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Abstract: PO1089

Chemogenetic Activation of the Distal Convoluted Tubule Enhances Sodium Excretion Through Rapid Dephosphorylation of the Sodium-Chloride Cotransporter

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Martz, Kevin J., Oregon Health & Science University, Portland, Oregon, United States
  • Su, Xiao-Tong, Oregon Health & Science University, Portland, Oregon, United States
  • Maeoka, Yujiro, Oregon Health & Science University, Portland, Oregon, United States
  • Sharma, Avika, Oregon Health & Science University, Portland, Oregon, United States
  • McCormick, James A., Oregon Health & Science University, Portland, Oregon, United States
  • Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
  • Nelson, Jonathan W., Oregon Health & Science University, Portland, Oregon, United States
Background

The distal convoluted tubule (DCT) plays a crucial role in the regulation of sodium and potassium balance, predominantly through its apical sodium chloride entry pathway, NCC, which is activated by N-terminal phosphorylation. This nephron segment is rich in G protein-coupled receptors (GPCR), including ptgfr (prostacyclin F), avpr2 (arginine vasopressin), and others. The role that these GPCRs play regulating DCT function has been a challenge to investigate as they are expressed within multiple cell types that alter kidney physiology. Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology can be used to explore the physiological role of GPCR activation.

Methods

To explore the role of Gq-coupled GPCRs along the DCT, we bred DCT-specific inducible Cre Recombinase mice (NCC-creERT2) to Gq-coupled DREADD mice to create DCT-DREADD mice. We verified the localization of the Gq DREADD protein using immunohistochemistry. We then activated Gq signaling in DCT cells with i.p. injection of DREADD-specific agonist deschloroclozapine (DCZ) and determined the abundance of phosphorylated NCC by Western blot. Lastly, we measured sodium excretion in metabolic cages for 4 hours following DCZ administration in WT compared to DCT-DREADD mice.

Results

We found that the Gq-DREADD protein was specifically expressed within the basolateral membrane of the DCT (Figure A). DCZ injection caused rapid dephosphorylation of NCC within 30 minutes to 15% of the abundance observed in DCT-DREADD mice not treated with DCZ (Figure B, 100 ± 15 vs. 15 ± 3, t-test: p<.001). Injection of DCZ increased sodium excretion (UNaV) by 215% in DCT-DREADD mice compared to wildtype controls (Figure C, 100 ± 13 vs. 215 ± 24, t-test: p<.001).

Conclusion

We conclude that chemogenetic activation of the DCT enhances sodium excretion through rapid dephosphorylation of NCC. These findings support a role for Gq GPCR-mediated regulation of NCC. The DCT-DREADD mouse is a novel model for exploring the molecular mechanisms that underlie the regulation of NCC activity by GPCRs absent the confounding effects of other epithelial cells along the nephron.

Funding

  • NIDDK Support