Adrenergic regulation of salt and fluid secretion in human medullary collecting duct cells

2004 ◽  
Vol 287 (4) ◽  
pp. F639-F648 ◽  
Author(s):  
Darren P. Wallace ◽  
Gail Reif ◽  
Anne-Marie Hedge ◽  
J. Brantley Thrasher ◽  
Paul Pietrow
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Intracellular Camp ◽  
Anion Secretion ◽  
Cell Monolayers ◽  
Urine Composition ◽  
Camp Levels

Transepithelial salt and fluid secretion mediated by cAMP in initial inner medullary collecting ducts (IMCDi) may be important for making final adjustments to urine composition. We examined in primary cultures of human IMCDi cells the effects of adrenergic receptor (AR) agonists and antagonists on intracellular cAMP levels, short-circuit current ( ISC), and fluid secretion. Epinephrine (1 μM), norepinephrine (1 μM), and isoproterenol (10 nM) individually increased intracellular cAMP levels 57-, 2-, and 25-fold, respectively, and stimulated ISC 3.3-, 2.9-, and 3.4-fold, respectively. β-AR activation increased net fluid secretion by cultured human IMCDi cell monolayers from 0.09 ± 0.04 to 0.26 ± 0.05 μl·h−1·cm−2 and freshly isolated rat IMCDi from 0.02 ± 0.01 to 0.09 ± 0.02 nl·h−1·mm−1. In monolayers, these effects were eliminated by blocking β2-AR, but not β1-AR. Activation of α2-AR with guanabenz inhibited isoproterenol-induced ISC by 37% in human IMCDi monolayers and fluid secretion by 91% in rat IMCDi. Immunohistochemistry of human medullary tissue sections revealed greater expression of β2-AR than β1-AR; β2-AR was localized to the basolateral membranes of human IMCDi. Immunoblots identified α2A-AR and α2B-AR in cultured human IMCDi cell monolayers. We conclude that 1) catecholamines stimulate cAMP-dependent anion and fluid secretion by IMCDi cells primarily through β2-AR activation and 2) α2-AR activation attenuates cAMP-dependent anion secretion.

Modulation of aldosterone-induced stimulation of ENaC synthesis by changing the rate of apical Na+ entry

2001 ◽  
Vol 281 (4) ◽  
pp. F687-F692 ◽  
Author(s):  
Lisette Dijkink ◽  
Anita Hartog ◽  
Carel H. Van Os ◽  
René J. M. Bindels
Keyword(s):  
Feedback Inhibition ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Entry Rate ◽  
Protein Levels ◽  
Stimulation Of

Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+ entry rate on aldosterone-induced transepithelial Na+ transport, which was measured as benzamil-sensitive short-circuit current ( I sc). Stimulation of the apical Na+ entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive I sc from 320 ± 49 to 117 ± 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 ± 74%). Immunoprecipitation of [35S]methionine-labeled β-rabbit epithelial Na+ channel (rbENaC) revealed that the effects of modulation of apical Na+ entry on transepithelial Na+ transport are exactly mirrored by β-rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced β-rbENaC protein synthesis from 310 ± 51 to 56 ± 17%. Exposure to benzamil doubled the β-rbENaC protein level to 281 ± 68% in control cells but had no significant effect on aldosterone-stimulated β-rbENaC levels (282 ± 68%). In conclusion, stimulation of apical Na+ entry suppresses the aldosterone-induced increase in transepithelial Na+transport. This negative-feedback inhibition is reflected in a decrease in β-rbENaC synthesis or in an increase in β-rbENaC degradation.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1337-F1350 ◽  
Author(s):  
Darren P. Wallace ◽  
Marcy Christensen ◽  
Gail Reif ◽  
Franck Belibi ◽  
Brantley Thrasher ◽  
...  
Keyword(s):  
Fluid Transport ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Transport Studies ◽  
Collecting Ducts ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCDi) and terminal (hIMCDt) inner medullary regions of human kidneys. AVP, PGE2, and forskolin increased cAMP in both hIMCDi and hIMCDt cells. The effects of AVP and PGE2 were greatest in hIMCDi; however, forskolin increased cAMP to the same extent in hIMCDi and hIMCDt. Basal short-circuit current ( I SC) of hIMCDi monolayers was 1.4 ± 0.5 μA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE2, and forskolin increased I SC; the current was reduced by blocking PKA, apical Cl− channels, basolateral NKCC1 (a Na+-K+-2Cl−cotransporter), and basolateral Cl−/HCO[Formula: see text]exchangers. In fluid transport studies, hIMCDi monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCDt monolayers, basal current was not different from zero and cAMP had no effect on I SC. We conclude that AVP and PGE2stimulate cAMP-dependent Cl− secretion by hIMCDi cells, but not hIMCDt cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

