Kidney Week

Abstract: FR-PO568

GPR39 Activation Alters Cytoskeleton Integrity and AQP2 Trafficking in Collecting Duct Cells

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Kui, Mackenzie, Johns Hopkins University, Baltimore, Maryland, United States

Mackenzie Kui

• Praetorius, Helle A., Aarhus Universitet, Aarhus, Midtjylland, Denmark

Helle A. Praetorius, MD, PhD, FASN

• Pluznick, Jennifer L., Johns Hopkins University, Baltimore, Maryland, United States

Jennifer L. Pluznick, PhD

Background

Despite being highly expressed in the kidney, the G-protein coupled receptor, GPR39, has no known role in renal physiology.

Methods

A Gpr39 RNAScope probe was used to localize renal Gpr39. Activation by a GPR39-specific agonist, cpd1324, was used in murine principal kidney cortical collecting duct cells (mpkCCDs); we also utilized GPR39 knockout (KO) cells (made via CRISPR). mpkCCD respond to vasopressin (dDAVP) by expressing and trafficking AQP2 to the apical membrane. Endpoint assays included immunofluorescence (IF), transepithelial resistance (TER), Western Blots (WB) and cAMP assays.

Results

RNAScope for Gpr39 and IF for aquaporin 2 (AQP2) revealed colocalization of Gpr39 transcripts in AQP2-positive cells of the inner medullary and cortical collecting ducts. To study the effect of GPR39 activation, mpkCCD cells were treated with 1µM GPR39 agonist cpd1324 with 10µM cofactor ZnCl_2, or ZnCl₂ alone. After 4 hours, cpd1324 impaired TER (WT/veh=12.5±0.7, WT/cpd = 0.92±0.35, KO/veh = 7.9±0.2, KO/cpd = 8.9±0.3, n=3 per condition (kΩxcm²), disrupted tight junctions (zona occludens-1 (ZO-1), Fig 1A), altered apical membrane morphology (apical marker wheat germ agglutinin (WGA), Fig 1B), targeted F-actin to the basolateral compartment (phalloidin, Fig 1C), and induced AQP2 internalization (Fig 2). After 24 hours of treatment, AQP2 was degraded (by WB), but the cytoskeleton morphology and TER recovered. These effects were not seen in GPR39 KO cells. In cAMP assays, 15-minute dDAVP treatment induced cAMP accumulation from 206.5±0.3 pmol/mL to 213.11±1.3 pmol/mL; however, 4h and 24h pretreatment with cpd1324 only reduced dDAVP-mediated cAMP pools to 211.3±0.6 and 210.0±1.5 pmol/mL, respectively. These data suggest that the effect on AQP2 and the cytoskeleton are only minimally affected by reductions in V2-receptor signaling.

Conclusion

These data suggest that the effect on AQP2 and the cytoskeleton are only minimally affected by reductions in V2-receptor signaling.

Funding

• NIDDK Support