CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

Background/Objective Kidney stones are a globally prevalent urological disease, with calcium oxalate stones being the most common type. Their pathogenesis is complex, and postoperative recurrence rates are high. The calcium-sensing receptor (CaSR) and solute carrier family 26 member 6 (SLC26A6) play key roles in hypercalciuria and hyperoxaluria, respectively. However, the intrinsic relationship and regulatory mechanism between them in kidney stone formation remain unclear. This study aims to investigate whether CaSR regulates SLC26A6 expression through a specific signaling pathway, thereby playing a role in experimental calcium oxalate kidney stone formation in rats. Methods In vivo , a calcium oxalate kidney stone model was established in Wistar rats by intragastric administration of 1% ethylene glycol (E.G.,) and 1% ammonium chloride. Rats were divided into six groups: blank control (NC), E.G., model (E.G.,), CaSR agonist (CaSR-a), CaSR inhibitor (CaSR-i), protein kinase A inhibitor (PKA-i), and Forkhead box protein O4 inhibitor (FOXO4-i) groups. Urinary calcium and oxalate levels were measured. Kidney crystal formation was observed via Hematoxylin and Eosin (HE) staining and Pizzolato’s staining. Protein and mRNA expression of CaSR, p-protein kinase A (PKA) substrate, p-FOXO4 (Thr451), and SLC26A6 in kidney tissues were detected by Western blotting, immunohistochemistry, and Real-Time quantitative PCR (RT-qPCR). In vitro , rat renal tubular epithelial cells (NRK-52E) were intervened with calcium oxalate monohydrate (COM) crystals and treated with agonists or inhibitors of CaSR, PKA, and Forkhead box protein O4 (FOXO4). Pathway-related protein expression was detected by Western blotting. A dual-luciferase reporter gene assay was used to validate the transcriptional regulation of the SLC26A6 promoter by FOXO4. Results Compared to the NC group, the, E.G., group showed significantly increased urinary calcium and oxalate concentrations, increased renal crystal deposition, and upregulated expression of CaSR, p-PKA substrate, p-FOXO4, and SLC26A6. Activating CaSR (CaSR-a group) further exacerbated these phenomena, whereas inhibiting CaSR (CaSR-i group), PKA (PKA-i group), or FOXO4 (FOXO4-i group) significantly alleviated crystal formation and reduced SLC26A6 expression. Cell experiments confirmed that activating CaSR enhanced PKA and FOXO4 phosphorylation and SLC26A6 expression; activating PKA enhanced FOXO4 phosphorylation and SLC26A6 expression; activating FOXO4 upregulated SLC26A6 expression. The dual-luciferase reporter gene assay showed that FOXO4 functionally regulates SLC26A6 promoter and regulates its transcriptional activity. Conclusion During the formation of experimental calcium oxalate kidney stones in rats, CaSR activation promotes PKA-mediated FOXO4 phosphorylation, leading to upregulation of SLC26A6 expression through transcriptional mechanisms. This signaling axis promotes urinary oxalate excretion and contributes to kidney stone formation. This study reveals the potential role of the novel CaSR-PKA-FOXO4-SLC26A6 signaling pathway in the pathogenesis of kidney stones.