Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro.
ABSTRACT P2Y receptors couple to G proteins and either mobilize intracellular Ca(2+) or alter cAMP levels to modulate the activity of Ca(2+)- and cAMP-sensitive ion channels. We hypothesize that increased ion transport into the lumen of MDCK cysts can osmotically drive fluid movement and increase cyst size. Furthermore, activation of the adenylate cyclase/cAMP pathway may trigger cell proliferation via an extracellular signal-related kinase cascade. To test this hypothesis, several P2Y receptor inhibitors were used on the MDCK in vitro model of renal cyst formation. The nonspecific P2 receptor inhibitors reactive blue 2 and suramin reduced cyst growth significantly, as did PPADS and, to a lesser extent, the P2Y(1)-specific antagonist MRS2179. Cyst growth was reduced by approximately 50% when ATP was removed from the culture medium with apyrase, although stable analogs of ATP failed to increase cyst size. The nonselective P2X receptor inhibitor Coomassie brilliant blue G was ineffective at reducing cyst growth, suggesting no involvement of P2X receptors. Finally, the presence of selective inhibitors of ERK activation (either PD98059 or U0126) greatly reduced cyst growth, whereas in untreated cysts ERK activity was observed to increase with time. We conclude that stimulation of endogenous P2Y receptors by extracellular ATP increases growth of MDCK cysts via cAMP-dependent activation of the ERK pathway. P2Y receptor antagonists may have therapeutic potential in reducing cyst size and slowing disease progression; although further studies in vitro and in vivo are needed to investigate the specificity and role of these P2Y receptors in renal cystic diseases.
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ABSTRACT: It has been suggested that more than 70% of the renal cysts in patients with autosomal dominant polycystic kidney disease (ADPKD) arise from the collecting duct and that within this segment cysts originate almost exclusively from principal rather than intercalated cells. The mechanisms for this predisposition of principal cells have so far remained elusive. We, therefore, used Madin-Darby canine kidney (MDCK) subclones resembling principal cells and alpha-intercalated cells in a three-dimensional in vitro model to determine differences in cystogenesis and cyst growth, including the response to cyclic adenosine monophosphate (cAMP) elevation and the dependence on ATP signaling. We found that in vitro cysts developed only from principal-like but not from intercalated-like MDCK cell clones. This specificity could be verified in mixed MDCK cultures enriched for principal- or intercalated-like cells. In vitro cyst growth upon elevation of intracellular cAMP was mainly driven by fluid secretion, rather than increased cell proliferation. The cAMP-dependent fluid secretion was found to depend on extracellular adenosine-5'-triphosphate (ATP) and to act synergistically with purinergic signaling, as the use of the ATP scavenger apyrase, as well as the P2 receptor inhibitor suramin, reduced cAMP-driven fluid secretion, while increasing extracellular ATP potentiated cAMP-mediated cyst growth. In conclusion, we provide in vitro evidence for the ability of principal rather than intercalated cells to form cysts, based on a synergism of cAMP and ATP signaling in enhancing apical fluid secretion.Journal of Molecular Medicine 03/2011; 89(3):251-61. · 4.77 Impact Factor
- Journal of Molecular Medicine 03/2011; 89(3):247-50. · 4.77 Impact Factor
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ABSTRACT: The P2X7 receptor participates in purinergic signaling, which may promote the progression of ADPKD. We examined the effects of a P2X7 receptor antagonist and a P2X7 receptor agonist on cyst development in a zebrafish model of polycystic kidney disease in which we knocked down pkd2 by morpholinos. We used live wt-1b pronephric-specific GFP-expressing zebrafish embryos to directly observe changes in the pronephros. Exposure of pkd2-morphant zebrafish to a P2X7 receptor antagonist (oxidized ATP [OxATP]) significantly reduced the frequency of the cystic phenotype compared with either exposure to a P2X7 receptor agonist (BzATP) or with no treatment (P < 0.01). Histology confirmed improvement of glomerular cysts in OxATP-treated pkd2 morphants. OxATP also reduced p-ERK activity and cell proliferation in pronephric kidneys in pkd2 morphants. Inhibition of P2X7 with an additional specific antagonist (A-438079), and through morpholino-mediated knockdown of p2rx7, confirmed these effects. In conclusion, blockade of the P2X7 receptor reduces cyst formation via ERK-dependent pathways in a zebrafish model of polycystic kidney disease, suggesting that P2X7 antagonists may have therapeutic potential in ADPKD.Journal of the American Society of Nephrology 06/2011; 22(9):1696-706. · 8.99 Impact Factor