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.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

An Investigation of the Role of Possible Neural Mechanisms in Cholera Toxin-Induced Secretion in Rabbit Ileal Mucosa in Vitro

1989 ◽  
Vol 77 (2) ◽  
pp. 161-166 ◽  
Author(s):  
K. J. Moriarty ◽  
N. B. Higgs ◽  
M. Woodford ◽  
L. A. Turnberg
Keyword(s):  
Cholera Toxin ◽  
Fluid Transport ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Rabbit Ileum ◽  
Ileal Mucosa

1. Cholera toxin stimulates intestinal secretion in vitro by activation of mucosal adenylate cyclase. However, it has been proposed that cholera toxin promotes secretion in vivo mainly through an indirect mechanism involving enteric neural reflexes. 2. We examined this hypothesis further by studying the influence of neuronal blockade on cholera toxin-induced changes in fluid transport across rabbit ileum in vitro. Mucosa, stripped of muscle layers, was mounted in flux chambers and luminal application of crude cholera toxin (2 μg/ml) caused a delayed but sustained rise in the short-circuit current, electrical potential difference and Cl− secretion. Pretreatment with the nerve-blocking drug, tetrodotoxin (5 × 10−6 mol/l serosal side), failed to influence the secretory response to cholera toxin, and addition of tetrodotoxin at the peak response to cholera toxin also had no effect. 3. That tetrodotoxin could block neurally mediated secretagogues was confirmed by the demonstration that the electrical responses to neurotensin (10−7 mol/l and 10−8 mol/l) were blocked by tetrodotoxin (5 × 10−6 mol/l). Furthermore, the response to cholera toxin of segments of ileum, which included the myenteric, submucosal and mucosal nerve plexuses, was not inhibited by tetrodotoxin. 4. We conclude that cholera toxin-induced secretion in rabbit ileum in vitro is not mediated via a neurological mechanism.

scholarly journals Soluble (pro)renin receptor regulation of ENaC involved in aldosterone signaling in cultured collecting duct cells

2020 ◽  
Vol 318 (3) ◽  
pp. F817-F825 ◽  
Author(s):  
Fei Wang ◽  
Renfei Luo ◽  
Kexin Peng ◽  
Xiyang Liu ◽  
Chuanming Xu ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Α Subunit ◽  
Duct Cells ◽  
Nadph Oxidase 1 ◽  
Collecting Duct Cells ◽  
Two Phases

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na+ channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the β-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not β- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na+ transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.

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.

Abstract P229: Site-1 Protease-derived Soluble (pro) Renin Receptor Mediates Aldosterone-induced Enac Activation In The Collecting Duct

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Fei Wang ◽  
Renfei Luo ◽  
KEXIN PENG ◽  
Peng Wu ◽  
Xiyang Liu ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Vehicle Group ◽  
Α Subunit ◽  
Collecting Duct Cells

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na + channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), generated by site-1 protease (S1P), a newly identified PRR cleavage protease, in ENaC regulation, and further tested its relevance to Aldo signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM for 24 h induced a significant increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique ( I eq : 7.5 ± 0.7 μA/cm 2 in sPRR group vs. 3.5 ± 0.5 μA/cm 2 in vehicle group, n = 6, p < 0.01) . In primary cultured rat IMCD cells, the same sPRR-His treatment induced a 1.7 fold increase in protein expression of the α-subunit but not β- or γ-subunit of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The upregulation of α-ENaC transcription depended on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Using chamber determination of short-circuit current showed that Aldo-induced ENaC activity was almost completely abolished by PF-429242 (PF), a S1P inhibitor, and the response was restored by supplement of sPRR-His ( I eq : 7.2 ± 0.7 μA/cm 2 in Aldo group vs. 5.0 ± 0.3 μA/cm 2 in Aldo/PF group vs. 6.8 ± 0.3 μA/cm 2 in Aldo/PF/sPRR-His group, n = 5, p < 0.05). Medium sPRR was elevated by Aldo and inhibited by PF. Male C57BL/6 mice were pretreated with PF (30 mg/kg/day) or vehicle via minipump, followed by 3 days of aldosterone (0.2 mg/kg/day via a second minipump). Amiloride-sensitive Na+ current in freshly isolated CCD as measured by using patch clamp lower in Aldo + PF group than in Aldo group. Together, these results support an essential role of S1P-derived sPRR in mediating Aldo-induced ENaC activation.

scholarly journals Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

2012 ◽  
Vol 302 (7) ◽  
pp. F801-F808 ◽  
Author(s):  
Takamitsu Saigusa ◽  
Ryan Reichert ◽  
Jennifer Guare ◽  
Brian J. Siroky ◽  
Monika Gooz ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Renal Cysts ◽  
Short Circuit Current ◽  
Polycystic Kidney ◽  
Collecting Duct Cells ◽  
And Function ◽  
Apical Membranes ◽  
Apical Localization

