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High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na+ Channel (ENaC) is the final arbiter of Na+ excretion in the kidneys. Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure. We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII-dependent ENaC-Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split-open collecting ducts from principal cell-specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank-3 binding to β-ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation. This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816. This is the full abstract presented at the American Physiology Summit 2023 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.

Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.

The article is devoted to the formation of the Zemstvo’s pedagogical intelligentsia in Laishevsky district of Kazan province. Based on sources of personal origin and records of County self-government bodies, a comprehensive analysis of the personnel composition of Zemstvo’s primary schools was conducted. The author established the number of teachers, sources of their replenishment, the level of education, and also considered the socioeconomic situation of teachers. The teaching staff included law teachers, secular teachers, and assistants. Some former priests-teachers of Church schools also joined the work of Zemstvo’s educational institutions as experienced teachers. The available materials clearly show the evolution of the educational level of teachers in favor of specialists with secondary education. As a result, thanks to the Zemstvo’s school, a new social stratum of rural intelligentsia formed in the district, including people with secondary education. Measures taken by the Zemstvo’s to improve the working and living conditions of teachers are also shown. The principle of determining the amount of salary based on the number of students contradicted the principle of assigning salaries depending on the professional training and education of the teacher. It is concluded that despite the support of experienced and responsible teachers, the profession of a Zemstvo’s teacher remained unattractive, but at the same time women became teachers, which corresponded to the emancipation movement that was gaining momentum at that time.

The article describes the diplomatic activity of Mikhail Nikolayevich von Giers. He made a brilliant career in the Russian Empire; during the October Revolution he was the Russian ambassador to Italy and the doyen of the Russian foreign diplomatic corps. Throughout the pre-revolutionary period Mikhail Nikolayevich did a lot to improve the activity of the diplomatic corps. He became one of the founders of the unique “state outside borders” — the Russia Аbroad, and took an active part in working out the new statute of a stateless person and the so-called “Nansen certificate”. On a parallel with that, he participated in solving all issues of importance to the refugee community. He did all in his power to help his compatriots so that they could eventually return to their homeland and contribute to the rebirth of Russia.

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.

Stimulation of metabotropic Gq-coupled purinergic P2Y2receptors decreases activity of the epithelial Na+channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2receptor signaling disruption in the P2Y2receptor knockout mouse are decreased Na+excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2receptor, we test the sufficiency of targeted stimulation of Gqsignaling in principal cells of the distal nephron and P2Y2receptors to increase UNaV. Selective stimulation of the P2Y2receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2receptor and Gqsignaling in principal cells is sufficient to promote renal salt excretion.

Studies on the influence of biologically active substances with immunomodulatory properties on the productivity of greenhouse cucumber were conducted in the winter-spring turnover of the sixth light zone in 2019-2020. The paper analyzes data on the prevalence of major diseases in cucumber plantings, the intensity of respiration and photosynthesis in plants, the level of yield and yielding of standard crop products, depending on the use of immunomodulating substances against the background of the main nutrition scheme. As a result of scientific research, it was found that the use of immunomodulators (arachidonic acid, hydroxycinnamic acids, triterpenic acids, salicylic acid) in the technology of cucumber cultivation activated the protective properties of plants against pathogens, contributed to the activation of the intensity of respiration, photosynthesis, obtaining an increased yields and the output of standard products.

Vegetable raw materials serve as a source of a large number of organic compounds of various structures with a variety of preventive and curative nutritional properties. The article presents the results of a study on the establishment of parameters and modes of extraction of biologically active substances from fruit and berry raw materials in order to obtain extracts for the preparation of functional beverages. The work was carried out on the basis of the educational and scientific laboratory of technology of winemaking and food from vegetable raw materials of the Stavropol State Agrarian University according to generally accepted methods in accordance with GOST (State standards). When preparing the extracts, water treatment of the crushed mass of feijoa and blackberries was used. The extraction process was controlled by changing the mass fraction of dry substances. As a result of the research, the optimal conditions for the process of extracting dry substances from feijoa and blackberries were established: hydromodule – 1:1, the duration of the process – 24 hours; the extraction temperature is 25 degrees C. Such conditions of the process provided the greatest extraction of dry substances without destroying the functional ingredients, allowed the extraction to be carried out within a short period of time and reduced the cost of temperature treatment of the medium. The paper presents the results of tasting evaluation of the obtained extracts.

