previous work unveiled that KvLQT1 K+ channels also participate in the control of Na+/liquid absorption in alveolar epithelial cells. Our aim was to further investigate the function of KvLQT1 channels and their interplay with other channels/transporters involved in ion/liquid transport in vivo using adult wild-type (WT) and KvLQT1 knock-out (KO) mice under physiological conditions and after thiourea-induced lung edema. A slight but significant increase in water lung content (WLC) was observed in naïve KvLQT1-KO mice, relative to WT littermates, whereas lung function was generally preserved and histological structure unaltered. Following thiourea-induced lung edema, KvLQT1-KO did not worsen WLC or lung function. Similarly, lung edema was not aggravated by the administration of a KvLQT1 inhibitor (chromanol). However, KvLQT1 activation (R-L3) significantly reduced WLC in thiourea-challenged WT mice. The benefits of R-L3 were prevented in KO or chromanol-treated WT mice. Furthermore, R-L3 treatment had no effect on thiourea-induced endothelial barrier alteration but restored or enhanced the levels of epithelial alveolar AQP5, Na+/K+-ATPase, and ENaC expressions. Altogether, the results indicate the benefits of KvLQT1 activation in the resolution of lung edema, probably through the observed up-regulation of epithelial alveolar channels/transporters involved in ion/water transport.

Cutaneous Microvascular Function Following Ischemia-reperfusion Injury During Passive Heat Stress

Increased kidney injury risk during heat stress is mediated, in part, by renal ischemia-reperfusion (IR) injury. However, in vivo studies are not possible in humans. Eccrine sweat glands and the cutaneous circulation have been proposed as a model of the nephron given the physiological similarities (e.g., sodium reabsorption). We previously reported impairments in sweat gland sodium reabsorption following IR injury during passive heat stress, which could be contributed to by alterations in cutaneous microvascular function (i.e., oxygen supply). PURPOSE: Test the hypothesis that forearm cutaneous microvascular function is impaired following IR injury during passive heat stress. METHODS: Fifteen healthy subjects (10 women; age: 25 ± 3 y; body mass index: 23.7 ± 3.5 kg·m2) completed a cross-body design experiment consisting of ~160 min of passive heat stress (core body temperature: +1.5 ± 0.2 °C) utilizing a suit perfused with 50 °C water. At 60 min of whole-body heating, one forearm arm (randomized) was occluded (220 mmHg) for 20 min followed by reperfusion. Local thermal hyperemia tests were performed 20 min following reperfusion using a standard local heating protocol (39 °C for 40 min + 44 °C for 20 min). Forearm skin blood flow was measured continuously at two sites (one heated, one control) on each forearm using laser-Doppler flowmetry. Cutaneous vascular conductance (CVC) was calculated as red blood cell flux/mean arterial pressure (PU/mmHg) and normalized to maximal CVC during local heating to 44 °C. Data are presented as mean ± SD. RESULTS: Maximal CVC was not different between IR and control arms (3.2 ± 0.7 vs. 3.4 ± 0.8 PU/mmHg, respectively; p = 0.48). Approximately 60 min after reperfusion, CVC at the control sites were not different between IR and control arms (78 ± 11 vs. 76 ± 11%, respectively; p = 0.65). Following 40 min of local heating (39 °C), CVC was not different between IR and control arms (80 ± 10 vs. 78 ± 10%, respectively; p = 0.59). CONCLUSION: Cutaneous microvascular function was maintained following IR during passive heat stress indicating that impaired sodium reabsorption is not mediated by reductions in oxygen supply necessary for active transport of sodium. Rather, a disruption in sodium transporter activity may mediate increased sodium excretion, but this remains unknown.

The Immune System in Hypertension: a Lost Shaker of Salt 2021 Lewis K. Dahl Memorial Lecture.

