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Immunoperoxidase microscopy of-ENaC in DCT2, CNT, and CCD from CON rats (A, C, and E) and rats infused with ANP for 90 min (B, D, and F). Immunoperoxidase labeling of-ENaC is mainly associated with intracellular vesicles dispersed in the entire cytoplasm of principal cells of the DCT2 (A), CNT (C), and CCD (E) in CON rats. Similarly, immunoperoxidase staining for-ENaC revealed diffuse cytoplasmic labeling throughout the DCT2 (B), CNT (D), and CCD (F) in ANP-infused rats, indicating unchanged subcellular localization. Arrowheads indicate intercalated cells. Magnification: 1,000.
Source publication
Atrial natriuretic peptide (ANP) acutely promotes water and sodium excretion, whereas subchronic effects involve water retention. Renal hemodynamics, water and sodium excretion, and aquaporin-2 (AQP2) and epithelial Na channel (ENaC) subcellular trafficking were determined in response to continuous ANP infusion in conscious rats, where body sodium...
Contexts in source publication
Context 1
... studies showing ENaC expression from the second half of the distal convoluted tubules (DCT2) to the IMCD, with the expression being most prominent in DCT2, connecting tubules (CNT), and CCD, with decreased labeling in medullary collecting duct segments (17). In control rats, immunoperoxidase microscopy demonstrated that immu- nolabeling of (Fig. 6)-, (Fig. 7)-, and (Fig. 8)-ENaC subunits was exclusively associated with the apical plasma membrane and the intracellular vesicles in DCT2, CNT, and CCD, consistent with previous findings (17) (data not shown for medullary collecting duct). After 10 min of ANP infusion, no significant changes were observed in the subcellular local- Fig. 4. ...
Context 2
... Fig. 8, B, D, and F, respectively) compared with control rats (Fig. 6, A, C, and E, and Fig. 8, A, C, and E, respectively). No changes were observed in the IMCD (not shown). In contrast, the labeling of -ENaC was unchanged and showed both apical plasma membrane and dispersed cytoplasmic labeling in DCT2, CNT, CCD, and IMCD cells in both control (Fig. 7, A, C, and E) and ANP- infused rats (Fig. 7, B, D, and F). Therefore, the increased apical ENaC expression may suggest a possible compensation for the natriuresis induced by the continuous infusion of ANP. On the other hand, plasma aldosterone levels at period 3 did not show any significant difference between the two groups (1,608 370 ...
Context 3
... with control rats (Fig. 6, A, C, and E, and Fig. 8, A, C, and E, respectively). No changes were observed in the IMCD (not shown). In contrast, the labeling of -ENaC was unchanged and showed both apical plasma membrane and dispersed cytoplasmic labeling in DCT2, CNT, CCD, and IMCD cells in both control (Fig. 7, A, C, and E) and ANP- infused rats (Fig. 7, B, D, and F). Therefore, the increased apical ENaC expression may suggest a possible compensation for the natriuresis induced by the continuous infusion of ANP. On the other hand, plasma aldosterone levels at period 3 did not show any significant difference between the two groups (1,608 370 in ANP-infused rats vs. 1,565 530 pg/ml in controls; not ...
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Background
Tolvaptan slows progression of autosomal dominant polycystic kidney disease (ADPKD) by antagonizing the vasopressin-cAMP axis. Nitric oxide (NO) stimulates natriuresis and diuresis, but its role is unknown during tolvaptan treatment in ADPKD.
Methods
Eighteen patients with ADPKD received tolvaptan 60 mg or placebo in a randomized, place...
Renal handling of sodium and water is abnormal in chronic kidney disease (CKD). The aim of this study was to test the hypothesis that abnormal activity of the aquaporin-2 water channels (AQP2), the sodium-potassium-2chloride transporter (NKCC2) and/or the epithelial sodium channels (ENaC) contribute to this phenomenon.
23 patients with CKD and 24 h...
Large shifts of osmolality occur in the kidney medulla as part of the urine concentrating mechanism. Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress. Here, we exam-ined the unfolded protein response (UPR) in hyperosmotically-challenged principal cells of the kidney collecting duct (CD) an...
