Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro

Abstract

Turner CM, King BF, Srai KS, Unwin RJ. Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro. Am J Physiol Renal Physiol 292: F15-F25, 2007. First published July 18, 2006; doi:10.1152/ajprenal.00103.2006.-P2Y receptors couple to G proteins and either mobilize intracellular Ca 2ϩ or alter cAMP levels to modulate the activity of Ca 2ϩ -and cAMPsensitive 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 P2Y1-specific antagonist MRS2179. Cyst growth was reduced by ϳ50% 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. ATP; polycystic kidney disease; Madin-Darby canine kidney; extracellular signal related kinase; renal cyst growth; ADPKD AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) affects 1 in 1,000 births and accounts for 5% of hemodialysis patients worldwide. Most cases of ADPKD are caused by loss-of-function mutations in the PKD1 and PKD2 genes that encode the proteins polycystin-1 (TRPP1) and polcystin-2 (TRPP2), respectively. The disease is characterized by the progressive development and enlargement of multiple cysts in both kidneys and is the most common inherited cause of renal failure.During cystogenesis, the normal and mainly reabsorptive role of the renal epithelium is reversed to a predominantly secretory one, although the mechanisms underlying this change are poorly understood. However, it is generally thought that cyst expansion is the result of proliferation of cyst wall epithelial cells and accumulation of fluid within the cyst cavity. It is known that the rate of ATP-release is elevated in isolated epithelial cells cultured from human and mouse polycystic kidneys and that the ATP concentration in cyst luminal fluid is signific...

The Gretchen question in autosomal-dominant polycystic kidney disease research

“…The cAMP-dependent fluid secretion was found to depend on extracellular ATP and to act synergistically with purinergic signaling, since the ATP scavenger apyrase and the P2Y receptor inhibitor suramin-reduced cAMP-driven fluid secretion, while increasing extracellular ATP potentiated cAMP-mediated cyst growth. These findings are in accord with the earlier report by Turner et al [6], who showed that stimulation of endogenous P2Y receptors by extracellular ATP increases growth of MDCK cysts via cAMP-dependent activation of the ERK pathway. They suggested that P2Y receptor antagonists could have therapeutic potential in reducing cyst size and slowing disease progression.…”

“…The principal cells mediate the collecting duct's influence on sodium and potassium balance via sodium and potassium channels located on the cell's basolateral membrane. [6] showed years ago that MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3′,5′-cyclic monophosphate. They also observed that 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor, potentiated all agonists.…”

Purinergic signalling in the kidney in health and disease

“…Blockade of the P2X7 receptor with oxidised ATP (or A-438079) reduced cyst formation via ERK-dependent pathways in a zebrafish model of PKD [52]. It was suggested that nucleotides present in the cyst lumen fluid could activate P2Y receptors to increase the growth of MDCK-derived cysts [377]. Attenuated flow-induced ATP release contributes to the absence of flow-sensitive, purinergic [Ca 2+ ] i signalling in human ADPKD cyst epithelial cells [421].…”

Purinergic signaling in the lumen of a normal nephron and in remodeled PKD encapsulated cysts

“…More ideal purinergic microenvironments in remodeled polycystic kidney tissue: a normal purinergic signaling paradigm in an open system becomes detrimental in a closed system Again, in first-principle terms, purinergic ligands are most potent as local mediators or autocoids [111][112][113][114][115][116][117][118][119][120][121][122][123]. They can be potent as paracrine factors; however, diffusion away from the source as well as degradation limits their potency.…”

“…They can be potent as paracrine factors; however, diffusion away from the source as well as degradation limits their potency. Purinergic agonists in vivo are not efficient as blood-borne mediators [111][112][113][114][115][116][117][118][119][120][121][122][123][124]. ATP is degraded rapidly in the general circulation via multiple membrane-bound and secreted ecto-ATPases [111][112][113][114][115][116][117][118][119][120][121][122][123][124].…”