Insights into the apelin-13 effects on renal function and NHE3 activity following ischemia/reperfusion-induced acute kidney injury

Guilherme Lopes Gonçalves ^* Juliana Martins da Costa Pessoa Mariana Charleaux de Ponte Heitor Macedo Braz Maria Oliveira-Souza ^*

• University of São Paulo, São Paulo, Brazil

Introduction: Acute kidney injury (AKI) is recognized as a clinical syndrome characterized by a rapid decline in renal function with severity variations. In this context, tubular function is impaired in ischemic AKI. Although there are no effective therapies for AKI, many compounds have been described to lessen kidney injury, such as apelin-13. Considering the relevance of proximal tubular cells in maintaining fluid and electrolyte homeostasis, the actions of apelin-13 on tubular injury or sodium proximal transport are still unclear. Thus, this study aimed to evaluate the effects of exogenous administration of apelin-13 in the renal ischemia/reperfusion (I/R) model, with a particular interest in renal function, injury markers and tubular proliferation. Methods: Male C57BL/6 mice were previously treated with vehicle or a high dose of apelin-13 (200 µg/kg/day) and submitted to kidney bilateral ischemia procedure for 30 min or sham surgery. Mice were euthanized two days after the ischemic procedure by exsanguination. Renal function was assessed by plasma urea levels and creatinine clearance. Tubular injury was evaluated by hematoxylin-eosin-stain. Kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), megalin, Ki67, and phospho ERK/1/2 (Thr202/Tyr204) were evaluated by immunohistochemistry or immunoblotting experiments. Also, murine proximal tubular cells (TKPTS) were treated with apelin-13 (100 nM) to evaluate the activity of Na+/H+ exchanger isoform 3 (NHE3) by intracellular pH measurements. Results: The previous administration of apelin-13 did not improve tubular injury, creatinine clearance or plasma urea levels after the renal I/R. Moreover, KIM-1 and NGAL markers were mainly increased by renal I/R and were not reduced in the apelin-13 + I/R group. Also, the downregulation of megalin by renal I/R was not prevented by apelin-13. Interestingly, apelin-13 worsens renal response to tubular proliferation after renal I/R, as Ki67 and phosphorylation of ERK 1/2 (Thr202/Tyr204) were sharply reduced in the apelin-13 + I/R group. Furthermore, in vitro experiments demonstrated that apelin-13 inhibited NHE3 activity in murine proximal tubular cells. Overall, these findings suggest that apelin-13 suppresses tubular proliferation, potentially impairing the adaptive response to renal I/R injury, thereby highlighting its relevance to ischemic AKI.