Aldosterone-dependent regulation of Na-K-ATPase subunit mRNA in the rat CCD: competitive PCR analysis

1996 ◽  
Vol 271 (1) ◽  
pp. F7-F15 ◽  
Author(s):  
K. Tsuchiya ◽  
G. Giebisch ◽  
P. A. Welling
Keyword(s):  
Collecting Duct ◽  
Mrna Abundance ◽  
Pcr Analysis ◽  
Cortical Collecting Duct ◽  
Wild Type ◽  
Competitive Pcr ◽  
Atpase Subunit ◽  
Subunit Mrna ◽  
Pcr Products

In the cortical collecting duct (CCD), aldosterone increases the number of functionally active Na-K-adenosin-etriphosphatase (Na-K-ATPase) molecules by a mechanism involving an isoform-specific increase in the abundance of the Na-K-ATPase alpha 1- and beta 1-subunit protein. However, the molecular basis for the response, particularly in the mammalian CCD in vivo, has remained unclear. To resolve this issue, reverse transcription (RT) and a competitive polymerase chain reaction (PCR) were employed to study mineralocorticoid-dependent regulation of alpha 1- and beta 1-subunit mRNA in the rat CCD. Na-K-ATPase subunit-specific oligonucleotides primers were used in the PCR to amplify reverse-transcribed subunit mRNA (RT-mRNA) from single microdissected CCD. Control templates were constructed (84-bp deletion mutation of the rat Na-K-ATPase alpha 1-subunit cDNA and 70-bp deletion of the beta 1-subunit cDNA), serially diluted, and coamplified with the wild-type Na-K-ATPase subunit RT-mRNA from single CCD. PCR products of predicted size were observed by ethidium bromide staining. Southern blots with an internal subunit-specific oligonucleotide confirmed Na-K-ATPase alpha 1- and beta 1-subunit identity. The ratio of the amplified wild-type to mutant PCR products was found to be linear over the range of input control cDNA tested so that the amount of subunit mRNA could be determined. A chronic reduction in corticosteroid levels by bilateral adrenalectomy (7 days) reduced the apparent level of alpha 1-subunit transcript by 54.0 +/- 6.3% but not the beta 1-subunit. Administering aldosterone to physiological levels is sufficient to restore CCD alpha 1-subunit mRNA abundance toward control levels within 6 h. We conclude the following: 1) regulation of Na-K-ATPase of CCD in vivo can be attributed, at least in part, to mineralocorticoid-dependent control of Na-K-ATPase alpha 1-subunit mRNA abundance; and 2) competitive PCR may provide a sensitive and quantitative tool for determining hormone-dependent regulation of mRNA abundance in nephron segments.

scholarly journals Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

2013 ◽  
Vol 304 (5) ◽  
pp. F522-F532 ◽  
Author(s):  
Luca Vedovelli ◽  
John T. Rothermel ◽  
Karin E. Finberg ◽  
Carsten A. Wagner ◽  
Anie Azroyan ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Collecting Duct ◽  
Egfp Expression ◽  
Intercalated Cells ◽  
Wild Type ◽  
Western Blots ◽  
Atpase Subunit ◽  
Protein Levels ◽  
Baseline Conditions ◽  
Deficient Mice

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1−/−) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1−/− and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915–F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1−/− mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1+/+ mice) with Atp6v1b1−/− mice to generate novel EGFP-B1−/− mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1+/+ and EGFP-B1−/− mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1−/− mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.