scholarly journals Regulation of CFTR Expression and Arginine Vasopressin Activity Are Dependent on Polycystin-1 in Kidney-Derived Cells

2016 ◽  
Vol 38 (1) ◽  
pp. 28-39 ◽  
Author(s):  
Carolina Monteiro de Lemos Barbosa ◽  
Jackson Souza-Menezes ◽  
Andressa Godoy Amaral ◽  
Luiz Fernando Onuchic ◽  
Liudmila Cebotaru ◽  
...  
Keyword(s):  
Arginine Vasopressin ◽  
Chloride Channel ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Renal Cysts ◽  
Fluid Secretion ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Cortical Collecting Duct

Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple, progressive, fluid-filled renal cysts that distort the renal parenchyma, leading to end-stage renal failure, mainly after the fifth decade of life. ADPKD is caused by a mutation in the PKD1 or PKD2 genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. PC-1 is an important regulator of several signaling pathways and PC-2 is a nonselective calcium channel. The CFTR chloride channel is responsible for driving net fluid secretion into the cysts, promoting cyst growth. Arginine vasopressin hormone (AVP), in turn, is capable of increasing cystic intracellular cAMP, contributing to cell proliferation, transepithelial fluid secretion, and therefore to disease progression. The aim of this study was to assess if AVP can modulate CFTR and whether PC-1 plays a role in this potential modulation. Methods: M1 cells, derived from mouse cortical collecting duct, were used in the current work. The cells were treated with 10-7 M AVP hormone and divided into two main groups: transfected cells superexpressing PC-1 (Transf) and cells not transfected (Ctrl). CFTR expression was assessed by immunodetection, CFTR mRNA levels were quantified by quantitative reverse transcription-polymerase chain reaction, and CFTR net ion transport was measured using the Ussing chamber technique. Results: AVP treatment increased the levels of CFTR protein and mRNA. CFTR short-circuit currents were also increased. However, when PC-1 was overexpressed in M1 cells, no increase in any of these parameters was detected. Conclusions: CFTR chloride channel expression is increased by AVP in M1 cells and PC-1 is capable of regulating this modulation.

scholarly journals Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

2008 ◽  
Vol 294 (6) ◽  
pp. C1443-C1453 ◽  
Author(s):  
Dong Wang ◽  
Ying Sun ◽  
Wei Zhang ◽  
Pingbo Huang
Keyword(s):  
Epithelial Cells ◽  
Adenosine Receptors ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Specific Inhibitor ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Intracellular Camp ◽  
Anion Secretion ◽  
Human Airway

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current ( Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.