The renal collecting duct is the nephron segment where the final urine content of acid equivalents and inorganic ions are determined. The role of two different cell types present in this nephron segment has been determined many years ago: principal cells that express the epithelial sodium channel ENaC and aquaporin 2, regulate electrolyte reabsorpt...
Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat...
Citations
... Since ClC-6 is also found in the proximal tubule, thick ascending limb, distal convoluted tubule, and collecting duct according to a nephron segment-specific deep sequencing database (Lee et al., 2015;Zhao et al., 2016), it would be of interest to more closely examine the expression of Na + transporters and channels in these regions. ANP inhibits several Na + -dependent transport systems, including the Na + -H + exchanger (Winaver et al., 1990), type IIa Na-Pi cotransporter (Bacic et al., 2001), Na + -K + -ATPase (Aperia et al., 1994), Na + -K + -2Cl − cotransporter 2 (Ares et al., 2011), and the epithelial sodium channel (ENaC) (Guo et al., 2013;Wang et al., 2006). In the kidney, the collecting duct is a main target of ANP (Inoue et al., 2001), which acutely reduces ENaC activity through a reduction in open probability through a cGMP specific signaling pathway and increases surface expression of the αand γ-ENaC subunits within 90 minutes of ANP stimulation (Guo et al., 2013;Wang et al., 2006). ...
... ANP inhibits several Na + -dependent transport systems, including the Na + -H + exchanger (Winaver et al., 1990), type IIa Na-Pi cotransporter (Bacic et al., 2001), Na + -K + -ATPase (Aperia et al., 1994), Na + -K + -2Cl − cotransporter 2 (Ares et al., 2011), and the epithelial sodium channel (ENaC) (Guo et al., 2013;Wang et al., 2006). In the kidney, the collecting duct is a main target of ANP (Inoue et al., 2001), which acutely reduces ENaC activity through a reduction in open probability through a cGMP specific signaling pathway and increases surface expression of the αand γ-ENaC subunits within 90 minutes of ANP stimulation (Guo et al., 2013;Wang et al., 2006). Additionally, ANP acts as a counterbalance to the RAAS system, acting as an antagonist of Angiotensin II signaling and exerting oppositional effects systemically. ...
The AGTRAP-PLOD1 locus is a conserved gene cluster containing several blood pressure regulatory genes, including CLCN6, MTHFR, NPPA, and NPPB. Previous work revealed that knockout of Clcn6 on the Dahl Salt-Sensitive (SS) rat background (SS-Clcn6) resulted in lower diastolic blood pressure compared to SS-WT rats. Additionally, a recent study found sickle cell anemia patients with mutations in CLCN6 had improved survival and reduced stroke risk. We investigated whether loss of Clcn6 would delay the mortality of Dahl SS rats on an 8% NaCl (HS) diet. No significant difference in survival was found. The ability of Clcn6 to affect mRNA expression of nearby Mthfr, Nppa, and Nppb genes was also tested. On normal salt (0.4% NaCl, NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS-Clcn6 rats. MTHFR reduces homocysteine to methionine, but no differences in circulating homocysteine levels were detected. Nppa mRNA levels in cardiac tissue from SS-Clcn6 rat in both normotensive and hypertensive conditions were significantly reduced compared to SS-WT. Nppb mRNA expression in SS-Clcn6 rats on a NS diet was also substantially decreased. Heightened Mthfr expression would be predicted to be protective; however, diminished Nppa and Nppb expression could be deleterious and by preventing or blunting vasodilation, natriuresis, and diuresis that ought to normally occur to offset blood pressure increases. The conserved nature of this genetic locus in humans and rats suggests more studies are warranted to understand how mutations in and around these genes may be influencing the expression of their neighbors.
