Activation of Basolateral Gq-Coupled Receptors in the Distal Convoluted Tubule Induces Acute Dephosphorylation of the Sodium-Chloride Cotransporter (NCC)

Background: Hypertension remains a significant global health issue, contributing to widespread morbidity and mortality, and emphasizing the need for novel treatment strategies (PMID 32024986). At the core of blood pressure regulation lies the distal convoluted tubule (DCT) of the nephron, where sodium balance is carefully controlled by the NaCl cotransporter (NCC). NCC activity is regulated through phosphorylation: when phosphorylated, NCC enhances sodium reabsorption, contributing to higher blood pressure, whereas dephosphorylation promotes sodium excretion, lowering blood pressure. This fine-tuned balance is orchestrated by kinases such as WNK4 and SPAK (PMID 24855283). Current understanding highlights the impact of diet on NCC regulation, with Western diets—often low in potassium—leading to increased NCC activity and phosphorylation, thus contributing to hypertension (PMID 34788509). This highlights the complexity of hypertension and the need to explore additional regulatory mechanisms. In this search for new therapeutic targets, G-protein-coupled receptors (GPCRs), which are the focus of a third of FDA-approved drugs (PMID 29075003), emerge as promising candidates. Recent transcriptomic studies have identified multiple GPCRs in the DCT, suggesting they could play a key role in regulating NCC and, by extension, blood pressure (PMID 34029142). Notably, in vitro studies have shown that GPCRs, such as the calcium-sensing receptor (CaSR), can inhibit Kir4.1 channels (PMID 21084311). In the DCT, Kir4.1 channels function as K+ sensors that modulate NCC activity (PMID 28052988), raising the possibility that GPCR signaling might influence blood pressure regulation through their effects Kir4.1. These findings highlight the importance of exploring GPCR signaling modulation in the DCT as a potential novel therapeutic target for controlling sodium balance and treating hypertension. Methods: To investigate the physiological role of Gq-coupled GPCR activation in the DCT, we used chemogenetic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology (PMID 29351511). This approach enables selective GPCR activation in specific cell types, such as DCT cells, via Cre recombinase technology. We bred DCT-specific inducible Cre recombinase mice (NCC-creERT2) with Gq-coupled DREADD mice to generate DCT-specific DREADD mice. The DREADD receptors can be activated in vivo with the designer drug deschloroclozapine (DCZ), providing a precise tool to study GPCR signaling in the DCT. Results: Immunofluorescence (IF), western blot (WB), and hydrochlorothiazide (HCT) challenge confirmed DREADD expression on the basolateral membrane of DCT cells and preserved NCC function. WB and IF showed that Gq-GPCR activation with DCZ reduced NCC phosphorylation independently of WNK4-SPAK kinase pathway changes. This dephosphorylation was observed even during increased NCC activity/phosphorylation caused by a low-potassium (LK) diet. We then tested whether the effects are direct using ex vivo tubule suspensions from DREADD-expressing mice. These confirmed that DCZ rapidly reduced phosphorylated NCC abundance in normal and LK conditions, consistent with in vivo findings. Conclusions: Gq-coupled GPCR activation in the DCT downregulates NCC activity via a mechanism independent of the WNK4-SPAK pathway, suggesting an alternative GPCR-mediated regulatory pathway. We hypothesize that basolateral Gq-GPCRs in the DCT dephosphorylate NCC through a Kir4.1-involved mechanism. These findings highlight the potential for antihypertensive therapies by targeting GPCR signaling within the DCT, offering innovative strategies for blood pressure management. NIH R01 DK133220, DK51496, U54TR001628.Fondation LeDucq 17CVD05. JBL is supported by a Benjamin J. Lipps Research Fellowship, KidneyCure. JWN is supported by NIDDK DK121737. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.