The seminal observations of Dr Lewis Dahl regarding renal mechanisms of hypertension remain highly relevant in light of more recent experiments showing that immune system dysfunction contributes to hypertension pathogenesis. Dr Dahl established that inappropriate salt retention in the kidney plays a central role via Ohm's Law in permitting blood pressure elevation. Nevertheless, inflammatory cytokines whose expression is induced in the early stages of hypertension can alter renal blood flow and sodium transporter expression and activity to foster renal sodium retention. By elaborating these cytokines and reactive oxygen species, myeloid cells and T lymphocytes can connect systemic inflammatory signals to aberrant kidney functions that allow sustained hypertension. By activating T lymphocytes, antigen-presenting cells such as dendritic cells represent an afferent sensing mechanism triggering T cell activation, cytokine generation, and renal salt and water reabsorption. Manipulating these inflammatory signals to attenuate hypertension without causing prohibitive systemic immunosuppression will pose a challenge, but disrupting actions of inflammatory mediators locally within the kidney may offer a path through which to target immune-mediated mechanisms of hypertension while capitalizing on Dr Dahl's key recognition of the kidney's importance in blood pressure regulation.

A comparative study of endothelial cell transport in pig and human cornea

Purpose. To study the basic transport characteristics of human and pig corneal endothelial cells, including osmotic water permeability, activation of sodium transport from the cell after conservation, and the ability of the cells to restore their volume when transport mechanisms of the endothelial cells are activated at 37°C.Material and methods. The experiments were held on the primary cell culture of human and pig cornea endothelia. Changes in cell volume were determined by a method based on quenching of Calcein fluorescence probe by the cytosol proteins. Changes in intracellular sodium ion concentration were studied using Sodium Green as a fluorescent probe. Restoration dynamics of cell volume and intracellular sodium concentration were studied under medium temperature changes from 20 to 37°C. Osmotic water permeability was calculated from the rate of cell volume changes under medium osmolality decreasing from 560 to 280 mOsm/kg H2 O.Results. It was established that human endothelial cells plasma membrane has a significantly higher osmotic water permeability than pig endothelial cells (Pf = 1.90E-01 ± 4.66E-02 and 1.31E-01 ± 1.16E-02 cm/s, respectively; p < 0.01, n = 17). Human cells after the temperature restored to 37°C, sodium ions removal from human cells occurs more intensely than from pig cells (-3.2E-3 ± 3.1E-4 с-1 и -6.5E-4 ± 1.2E-5 s-1, respectively; p < 0.01, n = 6). The study of cell volume drop has shown that heat activation of cellular transport restores the endothelial cell volume in humans more slowly (-1,7E-4 ± 5,5E-5 с-1, n = 9) than that of pig cells (-1.7E-3 ± 4E-4 s-1, n = 4, p < 0.05).Conclusion. When using the endothelium of pig cornea as an experimental model of human endothelium, we need to take into account the significant difference in parameters that determine cell volume regulation.

Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice.

Aberrant activation of with-no-lysine kinase (WNK)-STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) kinase signaling in the distal convoluted tubule (DCT) causes unbridled activation of the thiazide-sensitive sodium chloride cotransporter (NCC), leading to familial hyperkalemic hypertension (FHHt) in humans. Studies in FHHt mice engineered to constitutively activate SPAK specifically in the DCT (CA-SPAK mice) revealed maladaptive remodeling of the aldosterone sensitive distal nephron (ASDN), characterized by decrease in the potassium excretory channel, renal outer medullary potassium (ROMK), and epithelial sodium channel (ENaC), that contributes to the hyperkalemia. The mechanisms by which NCC activation in DCT promotes remodeling of connecting tubule (CNT) are unknown, but paracrine communication and reduced salt delivery to the ASDN have been suspected. Here, we explore the involvement of prostaglandin E2 (PGE2). We found that PGE2 and the terminal PGE2 synthase, mPGES1, are increased in kidney cortex of CA-SPAK mice, compared to control or SPAK KO mice. Hydrochlorothiazide (HCTZ) reduced PGE2 to control levels, indicating increased PGE2 synthesis is dependent on increased NCC activity. Immunolocalization studies revealed mPGES1 is selectively increased in the CNT of CA-SPAK mice, implicating low salt-delivery to ASDN as the trigger. Salt titration studies in an in vitro ASDN cell model, mouse CCD cell (mCCD-CL1), confirmed PGE2 synthesis is activated by low salt, and revealed that response is paralleled by induction of mPGES1 gene expression. Finally, inhibition of the PGE2 receptor, EP1, in CA-SPAK mice partially restored potassium homeostasis as it partially rescued ROMK protein abundance, but not ENaC. Together, these data indicate low sodium delivery to the ASDN activates PGE2 synthesis and this inhibits ROMK through autocrine activation of the EP1 receptor. These findings provide new insights into the mechanism by which activation of sodium transport in the DCT causes remodeling of the ASDN.

Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells.

Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxia-inducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. We investigated HIF's effect on ENaC expression in mpkCCD cl4 cells (a model of collecting duct principal cells) using real-time PCR and western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of β ENaC and γ ENaC, as well as of Na,K-ATPase. HIF1 α silencing increased β ENaC and γ ENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1 α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γ ENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γ ENaC abundance under high sodium intake. This study reveals that γ ENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

Time course of renal sodium transport in the pregnant rat.

Progressive sodium retention and cumulative plasma volume expansion occur to support the developing fetus during pregnancy. Sodium retention is regulated by individual tubular transporters and channels. An increase or decrease in any single transporter could cause a change in sodium balance. Understanding the time-course for changes in each sodium transporter during pregnancy will enable us to understand progressive sodium retention seen in pregnancy. Here, we examined the activity of the major apical sodium transporters found in the nephron using natriuretic response tests in virgin, early pregnant, mid-pregnant, and late pregnant rats. We also measured renal and serum aldosterone levels. We found that furosemide sensitive sodium transport (NKCC2) is only increased during late pregnancy, thiazide sensitive sodium transport (NDCBE/pendrin) is increased in all stages of pregnancy, and that benzamil sensitive sodium transport (ENaC) is increased beginning in mid-pregnancy. We also found that serum aldosterone levels progressively increased throughout gestation and kidney tissue aldosterone levels increased only during late pregnancy. Here we have shown progressive turning on of specific sodium transport mechanisms to help support progressive sodium retention through the course of gestation. These mechanisms contribute to the renal sodium retention and plasma volume expansion required for an optimal pregnancy.

Aldosterone up-regulates voltage-gated potassium currents and NKCC1 protein membrane fractions

Na+–K+–2Cl− Cotransporter (NKCC1) is a protein that aids in the active transport of sodium, potassium, and chloride ions across cell membranes. It has been shown that long-term systemic treatment with aldosterone (ALD) can enhance NKCC1 protein expression and activity in the aging cochlea resulting in improved hearing. In the present work, we used a cell line with confirmed NKCC1 expression to demonstrate that in vitro application of ALD increased outward voltage-gated potassium currents significantly, and simultaneously upregulated whole lysate and membrane portion NKCC1 protein expression. These ALD-induced changes were blocked by applying the mineralocorticoid receptor antagonist eplerenone. However, application of the NKCC1 inhibitor bumetanide or the potassium channel antagonist Tetraethyl ammonium had no effect. In addition, NKKC1 mRNA levels remained stable, indicating that ALD modulates NKCC1 protein expression via the activation of mineralocorticoid receptors and post-transcriptional modifications. Further, in vitro electrophysiology experiments, with ALD in the presence of NKCC1, K+ channel and mineralocorticoid receptor inhibitors, revealed interactions between NKCC1 and outward K+ channels, mediated by a mineralocorticoid receptor-ALD complex. These results provide evidence of the therapeutic potential of ALD for the prevention/treatment of inner ear disorders such as age-related hearing loss.