Characterization of the Hsp110/SSE gene family response to hyperosmolality and other stresses

1998 ◽  
Vol 274 (6) ◽  
pp. F1054-F1061 ◽  
Author(s):  
Bento C. Santos ◽  
Alejandro Chevaile ◽  
Ryoji Kojima ◽  
Steven R. Gullans
Keyword(s):  
Heat Shock ◽  
Gene Family ◽  
Stress Proteins ◽  
Collecting Duct ◽  
Suppressive Effect ◽  
Nacl Stress ◽  
Pcr Analysis ◽  
Mouse Tissues

Hsp110, Osp94, and Hsp70RY are members of the recently described Hsp110/SSE subfamily of (heat and osmotic) stress proteins whose members are structurally related to the Hsp70/BiP gene superfamily. To date, little is known about the response of this gene family to stresses in vitro or in vivo. In this study, an analysis of mRNA expression showed that Hsp110 and Osp94, like Hsp70, are induced in renal murine inner medullary collecting duct (mIMCD3) epithelial cells by heat shock, hyperosmotic NaCl, and cadmium, whereas low pH had a suppressive effect on Osp94. H2O2decreased expression of Osp94 while inducing levels of Hsp110 and Hsp70 message. Tunicamycin, hypertonic urea, and tumor necrosis factor-α had no effects. Hsp70RY was responsive exclusively to cadmium chloride. Moreover, enhanced expression of Hsp110 and Osp94 was subsequent to induction of Hsp70 and was suppressed by inhibition of protein synthesis by cycloheximide. RT-PCR analysis showed Hsp110, Osp94, and Hsp70RY are ubiquitously expressed in mouse tissues. In murine kidney, there was a corticomedullary gradient of expression of Hsp110, Osp94, Hsp70RY, and Hsp70 but not Hsc70 or BiP. Furthermore, dehydration increased inner medullary expression of Hsp110 and Osp94. An analysis of stress tolerance in mIMCD3 cells showed that heat shock and hyperosmotic NaCl stress are cross-tolerant stresses, suggesting hyperosmolality is a physiological correlate of heat shock in mammalian kidney. Thus Hsp110 and Osp94 behave as heat shock proteins, although they are regulated differently than Hsp70.

scholarly journals Piquiá Shells (Caryocar villosum): A Fruit by-Product with Antioxidant and Antiaging Properties in Caenorhabditis elegans

2020 ◽  
Vol 2020 ◽  
pp. 1-19 ◽  
Author(s):  
Mariana Roxo ◽  
Herbenya Peixoto ◽  
Pille Wetterauer ◽  
Emerson Lima ◽  
Michael Wink
Keyword(s):  
Caenorhabditis Elegans ◽  
Lethal Dose ◽  
Pcr Analysis ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Wild Type ◽  
Fluorescent Reporter ◽  
Triterpenoid Saponins ◽  
Promising Source

In a context of rising demand for sustainable antiaging interventions, fruit processing by-products are a promising source of bioactive compounds for the production of antiaging dietary supplements. Piquiá (Caryocar villosum) is a native Amazonian fruit consisting of 65% nonedible shells. In the present study, the phytochemical profile of a hydroalcoholic extract of piquiá shells (CV) was characterized by LC-MS/MS analysis. Its antioxidant and antiaging activities were investigated using the nematode Caenorhabditis elegans as an in vivo model. CV is mainly composed by hydrolysable tannins and triterpenoid saponins. The extract enhanced stress resistance of wild-type and mutant worms by reducing the intracellular levels of reactive oxygen species (ROS) and by increasing their survival against a lethal dose of the prooxidant juglone. These effects involved the upregulation of sod-3 and downregulation of gst-4 and hsp-16.2, studied through the GFP fluorescent reporter intensity and at the transcriptional level by qRT-PCR analysis. CV extended the lifespan of wild-type worms in a DAF-16/FoxO- and SKN-1/Nrf-dependent manner. Taken together, our findings indicate piquiá shells as potential candidates for nutraceutical applications. Further studies are needed to validate the relevance of our findings to antiaging interventions in humans.

scholarly journals Marinobufagenin enhances cardiac contractility in mice with ouabain-sensitive α1 Na+-K+-ATPase

2009 ◽  
Vol 296 (6) ◽  
pp. H1833-H1839 ◽  
Author(s):  
Arshani N. Wansapura ◽  
Valerie Lasko ◽  
Zijian Xie ◽  
Olga V. Fedorova ◽  
Alexei Y. Bagrov ◽  
...  
Keyword(s):  
Binding Site ◽  
Functional Role ◽  
Cardiac Contractility ◽  
Cardiac Performance ◽  
Wild Type ◽  
Ouabain Binding ◽  
Atpase Subunit ◽  
Cardiac Inotropy ◽  
Rats And Mice

