Concomitant inhibition of TLR-4 and SGLT2 by phloretin and empagliflozin prevents diabetes-associated ischemic acute kidney injury

Abstract

Toll-like receptor-4 (TLR4) and sodium-glucose co-transporter 2 (SGLT2) signaling is involved in the pathogenesis of diabetes-associated kidney diseases. The purpose of this study was to explore the role and effect of phloretin, a TLR4 inhibitor, as an adjuvant therapy to empagliflozin, an SGLT2 inhibitor, in ischemic acute kidney injury (AKI) under diabetic conditions. To achieve this, firstly we induced type 1 diabetes using streptozotocin (55 mg per kg per intraperitoneally (i.p.)) followed by performing bilateral ischemia-reperfusion kidney injury to induce AKI in male Wistar rats. Treatment with phloretin (50 and 100 mg per kg per orally) and empagliflozin (10 mgper kg per orally) alone or in combination was administered to the diabetic rats for 4 days and 1 h before surgery. Moreover, a hypoxia-reperfusion injury was induced using sodium azide in NRK52E cells under a hyperglycemic environment to mimic the in vivo model. The cells were treated with phloretin (50 μM) and empagliflozin (100 nM) for 24 h. For biochemical analysis, plasma and urine samples were used. The kidney tissues were used to perform immunoblotting, histopathology, and immunohistochemistry. Other experiments like immunofluorescence, cell viability assay, and flow cytometry analysis were performed using the in vitro samples. The study outcomes revealed that compared to monotherapy, combination therapy of phloretin and empagliflozin was significantly effective. Phloretin and empagliflozin target the HMGB1/TLR4/MyD88/IK-β/α/NF-κB pathway to reduce inflammation and apoptosis, in addition to their antihyperglycemic effect. Thus, phloretin, a natural dietary supplement, as an adjuvant therapy to empagliflozin can be helpful to reduce empagliflozin-associated side effects, by reducing its clinical dose and increasing its therapeutic efficacy in AKI-diabetes comorbidity.