SAT-135 LOW-DOSE FUROSEMIDE IMPROVES RENAL MEDULLARY OXYGENATION IN OVINE SEPTIC ACUTE KIDNEY INJURY

Furosemide, the most commonly used diuretic, has been proposed to improve renal medullary oxygenation. However, studies in patients with acute kidney injury (AKI) demonstrated that treatment with high doses of furosemide worsens renal function. In experimental sepsis, the rapid development of renal medullary hypoxia precedes the development of AKI and may contribute to its pathogenesis. We therefore investigated whether inhibiting active sodium transport and oxygen consumption in the medullary thick ascending limb with low-dose furosemide attenuates the medullary hypoxia in experimental septic AKI.

Furosemide reverses medullary tissue hypoxia in ovine septic acute kidney injury.

In experimental sepsis, the rapid development of renal medullary hypoxia precedes the development of acute kidney injury (AKI) and may contribute to its pathogenesis. We investigated whether inhibiting active sodium transport and oxygen consumption in the medullary thick ascending limb with furosemide attenuates the medullary hypoxia in experimental septic AKI. Sheep were instrumented with flow probes on the pulmonary and renal arteries and fiber optic probes to measure renal cortical and medullary perfusion and oxygen tension (Po2). Sepsis and AKI were induced by infusion of live Escherichia coli. At 24 h of sepsis there were significant decreases in renal medullary tissue perfusion (1,332 ± 233 to 698 ± 159 blood perfusion units) and Po2 (44 ± 6 to 19 ± 6 mmHg) (both P < 0.05). By 5 min after intravenous administration of furosemide (20 mg), renal medullary Po2 increased to 43 ± 6 mmHg and remained at this normal level for 8 h. Furosemide caused transient increases in fractional excretion of sodium and creatinine clearance, but medullary perfusion, renal blood flow, and renal oxygen delivery were unchanged. Urinary F2-isoprostanes, an index of oxidative stress, were not significantly changed at 24 h of sepsis but tended to transiently decrease after furosemide treatment. In septic AKI, furosemide rapidly restored medullary Po2 to preseptic levels. This effect was not accompanied by changes in medullary perfusion or renal oxygen delivery but was accompanied by a transient increase in fractional sodium excretion, implying decreased oxygen consumption as a mechanism.

Model of fluid and solute shifts during hemodialysis with active transport of sodium and potassium.

BackgroundMathematical models are useful tools to predict fluid shifts between body compartments in patients undergoing hemodialysis (HD). The ability of a model to accurately describe the transport of water between cells and interstitium (Jv,ISIC), and the consequent changes in intracellular volume (ICV), is important for a complete assessment of fluid distribution and plasma refilling. In this study, we propose a model describing transport of fluid in the three main body compartments (intracellular, interstitial and vascular), complemented by transport mechanisms for proteins and small solutes.MethodsThe model was applied to data from 23 patients who underwent standard HD. The substances described in the baseline model were: water, proteins, Na, K, and urea. Small solutes were described with two-compartment kinetics between intracellular and extracellular compartments. Solute transport across the cell membrane took place via passive diffusion and, for Na and K, through the ATPase pump, characterized by the maximum transport rate, JpMAX. From the data we estimated JpMAX and two other parameters linked to transcapillary transport of fluid and protein: the capillary filtration coefficient Lp and its large pores fraction αLP. In an Expanded model one more generic solute was included to evaluate the impact of the number of substances appearing in the equation describing Jv,ISIC.ResultsIn the baseline model, median values (interquartile range) of estimated parameters were: Lp: 11.63 (7.9, 14.2) mL/min/mmHg, αLP: 0.056 (0.050, 0.058), and JpMAX: 5.52 (3.75, 7.54) mmol/min. These values were significantly different from those obtained by the Expanded model: Lp: 8.14 (6.29, 10.01) mL/min/mmHg, αLP: 0.046 (0.038, 0.052), and JpMAX: 16.7 (11.9, 25.2) mmol/min. The relative RMSE (root mean squared error)averaged between all simulated quantities compared to data was 3.9 (3.1, 5.6) %.ConclusionsThe model was able to accurately reproduce most of the changes observed in HD by tuning only three parameters. While the drop in ICV was overestimated by the model, the difference between simulations and data was less than the measurement error. The biggest change in the estimated parameters in the Expanded model was a marked increase of JpMAX indicating that this parameter is highly sensitive to the number of species modeled, and that the value of JpMAX should be interpreted only in relation to this factor.

