Regulation Of Sodium Transport Research Articles

Regulating distal nephron functions and salt sensitivity.

This review highlights the molecular basis of salt sensitivity in hypertension, with a focus on the regulation of sodium transport in the distal nephron. Sodium reabsorption in this region is often linked to the actions of aldosterone, although in recent years numerous findings have been reported on the aldosterone-independent pathway of acquiring salt sensitivity by potassium deficiency or potassium loading. The key to this discussion is the interplay, through extracellular potassium concentration, between the first part of the tubules expressing the Na+-Cl- cotransporter (NCC) and the second part expressing the epithelial Na+ channel (ENaC). The molecular pathways such as with-no-lysine 1 (WNK)-STE20/SPS1-related proline-alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) signaling, Kelch-like family member 3 (KLHL3)-cullin 3 (CUL3) complex, protein phosphatases, and mechanistic target of rapamycin complex 2 (mTORC2)-Nedd4L pathway are described as the mechanism by which salt sensitivity on blood pressure is acquired in response to changes in physiological conditions including potassium depletion or loading. This review highlights the potential for targeting these molecular pathways to develop novel therapeutic strategies for the treatment of salt-sensitive hypertension, the mechanism of which remains to be elucidated.

Global Deletion of DPP4 Prevents the Development of Salt-sensitive Hypertension in Obese Mice via Regulation of Sodium and Water Excretion

Salt-sensitivity of hypertension (HTN) occurs in up to 50% of hypertensive patients and is more common in the older population, Blacks and the obesity/diabetes/hypertension subsets. The renin-angiotensin aldosterone system (RAAS), in particular Angiotensin II (Ang II) and aldosterone known to regulate the Na+/H+ exchanger isoform 3 (NHE3) and ENaC respectively, is thought to be activated in the obesity/diabetes/hypertension subsets and thereby plays an important role in salt-sensitive HTN. RAAS blockade is an acceptable treatment for salt-sensitive HTN along with the use of diuretics. However, not every patient can tolerate RAAS blockade. Recently, dipeptidyl peptidase 4 (DPP4) has been shown to bind to NHE3, colocalizes to the microvillar fractions with active NHE3 and DPP4 inhibition reduces sodium uptake into proximal tubule cells and isolated rat proximal tubules. In addition, DPP4 may act on more distal transporters. Furthermore, data from our lab has revealed that DPP4 knockout (DPP4KO) mice have a lower sodium balance both under conditions of high salt (1.23%) and low salt (0.02%) ingestion suggesting that DPP4 regulates sodium transport and/or balance under physiological conditions. However, whether DPP4 has any effects on salt-sensitive HTN and the underlying mechanisms remain unknown. Therefore, we hypothesized that the absence of DPP4 could mitigate the development of salt-sensitive HTN in an obesity model, by improving the natriuretic and diuretic response mediated by NHE3 and/or distal mechanisms. To this end, 6-8wk old DPP4KO and C57Bl/6 littermate wild-type (WT) mice were fed a control (CD) or Western diet (WD, high in refined fats and sugars) to induce obesity. Circulating (175%) and kidney (125%) DPP4 activity were both elevated in the obese WT mice compared to the CD-fed WT mice (100%) and DPP4KO mice had minimal DPP4 activity as expected. The lack of DPP4 prevented the increase in systolic blood pressure (~15 mmHg) in the obese animals, and the absence of the development of cardiac hypertrophy in this group corroborated the pressure effect. The development of salt-sensitive HTN in the obese WT mice was associated with a lower natriuretic and diuretic response assessed by the salt overload experiment, suggesting higher tubular reabsorption of sodium and water by the nephrons. In turn, the lack of DPP4 prevented the lowering of natriuretic and diuretic response in obese mice, indicating that the beneficial effect of the absence of DPP4 on the development of salt-sensitive HTN is closely related to effects on renal tubule transport. Importantly, a high salt diet increased sodium balance and blood pressure in the obese WT mice and DPP4 deletion mitigated this increase. Furthermore, on low salt diet, the absence of DPP4 led to “salt wasting” in the obese mice similar to the CD-fed mice. Surprisingly, gene and protein expression measurements showed involvement of distal tubule sodium transporters and aquaporins and not NHE3. In contrast, assessment of the DPP4 interactome in obese mice showed binding to Myosin 6 and APN, which may regulate proximal tubule transporters and hypertension. Transdermal measurements showed no changes in the glomerular filtration rate. In conclusion, deletion of DPP4 protects against the development of salt-sensitive hypertension in obese mice by improving the natriuretic and diuretic response likely via regulation of sodium and water transporters. FAPESP, Brazil NIH K08DK115886, USA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC β- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Anti-Inflammatory Effects of Peripheral Dopamine.

