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

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.

Download Full-text

Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v4111855 ◽

1994 ◽

Vol 4 (11) ◽

pp. 1855-1860

Author(s):

B A Stoos ◽

O A Carretero ◽

J L Garvin

Keyword(s):

Nitric Oxide ◽

Maximum Rate ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Sodium Absorption ◽

Cortical Collecting Duct ◽

Duct Cells ◽

Collecting Duct Cells

Previously, it has been shown that the addition of bradykinin (Bk) to M-1 cortical collecting duct cells in the presence of endothelial cells decreased short-circuit current (Isc), a measure of net active transport. This effect is presumably due to the release of endothelium-derived nitric oxide (EDNO), because the decrease in Isc could be blocked with Nw-nitro-L-arginine. To show that the inhibition of Isc was due to EDNO rather than prostaglandins, the ability of a cyclooxygenase inhibitor to block the inhibition was examined. When Bk was added to cocultures in the presence of meclofenamate (10(-5) M), Isc decreased from 62 +/- 12 to 44.5 +/- 7 muA/cm2, not significantly different from that in the absence of meclofenamate. To determine if the effect was due to an alteration of sodium absorption, Bk (10(-9) M) was added to cocultures, resulting in a decrease in Na flux from 28 +/- 3.1 to 20 +/- 2.2 nEq/min (P < 0.05), with Isc decreasing from 25 +/- 2.4 to 20 +/- 3.6 nEq/min (P < 0.05). To examine if the inhibition was due to blockade at the apical membrane sodium channel or the basolateral Na+/K+ ATPase, the cation-selective ionophore nystatin was used. Nystatin reversed the effect of EDNO on Isc. The effects of EDNO on Na+/K+ ATPase were also measured directly. Under maximum rate conditions, the Na+/K+ ATPase activity of control and Bk-treated cocultures was 5.2 +/- 0.3 and 6.8 +/- 1.0 nmol/min per square centimeter, respectively (not significantly different).(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.4.f552 ◽

1999 ◽

Vol 277 (4) ◽

pp. F552-F559 ◽

Cited By ~ 37

Author(s):

David E. McCoy ◽

Amanda L. Taylor ◽

Brian A. Kudlow ◽

Katherine Karlson ◽

Margaret J. Slattery ◽

...

Keyword(s):

Plasma Membrane ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Extracellular Nucleotides ◽

P2x Receptor ◽

Apical Plasma Membrane ◽

Receptor Subtypes ◽

Nacl Transport

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current[Formula: see text])] and stimulated Cl− secretion [measured via Cl−short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.00165.2002 ◽

2002 ◽

Vol 283 (6) ◽

pp. F1337-F1350 ◽

Cited By ~ 27

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.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.00436.2019 ◽

2020 ◽

Vol 318 (3) ◽

pp. F817-F825 ◽

Cited By ~ 4

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.

Download Full-text

Characteristics of papillary collecting duct cells in primary culture

AJP Renal Physiology ◽

10.1152/ajprenal.1988.255.6.f1160 ◽

1988 ◽

Vol 255 (6) ◽

pp. F1160-F1169 ◽

Cited By ~ 9

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.

Download Full-text

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

Hypertension ◽

10.1161/hyp.76.suppl_1.p229 ◽

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.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.00548.2014 ◽

2015 ◽

Vol 308 (5) ◽

pp. F450-F458 ◽

Cited By ~ 12

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.

Download Full-text

Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

The Journal of General Physiology ◽

10.1085/jgp.200409124 ◽

2004 ◽

Vol 125 (1) ◽

pp. 81-101 ◽

Cited By ~ 130

Author(s):

Michael B. Butterworth ◽

Robert S. Edinger ◽

John P. Johnson ◽

Raymond A. Frizzell

Keyword(s):

Hormonal Regulation ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Membrane Capacitance ◽

Apical Surface ◽

Cortical Collecting Duct ◽

Ussing Chambers ◽

Camp Stimulation

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Download Full-text

Decreased amiloride-sensitive Na+ absorption in collecting duct principal cells isolated from BPK ARPKD mice

AJP Renal Physiology ◽

10.1152/ajprenal.00169.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. F244-F254 ◽

Cited By ~ 41

Author(s):

Elias I. Veizis ◽

Cathleen R. Carlin ◽

Calvin U. Cotton

Keyword(s):