Mycophenolate Mofetil Attenuates DOCA-Salt Hypertension: Effects on Vascular Tone.

Inflammation is increasingly recognized as a driver of hypertension. Both genetic and pharmacological inhibition of B and T cells attenuates most forms of experimental hypertension. Accordingly, the immunosuppressive drug mycophenolate mofetil (MMF) reduces blood pressure in the deoxycorticosterone acetate (DOCA-) salt model. However, the mechanisms by which MMF prevent hypertension in the DOCA-salt model remain unclear. Recent studies indicate that immunosuppression can inhibit sodium transporter activity in the kidney, but its effect on vascular tone is not well characterized. Therefore, the aim of the present study was to analyze the vascular and renal tubular effects of MMF in the DOCA-salt model in rats (4 weeks without uninephrectomy). Co-treatment with MMF attenuated the rise in blood pressure from day 11 onward resulting in a significantly lower telemetric mean arterial pressure after 4 weeks of treatment (108 ± 7 vs. 130 ± 9 mmHg, P < 0.001 by two-way analysis of variance). MMF significantly reduced the number of CD3+ cells in kidney cortex and inner medulla, but not in outer medulla. In addition, MMF significantly reduced urinary interferon-γ excretion. Vascular tone was studied ex vivo using wire myographs. An angiotensin II type 2 (AT2) receptor antagonist blocked the effects of angiotensin II (Ang II) only in the vehicle group. Conversely, L-NAME significantly increased the Ang II response only in the MMF group. An endothelin A receptor blocker prevented vasoconstriction by endothelin-1 in the MMF but not in the vehicle group. MMF did not reduce the abundances of the kidney sodium transporters NHE3, NKCC2, NCC, or ENaC. Together, our ex vivo results suggest that DOCA-salt induces AT2 receptor-mediated vasoconstriction. MMF prevents this response and increases nitric oxide availability. These data provide insight in the antihypertensive mechanism of MMF and the role of inflammation in dysregulating vascular tone.

Metformin increases urinary sodium excretion by reducing phosphorylation of the sodium-chloride cotransporter

ObjectiveMetformin is an antidiabetic drug that is widely used to treat patients with diabetes mellitus. Recent studies have reported that treatment with metformin not only improved blood glucose levels but also reduced blood pressure. However, it remains unclear how metformin reduces blood pressure. We hypothesized that metformin affects sodium reabsorption in the kidneys. MethodsUrinary sodium excretion and expression of renal sodium transporters were examined in 8-week-old male C57BL/6 mice with acute and chronic treatment of metformin. In addition, we examined metformin effects using ex vivo preparations of mice kidney slices. ResultsIn this study, we demonstrated that metformin increased urinary sodium excretion by reducing phosphorylation of the thiazide-sensitive Na-Cl cotransporter (NCC) in acute and chronic metformin administration. We also confirmed reduction of phosphorylated NCC in an ex vivo study. The activity of other renal sodium transporters, such as NKCC2, ENaC, and NHE3 did not show significant changes. WNK-OSR1/SPAK kinase signals were not involved in this inactivation effect of metformin on NCC. ConclusionMetformin increased urinary sodium excretion by reducing phosphorylation of NCC, suggesting its role in improving hypertension.

Epidermal epidemic: unravelling the pathogenesis of chytridiomycosis.

