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

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.

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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.

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The Lonidamine Derivative H2-Gamendazole Reduces Cyst Formation in Polycystic Kidney Disease

10.1101/2020.09.09.258160 ◽

2020 ◽

Author(s):

Shirin V. Sundar ◽

Xia Zhou ◽

Brenda S. Magenheimer ◽

Gail A. Reif ◽

Darren P. Wallace ◽

...

Keyword(s):

Cell Proliferation ◽

Kidney Disease ◽

Cell Motility ◽

Polycystic Kidney Disease ◽

Short Circuit ◽

Cyst Formation ◽

Polycystic Kidney ◽

Cyst Growth

ABSTRACTAutosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl− secretion. We have examined the effectiveness of the indazole carboxylic acid, H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes and cyst formation using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl−-mediated short-circuit currents in human ADPKD cells at 1 μM and it significantly inhibited both cAMP- and EGF-induced proliferation of ADPKD cells with an IC50 of 5-10 μM. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and hyperphosphorylated Rb levels. H2-GMZ treatment also decreased ErbB2, Akt, and Cdk4, consistent with inhibition of the chaperone Hsp90, and reduced the levels of the CFTR Cl− channel. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Studies using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ (20mg/kg) was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox:Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl− secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.

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Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via cAMP and EGFR-Src-MEK pathways

AJP Renal Physiology ◽

10.1152/ajprenal.00677.2011 ◽

2012 ◽

Vol 303 (7) ◽

pp. F982-F990 ◽

Cited By ~ 31

Author(s):

Kyle Jansson ◽

Anh-Nguyet T. Nguyen ◽

Brenda S. Magenheimer ◽

Gail A. Reif ◽

Lavakumar Reddy Aramadhaka ◽

...

Keyword(s):

Fluid Transport ◽

Egf Receptor ◽

Synergistic Effects ◽

Wild Type Mouse ◽

Renal Cysts ◽

Human Kidney ◽

Collagen Matrix ◽

Fluid Secretion ◽

Cyst Growth

In autosomal-dominant polycystic kidney disease (ADPKD), renal cysts develop by aberrant epithelial cell proliferation and transepithelial fluid secretion. We previously showed that ouabain increases proliferation of cultured human ADPKD cells via stimulation of the EGF receptor (EGFR)-Src-MEK/ERK signaling pathway. We examined whether ouabain affects fluid secretion and in vitro cyst growth of human ADPKD cell monolayers, ADPKD cell microcysts cultured in a three-dimensional collagen matrix, and metanephric organ cultures from Pkd1 m1Bei mice. Physiological concentrations of ouabain alone did not affect net transepithelial basal-to-apical fluid transport in ADPKD monolayers or growth of cultured ADPKD microcysts. In contrast, in the presence of forskolin or 8-bromo-cAMP, ouabain significantly enhanced ADPKD fluid secretion and microcyst expansion. Ouabain exerted this effect by enhancing cAMP-dependent Cl− secretion via the CFTR. Similarly, ouabain accelerated cAMP-dependent cyst enlargement in Pkd1 m1Bei mice metanephroi, with a more prominent response in homozygous than heterozygous mice. Ouabain had no effect on fluid secretion and cystogenesis of normal human kidney cells and caused only slight cystic dilations in wild-type mouse kidneys. The effects of ouabain in ADPKD cells and Pkd1 m1Bei metanephroi were prevented by inhibitors of EGFR (AG1478), Src (PP2), and MEK (U0126). Together, our results show that ouabain, used in physiological concentrations, has synergistic effects on cAMP-mediated fluid secretion and cyst growth, via activation of the EGFR-Src-MEK pathway. These data provide important evidence for the role of ouabain as an endogenous hormone that exacerbates ADPKD cyst progression.

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Characterization of Fibroblasts with Son of Sevenless-1 Mutation

Journal of Dental Research ◽

10.1177/154405910608501115 ◽

2006 ◽

Vol 85 (11) ◽

pp. 1050-1055 ◽

Cited By ~ 13

Author(s):

E.J. Lee ◽

S.I. Jang ◽

D. Pallos ◽

J. Kather ◽

T.C. Hart

Keyword(s):

Cell Proliferation ◽

Three Dimensional ◽

Collagen Matrix ◽

Histological Assessment ◽

M Phase ◽

Son Of Sevenless ◽

Gingival Fibromatosis

Although non-syndromic hereditary gingival fibromatosis (HGF) is genetically heterogeneous, etiologic mutations have been identified only in the Son of Sevenless-1 gene ( SOS1). To test evidence of increased cell proliferation, we studied histological, morphological, and proliferation characteristics in monolayer and three-dimensional cultures of fibroblasts with the SOS1 g.126,142–126,143insC mutation. Histological assessment of HGF gingiva indicated increased numbers of fibroblasts (30%) and increased collagen (10%). Cell proliferation studies demonstrated increased growth rates and 5-bromo-2-deoxyuridine incorporation for HGF fibroblasts. Flow cytometry showed greater proportions of HGF fibroblasts in the G2/M phase. Attachment of HGF fibroblasts to different extracellular matrix surfaces demonstrated increased formation of protrusions with lamellipodia. HGF fibroblasts in three-dimensional culture showed greater cell proliferation, higher cell density, and alteration of surrounding collagen matrix. These findings revealed that increased fibroblast numbers and collagen matrix changes are associated with mutation of the SOS1 gene in vitro and in vivo.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00448.2003 ◽

2004 ◽

Vol 287 (4) ◽

pp. F639-F648 ◽

Cited By ~ 14

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.

