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Characterization of purinergic receptor expression in ARPKD cystic epithelia

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Abstract

Polycystic kidney diseases (PKDs) are a group of inherited nephropathies marked by formation of fluid-filled cysts along the nephron. Growing evidence suggests that in the kidney formation of cysts and alteration of cystic electrolyte transport are associated with purinergic signaling. PCK/CrljCrl-Pkhd1pck/CRL (PCK) rat, an established model of autosomal recessive polycystic kidney disease (ARPKD), was used here to test this hypothesis. Cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley rat kidney cortical interstitium as assessed by highly sensitive ATP enzymatic biosensors. 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 profound reduction of the ATP evoked transient, which suggests calcium entry into the cyst-lining cells is occurring via the extracellular (ionotropic) P2X channels. Further use of pharmacological agents (α,β-methylene-ATP, 5-BDBD, NF449, isoPPADS, AZ10606120) and immunofluorescent labeling of isolated cystic epithelia allowed us to narrow down potential candidate receptors. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is shifted in ARPKD cystic epithelia in an age-related manner towards prevalence of P2X4 and/or P2X7 receptors, which opens new avenues for the treatment of this disease.

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Acknowledgements

Christine Duris (Children’s Hospital of Wisconsin) and Elena Sorokina (Department of Pediatrics, Medical College of Wisconsin) are recognized for excellent technical assistance with immunostaining and RNA isolation, respectively.

Funding

This research was supported by the National Institute of Health grants: R35 HL135749 (to A.S.); R00 HL116603 and P30 DK090868 via Baltimore PKD Center P&F Grant (to TSP); R00 DK105160 and PKD Foundation (221G18a) award (to DVI); T32 HL134643 and CVC A.O. Smith Fellowship (to C.A.K); and American Heart Association grants: 16EIA26720006 (to A.S.) and 17SDG33660149 (to OP); and Department of Veteran Affairs I01 BX004024 (AS).

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Author notes

  1. Daria V. Ilatovskaya

    Present address: Division of Nephrology, Department of Medicine, Medical University of South Carolina, Charleston, SC, USA

  2. Tengis S. Pavlov

    Present address: Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Boulevard, E&R Bldg, 7045, Detroit, MI, 48202, USA

  3. Oleg Palygin and Daria V. Ilatovskaya contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Oleg Palygin, Daria V. Ilatovskaya, Vladislav Levchenko, Christine A. Klemens, Lashodya Dissanayake, Anna Marie Williams, Tengis S. Pavlov & Alexander Staruschenko

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  1. Oleg Palygin
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  2. Daria V. Ilatovskaya
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  3. Vladislav Levchenko
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Correspondence to Tengis S. Pavlov or Alexander Staruschenko.

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Oleg Palygin declares that he/she has no conflict of interest.

Daria V. Ilatovskaya declares that he/she has no conflict of interest.

Vladislav Levchenko declares that he/she has no conflict of interest.

Christine A. Klemens declares that he/she has no conflict of interest.

Lashodya Dissanayake declares that he/she has no conflict of interest.

Anna Marie Williams declares that he/she has no conflict of interest.

Tengis S. Pavlov declares that he/she has no conflict of interest.

Alexander Staruschenko declares that he/she has no conflict of interest.

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Animal use and welfare adhered to the NIH Guide for the Care and Use of Laboratory Animals following a protocol reviewed and approved by the IACUC at the Medical College of Wisconsin.

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Palygin, O., Ilatovskaya, D.V., Levchenko, V. et al. Characterization of purinergic receptor expression in ARPKD cystic epithelia. Purinergic Signalling 14, 485–497 (2018). https://doi.org/10.1007/s11302-018-9632-5

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