Chytridiomycosis, a lethal fungal skin disease of amphibians, fatally disrupts ionic and osmotic homeostasis. Infected amphibians increase their skin shedding rate (sloughing) to slow pathogen growth, but the sloughing process also increases skin permeability. Healthy amphibians increase active ion uptake during sloughing by increasing ion transporter abundance to offset the increased skin permeability. How chytridiomycosis affects the skin function during and between sloughing events remains unknown. Here, we show that non-sloughing frogs with chytridiomycosis have impaired cutaneous sodium uptake, in part because they have fewer sodium transporters in their skin. Interestingly, sloughing was associated with a transient increase in sodium transporter activity and abundance, suggesting that the newly exposed skin layer is initially fully functional until the recolonization of the skin by the fungus again impedes cutaneous function. However, the temporary restoration of skin function during sloughing does not restore ionic homeostasis, and the underlying loss of ion uptake capacity is ultimately detrimental for amphibians with chytridiomycosis.

Collecting Duct Nitric Oxide Synthase 1ß Activation Maintains Sodium Homeostasis During High Sodium Intake Through Suppression of Aldosterone and Renal Angiotensin II Pathways.

BackgroundDuring high sodium intake, the renin‐angiotensin‐aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1β is critical for fluid‐electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1β pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin‐angiotensin‐aldosterone system–epithelial sodium channel axis.Methods and ResultsMale control and collecting duct nitric oxide synthase 1β knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin‐converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin‐angiotensin‐aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls.ConclusionsThese data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1β signaling induces suppression of systemic and intrarenal renin‐angiotensin‐aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis.

Abstract P336: Excreted Living Renal Proximal Tubule Cells (RPTC) Demonstrate Increased Sodium and Bicarbonate Transport in Inverse Salt Sensitive Individuals Using a Patch Clamp Instrument

In our previous clinical studies of salt sensitivity of blood pressure, we have demonstrated that approximately 11% of study participants have a paradoxical increase in ≥7mm of Hg blood pressure on a low NaCl diet (5 meq/day) vs a high NaCl diet (300 meq/day). We define these participants as having inverse salt sensitivity (ISS) while salt resistant (SR) participants demonstrate ≤7mm Hg. RPTCs from 2 ISS and 2 salt resistance (SR) participants were patch clamped and ramp currents were recorded using a voltage ramp protocol from -100 to +45 mV within 200 ms in at least 5 experiments for each cell line. Activity of sodium transporters was probed by exchanging an 80 mM Na + with a 140 mM Na + buffer. Na + transport was significantly increased during the low salt to high salt exchange (ISS1: 80mM Na+ 90.6 ± 17.78 pA VS 140 mM Na, 192.8 ± 47.67 pA, n=9, P&lt;0.05 paired t-test; ISS2: 126.5 ± 41.14 pA VS 198.3 ± 47.45 pA, n=6 P&lt;0.05 paired t-test). The fold change in current from 80 to 140 mm Na + buffer in ISS cells was significantly increased when compared to salt resistant (SR) (normal) participants indicating ISS cells have more sodium channel activity than SR cells (ISS 2.07 ± 0.01, n=2 VS SR 1.3 ± 0.1, n=2, P&lt;0.05, t-test). Activity of bicarbonate transporters was probed by exchanging 25mM Na gluconate with 25mM NaHCO3. A similar trend was found for bicarbonate transport which showed increased current when we exchanged the Na + gluconate to NaHCO3, while SR cells didn’t show any difference when switching Na + gluconate to NaHCO3 (ISS1: Gluconate 461.5 ± 226.6 pA VS 1932 ± 658.7 pA, n=8, P&lt;0.05 paired t-test; ISS2: 284.1 ± 83.02 pA VS 1102 ± 295.4 pA, n=6, P&lt;0.05 paired t-test; SR1: 144.1 ± 20.93 pA VS 164.7 ± 27.22 pA, n=12; SR2: 256.5 ± 38.67 pA VS 277.8 ± 38.16 pA, n=5). Comparing bicarbonate / gluconate ratios, ISS cells demonstrated an increased ratio over the SR cells (ISS 8.65 ± 2.35, n=2 VS SR 1.11 ± 0.01, n=2, p=0.08) indicating a sodium bicarbonate transporter is hyperactive in ISS cells. Many transporters can be contribute to ISS, therefore we will selectively silence the sodium and bicarbonate transporters to determine which one(s) contribute to the etiology of ISS which then could be targeted for therapeutic intervention.