Dopamine is synthesized in the nervous system where it acts as a neurotransmitter. Dopamine is also synthesized in a number of peripheral organs as well as in several types of cells and has organ-specific functions and, as demonstrated more recently, is involved in the regulation of the immune response and inflammatory reaction. In particular, the renal dopaminergic system is very important in the regulation of sodium transport and blood pressure and is particularly sensitive to stimuli that cause oxidative stress and inflammation. This review is focused on how dopamine is synthesized in organs and tissues and the mechanisms by which dopamine and its receptors exert their effects on the inflammatory response.

Regulation of Epithelial Sodium Transport by SARS-CoV-2 Is Closely Related with Fibrinolytic System-Associated Proteins.

Dyspnea and progressive hypoxemia are the main clinical features of patients with coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary pathology shows diffuse alveolar damage with edema, hemorrhage, and the deposition of fibrinogens in the alveolar space, which are consistent with the Berlin Acute Respiratory Distress Syndrome Criteria. The epithelial sodium channel (ENaC) is a key channel protein in alveolar ion transport and the rate-limiting step for pulmonary edema fluid clearance, the dysregulation of which is associated with acute lung injury/acute respiratory distress syndrome. The main protein of the fibrinolysis system, plasmin, can bind to the furin site of γ-ENaC and induce it to an activation state, facilitating pulmonary fluid reabsorption. Intriguingly, the unique feature of SARS-CoV-2 from other β-coronaviruses is that the spike protein of the former has the same furin site (RRAR) with ENaC, suggesting that a potential competition exists between SARS-CoV-2 and ENaC for the cleavage by plasmin. Extensive pulmonary microthrombosis caused by disorders of the coagulation and fibrinolysis system has also been seen in COVID-19 patients. To some extent, high plasmin (ogen) is a common risk factor for SARS-CoV-2 infection since an increased cleavage by plasmin accelerates virus invasion. This review elaborates on the closely related relationship between SARS-CoV-2 and ENaC for fibrinolysis system-related proteins, aiming to clarify the regulation of ENaC under SARS-CoV-2 infection and provide a novel reference for the treatment of COVID-19 from the view of sodium transport regulation in the lung epithelium.

Thick ascending limb specific inactivation of Myh9and Myh10 results in progressive kidney disease with sexually dimorphic sodium transport regulation

