Purinergic receptors profile in the ARPKD cystic epithelia

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Conference Proceeding

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FASEB Journal


Polycystic kidney diseases (PKD) are a group of inherited nephropathies marked with the formation of fluid-filled cysts along the nephron. Growing evidence suggests that autocrine and paracrine effects of purinergic signaling via P2 receptors could detrimentally contribute to cyst expansion. However, little is known about purinergic signaling in renal cyst epithelium, which is characterized by loss of polarity, dedifferentiation and other abnormalities which can lead to purinergic signaling remodeling. We have proposed that ATP via associated intracellular signaling can contribute to cystogenesis by modulation of calcium influx. PCK/CrljCrl-Pkhd1pck/CRL (PCK) rat, an established model of ARPKD, was used here to test this hypothesis. The cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley (SD) rat kidney cortical interstitium as assessed by highly sensitive for ATP enzymatic biosensors (211±55 vs 1082±147 nM for SD and PCK cortex correspondingly, and 2,078±391 nM for cystic fluid). Confocal calcium imaging of the freshly isolated cystic monolayers revealed a stronger response to ATP in a higher range of concentrations (above 100 μM). The removal of extracellular calcium results in the absence of ATP evoked transient, which pointed towards the extracellular (ionotropic) calcium entry in cyst-lining cells rather than the metabotropic P2Y-mediated internal depot. Application of iso-PPADS (a non-selective P2X antagonist) resulted in partial blockade of ATP response (calcium release after ATP was 30.1 ± 0.9, 22.2 ± 1.8 and 19.5 ± 3.2 a.u. in control, after incubation with iso-PPADS, and after washout, respectively) indicating the contribution of P2X4 purinoreceptor in the cystic monolayer. Next, to specifically assess the role of P2X7 in the ATP-mediated calcium influx, we employed AZ10606120, a potent P2X7 receptor antagonist. Application of AZ10606120 (5 μM) resulted in a bunted calcium response to ATP (32.4 ± 2.2, 13.5 ± 6.4, and 11.1 ± 3.1 a.u. of total calcium release in control, after incubation with AZ10606120, and after washout, respectively), which corroborates the commonly hypothesized role of P2X7 in cyst development. Further use of pharmacological agents (α,β-methylene-ATP, 5-BDBD, and NF449) allowed to narrow down potential candidate receptors and suggested a significant involvement of the P2X4 and/or P2X7 signaling axis in the regulation of cytosolic calcium level in the cystic epithelia. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is altered in the ARPKD cystic epithelia towards the prevalence of P2X4 and/or P2X7 receptors, which opens new avenues for the treatment of this disease.




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