Genome-wide DNA methylation analysis in permanent atrial fibrillation.

Atrial fibrillation (AF) is a highly heterogeneous genetic disease; however, the pathogenesis of AF cannot be explained by genetic variants alone. DNA methylation is a heritable method of gene expression regulation, and may be a potential regulatory mechanism in AF. Therefore, in the present study, the genome‑wide DNA methylation pattern in cells derived from the left atrium of patients with permanent AF (n=7) was compared with that of healthy heart donors (n=4) with a normal sinus rhythm (SR). Enriched biological functions of the differentially methylated genes were assessed. Integrated analysis of genome‑wide methylation and mRNA expression profiles was performed, and reverse transcription quantitative‑polymerase chain reaction (RT‑qPCR) was used to determine the expression levels of four selected genes. A total of 417 differentially methylated CpG sites were identified in the fibrillating atrium (P<0.05; |β|>0.17); the majority of which were located in gene‑body and intergenic regions outside of CpG islands. Aberrantly methylated genes participated in the activation of inflammation, sodium and potassium ion transport, fibrosis and the reduction of lipid metabolism. Hypermethylation in the AF susceptible loci, paired‑like homeodomain transcription factor2 (chromosome 4q25) and coiled‑coil domain containing 141 (chromosome 2q31), aswellas hypomethylation in the calcium voltage‑gated channel subunitα1C (chromosome 12p13) locus, were identified in all patients with AF. Of the 420 upregulated and 567downregulated genes previously identified in patients with AF relative to those with normal SR (fold‑change >2.0; P≤0.05), 12 genes were hypomethylated and eight genes were hypermethylated in each group, respectively (|β|>0.2: P<0.05). RT‑qPCR analysis of four of these genes supported the modulatory effect of DNA methylation on gene expression. These results suggest that DNA methylation‑mediated regulation of gene expression may serve an important role in AF pathogenesis, and several susceptible AF CpG loci were identified which may be involved in the initiation of AF.

Living with a leaky skin: upregulation of ion transport proteins during sloughing.

Amphibian skin is a multifunctional organ providing protection from the external environment and facilitating the physiological exchange of gases, water and salts with the environment. In order to maintain these functions, the outer layer of skin is regularly replaced in a process called sloughing. During sloughing, the outermost layer of the skin is removed in its entirety, which has the potential to interfere with skin permeability and ion transport, disrupting homeostasis. In this study, we measured, in vivo, the effects of sloughing on the cutaneous efflux of ions in toads Rhinella marina kept in freshwater conditions. We also measured transepithelial potential, cutaneous resistance, active ion transport and the distribution, abundance and gene expression of the key ion transport proteins sodium-potassium ATPase (NKA) and epithelial sodium channel (ENaC) during sloughing. We hypothesised that the increase in transepithelial efflux of ions during sloughing is a consequence of increased permeability and/or a reduction in the abundance or expression of cutaneous ion transport proteins, resulting in disruption of internal ion homeostasis. There was a significant increase in sodium and chloride efflux during sloughing in R. marina However, although in vitro skin resistance decreased after sloughing, active sodium transport increased commensurate with an increase in NKA and ENaC protein abundance in the skin. These changes in skin function associated with sloughing did not affect the maintenance of internal electrolyte homeostasis. These results suggest that during sloughing, amphibians actively maintain internal homeostasis by increasing cutaneous rates of ion uptake.