Mutations in MYH9 gene that encodes for actin associated molecular motor nonmuscle myosin II (NM2) result in kidney disease in some patients. Based on our previous work that demonstrated a novel role for NM2 motors, we hypothesized that NM2 genes might play a critical role in renal epithelial transport. To test our hypothesis, we generated pan renal tubular conditional knockout (cKO) of Myh9&10 in adult mice that resulted in progressive kidney disease of TAL origin with aberrant transport of TAL cargoes UMOD and NKCC2 (Otterpohl et al; 2020). To confirm the cell‐autonomous role for NM2 proteins in adult TAL epithelium, we generated a TAL‐specific cKO mouse model of Myh9&10using a TAL specific cre transgenic mice, UMOD‐>CreERT2. Cre transgene was induced at 5 weeks of age with tamoxifen injections, age matched control and cKO littermates were part of every cohort. The mice underwent metabolic, histologic, immunohistochemical, and biochemical analysis at varying time points until moribund. The TAL‐specific Myh9&10 cKO mice develop progressive kidney disease indicated by increase in BUN and serum creatinine as well as increasing levels of the tubular injury marker, NGAL. Our results show that Myh9&10 TAL‐ cKO females had significant loss of weight at 12 weeks, the female cKO animals became moribund between 13 – 14 weeks, while the male cohorts survive up to 25 weeks. Histology confirmed the tubulointerstitial disease in Myh9&10 TAL‐cKO with dilated tubules and cellular infiltration. Urine pH declined starting at 9‐weeks of age in females and 12‐week in males. Interestingly, female cKO mice have hypernatremia at 12‐weeks of age compared to male mice which maintain normal sodium levels. Since the pan renal tubular Myh9&10 cKO exhibited loss of NKCC2 leading to salt wasting, this result was unexpected. We analyzed NKCC2 protein levels in kidney lysates, loss of NKCC2 was evident in both female and male Myh9&10 TAL‐cKO. We also observe significant decline in serum potassium levels in 7.5‐week females. We speculated that the hypernatremia in TAL‐Myh9&10 cKO females is due to tubular adaptation in the distal nephron and the collecting duct. Characterization of sodium cotransporters and channels in distal nephron and collecting duct segment revealed an increase in NCC/pNCC levels in males and a decrease in females. Immunostaining experiments showed increase in ENaC gamma expression in the cortical collecting ducts of the females. This could potentially explain the hypernatremia and early morbidity observed in Myh9&10 TAL‐cKO females. We speculate that females upregulate ENaC gamma to compensate for loss of NKCC2, instead males upregulate NCC/pNCC that prevents their early lethality. These experiments confirm the critical role for Myh9&10 proteins in TAL tubule and identify a novel, sex specific tubular adaptation mechanisms. NM2 motor regulating the unique, TAL‐specific sodium cotransporter is intriguing and warrants further analysis. Our model also provides a good resource for understanding the sex‐specific differences of sodium handling and potentially clarify mechanisms underlying Type I Bartter Syndrome.

Visualization of Ectopic Serine Protease Activity by Förster Resonance Energy Transfer-Based Reporters.

Channel-activating proteases (CAPs) play a fundamental role in the regulation of sodium transport across epithelial tissues mainly via cleavage-mediated fine-tuning of the activity of the epithelial sodium channel (ENaC). Hyperactivity of CAPs and subsequently increased ENaC activity have been associated with various diseases, including cystic fibrosis (CF). To date, there is only a limited number of tools available to investigate CAP activity. Here, we developed ratiometric, peptide-based Förster resonance energy transfer (FRET) reporters useful to visualize and quantify the activity of ectopic serine proteases including the CAPs prostasin and matriptase in human and murine samples in a temporally and spatially resolved manner. Lipidated varieties were inserted into the outer leaflet of the plasma membrane to detect enzyme activity on the surface of individual cells, that is, close to the protease substrates. The FRET reporters (termed CAPRee) selectively detected the activity of ectopic serine proteases such as CAPs in solution and on the surface of human and murine cells. We found increased CAP activity on the surface of cells with a genetic background of CF. The new reporters will contribute to a better understanding of ectopic serine protease activity and their regulation under physiological and pathophysiological conditions.

Angiotensin II Elicits Intracellular Ca 2+ Responses in Rat Cortical Collecting Ducts

Regulation of sodium transport in the cortical collecting duct (CCD) is pivotal for the long-term blood pressure control and excessive Na+ retention in this nephron segment is a known pathogenic determinant of hypertension. Sodium reabsorption in the CCD cells can be stimulated by volume-regulatory hormones, such as angiotensin II (AngII). AngII reportedly binds to AT1 receptors and increases the activity of the epithelial sodium channel (ENaC) via generation of reactive oxygen species (ROS). Yet, our understanding of the exact molecular determinants mediating AngII-induced intracellular signaling in the CCD cells remains limited. Numerous studies show that AngII induces intracellular Ca2+ mobilization in the vasculature, cardiomyocytes and glomerular podocytes. Here, we used ratiometric Fura-2 Ca2+-imaging to test if AngII employs Ca2+ as a second messenger in the CCDs freshly isolated from male and female Sprague-Dawley (SD) rats. We found that application of 500 nM of AngII to a split-opened CCD results in a transient elevation of [Ca2+]i in the CCD cells. The amplitude of the observed [Ca2+]i increase is about 150 nM and is not significantly different in the CCDs isolated from male and female rats. AngII-dependent [Ca2+]i signal is not affected by removal of Ca2+ from the extracellular bathing solution, but is practically abolished, when the tubules are pre-incubated in 3 μM thapsigargin, indicative of Ca2+ release from the intracellular stores. Inhibition of PLC with 10 μM U73122 markedly diminishes AngII-induced [Ca2+]i transient. Chronic AngII infusion moderately impairs [Ca2+]i responses to AngII in the CCDs isolated from male rats by approximately 30%. In contrast, AngII-induced [Ca2+]i elevation is attenuated by 70% in the CCDs from AngII-infused females. We have previously reported a stronger antihypertensive effect of aldosterone antagonism in AngII-infused females, when compared to males. Here, we found that systemic administration of eplerenone prevents the decline of AngII-stimulated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension, but not in males. Overall, our data demonstrate that AngII triggers Ca2+ release from the intracellular stores of the CCD cells in a PLC-dependent manner. This AngII-activated [Ca2+]i mobilization is impaired in AngII-infused SD rats of both sexes, but the attenuation is remarkably more pronounced in females. Systemic aldosterone antagonism with eplerenone restores the AngII-mediated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension. Future studies are granted to investigate the interplay between AngII-dependent [Ca2+]i signal and ROS generation in the CCD cells.

