Dapagliflozin Attenuates NKCC2 Protein Expression and Alleviates Diabetic Kidney Disease in Salt Loaded-Hypertensive Diabetic Db/Db Mice.

Dapagliflozin, a potent sodium-glucose cotransporter 2 (SGLT2) inhibitor, slows diabetic kidney disease (DKD) progression and reduces blood pressure in diabetic mice, potentially via modulation of tubular sodium transporters and inflammatory pathways. RING-type E3 ligases, including cullin-based complexes, regulate the stability of with-no-lysine kinases (WNK1/4), key upstream activators of the sodium-potassium-chloride cotransporter 2 (NKCC2). Twelve-week-old male db/db mice were placed on a high-salt diet to induce hypertension and accelerate DKD, then randomized to receive dapagliflozin or vehicle by oral gavage for 14 days. Metabolic cages were used for 24-h urine collection, and glomerular filtration rate was measured using FITC-sinistrin clearance. Blood pressure was assessed by tail-cuff plethysmography. Kidneys were processed for histology, immunohistochemistry, Western blotting, and label-free proteomics. Dapagliflozin markedly reduced systolic blood pressure and improved renal injury on Periodic acid-Schiff staining, accompanied by reduced lactic acid accumulation and a lower urinary albumin-to-creatinine ratio. Proteomic profiling demonstrated differential abundance of multiple signaling proteins between groups. Western blotting showed increased expression of cullin 1, 2, and 3 family members, with a significant reduction in NKCC2 and aquaporin-2 protein levels in dapagliflozin-treated mice. In cultured mouse thick ascending limb cells, dapagliflozin inhibited high glucose-induced activation of the NLRP3 inflammasome, indicating an additional anti-inflammatory effect. Dapagliflozin confers renoprotection in salt-loaded hypertensive db/db mice by lowering blood pressure, attenuating tubular and glomerular injury, reducing NKCC2 abundance possibly via enhanced cullin-mediated degradation, and attenuating high-glucose-induced NLRP3 protein expression.