SAT-141 DISTINCT MECHANISMS OF ACTION OF CYCLOPHILIN A VERSUS CYCLOPHILIN D IN PROMOTING ISCHAEMIA/REPERFUSION INJURY

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Cyclophilins are enzymes that regulate protein folding. Within this enzyme family, cyclophilin A (CypA) promotes leukocyte recruitment and inflammation, while cyclophilin D (CypD) facilitates mitochondrial-dependent cell death. CypD-/- mice are known to be protected from ischaemia/reperfusion injury (IRI). We investigated: (1) whether a novel pan-cyclophilin inhibitor (CYPi), which does not block calcineurin function, could prevent anticipated renal IRI, (2) the contributions of CypA and CypD in renal IRI. Aims: To determine if cyclophilin (Cyp) inhibition can prevent renal IRI, and examine the specific contributions of CypA and CypD in promoting renal IRI. Groups of 10 mice underwent bilateral renal ischaemia and were killed 24 hours after reperfusion: (1) C57BL/6J mice were treated with 30mg/kg/BID CYPi or vehicle by oral gavage; (2) CypA-/- versus wild type (WT) mice on the 129 background. Controls were sham operated. (3) CypD-/- vs WT cultured proximal tubular epithelial cells (PTEC) were exposed to 0.5 mM of H2O2 for 24hr, with or without CYPi. Renal IRI caused acute renal failure in C57BL/6J mice (179.0±19.1 vs 12.2±1.4umol/L serum creatinine (sCr) in sham; P<0.0001). CYPi protected against renal failure (sCr 36.4±6.7umol/L; P<0.0001). CYPi treatment reduced histologic tubular damage (P<0.0001). CYPi treatment also reduced apoptotic tubular cells (TUNEL+ cells), TNF-α mRNA levels and neutrophil and macrophage infiltration (all P<0.005 vs vehicle). Renal IRI induced acute renal failure in 129 mice (sCr 41±13.76 vs 6.25±1.66umol/L in sham; P<0.0001). CypA-/- mice were protected from renal dysfunction (sCr 20.7±3.53umol/L; P<0.0001). CypA-/- mice had less histologic tubular damage (P<0.005) and lower KIM-1 mRNA levels (P<0.005). CypA-/- mice also had less tubular cell death (TUNEL+ cells; P<0.001), inflammatory cytokines (TNF-α and IL-36-α PCR; P<0.05), and reduced neutrophil infiltration (P<0.001). In addition, cell culture studies showed that CYPi treatment significantly reduced H2O2 induced cell death in WT PTEC (P<0.0001). CypD-/- PTEC also showed reduced H2O2 induced cell death which was not further reduced by CYPi treatment, indicating that it is CypD and not the other cyclophilins that mediate oxidant-induced cell death. Pan-cyclophilin inhibition prevented anticipated IRI-induced acute renal failure by suppressing tubular cell death and inflammation. Our data suggest that CypA promotes leukocyte infiltration and inflammation leading to acute kidney injury following renal IRI, whilst CypD specifically contributes to cell death from the ischaemic/hypoxic insult.