Sodium transport regulation in the kidney collecting duct cells in mice with the Agouti yellow (Ay) mutation

Sodium transport regulation in the kidney collecting duct cells in mice with the Agouti yellow (Ay) mutation

Impaired Intestinal Sodium Transport in Inflammatory Bowel Disease: From the Passenger to the Driver's Seat.

Although impaired intestinal sodium transport has been described for decades as a ubiquitous feature of inflammatory bowel disease (IBD), whether and how it plays a pivotal role in the ailment has remained uncertain. Our identification of dominant mutations in receptor guanylyl cyclase 2C as a cause of IBD-associated familial diarrhea syndrome brought a shift in the way we envision impaired sodium transport. Is this just a passive collateral effect resulting from intestinal inflammation, or is it a crucial regulator of IBD pathogenesis? This review summarizes the mutational spectrum and underlying mechanisms of monogenic IBD associated with congenital sodium diarrhea. We constructed a model proposing that impaired sodium transport is an upstream pathogenic factor in IBD. The review also synthesized emerging insights from microbiome and animal studies to suggest how sodium malabsorption can serve as a unifying mediator of downstream pathophysiology. Further investigations into the mechanisms underlying salt and water transport in the intestine will provide newer approaches for understanding the ion–microbiome–immune cross-talk that serves as a driver of IBD. Model systems, such as patient-derived enteroids or induced pluripotent stem cell models, are warranted to unravel the role of individual genes regulating sodium transport and to develop more effective epithelial rescue and repair therapies.

Empagliflozin Contributes to Polyuria via Regulation of Sodium Transporters and Water Channels in Diabetic Rat Kidneys.

Besides lowering glucose, empagliflozin, a selective sodium-glucose cotransporter-2 (SGLT2) inhibitor, have been known to provide cardiovascular and renal protection due to effects on diuresis and natriuresis. However, the natriuretic effect of SGLT2 inhibitors has been reported to be transient, and long-term data related to diuretic change are sparse. This study was performed to assess the renal effects of a 12-week treatment with empagliflozin (3 mg/kg) in diabetic OLETF rats by comparing it with other antihyperglycemic agents including lixisenatide (10 μg/kg), a glucagon-like peptide receptor-1 agonist, and voglibose (0.6 mg/kg), an α-glucosidase inhibitor. At 12 weeks of treatment, empagliflozin-treated diabetic rats produced still high urine volume and glycosuria, and showed significantly higher electrolyte-free water clearance than lixisenatide or voglibose-treated diabetic rats without significant change of serum sodium level and fractional excretion of sodium. In empagliflozin-treated rats, renal expression of Na+-Cl- cotransporter was unaltered, and expressions of Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter, and epithelial Na+ channel were decreased compared with control diabetic rats. Empagliflozin increased an expression of aquaporin (AQP)7 but did not affect AQP3 and AQP1 protein expressions in diabetic kidneys. Despite the increased expression in vasopressin V2 receptor, protein and mRNA levels of AQP2 in empagliflozin-treated diabetic kidneys were significantly decreased compared to control diabetic kidneys. In addition, empagliflozin resulted in the increased phosphorylation of AQP2 at S261 through the increased cyclin-dependent kinases 1 and 5 and protein phosphatase 2B. These results suggest that empagliflozin may contribute in part to polyuria via its regulation of sodium channels and AQP2 in diabetic kidneys.

