Defense Date

2022

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Dr. Joseph Ritter

Abstract

Renal handling of sodium (Na+) is important for the regulation of extracellular fluid volume and blood pressure. Disruption in Na+ balance due to increased Na+ reabsorption by the kidney may contribute to mechanisms of salt-sensitive hypertension, which is observed in ~50% of patients with essential hypertension. The renal proximal tubules are responsible for a majority of Na+ reabsorption primarily occurring by Na+ transporters in the Na+-H+ exchanger (NHE) and Na+-glucose cotransporter (SGLT) families. Marijuana and other exogenous cannabinoids are known diuretic and natriuretic agents, and renal tubules in the cortex and outer medulla possess an endogenous cannabinoid signaling system. Cannabinoid type 1 receptors (CB1) are localized both apically and intracellularly in mouse proximal tubule cells. Based on these relationships, we hypothesized that activation of CB1 receptors in renal proximal tubule cells will decrease Na+ reabsorption by inhibiting NHE isoform 3 (NHE3). To investigate the effects on Na+ influx in the proximal tubule, NRK-52E cells were loaded with SBFI-AM, a Na+-sensing fluorometric dye, and the uptake of Na+ in response to increasing bath Na+ concentration from 0 to 150 mM was measured. Treatment with the CB1-selective agonist, arachidonoyl-2-chloroethylamine (ACEA), decreased the flux of Na+ into NRK-52E cells based on both maximum Na+ response and total Na+ flux measured by the area under the curve (AUC) of the SBFI-FIR (fluorescence intensity ratio; F340/F380) vs. time curve. In contrast, rimonabant (Rim), a CB1-selective inverse agonist, had an opposite effect with a larger magnitude, increasing the Na+ influx in both measurements. Analysis of the slope of the rising phase of intracellular Na+ suggested that CB1 blockade activates a Na+ entry mechanism. Using inhibitors of SGLT2 (empagliflozin, Empag), NHE1 (zoniporide, Zon) and NHE3 (tenapanor, Ten), we found that total Na+ influx was significantly inhibited by Empag (~9%) and Zon (25%), but Ten had no effect. In contrast, Ten was the only Na+ transport inhibitor to significantly inhibit the Rim response, although its effect was weak (22% inhibition). To determine if Rim acts by activating NHE activity, its effect on intracellular pH (pHi) was monitored in response from 0-150 mM using the pH-sensitive fluorometric dye BCECF-AM. The presence of functional NHE activity was supported by the observed alkalinization of the pHi after addition of Na+ and by the inhibition of this response by either Zon or Ten. However, the effect of Zon was significantly stronger, indicating that the H+ efflux is predominantly via NHE1 (remember Na+ goes in and H+ comes out via both NHE3 and NHE1). The lack of the enhancing effect by Rim on the alkalinization response, instead inhibiting it, does not support general activation of NHEs as the primary Na+-elevating mechanism of Rim. The interaction of Rim with insulin, a known NHE3 inducer, was studied. Insulin increased Na+ influx by a magnitude similar to that of Rim, and the combination of Rim + insulin appeared to be additive. Rim and insulin were also noted to each decrease extracellular pH (pHo) and glucose levels, and these effects were additive when cells were treated with both Rim + insulin. Addition of pyruvate inhibited the enhancing effect of Rim on Na+ flux and the reduced pHo and glucose responses to Rim and insulin both individually and in combination. Our data support that CB1 signaling regulates NHE3 activity in proximal tubule cells. However, given that NHE3 inhibition does not completely block Rim’s effect, some additional mechanism may account for most of Rim’s enhancing effect on Na+ uptake, involving another Na+ entry mechanism or possibly Na+ exit. A better understanding of these mechanisms may provide fresh insights into the possible therapeutic use of CB1 inverse agonists for hypertension and obesity- and diabetic-nephropathy.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-10-2022

Available for download on Sunday, May 09, 2027

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