... The epithelial Na + channels (ENaC) in the distal nephron are the limiting step of sodium reabsorption, and it was shown that inhibiting ENaC can attenuate salt sensitivity in animal models (20)(21)(22). There is a variety of endogenous stimuli that can regulate ENaC, including ANP (23)(24)(25). Importantly, the SS phenotype in Nppa -/mice was rescued by an ENaC inhibitor amiloride (26). ...
Atrial natriuretic peptide (ANP), encoded by Nppa, is a vasodilatory hormone that promotes salt excretion. Genome-wide association studies identified Nppa as a causative factor of blood pressure development, and in humans, ANP levels were suggested as an indicator of salt sensitivity. This study aimed to provide insights into the effects of ANP on cardiorenal function in salt-sensitive hypertension. To address this question, hypertension was induced in SSNPPA-/- (knockout of Nppa in the Dahl Salt-Sensitive (SS) rat background) or SSWT (wild type Dahl SS) rats by a high salt diet challenge (HS, 4% NaCl for 21 days). Chronic infusion of ANP in SSWT rats attenuated the increase in blood pressure and cardiorenal damage. Overall, SSNPPA-/- strain demonstrated higher blood pressure and intensified cardiac fibrosis (with no changes in ejection fraction) compared to SSWT rats. Furthermore, SSNPPA-/- rats exhibited kidney hypertrophy and higher glomerular injury scores, reduced diuresis, and lower sodium and chloride excretion than SSWT when fed a HS diet. Additionally, the activity of epithelial Na+ channel (ENaC) was found to be increased in the collecting ducts of the SSNPPA-/- rats. Taken together, these data show promise for the therapeutic benefits of ANP and ANP-increasing drugs for treating salt-sensitive hypertension.
... Urine volume is primarily regulated by vasopressin and aquaporin-2 water channels in the apical membrane of principal cells in the kidney [1]. The renin-angiotensinaldosterone system, the natriuretic peptide system and the sympathetic adrenergic nervous system also play a role in the opening degree of the AQP2 channels and therefore affect urine concentration [2][3][4][5][6][7]. The ability to concentrate urine can be measured by a water deprivation test. ...
Background:
Concentration of the urine is primarily regulated via vasopressin dependent aquaporin-2 water channels in the apical membrane of kidney principal cells. It is unclear whether urine concentration ability in ADPKD differs from other patients with similar degree of impaired renal function (non-ADPKD patients). The purpose of this case control study was to measure urine concentration ability in ADPKD patients compared to non-ADPKD patients and healthy controls.
Methods:
A seventeen hour long water deprivation test was carried out in 17 ADPKD patients (CKD I-IV), 16 non-ADPKD patients (CKD I-IV), and 18 healthy controls. Urine was collected in 4 consecutive periods during water deprivation (12 h, 1 h, 2 h and 2 h, respectively) and analyzed for osmolality (u-Osm), output (UO), fractional excretion of sodium (FENa), aquaporin2 (u-AQP2) and ENaC (u-ENaC). Blood samples were drawn trice (after 13-, 15-, and 17 h after water deprivation) for analyses of osmolality (p-Osm), vasopressin (p-AVP), and aldosterone (p-Aldo).
Results:
U-Osm was significantly lower and FENa significantly higher in both ADPKD patients and non-ADPKD patients compared to healthy controls during the last three periods of water deprivation. During the same periods, UO was higher and secretion rates of u-AQP2 and u-ENaC were lower and at the same level in the two groups of patients compared to controls. P-AVP and p-Osm did not differ significantly between the three groups. P-Aldo was higher in both groups of patients than in controls.
Conclusions:
Urine concentration ability was reduced to the same extent in patients with ADPKD and other chronic kidney diseases with the same level of renal function compared to healthy controls. The lower urine excretion of AQP2 and ENaC suggests that the underlying mechanism may be a reduced tubular response to vasopressin and aldosterone.
Trial registration:
Current Controlled Trial NCT04363554 , date of registration: 20.08.2017.