Antiviral activity of the mineralocorticoid receptor NR3C2 against Herpes simplex virus Type 1 (HSV-1) infection

Analysis of a genome-scale RNA interference screen of host factors affecting herpes simplex virus type 1 (HSV-1) revealed that the mineralocorticoid receptor (MR) inhibits HSV-1 replication. As a ligand-activated transcription factor the MR regulates sodium transport and blood pressure in the kidney in response to aldosterone, but roles have recently been elucidated for the MR in other cellular processes. Here, we show that the MR and other members of the mineralocorticoid signalling pathway including HSP90 and FKBP4, possess anti-viral activity against HSV-1 independent of their effect on sodium transport, as shown by sodium channel inhibitors. Expression of the MR is upregulated upon infection in an interferon (IFN) and viral transcriptional activator VP16-dependent fashion. Furthermore, the MR and VP16, together with the cellular co-activator Oct-1, transactivate the hormone response element (HRE) present in the MR promoter and those of its transcriptional targets. As the MR induces IFN expression, our data suggests the MR is involved in a positive feedback loop that controls HSV-1 infection.

Rapid therapeutic advances in CFTR modulator science.

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by variants in the gene encoding the cystic fibrosis transmembrane conduction regulator (CFTR) protein. Loss of CFTR function disrupts chloride, bicarbonate and regulation of sodium transport, producing a cascade of mucus obstruction, inflammation, pulmonary infection, and ultimately damage in numerous organs. Established CF therapies treat the downstream consequences of CFTR dysfunction and have led to steady improvements in patient survival. A class of drugs termed CFTR modulators has recently entered the CF therapeutic landscape. These drugs differ fundamentally from prior therapies in that they aim to improve the function of disease‐causing CFTR variants. This review summarizes the science behind CFTR modulators, including their targets, mechanism of action, clinical benefit, and future directions in the field. CFTR modulators have dramatically changed how CF is treated, validated CFTR as a therapeutic target, and opened the door to truly personalized therapies and treatment regimens.

DUOX1-mediated hydrogen peroxide release regulates sodium transport in H441 bronchiolar epithelial cells.

Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase-1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H2 O2 is instrumental for the sodium channel upregulation observed in H441 cells. In vitro cell culture, nystatin-perforated whole-cell patch-clamp technique, immunocytochemistry and RT-PCR methods have been used. Cells forming epithelial domes induced by dexamethasone (0.1μmolL-1 , 24hours) and by 5-aza-2'-deoxytidine (1μmolL-1 , 48hours) expressed more DUOX1 protein compared with other cells in the monolayer. Dome formation could be inhibited by exogenous catalase in a concentration-dependent manner and by the NADPH oxidase inhibitor diphenyliodonium, which suggested the involvement of H2 O2 . While single application of 0.2mmolL-1 H2 O2 induced transient dome formation, lower doses were ineffective and higher doses disrupted the cell monolayer. Hydrogen peroxide (0.1mmolL-1 ) activated acutely amiloride-sensitive whole-cell currents from 3.91±0.79pApF-1 to 4.76±0.98pApF-1 in dome-forming cells and had no effect in cells outside of domes. ENaC but not DUOX1 transcription was potentiated by catalase in the presence of dexamethasone, which suggested negative feedback of H2 O2 on ENaC gene expression. Our observations suggest that tonic production of H2 O2 by DUOX1 participates in maintaining the level of vectorial sodium transport by lung epithelial cells. Moreover, the system appears to be well tuned as it would allow H2 O2 -dependent innate immunity without inducing airway/alveolar sodium and fluid hyperabsorption.

Molecular Mechanisms of Sodium-Sensitive Hypertension in the Metabolic Syndrome.

