Epac-Rap Signaling Reduces Oxidative Stress in the Tubular Epithelium

Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-2′-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-2′-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate–induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-2′-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-2′-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.Renal ischemia-reperfusion (IR) injury is an important cause of AKI¹ and a significant risk factor for the development of renal dysfunction after kidney transplantation.² During IR injury, morphologic and functional alterations of the proximal tubular epithelium occur that are linked to the development of renal failure and activation of immune cells via release of proinflammatory cytokines.³Exchange protein activated by cAMP (Epac) is a guanine nucleotide exchange factor for the small GTPase Rap1.⁴ Activation of Epac by cAMP or by the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP (also referred to as 007) induces functional activation of Rap1.⁵ Initial studies showed that Epac-Rap signaling enhances cell adhesion by supporting maturation of cell-cell junctions^6,7 and promoting integrin-mediated cell-matrix adhesion.^8,9 In line with these studies, we recently demonstrated that selective activation of Epac reduces proximal tubular epithelial cell (PTEC) detachment during IR injury using in vitro and in vivo models.¹⁰ Activation of Epac-Rap was associated with reduced expression of markers for cellular stress in PTECs. In addition, in vitro cisplatin-induced apoptosis of PTECs could be significantly reduced by activation of Epac and this was also associated with improved adhesion of cells.¹¹ On the basis of these findings, we hypothesized that activation of Epac-Rap signaling may protect against a common cytotoxic event in these injury models.Unbalanced and uncontrolled production of reactive oxygen species (ROS) is an important mediator of cell injury and occurs during cisplatin nephrotoxicity,¹² IR injury,¹³ and renal fibrosis.¹⁴ In renal pathology, intracellular ROS can be produced enzymatically such as by NADPH oxidase (NOX) complexes or derive from dysfunctional mitochondrial activity. Mitochondrial ROS production appears to be the driving force behind hypoxia-reoxygenation cell injury¹⁵ and cisplatin cytotoxicity.¹⁶Here we studied the role of specific proximal tubular activation of Epac and how this protects against renal injury in both in vitro and in vivo models for IR injury. We found that ROS production during reoxygenation after hypoxia was decreased by activation of Epac. Selective proximal tubular activation of Epac by renal targeting of 8-pCPT-2′-O-Me-cAMP conjugated to lysozyme (LZM-007) reduced oxidative stress in an in vivo model for IR injury and significantly decreased IR injury–associated renal failure and tubular damage. Our data show that Epac activation reduces ROS-mediated cellular injury in renal disease and may be a therapeutic strategy for modulation of oxidative stress.