Solid organ transplantation necessitates the use of lifelong immunosuppression. In particular, the calcineurin inhibitors (CNIs) are utilized in nearly all transplant recipients as they are potent immunosuppressants. In kidney transplantation, the use of CNIs has led to the very low rejection rates of ~7-10% per year. However, long term graft survival has not improved substantially with their utilization, with a mean half-life of 10 years for kidney transplants. A key contributor to late graft injury is CNI-nephrotoxicity which is manifested by declining kidney function and the histological features of interstitial fibrosis, tubular atrophy, and arteriolar hyalinosis. The mechanism by which CNI induce kidney injury and failure is multifactorial, and to date, there are no specific therapeutic strategies to mitigate this injury. The goal of this submission is to identify a novel and clinically applicable strategy to ameliorate CNI-associated kidney injury. Recently, AMP-Activated Protein Kinase (AMPK), a key regulator of cell metabolism, autophagy, and mitochondrial biogenesis, has been linked to modulating kidney injury. Further, loss of AMPK activation is associated with organ inflammation and fibrosis. We hypothesize that AMPK activation could ameliorate some of the adverse metabolic consequences in renal tubular epithelium and as a corollary, AMPK activation could be a clinically relevant intervention to mitigate long term CNI nephrotoxicity. Our study will focus on cyclosporine A (CsA), a classic CNI therapeutic agent. We will utilize a complementary series of studies, both in vitro and in vivo, using a mouse model of CsA injury that we have used successfully in our laboratory. In vitro, we will focus on renal tubular epithelium, the primary target of in vivo injury. We will study both the impact of AMPK activation as well as knock down using pharmacologic agents and silencing RNAs. Outcomes will include assessments of bioenergetics, mitochondrial function, pro-inflammatory markers and the DAMP HMGB1, and paracrine impact on macrophages and their differentiation. In vivo, we will test the impact of AMPK activation on ameliorating CsA renal injury by assessing kidney function (serum creatinine), mRNA expression of inflammatory mediators within the kidney, and biochemical, cellular and histologic assessments of injury and fibrosis. We will assess both pre-emptive inhibition as well as determine the impact of AMPK activation after established CsA nephrotoxicity. To complement these pharmacologic studies, we will also employ the novel use of AMPK 1 and 2 deficient mice. Cross-transplant studies will allow us to dissect the role of systemic versus renal expression of AMPK in injury. We believe that this comprehensive approach will provide key mechanistic insights into ameliorating or mitigating CNI nephrotoxicity. The use of a clinically relevant activator, metformin, will provide an opportunity for rapid translation into our human recipients of ...