The collecting duct is the predominant nephron site of prorenin and prorenin receptor (PRR) expression. We previously demonstrated that the collecting duct PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which cell type is involved remains unclear. Herein, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR knockout (KO) mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B1 subunit of the H+ ATPase promoters, respectively. PC KO mice had reduced renal medullary ENaC-α abundance and increased urinary Na+ losses on a low-Na+ diet compared with controls. Conversely, IC KO mice had no apparent differences in Na+ balance or ENaC abundance compared with controls. Acute treatment with prorenin increased ENaC channel number and open probability in acutely isolated cortical collecting ducts from control and IC PRR KO, but not PC PRR KO, mice. Furthermore, compared with controls, PC KO, but not IC KO mice, had increased urine volume, reduced urine osmolality, and reduced abundance of renal medullary AQP2. Taken together, these findings indicate that PC, but not IC, PRR modulates ENaC activity, urinary Na+ excretion, and water transport.

Fluid flow along the renal nephron, particularly within the inner-medullary collecting duct (IMCD), increases in response to high salt intake. We previously showed in a series of reports that high salt intake increases nitric oxide synthase 1 (NOS1) activity in the CD mediating inhibition of the epithelial sodium channel (ENaC) to promote natriuresis. However, the mechanism of how high salt increases NOS1 activity in the CD is not known. Histone deacetylase 1 (HDAC1) is known to mediate post-translational deacetylation in response to flow in other cell types. Hence, we hypothesized that HDAC1 activates renal medullary NOS1-dependent NO production in response to high salt intake. Renal medullas were isolated from C57bl/6J mice after one week of normal (NS, 0.4% NaCl) or high salt (HS, 4% NaCl) diet. Inner-medullary HDAC1 expression (NS: 1.00±0.1, HS: 1.61±0.1 RDU; n=5/group, p=0.001), but not activity (NS: 0.07±0.02, HS: 0.05±0.01μM/μg, n=5/group, p>0.05), was significantly elevated in mice on HS compared to NS. HDAC1-selective inhibitor, MS-275 (300 nM), was utilized in in vitro and ex vivo experiments to determine the contribution of HDAC1 to NOS activity. Nitrite production, analyzed by HPLC, increased >5-fold (static: 231±31.1, flow: 1291±183 pmol/mg pr/hr; n=7/group, p2 fluid-flow. MS-275 significantly blunted flow-responsive NO production (MS-275: 342±44.0 pmol/mg pr/hr; n=9/group, pex vivo MS-275 treatment from mice on HS (HS: 0.34±0.11 NP o , HS+MS-275: 0.97±0.16 NP o ; n=22/group, po , HS+MS-275+DETA: 0.46±0.14 NP o ; n=10/group, pin vitro did not increase NOS1 acetylation status as determined by co-immunoprecipitation, suggesting that HDAC1 may not directly deacetylate NOS1. Taken together, these data indicate that HS intake increases renal medullary HDAC1 to positively regulate NOS1-derived NO production.

The modern development of the food industry is accompanied by an increasing use of automated process control systems. The widespread use of automated control systems is caused by a significant economic effect, which is achieved by ensuring the specified qualities of the products produced regardless of subjective factors, reducing the loss of valuable products, reducing the labor intensity of production processes, and increasing the culture of production. The article presents a scheme of automation and energy saving of the technological process of production of functional beverages based on direct-pressed grape juice with increased nutritional and biological value due to the inclusion of feijoa and blackberry extracts in their composition. Monitoring of the technological process was carried out using a multi-channel stand. The main parameters of monitoring were the consumption of basic and auxiliary products; the temperature of pasteurization and cooling of direct-pressed juice and extracts from fruit and berry raw materials; the level of pulp and product in the tanks. The obtained experimental data and developed technological methods allowed us to offer an effective energy-saving technology for the production of high-quality functional beverages in the conditions of energy-saving management of the production process. To implement the proposed technology, a hardware and technological scheme for the production of functional beverages has been developed. Process monitoring allows you to reduce energy consumption by up to 30% by using modern automation and control tools.