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Lithocholic acid attenuates cAMP-dependent Cl− secretion in human colonic epithelial T84 cells

AJP Cell Physiology ◽

10.1152/ajpcell.00350.2015 ◽

2016 ◽

Vol 310 (11) ◽

pp. C1010-C1023 ◽

Cited By ~ 4

Author(s):

Mei Ao ◽

Jada C. Domingue ◽

Nabihah Khan ◽

Fatima Javed ◽

Kashif Osmani ◽

...

Keyword(s):

Lithocholic Acid ◽

Short Circuit ◽

Muscarinic Acetylcholine Receptor ◽

Short Circuit Current ◽

Fluid Secretion ◽

Mek Inhibitor ◽

Inhibitory Effect ◽

Farnesoid X Receptor ◽

Intracellular Camp ◽

T84 Cells

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl− secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447–C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current ( Isc), it did not alter carbachol-stimulated Isc. LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca2+ concentration ([Ca2+]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc. LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc. Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl− current and inhibits a basolateral K+ current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl− secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.

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Sorafenib inhibits cAMP-dependent ERK activation, cell proliferation, and in vitro cyst growth of human ADPKD cyst epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00387.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. F944-F951 ◽

Cited By ~ 44

Author(s):

Tamio Yamaguchi ◽

Gail A. Reif ◽

James P. Calvet ◽

Darren P. Wallace

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Epithelial Cells ◽

Small Molecule ◽

Therapeutic Option ◽

Three Dimensional ◽

Collagen Gel ◽

Erk Signaling ◽

Cyst Growth

In autosomal dominant polycystic kidney disease (ADPKD), aberrant proliferation of the renal epithelial cells is responsible for the formation of numerable fluid-filled cysts, massively enlarged kidneys, and progressive loss of renal function. cAMP agonists, including arginine vasopressin, accelerate cyst epithelial cell proliferation through protein kinase A activation of the B-Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway. The mitogenic effect of cAMP is equally potent and additive to growth factor stimulation. Here, we determined whether Sorafenib (BAY 43–9006), a small molecule Raf inhibitor, inhibits proliferation of cells derived from the cysts of human ADPKD kidneys. We found that nanomolar concentrations of Sorafenib reduced the basal activity of ERK, inhibited cAMP-dependent activation of B-Raf and MEK/ERK signaling, and caused a concentration-dependent inhibition of cell proliferation induced by cAMP, epidermal growth factor, or the combination of the two agonists. Sorafenib completely blocked in vitro cyst growth of human ADPKD cystic cells cultured within a three-dimensional collagen gel. These data demonstrate that cAMP-dependent proliferation of human ADPKD cyst epithelial cells is blocked by Sorafenib and suggest that small molecule B-Raf inhibitors may be a therapeutic option to reduce the mitogenic effects of cAMP on cyst expansion.

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Vibrio choleraeACE stimulates Ca2+-dependent Cl−/HCO3−secretion in T84 cells in vitro

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.3.c567 ◽

2000 ◽

Vol 279 (3) ◽

pp. C567-C577 ◽

Cited By ~ 33

Author(s):

Michele Trucksis ◽

Timothy L. Conn ◽

Steven S. Wasserman ◽

Cynthia L. Sears

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Fluid Secretion ◽

Apical Surface ◽

Rabbit Ileum ◽

T84 Cells ◽

Ussing Chambers ◽

Intracellular Ca ◽

Vitro Model

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current ( Isc) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in Iscand potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl−secretory cell. ACE added to the apical surface alone stimulated a rapid increase in Iscand PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl−and HCO3−. ACE acted synergistically with the Ca2+-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca2+but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca2+as a second messenger and that this toxin stimulates a novel Ca2+-dependent synergy.

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Calcium-stimulated Cl−secretion in Calu-3 human airway cells requires CFTR

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.6.l1208 ◽

1997 ◽

Vol 273 (6) ◽

pp. L1208-L1219 ◽

Cited By ~ 23

Author(s):

Samina Moon ◽

Meetpaul Singh ◽

Mauri E. Krouse ◽

Jeffrey J. Wine

Keyword(s):

Short Circuit ◽

Basolateral Membrane ◽

Short Circuit Current ◽

Fluid Secretion ◽

Disulfonic Acid ◽

Intracellular Camp ◽

Human Airway ◽

Cell Layers ◽

Confluent Cell ◽

Cf Transmembrane Conductance Regulator

Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl−-dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl−channels. Typically, CF disrupts adenosine 3′,5′-cyclic monophosphate (cAMP)-mediated Cl− secretion but spares Ca2+-mediated secretion. However, in CF airway glands, Ca2+-mediated secretion is also greatly reduced. To determine the basis of Ca2+-mediated Cl− secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+ concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current ( I sc) and transepithelial conductance ( G te) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (Δ) in I sc and G te, whereas forskolin stimulated variable and smaller increases. Δ I sc was decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 4,4′-dinitrostilbene-2,2′-disulfonic acid, and calixarene. I sc was measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl− conductance ( G Cl) that was decreased by DPAC but not by DIDS. Apical G Cl could be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive G Cl pathway in the apical membrane and display ∼60% of maximum conductance at rest. Thus elevated [Ca2+]iincreases K+ conductance to force Cl− through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.

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OSMOTIC INHIBITION OF THE NATRIFERIC RESPONSE OF THE TOAD URINARY BLADDER TO VASOPRESSIN

Journal of Endocrinology ◽

10.1677/joe.0.0670119 ◽

1975 ◽

Vol 67 (1) ◽

pp. 119-125

Author(s):

P. J. BENTLEY

Keyword(s):

Urinary Bladder ◽

Water Movement ◽

Short Circuit ◽

Electrical Potential ◽

Short Circuit Current ◽

Toad Bladder ◽

Toad Urinary Bladder ◽

Electrical Potential Difference ◽

Osmotic Gradient

SUMMARY The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.

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