Abstract: TH-PO323
Utilizing Chemogenetic Tools to Study Sodium-Chloride Cotransporter Regulation
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders
- 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic
Authors
- Bradford, Tanner, Oregon Health & Science University, Portland, Oregon, United States
- Martz, Kevin J., 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
In the Distal Convoluted Tubule (DCT), sodium balance is primarily determined by the sodium-chloride cotransporter (NCC). When phosphorylated, NCC reabsorbs sodium away from the lumen of the nephron. Dephosphorylation of NCC inactivates the transporter, resulting in increased sodium excretion. A similar effect is leveraged by a class of anti-hypertensive medications called thiazides, which block NCC activity. To determine whether GPCRs play a role in NCC regulation, we implemented a cell-specific chemogenetic approach with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). DREADDs stimulate GPCR (G-protein-coupled receptor) signaling within a targeted cell and have been previously used to activate neurons through calcium release. We hypothesize that DREADD activation within the DCT will change NCC activity.
Methods
We bred DCT-specific inducible Cre Recombinase mice (NCC-creERT2) to conditional Gq-GPCR coupled mice (Gq-DREADD) to create DCT-DREADD mice. First, we performed metabolic cage experiments examining thiazide responses within these animals. Next, we activated Gq-GPCR signaling in DCT cells by intraperitoneal injection of the DREADD-specific agonist deschloroclozapine (DCZ). To confirm that DCZ-induced DREADD activation alters electrolyte handling along the nephron we measured sodium excretion (UNaV) following DCZ administration. To investigate kinetics, we performed a timecourse experiment measuring pNCC from 15 minutes to 24 hours after DREADD activation using western blots.
Results
DREADD expression confirmed along the DCT in transgenic mice.
DCT-DREADD mice maintain an intact thiazide response before DREADD activation, indicating DREADD expression does not alter DCT function at baseline.
Intraperitoneal injection of the DREADD-specific agonist deschloroclozapine (DCZ) reduced NCC phosphorylation by more than 80% within 1 hour, with recovery of pNCC between 4-6 hours in both males and females.
Concomitant with the effects on NCC, DCT-DREADD activation increased UNaV by 215% compared to controls.
Conclusion
We posit that the chemogenetic activation of DCT leads to a release of intracellular calcium and rapid activation of the phosphatase required for the dephosphorylation of NCC. We are currently using HEK293 cells co-transfected with DREADD and NCC to test various phosphatase inhibitors, to determine the identity of the DREADD-induced phosphatase.
Funding
- NIDDK Support