We review the known mechanisms of sodium-sensitive hypertension in the metabolic syndrome with a focus on preclinical models, differences between these models, and methodological limitations. We also identify future directions for a better understanding and treatment of this common condition. Rigorous methodologies to measure blood pressure in preclinical models may clarify some of the inconsistencies in the literature. Renal, neural, hormonal, and cardiovascular systems are dysregulated and contribute to elevated blood pressure. Local renin-angiotensin systems enhance systemic hormone signaling to increase blood pressure. Since the original description of metabolic syndrome, investigators from many fields have contributed to an increasingly complex and mechanistic understanding of this common condition. These systems integrate to regulate sodium transport in the kidney leading to hypertension and enhanced sodium sensitivity. An array of non-uniform preclinical models are used and support clinical studies to inform which models are pathophysiologically relevant for further mechanistic studies to guide targeted therapy.

GPR37L1 Expressed in Renal Proximal Tubule Cells Regulates Sodium Transport

GPR37L1 is a G protein‐coupled receptor that is expressed mainly in brain glial cells and muscle‐myenteric nerve layers in the GI tract. GPR37L1 knockout (KO) mice have increased systolic blood pressure. Prosaposin (PSAP) is a precursor for saposins A, B, C, and D. PSAP was identified as a ligand for GPR37L1 although recent reports showed conflicting results on the role of PSAP as ligand for GPR37L1. There is no report on the expression or function of GPR37L1 in the kidney. Our studies using immunostaining and immunoblotting showed that GPR37L1 is expressed in the apical membrane of mouse renal proximal tubules but not collecting duct cells; RT‐PCR on proximal tubule and collecting duct cells obtained by laser‐capture microdissection of mouse kidney sections, confirmed these findings. Live‐cell imaging of human renal proximal tubule cells (hRPTC) expressing GPR37L1‐GFP protein showed the internalization of the membrane GPR37L1‐GFP following exposure to prosaptide, a 14 amino acid peptide derived from the neurotropic region in the N‐terminal portion of saposin C. cAMP production was increased in hRPTC expressing GPR37L1 treated with prosaptide (4.2 ± 1.4‐fold, P<0.05). In addition, chronic high salt intake increased the renal expression of PSAP suggesting that PSAP is a ligand for GPR37L1 and has a role in renal function. To determine the role of GPR37L1 in renal Na+ transport, we over‐expressed or silenced the expression of GPR37L1 in hRPTC. Over‐ expression of GPR37L1 in hRPTC increased intracellular Na+ (3.2 ± 0.6‐fold, P<0.001) compared with mock‐transfected cells. Treatment with prosaptide blunted the GPR37L1‐ enhanced increase in intracellular Na+, suggesting that prosaptide binding to GPR37L1 may inhibit Na+ entry into the cell. Treatment with a selective NHE3 inhibitor (S3226) decreased intracellular Na+ in GPR37L1‐over expressing, as well as mock‐transfected hRPTC. Treatment with Na+, K+‐ATPase inhibitor (ouabain) showed no effect on the GPR37L1 enhancement of intracellular Na+ in GPR37L1 over‐expressing hRPTC. Silencing of GPR37L1 expression decreased the basal level of intracellular Na+ in the hRPTC. Immunoblot analyses showed increased phosphorylation of Erk1/2 (1.73 ± 0.11‐fold, P<0.05) and ribosomal S6 protein (1.49 ± 0.08‐fold, P<0.01) in hRPTC over‐expressing GPR37L1, compared with mock‐transfected hRPTC. Taken together, these results show that GPR37L1 is expressed in the renal proximal tubule cells, signals via the Erk1/2 pathway, and plays a role in renal Na+ transport and may be a novel target to design drugs to treat patients with hypertension.

Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis.

The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure.

030 - Potential Role for Plasmin Mediated Proteolytic Activation of ENaC in Diuretic Resistance in Heart Failure