Fluorescent Protein ◽

Kidney Diseases ◽

Collecting Duct ◽

Short Circuit ◽

Expression Patterns ◽

Renal Cysts ◽

Short Circuit Current ◽

Sodium Absorption ◽

Principal Cells ◽

Green Fluorescent

The main feature of polycystic kidney diseases (PKD) is formation and progressive enlargement of renal cysts. Alterations in epithelial cell proliferation, extracellular matrix, and ion transport are thought to contribute to cyst enlargement and loss of renal function. Abnormal Cl- secretion is implicated in cyst enlargement in autosomal dominant PKD (ADPKD), but little is known about transport abnormalities in autosomal recessive PKD (ARPKD). We developed a method to isolate collecting duct (CD) principal cells (site of the lesion in ARPKD) from normal and ARPKD mice. A transgenic mouse (Hoxb7/GFP) in which enhanced green fluorescent protein (GFP) is expressed in CDs was bred with an ARPKD mouse (BPK), and GFP-positive cells from normal and cystic mice were selected by fluorescence-activated cell sorting. GFP-positive CD cells (>95 ± 3%) obtained from either normal or cystic mice formed high-resistance, polarized epithelial monolayers. Expression patterns for marker proteins and the presence of a central cilium confirmed that the monolayers are composed of principal cells. Under basal conditions, the Cl- secretory responses elicited by elevation of cAMP or calcium were not significantly different between normal and cystic monolayers. In contrast, the amiloride-sensitive short-circuit current was significantly reduced in monolayers of cells isolated from cystic mice (12.9 ± 1.6 μA/cm2; n = 10) compared with monolayers of cells isolated from normal mice (27.3 ± 3.4 μA/cm2; n = 12). The results of these studies suggest that epithelial sodium channel-mediated sodium absorption is decreased in principal cells of ARPKD CD cysts and that the reduction in sodium absorption may contribute to the accumulation of luminal fluid.

Download Full-text

Heightened epithelial Na+ channel-mediated Na+ absorption in a murine polycystic kidney disease model epithelium lacking apical monocilia

AJP Cell Physiology ◽

10.1152/ajpcell.00339.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. C952-C963 ◽

Cited By ~ 34

Author(s):

Dragos Olteanu ◽

Bradley K. Yoder ◽

Wen Liu ◽

Mandy J. Croyle ◽

Elisabeth A. Welty ◽

...

Keyword(s):

Kidney Disease ◽

Polycystic Kidney Disease ◽

Collecting Duct ◽

Short Circuit ◽

Na Channel ◽

Cortical Collecting Duct ◽

Polycystic Kidney ◽

Luminal Membrane ◽

And Function ◽

Amiloride Analogs

The Tg737° rpk autosomal recessive polycystic kidney disease (ARPKD) mouse carries a hypomorphic mutation in the Tg737 gene. Because of the absence of its protein product Polaris, the nonmotile primary monocilium central to the luminal membrane of ductal epithelia, such as the cortical collecting duct (CCD) principal cell (PC), is malformed. Although the functions of the renal monocilium remain elusive, primary monocilia or flagella on neurons act as sensory organelles. Thus we hypothesized that the PC monocilium functions as a cellular sensor. To test this hypothesis, we assessed the contribution of Polaris and cilium structure and function to renal epithelial ion transport electrophysiology. Properties of Tg737° rpk mutant CCD PC clones were compared with clones genetically rescued with wild-type Tg737 cDNA. All cells were grown as polarized cell monolayers with similarly high transepithelial resistance on permeable filter supports. Three- to fourfold elevated transepithelial voltage ( Vte) and short-circuit current ( Isc) were measured in mutant orpk monolayers vs. rescued controls. Pharmacological and cell biological examination of this enhanced electrical end point in mutant monolayers revealed that epithelial Na+ channels (ENaCs) were upregulated. Amiloride, ENaC-selective amiloride analogs (benzamil and phenamil), and protease inhibitors (aprotinin and leupeptin) attenuated heightened Vte and Isc. Higher concentrations of additional amiloride analogs (ethylisopropylamiloride and dimethylamiloride) also revealed inhibition of Vte. Cell culture requirements and manipulations were also consistent with heightened ENaC expression and function. Together, these data suggest that ENaC expression and/or function are upregulated in the luminal membrane of mutant, cilium-deficient orpk CCD PC monolayers vs. cilium-competent controls. When the genetic lesion causes loss or malformation of the monocilium, ENaC-driven Na+ hyperabsorption may explain the rapid emergence of severe hypertension in a majority of patients with ARPKD.

Download Full-text