Effect of D4 Dopamine Receptor on Na+-K+-ATPase Activity in Renal Proximal Tubule Cells

**Background:** Essential hypertension is a complex trait influenced by genetic, epigenetic, and environmental factors. The kidney plays a central role in blood pressure regulation, with renal proximal tubules (RPTs) responsible for approximately 70% of renal sodium reabsorption. Dopamine acts as an autocrine/paracrine substance in the kidney, regulating sodium transport through dopamine receptors. D2-like receptors (including D4 receptors) work in concert with D1-like receptors to decrease the activities of Na+-K+-ATPase (NKA) and other sodium transporters. While D4 dopamine receptors are widely expressed in the kidney including proximal tubules, their role in regulating NKA activity was previously unclear. This study investigated whether D4 receptor activation directly inhibits NKA activity in RPT cells and explored the underlying signaling mechanisms.

**Methods:** Immortalized RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were cultured at 37°C in 95% air and 5% CO2 atmosphere. NKA activity was measured as the difference between total and ouabain-insensitive ATPase activity, normalized to total cell protein content. RPT cells from WKY rats were treated with varying concentrations (10^-9 to 10^-5 mol/L) of the D4 receptor agonist PD168077 for 15 minutes (n=8) to establish concentration-response relationships, and with PD168077 (10^-6 mol/L) over time (n=6) for time-course experiments. Specificity was confirmed using the D4 receptor antagonist L745870 (10^-6 mol/L, n=6). To investigate signaling mechanisms, cells were treated with the NO synthase inhibitor L-NAME (10^-4 mol/L, n=6) or the soluble guanylyl cyclase inhibitor ODQ (10^-5 mol/L, n=5). NO levels in culture medium were measured using the Griess test (n=8), and cGMP levels were measured by ELISA (n=13). D4 receptor expression in total cell lysates (n=4) and plasma membrane fractions (n=3) was assessed by immunoblotting using a polyclonal rabbit anti-rat D4 receptor antibody (1:400), with normalization to GAPDH or NKA.

**Key Results:** Activation of D4 receptors with PD168077 inhibited NKA activity in WKY RPT cells in a concentration-dependent manner, with significant inhibition observed beginning at 10^-7 mol/L. The inhibition was also time-dependent, evident as early as 10 minutes after stimulation and maintained for at least 30 minutes. The D4 receptor antagonist L745870 (10^-6 mol/L) by itself had no effect but completely blocked PD168077-induced NKA inhibition. The NO synthase inhibitor L-NAME (10^-4 mol/L) and the soluble guanylyl cyclase inhibitor ODQ (10^-5 mol/L), each having no effect on NKA activity alone, both eliminated the inhibitory effect of PD168077 on NKA activity. PD168077 treatment significantly increased NO levels in the culture medium and cGMP levels in RPT cells (both P < 0.05 vs. control). In contrast to WKY cells, PD168077 (10^-6 mol/L) had no effect on NKA activity in SHR RPT cells, and this lack of effect was consistent across a range of concentrations (10^-9 to 10^-5 mol/L). Total D4 receptor expression was higher in SHR RPT cells than in WKY RPT cells (P < 0.05), but plasma membrane D4 receptor expression was significantly lower in SHR cells compared to WKY cells (P < 0.05).

**Clinical Implications:** This study identifies a novel mechanism by which D4 dopamine receptors regulate renal sodium transport through NO/cGMP-mediated inhibition of NKA activity. The finding that this regulatory pathway is impaired in SHRs, due to reduced D4 receptor localization at the plasma membrane despite increased total expression, suggests that defective D4 receptor signaling may contribute to enhanced renal sodium reabsorption and the development of hypertension. These results add to the growing evidence that D4 receptors play an important role in blood pressure regulation and may represent a potential therapeutic target for hypertension. However, as the authors note, most evidence to date relies on pharmacological agonists and antagonists, and further studies using D4 receptor knockout models and RPT cells with manipulated D4 receptor expression are needed to confirm these findings and establish causality.