Background: The amiloride sensitive Epithelial Na + channel (ENaC) plays a major role in regulation of sodium transport in the collecting duct of the kidney. Recent studies have shown that proteolytic cleavage of α and γ subunits by urinary proteases like plasmin play a major role in ENaC activation. Activation of ENaC in collecting tubules is thought to be an important mechanism driving renal Na + retention in several edematous disease states. Our aim was to probe if plasmin mediated ENaC activation is a potential mechanism of diuretic resistance in heart failure. Hypothesis: Increased urinary levels of plasminogen/plasmin will predict poor diuretic response in HF patients receiving loop diuretics. Methods: Heart failure outpatients in the Yale Transition Care Center treated with loop diuretics were prospectively studied (N = 131). Blood and urine chemistries were collected at baseline and at 1 to 1.5 hours post diuretic administration. Diuretic efficiency (DE) was defined as total sodium excreted per doubling of the diuretic dose. Total plasminogen was measured using the ELISA kit from Molecular Innovations (Novi, MI, USA). This assay kit measures both plasminogen and plasmin in biological fluids. Plasmin/plasminogen was indexed to urinary creatinine to correct for changes in urinary dilution. Results: The median dose of diuretic administered was 160 mg (40–280 mg) of furosemide equivalents, which resulted in an average of 987 ± 575 ml of urine and 97.4 ± 71.4 mmol of sodium output. There was good correlation between urinary plasminogen levels prior to and after administration of the loop diuretic (r = 0.74, P < .001), but interestingly plasmin levels increased by 190% ± 307% (P < .0001) with diuretic administration. Higher urinary plasminogen (indexed to urinary creatinine) levels at baseline were correlated with lower DE (r = −0.32, P = .001), as was the increase following administration of diuretics (r = −0.30, P = .004). In line with activation of ENaC, the fractional excretion of potassium at baseline (r = 0.26, P = .014) and post diuretic (r = 0.24, P = .012) was correlated with the corresponding urinary plasminogen concentration. Furthermore, the change in plasminogen was correlated with the post diuretic potassium to sodium ratio (a marker of distal tubular sodium/potassium exchange activity, r = 0.31, P = .002). Conclusion: In patients with heart failure receiving loop diuretics, urinary levels of plasmin/plasminogen were correlated with the degree of diuretic responsiveness and metrics of distal tubular potassium wasting. These hypothesis generating results suggest that ENaC activation via luminal plasmin may be a relevant and therapeutically modifiable target in HF. Additional research is warranted.

Loss of Protein Kinase C Alpha Disrupts Mean Arterial Pressure and Diurnal Rhythm

The sodium‐chloride cotransporter (NCC) is an essential regulator of sodium transport in the distal convoluted tubule (DCT). As such, functional aberrations will lead to dysregulation of sodium homeostasis and blood pressure. Although this role is well accepted, the mechanisms accounting for increased distal nephron sodium reabsorption is not completely understood. Previously published data from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study revealed that a single nucleotide polymorphism (SNP) in PKC‐α increases sensitivity to thiazide diuretics. To date, it is unknown whether this SNP is a gain or loss of function of PKC‐α. To investigate whether PKC‐α may be altering blood pressure via NCC, PKC‐α KO (PKC KO) and PKC Control (CTL) mice were used to measure blood pressure via radiotelemetry after normal and high salt (HS, 4%) dietary manipulations and Western blot analysis performed using kidney lysates. Using radiotelemetry (DSI) measurements obtained over an active and sleeping time periods, we report a significant decrease in mean arterial pressure (MAP) in the PKC KO mice as compared to control at baseline (111±1.3mmHg vs 120±2.4mmHg, n=12–16, p&lt;0.001). When comparing active vs. sleeping MAP, PKC KO mice had depressed MAP during both time periods, yet blood pressure differences (day vs. night) double during active periods (5.4±1.3mmHg PKC KO vs. 10.9±3.9mmHg PKC CTL, n=6–7). These differences were abolished with HS chow feeding (17.3mmHg PKC KO vs. 16.1mmHg PKC CTRL, n=4). To determine a possible mechanism for this phenomenon, whole kidney lysate was used to assay for total NCC protein. Total NCC protein was significantly increased in PKC KO mice at baseline, and continued during HS chow feeding, as compared to PKC CTL. In addition, PKC KO mice exhibited a diuresis with both normal and HS feeding. Our data suggest that lack of PKC‐α leads to decreased MAPs and diuresis, which may lead to increased total NCC expression to compensate. In addition, PKC KO mice exhibited an aberrant diurnal rhythm, which was recovered with HS diet suggesting that PKC‐α may be necessary to maintain sodium homeostasis.Support or Funding InformationNIDDK085097‐ RSH 2T32DK7656‐21 Emory Renal Division‐BMW