The purpose of the research is to study the effect of biologically active substances in organic fertilizers on the productivity of tomato Sherami F1 in a winter greenhouse. The production experience was laid in a winter glazed greenhouse, which is located in the sixth light zone.The objects of research were cherry tomato plants Sherami F1, organic fertilizers 5AP, Maxifol Rutfarm, Nutrimir 4-4-10, Quick-Link. In the experiment, the average plant growth per week, the height of the plants, the number of leaves, the number of brushes collected and the average weight of the fetus, the chemical composition of the plants and yield were determined. The largest growth in a week and the tallest plants were obtained with Quick-Link; the most brushes collected and the largest fruits - when applying fertilizer 5AP. The use of organic fertilizers contributed to the better absorption of macronutrients by tomato plants from a nutrient solution: the content of nitrogen, phosphorus and potassium in the leaves and fruits increased relative to the control. When using biologically active substances in organomineral fertilizers, tomato productivity increased by 1.1-2.7 kg/m2 compared to the control, and the biochemical composition of the fruits improved.

With an increase in urine flow there is a significant increase in shear stress against the renal epithelium including the inner medullary collecting duct, resulting in an increase in nitric oxide (NO) production. The mechanisms of the shear stress-mediated increases in NO are undetermined. Previous studies found that shear stress increases epithelial sodium channel (ENaC) open probability and endothelin (ET)-1 production in an ENaC-dependent mechanism in the collecting duct (CD). Given that ET-1 stimulates NO production in the CD, we hypothesized that shear stress-induced NO production is downstream of shear stress-induced ENaC activation and ET-1 production in a negative feedback loop. We determined that nitric oxide synthase 1 (NOS1) and NOS3 contribute to shear stress-mediated NO production in the CD, that is attenuated by low doses of the ENaC inhibitors amiloride and benzamil. Moreover, ETBreceptor blockade significantly blunted the shear stress-mediated NO production. We further elucidated whether mice lacking NOS1 in the collecting duct (CDNOS1KO) have an impaired renal ET-1 system in the CD. Although urinary ET-1 production and inner medullary ET receptor expression were similar between flox control and CDNOS1KO mice, acute ET-1 treatment significantly reduced ENaC open probability in CDs from flox mice but not CDNOS1KO mice compared with basal. Basal ENaC activity in CDs was similar between the genotypes. We conclude that during acute shear stress across the CD, ENaC acts in a negative feedback loop to stimulate NO production in an ETB/NOS1-dependent manner resulting in a decrease in ENaC open probability and promoting natriuresis.

The necessity of the creation of a multispindle nutrunner ensuring a relatively stable moment of tightening in group threaded connections is substantiated. A kinematic scheme of a multispindle nutrunner with passive means of adaptation on a moment on the basis of a differential mechanism use is offered. The analysis is given of a moment error at multispindle nutrunner tightening on the basis of passive adaptation.

epithelial Na(+) channel, ENaC, is the final arbiter of sodium excretion in the kidneys. As such, discretionary control of ENaC by hormones is critical to the fine-tuning of electrolyte and water excretion and, consequently, blood pressure. Casein kinase 2 (CK2) phosphorylates ENaC. Phosphorylation by CK2 is necessary for normal ENaC activity. We tested the physiological importance of CK2 regulation of ENaC as the degree to which ENaC activity is dependent on CK2 phosphorylation in the living organism is unknown. This was addressed using patch-clamp analysis of ENaC in completely split-open collecting ducts and whole animal physiological studies of sodium excretion in mice. We also used ENaC-harboring CK2 phosphorylation site mutations to elaborate the mechanism. We found that ENaC activity in ex vivo preparations of murine collecting duct had a significant decrease in activity in response to selective antagonism of CK2. In whole animal experiments selective antagonism of CK2 caused a natriuresis similar to benzamil, but not additive to benzamil, suggesting an ENaC-dependent mechanism. Regulation of ENaC by CK2 was abolished by mutation of the canonical CK2 phosphorylation sites in beta and gamma ENaC. Together, these results demonstrate that the appropriate regulation of ENaC by CK2 is necessary for the normal physiological role played by this key renal ion channel in the fine-tuning of sodium excretion.

Background During 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 Results Male control and collecting duct nitric oxide synthase 1β knockout ( CDNOS 1 KO ) 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 CDNOS 1 KO mice. CDNOS 1 KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT 1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS 1 KO 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 CDNOS 1 KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls. Conclusions These 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.