... Atrial natriuretic peptide stimulates sodium and water excretion from the inner medullary collecting duct thereby opposing the interstitial volume expansion (Baxter et al., 1988;Light et al., 1989). After prolonged exposure to ANP there is an increase in apical expression of aquaporin-2 and the gamma subunit of ENaC (Wang et al., 2006). However, systemic infusion of synthetic ANP or ANP extract in experimental studies in nephrotic individuals had a diminished natriuretic and diuretic response compared to healthy controls (Koepke and DiBona, 1987;Perico et al., 1989) despite the prediction from animal models for the opposite (Wang et al., 2006). ...
... After prolonged exposure to ANP there is an increase in apical expression of aquaporin-2 and the gamma subunit of ENaC (Wang et al., 2006). However, systemic infusion of synthetic ANP or ANP extract in experimental studies in nephrotic individuals had a diminished natriuretic and diuretic response compared to healthy controls (Koepke and DiBona, 1987;Perico et al., 1989) despite the prediction from animal models for the opposite (Wang et al., 2006). ...
Oedema is a defining element of the nephrotic syndrome. Its’ management varies considerably between clinicians, with no national or international clinical guidelines, and hence variable outcomes. Oedema may have serious sequelae such as immobility, skin breakdown and local or systemic infection. Treatment of nephrotic oedema is often of limited efficacy, with frequent side-effects and interactions with other pharmacotherapy. Here, we describe the current paradigms of oedema in nephrosis, including insights into emerging mechanisms such as the role of the abnormal activation of the epithelial sodium channel in the collecting duct. We then discuss the physiological basis for traditional and novel therapies for the treatment of nephrotic oedema. Despite being the cardinal symptom of nephrosis, few clinical studies guide clinicians to the rational use of therapy. This is reflected in the scarcity of publications in this field; it is time to undertake new clinical trials to direct clinical practice.
... Regarding the diuretic effects of pGC-A activators, studies have investigated diuretic actions of ANP in which exogenous cGMP directly reduced vasopression stimulated osmatic water permeability in rat IMCD cells [15]. ANP was also reported to modulate water-channel protein aquaporin-2 (AQP2) translocation during its diuretic actions [16]. In the study by Wang et al., diuresis induced by ANP infusion was not accompanied by changes in subcellular localization of AQP2 in the IMCD, whereas there was a marked increase in apical targeting of AQP2 in IMCD after ANP infusion, indicating a compensatory effect to reduce volume depletion in response to prolonged ANP infusion [16]. ...
... ANP was also reported to modulate water-channel protein aquaporin-2 (AQP2) translocation during its diuretic actions [16]. In the study by Wang et al., diuresis induced by ANP infusion was not accompanied by changes in subcellular localization of AQP2 in the IMCD, whereas there was a marked increase in apical targeting of AQP2 in IMCD after ANP infusion, indicating a compensatory effect to reduce volume depletion in response to prolonged ANP infusion [16]. These studies suggested that the effect of ANP/cGMP on osmatic water permeability contributed largely to in vivo diuretic actions. ...
The particulate guanylyl cyclase A (pGC-A)/cGMP pathway plays important roles in regulating renal physiological function and as well as in counteracting pathophysiological conditions. Naturally occurring peptide pGC-A activators consist of atrial natriuretic peptide (ANP), b-type NP (BNP), and urodilatin (URO). These activators bind and activate pGC-A, generating the second messenger cyclic 3',5' guanosine monophosphate (cGMP). Cyclic GMP binds to downstream pathway effector molecules including protein kinase G (PKG), cGMP-gated ion channels, and phosphodiesterases (PDEs). These mediators result in a variety of physiological actions in the kidney, including diuresis, natriuresis, increased glomerular filtration rate (GFR) and organ protection, thus, opposing renal cellular injury and remodeling. Downstream proteins regulated by PKG include collagen 1 (Col-1), transforming growth factor beta (TGF-β) and apoptosis-related proteins. In addition to their physiological regulatory effects, pGC-A/cGMP signaling is critical for preserving renal homeostasis in different renal diseases such as acute kidney injury (AKI). Regarding therapeutic options, native pGC-A activators have short half-lives and their activity can be further enhanced by advances in innovative peptide engineering. Thus, novel designer peptide pGC-A activators with enhanced renal activity are under development.
