S-27-4: OXIDIZED LDL POTENTIATES ANGIOTENSIN II-INDUCED GALPHAQ ACTIVATION THROUGH THE AT1-LOX1 RECEPTOR COMPLEX IN KIDNEY CELLS

Abstract

Objective: We have reported that the binding of oxidized LDL (oxLDL) to its receptor, LOX-1 activates the G protein-coupled angiotensin II(AII) type 1 receptor (AT1) through membrane complex of the receptors. oxLDL-induced activation of AT1 is characterized by the beta-arrestin-biased signaling with no activation of Galphaq, the main effector of AII-induced cellular response (2021 iScience). In this study, we performed cell experiments to investigate how the co-treatment of oxLDL and AII activates AT1 particularly by focusing on Galphaq-dependent pathway. Design and Methods: We used CHO-cells stably transfected with AT1 (CHO-AT1), LOX-1 and AT1 (CHO-LOX-1-AT1) and two rat kidney cells (NRK49F and NRK52E). Activation of Galphaq and the subsequent induction of Ca influx were quantified by accumulation of IP1 and Fura-2 AM assay, respectively. We also utilized Galphaq-BRET biosensor vector to detect the activity of Galphaq. We assessed mRNA expression of genes associated with oxidative stress, inflammation, and fibrosis in NRK49F and NRK52E. We also assessed protein expression of alphaSMA as a molecular marker of epithelial mesanchymal transition (EMT) after 3 days of the indicated stimulation in NRK49F and NRK52E.The proliferative activity was measured using BrdU assay in NRK49F. Results: The activation of Galphaq was not observed by oxLDL treatment alone but was enhanced by the co-treatment of oxLDL and AII compared to AII alone in CHO-LOX-1-AT1 but not in CHO-AT1. Ca influx was induced not by oxLDL alone but by the co-treatment of oxLDL and a low concentration of AII that was insufficient to induce Ca influx when treated alone in CHO-LOX-1-AT1. We also found that the activation of Galphaq and the induction of Ca influx were detected by the co-treatment of oxLDL and AII but not by single treatment in rat kidney cells. These phenomenons were inhibited by ARB, a Gq inhibitor or the siRNA knockdown of AT1 or LOX-1. Furthermore, combination treatment enhanced the expression of the majority of genes associated with renal injury compared to the single treatment, and EMT was induced only by the combination treatment. These phenomenons were inhibited either by ARB or the Gq inhibitor. The combination treatment enhanced the proliferative activity of kidney fibroblasts compared with single treatment, that was inhibited by ARB, the Gq inhibitor or the siRNA knockdown of AT1 or LOX-1. Conclusions: These findings suggest the synergistic role of the AT1-LOX-1 interaction whereby oxLDL potentiates Ang II-induced Galphaq signaling, leading to the enhancement of renal injury-associated cellular response.