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Kidney Week

Abstract: FR-PO619

Functional Substrates of Vasopressin-Responsive PARylation in Kidney Collecting Duct Cells

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Park, Eui-Jung, Kyungpook National University, Taegu, Korea (the Republic of)
  • Jung, Hyun Jun, University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Choi, Hyo-Jung, Kyungpook National University, Taegu, Korea (the Republic of)
  • Jang, Hyo-Ju, Kyungpook National University, Taegu, Korea (the Republic of)
  • Park, Hye-Jeong, Kyungpook National University, Taegu, Korea (the Republic of)
  • Kwon, Tae-Hwan, Kyungpook National University, Taegu, Korea (the Republic of)
Background

Poly (ADP-ribose) polymerases (PARPs) regulate cellular progress, such as cell cycle, cell proliferation and differentiation, through molecular regulation of protein-protein interactions and protein stability. In our previous study, cellular poly ADP-ribosylation (PARylation) was induced by vasopressin treatment in mpkCCD cells. The present study aimed to examine regulation of PARylation as a part of vasopressin signaling in renal collecting duct (CD) cells.

Methods

We examined 1) vasopressin-responsive PARylation in mpkCCD cells using pulldown assay of biotin-conjugated NAD+ and immunoprecipitation assay using PAR (poly ADP-ribose) antibody; 2) immunoblotting for PARP1 abundance in nuclei and cytoplasm. Substrate proteins of PARP1 in kidney CD cells were identified from data mining in multiple public databases. Functional enrichment of PARP1 substrates was analyzed using Metascape.

Results

dDAVP (10-9 M, 24 h) remarkably increased abundance of total PARylated proteins in mpkCCD cells in biotin-NAD+ pulldown and PAR immunoprecipitation assays. Interestingly, the cleavage of PARP1 was induced by both short-term (2 h, 6 h) and long-term (24 h, 48 h) dDAVP (10-9M) treatment, suggesting that vasopressin signaling affects PARP1 action. Immunoblots using subcellular fractions of mpkCCD cells confirmed the cleaved form of PARP1 produced by dDAVP treatment was exported to the cytosolic fraction. dDAVP-induced AQP2 mRNA and protein expression was significantly attenuated under siRNA-mediated PARP1 knockdown conditions in mpkCCD cells. From a data mining approach, we identified 752 substrate proteins of PARP1 and 171 proteins interacting with PARP1 in kidney CD cells. Among them, 72 proteins were found across all the matches, suggesting as putative targets of PARP1 in the kidney collecting duct. Functional enrichment analysis revealed that these PARP1 substrates are involved in DNA damage repair, gene transcription, insulator function and DNA methylation, which have been known as cellular functions of PARP1.

Conclusion

Vasopressin-responsive PARylation is accompanied with remarkable changes in protein abundance and cleavage of PARP1 in kidney CD cells. Bioinformatic approaches identified putative PARP1 substrates and functional clustering of the identified substrates that could be involved in vasopressin signaling in the kidney CD cells.

Funding

  • Government Support - Non-U.S.