... Consistent with previous finding, our data showed that the renal expression of sodium transporters including NHE3, NCC, NKCC2, and ENaCα were robustly decreased after ureteral obstruction while the expression of ENaCγ was not significantly changed in obstructed kidneys. Unlike other sodium transporters, the distinct regulation of ENaC subunits was reported previously by our and other groups [12,16,17]. ...
Ureteral obstruction is associated with reduced expressions of renal sodium transporters, which contributes to impaired urinary concentrating capacity. In this study, we employed a synthetic mitochondrial superoxide dismutase 2 (SOD2) mimic MnTBAP to investigate the role of mitochondrial oxidative stress in modulating the sodium transporters in obstructive kidney disease. Following unilateral ureteral obstruction (UUO) for 7 days, a global reduction of sodium transporters including NHE3, NCC, NKCC2, and ENaCa was observed as determined by qRT-PCR, Western Blotting or immunohistochemistry. Among these sodium transporters, the downregulation of NHE3, NCC, and NKCC2 was partially reversed by MnTBAP treatment. In contrast, the reduction of ENaCa was not affected by MnTBAP. The β and γ subunits of ENaC were not significantly altered by ureteral obstruction or MnTBAP therapy. To further confirm the anti-oxidant effect of MnTBAP, we examined the levels of TBARs in the urine collected from the obstructed ureters of UUO mice and bladder of sham mice. As expected, the increment of urinary TBARs in UUO mice was entirely abolished by MnTBAP therapy, indicating an amelioration of oxidative stress. Meantime, we found that three types of SOD were all reduced in obstructed kidneys determined by qRTPCR, which was unaffected by MnTBAP. Collectively, these results demonstrated an important role of mitochondrial oxidative stress in mediating the downregulation of sodium transporters in obstructive kidney disease.
... cGMP signaling-In addition to the canonical cAMP-induced pathway, the activation of the cGMP path way can modulate AQP2 trafficking [144][145][146]. ...
... Nitric oxide (NO) donors such as sodium nitroprusside (SNP) and NONOate, as well as the nitric oxide synthase (NOS) substrate L-arginine, induced AQP2 cell-surface localization by a cAMP-independent and cGMP-dependent pathway in rat kidney slices and LLC-PK1 cells expressing AQP2 [144]. In addition, atrial natriuretic peptide (ANP), infused in rats, stimulated AQP2 membrane insertion [146]. The mechanism by which cGMP induces AQP2 trafficking is still unclear. ...
Under physiological conditions, excessive loss of water through the urine is prevented by the release of the antidiuretic hormone arginine-vasopressin (AVP) from the posterior pituitary. In the kidney, AVP elicits a number of cellular responses, which converge on increasing the osmotic reabsorption of water in the collecting duct. One of the key events triggered by the binding of AVP to its type-2 receptor (AVPR2) is the exocytosis of the water channel aquaporin 2 (AQP2) at the apical membrane the principal cells of the collecting duct. Mutations of either AVPR2 or AQP2 result in a genetic disease known as nephrogenic diabetes insipidus, which is characterized by the lack of responsiveness of the collecting duct to the antidiuretic action of AVP. The affected subject, being incapable of concentrating the urine, presents marked polyuria and compensatory polydipsia and is constantly at risk of severe dehydration. The molecular bases of the disease are fully uncovered, as well as the genetic or clinical tests for a prompt diagnosis of the disease in newborns. A real cure for nephrogenic diabetes insipidus (NDI) is still missing, and the main symptoms of the disease are handled with s continuous supply of water, a restrictive diet, and nonspecific drugs. Unfortunately, the current therapeutic options are limited and only partially beneficial. Further investigation in vitro or using the available animal models of the disease, combined with clinical trials, will eventually lead to the identification of one or more targeted strategies that will improve or replace the current conventional therapy and grant NDI patients a better quality of life. Here we provide an updated overview of the genetic defects causing NDI, the most recent strategies under investigation for rescuing the activity of mutated AVPR2 or AQP2, or for bypassing defective AVPR2 signaling and restoring AQP2 plasma membrane expression.