Because of its cardio-protective effects, a low Na, high K diet (LNaHK) is often warranted in conjunction with diuretics to treat hypertensive patients. However, it is necessary to understand the renal handling of such diets in order to choose the best diuretic. Wild type (WT) or Renal Outer Medullary K channel (ROMK) knockout mice (KO) were given a regular (CTRL), LNaHK, or high K diet (HK) for 4–7 days. On LNaHK, mice treated with either IP furosemide for 12 hrs, or given furosemide in drinking water for 7 days, exhibited decreased K clearance. We used free-flow micropuncture to measure the [K+] in the early distal tubule (EDT [K+]) before and after furosemide treatment. Furosemide increased the EDT [K+] in WT on CTRL but decreased that in WT on LNaHK. Furosemide did not affect the EDT [K+] of KO on LNaHK or WT on HK. Furosemide-sensitive Na+ excretion was significantly greater in mice on LNaHK than those on CTRL or HK. Patch clamp analysis of split-open TALs revealed that 70-pS ROMK exhibited a higher open probability (Po) but similar density in mice on LNaHK, compared with CTRL. No difference was found in the density or Po of the 30 pS K channels between two groups. These results indicate mice on LNaHK exhibited furosemide-sensitive net K+ secretion in the TAL that is dependent on increased NKCC2 activity and mediated by ROMK. We conclude that furosemide is a K-sparing diuretic by decreasing the TAL net K+ secretion in subjects on LNaHK.

cAMP is a key mediator of connecting tubule and collecting duct (CD) Na+and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na+or K+excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na+channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na+and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na+and water excretion in general or vasopressin regulation of CD function in particular.

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na+ excretion and investigated the signaling mechanisms by which PRR regulates Na+ balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na+ diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na+ balance with normal- and low-Na+ intake. PRR KO mice had an attenuated hypertensive response and reduced Na+ retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na+ channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na+ wasting and reduces the hypertensive response to ANG II.

The epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK(Ca) channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K(+) efflux through BK(Ca) channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-β4 regulatory subunit, which is required for normal BK(Ca) channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-β4 null mice. ENaC in BK-β4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-β4 null mice. Consequently, sodium excretion is impaired in BK-β4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK(Ca) channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-β4 null mice likely contributes to their elevated blood pressure.

The epithelial Na + channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under negative-feedback regulation by the renin–angiotensin–aldosterone system in protection of sodium balance and blood pressure. We test here whether aldosterone is necessary and sufficient for ENaC expression and activity in the ASDN. Surprisingly, ENaC expression and activity are robust in adrenalectomized (Adx) mice. Exogenous mineralocorticoid increases ENaC activity equally well in control and Adx mice. Plasma [AVP] is significantly elevated in Adx vs. control mice. Vasopressin (AVP) stimulates ENaC. Inhibition of the V 2 AVP receptor represses ENaC activity in Adx mice. The absence of aldosterone combined with elevated AVP release compromises normal feedback regulation of ENaC in Adx mice in response to changes in sodium intake. These results demonstrate that aldosterone is sufficient but not necessary for ENaC activity in the ASDN. Aldosterone-independent stimulation by AVP shifts the role of ENaC in the ASDN from protecting Na + balance to promoting water reabsorption. This stimulation of ENaC likely contributes to the hyponatremia of adrenal insufficiency.

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na+ excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na+ regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na+ intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na+ regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na+ feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na+ retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.

Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.

Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na+ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y2-/- mice. Loss of collecting duct P2Y2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y2-/- mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.

Adenylyl cyclase (AC)-stimulated cAMP is a key mediator of collecting duct (CD) Na and water transport. AC isoforms 3, 4 and 6 are expressed in the CD; while AC6 is involved in modulating CD Na and...

Ion channels play key roles in physiology. They function as protein transducers able to transform stimuli and chemical gradients into electrical signals. They also are critical for cell signaling and play a particularly important role in epithelial transport acting as gateways for the movement of electrolytes across epithelial cell membranes. Experimental limitations, though, have hampered the recording of ion channel activity in many types of tissue. This has slowed progress in understanding the cellular and physiological function of these channels with most function inferred from in vitro systems and cell culture models. In many cases, such inferences have clouded rather than clarified the picture. Here, we describe a contemporary method for isolating and patch-clamping renal tubules for ex vivo analysis of ion channel function in native tissue. Focus is placed on quantifying the activity of the epithelial Na(+) channel (ENaC) in the aldosterone--sensitive distal nephron (ASDN). This isolated, split-open tubule preparation enables recording of renal ion channels in the close-to-native environment under the control of native cell signaling pathways and receptors. When combined with complementary measurements of organ and system function, and contemporary molecular genetics and pharmacology used to manipulate function and regulation, patch-clamping renal channels in the isolated, split-open tubule enables understanding to emerge about the physiological function of these key proteins from the molecule to the whole animal.