Endogenous Na+ pump inhibitors are thought to play important (patho)physiological roles and occur in two different chemical forms in the mammalian circulation: cardenolides, such as ouabain, and bufadienolides, such as marinobufagenin (MBG). Although all α Na+-K+-ATPase isoforms (α1-4) are sensitive to ouabain in most species, in rats and mice the ubiquitously expressed α1 Na+-K+-ATPase is resistant to ouabain. We have previously shown that selective modification of the putative ouabain binding site of either the α1 or α2 Na+-K+-ATPase subunit in mice substantially alters the cardiotonic influence of exogenously applied cardenolides. To determine whether the ouabain binding site also interacts with MBG and if this interaction plays a functional role, we evaluated cardiovascular function in α1-resistant/α2-resistant (α1R/Rα2R/R), α1-sensitive/α2-resistant (α1S/Sα2R/R), and α1-resistant/α2-sensitive mice (α1R/Rα2S/S, wild type). Cardiovascular indexes were evaluated in vivo by cardiac catheterization at baseline and during graded infusions of MBG. There were no differences in baseline measurements of targeted mice, indicating normal hemodynamics and cardiac function. MBG at 0.025, 0.05, and 0.1 nmol·min−1·g body wt−1 significantly increased cardiac performance to a greater extent in α1S/Sα2R/R compared with α1R/Rα2R/R and wild-type mice. The increase in LVdP/d tmax in α1S/Sα2R/R mice was greater at higher concentrations of MBG compared with both α1R/Rα2R/R and α1R/Rα2S/S mice ( P < 0.05). These results suggest that MBG interacts with the ouabain binding site of the α1 Na+-K+-ATPase subunit and can thereby influence cardiac inotropy.

Deoxycorticosterone-stimulated bicarbonate secretion in rabbit cortical collecting ducts: effects of luminal chloride removal and in vivo acid loading

1985 ◽  
Vol 249 (2) ◽  
pp. F205-F212 ◽  
Author(s):  
J. Garcia-Austt ◽  
D. W. Good ◽  
M. B. Burg ◽  
M. A. Knepper
Keyword(s):  
Collecting Duct ◽  
High Rate ◽  
Bicarbonate Secretion ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Urinary Ph ◽  
Collecting Ducts ◽  
Acid Loading

To assess the role of cortical collecting duct bicarbonate secretion in the regulation of net acid excretion, we have sought to identify what factors influence the secretion rate. Net and unidirectional bicarbonate fluxes were measured in isolated perfused cortical collecting ducts from deoxycorticosterone-treated rabbits. The collecting ducts secreted bicarbonate at 11-24 pmol X mm-1 X min-1, confirming the high rate seen in earlier studies. Oral acid loading (50 mM NH4Cl drinking water) completely inhibited the net bicarbonate secretion. The bath-to-lumen flux was markedly reduced with acid loading, but the lumen-to-bath flux changed very little. In tubules from rabbits treated with deoxycorticosterone (but not NH4Cl), luminal chloride replacement with either sulfate or gluconate completely and reversibly inhibited the net bicarbonate secretion. The bath-to-lumen flux was greatly inhibited, but there was little change in the lumen-to-bath flux. We conclude: 1) High rates of bicarbonate secretion can be induced in rabbit cortical collecting ducts by chronic treatment of the animals with deoxycorticosterone. 2) When deoxycorticosterone-treated rabbits were made acidotic by oral administration of NH4Cl, the bicarbonate secretion was prevented, indicating that the systemic acid-base state of the animal may be an important factor regulating bicarbonate secretion. 3) Replacement of chloride in the lumen with sulfate inhibits bicarbonate secretion in the cortical collecting duct, an effect which may explain in part the decrease in urinary pH in response to sulfate infusions in mineralocorticoid-stimulated animals.