... Vasopressin sensitive aquaporin2 water channels and epithelial sodium channels are expressed in the principal cells in the collecting ducts [1][2][3]. Stimulation of vasopressin receptors (V2) facilitates renal absorption of water and sodium [4][5][6][7]. This mechanism is antagonized by tolvaptan, a selective V2 receptor antagonist. ...
Background
Tolvaptan is a selective vasopressin receptor antagonist. Nitric Oxide (NO) promotes renal water and sodium excretion, but the effect is unknown in the nephron’s principal cells. In a dose-response study, we measured the effect of tolvaptan on renal handling of water and sodium and systemic hemodynamics, during baseline and NO-inhibition with L-NMMA (L-NG-monomethyl-arginine). Methods
In a randomized, placebo-controlled, double blind, cross over study, 15 healthy subjects received tolvaptan 15, 30 and 45 mg or placebo. L-NMMA was given as a bolus followed by continuous infusion during 60 min. We measured urine output (UO), free water clearance (CH2O), fractional excretion of sodium (FENa), urinary aquaporin-2 channels (u-AQP2) and epithelial sodium channels (u-ENaCγ), plasma vasopressin (p-AVP) and central blood pressure (cBP). ResultsDuring baseline, FENa was unchanged. Tolvaptan decreased u-ENaCγ dose-dependently and increased p-AVP threefold, whereas u-AQP2 was unchanged. During tolvaptan with NO-inhibition, UO and CH2O decreased dose-dependently. FENa decreased dose-independently and u-ENaCγ remained unchanged. Central BP increased equally after all treatments. Conclusions
During baseline, fractional excretion of sodium was unchanged. During tolvaptan with NO-inhibition, renal water excretion was reduced dose dependently, and renal sodium excretion was reduced unrelated to the dose, partly via an AVP dependent mechanism. Thus, tolvaptan antagonized the reduction in renal water and sodium excretion during NO-inhibition. Most likely, the lack of decrease in AQP2 excretion by tolvaptan could be attributed to a counteracting effect of the high level of p-AVP. Trial registrationClinical Trial no: NCT02078973. Registered 1 March 2014.
... The agonist of PKG, cGMP, has been shown to mediate translocation of AQP2 to the plasma membrane in AQP2transfected LLC-PK1 cells and in isolated kidney slices [22]. Activators of the cGMP pathway, such as atrial natriuretic peptide (ANP), L-arginine, cGMP phosphodiesterase type 5 (PDE5) inhibitors sildenafil citrate, elevated intracellular cGMP levels, resulting accumulation of AQP2 in plasma membrane [19,22,243]. These data suggests a positive role of PKG on AQP2 trafficking. ...
Aquaporins
(AQPs
)
are a family of membrane water
channels
that basically function
as regulators of intracellular and intercellular water flow. To date, thirteen AQPs
, which are distributed widely in specific cell types in various organs and tissues, have been characterized in humans. Four AQP
monomers, each of which consists of six membrane-spanning alpha-helices that have a central water-transporting pore, assemble to form tetramers, forming the functional units in the membrane. AQP
facilitates osmotic water transport
across plasma membranes
and thus transcellular fluid movement. The cellular functions
of aquaporins are regulated by posttranslational modifications
, e.g. phosphorylation, ubiquitination, glycosylation, subcellular distribution, degradation, and protein interactions. Insight into the molecular mechanisms responsible for regulated aquaporin trafficking and synthesis is proving to be fundamental for development of novel therapeutic targets or reliable diagnostic and prognostic biomarkers.
... Many strategies have been proposed that could bypass the defective V2R signaling and restore physiological AQP2 expression and/or trafficking [7,10,17]. These include activation of the intracellular cGMP pathway; activation of the intracellular cAMP pathway by stimulation of the Gαs-coupled calcitonin or secretin receptors; activation of E-prostanoid receptors EP2 and EP4 [18][19][20][21][22][23][24]. ...