Characteristics of papillary collecting duct cells in primary culture

1988 ◽  
Vol 255 (6) ◽  
pp. F1160-F1169 ◽  
Author(s):  
R. F. Husted ◽  
M. Hayashi ◽  
J. B. Stokes
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Calf Serum ◽  
Morphological Characteristics ◽  
Short Circuit Current ◽  
Papillary Collecting Duct ◽  
Collecting Duct Cells ◽  
Na Uptake

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

Effect of cAMP agonists on cell pH and anion transport by cultured rat inner medullary collecting duct cells

1996 ◽  
Vol 270 (1) ◽  
pp. F131-F140 ◽  
Author(s):  
C. Zhang ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Anion Transport ◽  
Disulfonic Acid ◽  
Anion Secretion ◽  
Inner Medullary Collecting Duct ◽  
Cell Ph ◽  
Medullary Collecting Duct

The rat inner medullary collecting duct is capable of secreting anions. We previously showed that adenosine 3',5'-cyclic monophosphate (cAMP) stimulates anion secretion; the apical membrane anion exit pathway activated by cAMP appears to be the cystic fibrosis transmembrane conductance regulator Cl- channel. The present experiments were designed to test the hypothesis that the entry pathway across the basolateral membrane is a Cl-/HCO3- exchanger operating in parallel with an Na+/H+ exchanger. We investigated the mechanism by measuring cell Cl-, cell pH, and short-circuit current under a variety of conditions designed to uncover these pathways. cAMP agonists caused little change in cell Cl-, but they produced a consistent intracellular acidification. This acidification was dependent on HCO3-, but not on Cl-, and was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The presence of the basolateral Cl-/HCO3- exchanger was demonstrated by several maneuvers, and its activity was inhibited by DIDS. Applied to the basolateral solution, DIDS did not inhibit the cAMP-dependent anion current but actually stimulated it. We conclude that cAMP-stimulated anion secretion does not require activation of the basolateral Cl-/HCO3- exchanger. The transporter responsible for Cl- entry across the basolateral membrane remains unknown and is not inhibited by a variety of anion transport inhibitors, including DIDS, bumetanide, and hydrochlorothiazide. The cell acidification induced by cAMP appears to be independent of acid secretion and is the result of activation of one or more HCO3- exit pathways that are resistant to DIDS but are inhibited by a nonspecific anion transport inhibitor, 5-nitro-2-(3-phenylpro-pylamino) benzoic acid. We present a revised model for anion transport by the rat inner medullary collecting duct.

CFTR disruption impairs cAMP-dependent Cl−secretion in primary cultures of mouse cortical collecting ducts

2001 ◽  
Vol 281 (3) ◽  
pp. F434-F442 ◽  
Author(s):  
Marcelle Bens ◽  
Jean-Paul Duong Van Huyen ◽  
Françoise Cluzeaud ◽  
Jacques Teulon ◽  
Alain Vandewalle
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Similar Degree ◽  
Transmembrane Conductance Regulator ◽  
Cortical Collecting Duct ◽  
Transmembrane Conductance ◽  
Nacl Transport ◽  
Cystic Fibrosis Transmembrane

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-d-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(−/−)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+ assessed by the short-circuit current ( I sc) method in both CFTR(+/+) and CFTR(−/−) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I sc induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I sc in BAm-treated CFTR(−/−) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of 36Cl− in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal36Cl− flux in CFTR(−/−) CCDs but not the basal-to-apical 36Cl− flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl− in mouse CCD.

AVP stimulates Na+ transport in primary cultures of rabbit cortical collecting duct cells

1992 ◽  
Vol 262 (3) ◽  
pp. F454-F461 ◽  
Author(s):  
C. M. Canessa ◽  
J. A. Schafer
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Cortical Collecting Duct ◽  
Na Transport ◽  
Permeable Membrane ◽  
Ussing Chambers ◽  
Principal And Intercalated Cells

