CERAMIDE ACCUMULATION DURING OXIDANT RENAL TUBULAR INJURY - MECHANISMS AND POTENTIAL CONSEQUENCES

Ceramide is an important signaling molecule that is typically generate d via sphingomyelinase (SMase)-mediated sphingomyelin (SM) hydrolysis. Although diverse forms of renal injury elicit ceramide accumulation, the molecular determinants of this change and its contribution to tiss ue damage are poorly defined. The present study uses iron (Fe/hydroxyq uinoline)-mediated injury of cultured human proximal tubular (HK-2) ce lls to gain additional insights into these issues. A 4-h Fe exposure d oubled ceramide levels in the absence of cell death. This was independ ent of de novo synthesis, since ceramide synthase inhibition (with fum onisin B-1) had no effect. Oxidant stress directly suppressed, rather than stimulated, SMase activity by: (I) decreasing SMase levels; (2) d epleting SMase-stimulating glutathione; and (3) increasing SM resistan ce to SMase attack. Fe suppressed cell sphingosine levels (3 to 4 time s ceramide/sphingosine ratio increments), suggesting a possible cerami dase block. Fe did not directly affect HK-2 ceramidase levels. However , arachidonic acid (C20:4) accumulation, a consequence of oxidant-indu ced phospholipase A, (PLA(2)) activation, markedly suppressed ceramida se and stimulated SMase activity. Exogenous C20:4, as well as PLA, (in doses simulating Fe-induced deacylation) recapitulated Fe's ceramide- generating effect. Because C20:4 is directly cytotoxic, it was hypothe sized that ceramide might offset some of C20:4's adverse effects. Supp orting this possibility were the following: (I) C20:4 exacerbated Fe t oxicity; (2) this was abrogated by ceramide treatment; and (3) ceramid e blunted Fe-mediated cell death. Conclusions: (1) ceramide accumulati on during acute cell injury can be an adaptive response to PLA, activa tion/C20:4 generation; (2) C20:4-induced ceramidase inhibition, couple d with SMase stimulation, may trigger this result; and (3) these ceram ide increments may exert a ''biostat'' function, helping to offset C20 :Lt/PLA(2)- and ''catalytic'' iron-mediated tubular cell death.