Effect of shigatoxin-1 on arachidonic acid release by human glomerular epithelial cells

Background. Altered arachidonic acid (AA) metabolism has been implicated in the pathogenesis of renal injury in the hemolytic uremic syndrome (HUS). H owever, there is very little information of the effect of shigatoxin (Stx; the putative mediator of renal damage in HUS) on AA release or metabolism b y renal cells. Since recent studies have demonstrated that glomerular epith elial cells (GECs) may be important early targets of Stx, the current study was undertaken to examine the effects of Stx on AA release and metabolism by GECs. Methods. Cultured human GECs were exposed to Stx1 +/- lipopolysaccharide (L PS)for 4 to 48 hours followed by determination of H-3-arachidonate release, thromboxane A(2) (TxA(2)) and prostacyclin (PGI,) production, cyclooxygena se (COX) activity, and Western and Northern analyses for phospholipase A, ( PLA(2)) and COX protein and mRNA levels, respectively. Results. Stx1 increased arachidonate release by GECs. LPS alone had no such effect, but increased arachidonate release in response to Stx1. Stx1-stimu lated arachidonate release correlated with elevations in cPLA(2) and sPLA(2 ) protein and cPLA(2) mRNA levels. Stx1 also increased both TxA(2) and PGI( 2) production by GECs; LPS alone did not alter eicosanoid production, but a ugmented Stx1 effects. Both Stx1 and LPS stimulated COX activity; however, these effects were not additive. Although there was an accompanying elevati on of COX-1 and COX-2 mRNA, Stx1 decreased and LPS did not change COX1 and COX2 protein levels. Conclusions. Stx1 alone or in conjunction with LPS increases arachidonate r elease and eicosanoid production by human GECs; this effect correlates with increased PLA2 protein and mRNA levels. To our knowledge, this is the firs t study identifying the mechanisms of Stx1-stimulated AA release. These res ults raise the possibility that arachidonate release and metabolism by GECs , and conceivably other renal cell types, are involved in renal injury in H US.