Epac-Rap Signaling Reduces Cellular Stress and Ischemia-induced Kidney Failure

Abstract

Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2Ј-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2Ј-O-MecAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2Ј-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2Ј-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-␣, and lateral expression of ␤-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation.

Epac-Rap Signaling Reduces Oxidative Stress in the Tubular Epithelium

“…We previously established that 8-pCPT-29-O-Me-cAMP-AM selectively activates Epac, whereas forskolin induces activation of Epac as well as protein kinase A (PKA). 10,11 The effect of long-term activation of PKA by forskolin may antagonize the protective outcome we observed in the acute exposures in monolayer assays and reflect PKAmediated differences in gene expression.…”

“…In our previous studies, we demonstrated that pharmacologic activation of Epac-Rap signaling provides protection against cell detachment during IR injury 10 and apoptosis during exposure to cisplatin. 11 Because excessive generation of ROS by mitochondria is a common feature in a diverse array of renal pathologies including IR injury and cisplatin nephrotoxicity, we hypothesized that Epac-Rap signaling influences mitochondrial function during injury.…”

“…10,11 IM-PTECs were preloaded with 5-(and-6)-carboxy-29,79-dichlorodihydrofluorescein diacetate (DCF-DA), which is converted into fluorescent DCF via oxidation by radicals. 18 After hypoxia, reoxygenation was induced by addition of fresh medium to the cells.…”

“…Selective and Prolonged Rap1 Activation in the Kidney after Systemic Administration of 8-pCPT-29-O-Me-cAMP Conjugated to Lysozyme Although local intrarenal administration of 8-pCPT-29-O-MecAMP is a feasible experimental approach to activate tubular Epac-Rap signaling, 10 systemic administration and renal targeting is preferable in the context of a renal ischemia model. To this end, an 8-pCPT-29-O-Me-cAMP-lysozyme conjugate was generated ( Figure 4A).…”

“…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.…”