Differential response to myocardial reperfusion injury in eNOS-deficient mice

2002 ◽  
Vol 282 (6) ◽  
pp. H2422-H2426 ◽  
Author(s):  
Brent R. Sharp ◽  
Steven P. Jones ◽  
David M. Rimmer ◽  
David J. Lefer
Keyword(s):  
Ischemia Reperfusion ◽  
Myocardial Necrosis ◽  
In Vivo Model ◽  
Pcr Analysis ◽  
Inos Mrna ◽  
Mrna Levels ◽  
Wild Type ◽  
Significant Difference ◽  
Deficient Mice

Two strains of endothelial nitric oxide synthase (eNOS)-deficient (−/−) mice have been developed that respond differently to myocardial ischemia-reperfusion (MI/R). We evaluated both strains of eNOS−/− mice in an in vivo model of MI/R. Harvard (Har) eNOS−/− mice ( n = 12) experienced an 84% increase in myocardial necrosis compared with wild-type controls ( P < 0.05). University of North Carolina (UNC) eNOS−/−( n = 10) exhibited a 52% reduction in myocardial injury versus wild-type controls ( P < 0.05). PCR analysis of myocardial inducible NO synthase (iNOS) mRNA levels revealed a significant ( P < 0.05) increase in the UNC eNOS−/− mice compared with wild-type mice, and there was no significant difference between the Har eNOS−/− and wild-type mice. UNC eNOS−/− mice treated with an iNOS inhibitor (1400W) exacerbated the extent of myocardial necrosis. When treated with 1400W, Har eNOS−/− did not exhibit a significant increase in myocardial necrosis. These data demonstrate that two distinct strains of eNOS−/− mice display opposite responses to MI/R. Although the protection seen in the UNC eNOS−/− mouse may result from compensatory increases in iNOS, other genes may be involved.

scholarly journals Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

2008 ◽  
Vol 295 (3) ◽  
pp. F780-F788 ◽  
Author(s):  
Genevieve Estilo ◽  
Wen Liu ◽  
Nuria Pastor-Soler ◽  
Phillip Mitchell ◽  
Marcelo D. Carattino ◽  
...  
Keyword(s):  
Splice Variants ◽  
Collecting Duct ◽  
Bk Channels ◽  
Bk Channel ◽  
Mrna Abundance ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
Channel Expression ◽  
Tubular Flow ◽  
Circulating Levels

Apical large-conductance Ca2+-activated K+ (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+ secretion. Dietary K+ loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+ secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+ (LS) or high-Na+ (HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+ concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+ (JNa) and K+ (JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+ and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNa was greater in LS vs. HS rabbits, yet the flow-stimulated increase in JK was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+ loading.

scholarly journals MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1

2014 ◽  
Vol 306 (1) ◽  
pp. F53-F60 ◽  
Author(s):  
Dao-Hong Lin ◽  
Peng Yue ◽  
Chengbiao Zhang ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Seed Sequence ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Luciferase Reporter Gene ◽  
High K ◽  
Low K

The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1–3′UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3′UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3′UTR but not with 3′UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity.

scholarly journals Effects of biomechanical forces on signaling in the cortical collecting duct (CCD)

2014 ◽  
Vol 307 (2) ◽  
pp. F195-F204 ◽  
Author(s):  
Rolando Carrisoza-Gaytan ◽  
Yu Liu ◽  
Daniel Flores ◽  
Cindy Else ◽  
Heon Goo Lee ◽  
...  
Keyword(s):  
Fluid Shear Stress ◽  
Ex Vivo ◽  
Collecting Duct ◽  
Cation Transport ◽  
Mapk Signaling ◽  
Collagen Type Iv ◽  
Cortical Collecting Duct ◽  
Type Iv ◽  
Biomechanical Forces

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm2 of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status

1994 ◽  
Vol 266 (4) ◽  
pp. F528-F535 ◽  
Author(s):  
C. Emmons ◽  
J. B. Stokes
Keyword(s):  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Acid Base Status

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.

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