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (−), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/ Ift88 gene). Interestingly, only cilia (−) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (−) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (−) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current ( Isc) in cilia (−) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of Isc in cilia (−) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

Fluorescent characterization of collecting duct cells: a second H+-secreting type

1988 ◽  
Vol 255 (5) ◽  
pp. F1003-F1014 ◽  
Author(s):  
G. J. Schwartz ◽  
L. M. Satlin ◽  
J. E. Bergmann
Keyword(s):  
Collecting Duct ◽  
Apical Surface ◽  
Cortical Collecting Duct ◽  
Cytosolic Ph ◽  
Duct Cells ◽  
Physiological Properties ◽  
Collecting Ducts ◽  
Outer Stripe ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

We have used three fluorescent probes to label acid-base transporting cells with specific physiological properties in the rabbit collecting duct. Rhodamine albumin identified cells active in luminal endocytosis; rhodamine peanut agglutinin (PNA) identified cells with apical surface PNA ligands; and 6-carboxyfluorescein (6-CF) diacetate identified cells with alkaline pH or acetazolamide-sensitive esterase activity. More than 90% of all cells identified by PNA or rhodamine albumin selectively concentrated 6-CF. Axial heterogeneity of the identified cells was clearly evident along the collecting duct. In the midcortical collecting duct the predominant labeled cell (108 +/- 15/mm) was positive for PNA and 6-CF. These cells were less prevalent (69 +/- 10/mm) in inner cortical collecting ducts and absent from the outer medullary collecting duct. Cells that labeled only with 6-CF (with no detectable luminal endocytosis or PNA binding) showed the opposite distribution. They were the predominant identified cell in the inner stripe of the outer medulla (126 +/- 20/mm), and were less common in the cortical collecting duct. Because the former segment secretes H+, it was likely that these cells were H+-secreting cells. We used excitation ratio microspectrofluorometry of 6-CF to measure cytosolic pH (pHi approximately 7.2) and found evidence for a basolateral DIDS-sensitive Cl- -HCO3- exchanger and a Na+-independent luminal H+ pump. The previously described endocytic H+-secreting cell was seen at its highest concentration in the outer stripe (39 +/- 6/mm). Finally, 5-10% of identified cells did not stain selectively with 6-CF in cortical collecting ducts (solely endocytic or PNA binding). The function of these latter types could not be established. These studies suggest that the distribution and number of these populations of cells may determine the direction and magnitude of H+ transport along the collecting duct.

Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors

2015 ◽  
Vol 308 (5) ◽  
pp. F450-F458 ◽  
Author(s):  
Morag K. Mansley ◽  
Winfried Neuhuber ◽  
Christoph Korbmacher ◽  
Marko Bertog
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Nerve Fibers ◽  
Good Evidence ◽  
Fine Tuning ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na+ absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na+ excretion takes place. Here, the appropriate regulation of transepithelial Na+ transport, mediated by the amiloride-sensitive epithelial Na+ channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na+ transport, we performed short-circuit current ( Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na + absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

Endothelin-1 inhibits AVP-stimulated osmotic water permeability in rat inner medullary collecting duct

1991 ◽  
Vol 261 (6) ◽  
pp. F951-F956 ◽  
Author(s):  
R. Oishi ◽  
H. Nonoguchi ◽  
K. Tomita ◽  
F. Marumo
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Renal Plasma Flow ◽  
Inhibitory Effect ◽  
Osmotic Water Permeability ◽  
Camp Generation ◽  
Osmotic Water ◽  
Collecting Ducts ◽  
Medullary Collecting Duct

Endothelin causes diuresis despite an accompanying decrease in glomerular filtration rate and renal plasma flow. Binding sites for endothelin are located not only in glomeruli but also in the inner medulla, possibly in inner medullary collecting ducts (IMCD). To determine whether endothelin has a direct tubular effect, effects of endothelin on water and urea transport were investigated using isolated microperfusion of rat IMCD segments in vitro. Endothelin, at 10(-10) and 10(-8) M, reversibly inhibited 10(-11) M arginine vasopressin (AVP)-stimulated osmotic water permeability (Pf) by 18 and 24%, respectively. Endothelin (10(-8) M) also inhibited Pf by 23% in the presence of a much higher dose of AVP (10(-9) M), whereas endothelin had no effect on Pf in the absence of AVP. On the other hand, 10(-8) M endothelin did not inhibit Pf stimulated by 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). Endothelin had no inhibitory effect on AVP-stimulated urea permeability. These data suggest that endothelin can cause diuresis by inhibiting AVP-stimulated Pf in IMCD and that the site of action is previous to cAMP generation.

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