Immunodissected rabbit cortical collecting duct (CCD) cells were grown in primary culture on permeable membrane supports. Transepithelial voltage, Na+, K+, and H+ gradients developed as expected for a mixed population of principal and intercalated cells. The amiloride-sensitive short-circuit current (Isc) was measured in Ussing chambers as an index of Na+ transport via apical membrane Na+ channels. Treatment of the cells in culture with 10 nM aldosterone for 48 h increased Isc from 7.4 +/- 1.4 to 19.3 +/- 3.2 microA/cm2. In contrast to the native rabbit CCD, 220 pM arginine vasopressin (AVP) produced a rapid and stable (greater than 60 min) increase in Isc to 15.8 +/- 2.0 and 29.0 +/- 3.8 microA/cm2 in untreated and aldosterone-treated cultures, respectively. Although prostaglandin E2 (PGE2) inhibits Na+ transport in the native rabbit CCD, it did not in the cultured cells, and it has previously been shown that PGE2 inhibition of AVP-dependent adenosine 3',5'-cyclic monophosphate production is lost in culture (W. K. Sonnenburg and W. L. Smith, J. Biol. Chem. 263: 6155-6160, 1988). We conclude that the development of a stable stimulation of Na+ transport by AVP is linked to the loss of the inhibitory effects of PGE2.

scholarly journals Tolvaptan inhibits ERK-dependent cell proliferation, Cl− secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

2011 ◽  
Vol 301 (5) ◽  
pp. F1005-F1013 ◽  
Author(s):  
Gail A. Reif ◽  
Tamio Yamaguchi ◽  
Emily Nivens ◽  
Hiroyuki Fujiki ◽  
Cibele S. Pinto ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Short Circuit ◽  
Three Dimensional ◽  
Short Circuit Current ◽  
Collagen Matrix ◽  
Fluid Secretion ◽  
Intracellular Camp ◽  
Erk Activity ◽  
Cyst Growth

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl−-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl− secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10−9 M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC50 of ∼10−10 M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl− channel blocker CFTRinh-172, consistent with AVP-induced transepithelial Cl− secretion. Tolvaptan inhibited AVP-induced Cl− secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl−-dependent fluid secretion by human ADPKD cystic cells.

Glucocorticoid receptors mediate mineralocorticoid-like effects in cultured collecting duct cells

1990 ◽  
Vol 259 (4) ◽  
pp. F672-F678 ◽  
Author(s):  
A. Naray-Fejes-Toth ◽  
G. Fejes-Toth
Keyword(s):  
Receptor Antagonist ◽  
Glucocorticoid Receptors ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Cortical Collecting Duct ◽  
Mineralocorticoid Receptor Antagonist ◽  
Ru 486 ◽  
K Secretion

To investigate the direct epithelial effects of corticosteroids on renal ion transport, we studied the influence of the pure glucocorticoid agonist RU 28362 and aldosterone on Na+ and K+ transport in primary cultures of immunodissected rabbit cortical collecting duct (CCD) cells. When grown on permeable supports in a steroid-free medium, CCD monolayers exhibited a lumen-negative transepithelial potential difference (PD) of 5.2 +/- 1.07 mV and a short-circuit current (SCC) of 8.54 +/- 2.2 microA/cm2. Transepithelial resistance averaged 660 +/- 49 omega/cm2. The cultures actively reabsorbed Na+ and secreted K+. Both aldosterone and RU 28362 significantly increased PD and SCC; the effects were time and dose dependent. The effect of RU 28362 was completely prevented by the glucocorticoid receptor antagonist RU 486, whereas ZK 91587, a specific mineralocorticoid receptor antagonist, did not block its effect. Both aldosterone and RU 28362 increased the bath-to-lumen concentration ratio of Na+ while lowering that of K+, indicating an increased Na+ reabsorption and K+ secretion. The number of Na(+)-K(+)-ATPase units was significantly enhanced (approximately 2-fold) by both RU 28362 and aldosterone. These results demonstrate that, in cultured CCD cells, not only aldosterone but also a pure glucocorticoid is able to exert mineralocorticoid-like effects, and this latter effect is mediated by glucocorticoid receptors. Because all parameters studied responded similarly to aldosterone and RU 28362, we speculate that in CCD cells glucocorticoids and mineralocorticoids might act by regulating the same gene(s).

Sign in / Sign up

Export Citation Format

Share Document