CONICET | Buscador de Institutos y Recursos Humanos

IntroductionHemolytic Uremic Syndromeassociated with Shiga toxin (Stx)-producing E.coli infection is the most common cause of acute renal failure (ARF) inchildren in Argentina. Stx type 2 (Stx2) damages human renal microvascularendothelial cells (HGEC) and tubular epithelial cells (HK-2) and also induces a broadinflammatory response. Subtilase cytotoxin (SubAB) may also contributeto this pathology. We developedHGEC/HK-2 coculture as a model of renal proximal tubules to clarify theinvolvement of endothelial-epithelial cross talk in kidney damage caused by toxins.In this work, we evaluated the response of selected pro-inflammatory mediatorsreleased by HGEC, HK-2 and HGEC/HK-2 exposed to Stx2, SubAB or Stx2+SubAB.MethodsHGEC, HK-2 and HGEC/HK-2 wereincubated with Stx2 (0.01 ng/ml), SubAB (1 ng/ml) or Stx2+SubAB [(0.01+1) ng/ml]for 24 h. Culture supernatants were collected and IL-6, IL-8 and TNF-α werequantified by ELISA. Stx2, SubAB, Stx2+SubAB vs. Ctrl, p<0.05, n=4. ResultsControls showed a differentialrelease of IL-6, IL-8 and TNF-α between monocultures and cocultures, suggestingthat secretion of soluble mediators depends on the cell type and cultureconditions (p<0.05). On HGEC/HK-2: Stx2, SubAB and Stx2+SubAB caused a significantincrease on IL-6 (4592 ± 258, 4254± 325, 4395± 317 vs. 3448 ± 179 pg/ml) and IL-8 release (1730 ± 161, 1746 ± 110,1830 ± 104 vs. 1272 ± 85 pg/ml) butthey did not modulate TNF-α. On HGEC: Stx2 increased IL-6 (5505 ± 170 vs. 4572 ± 101 pg/ml), IL-8 (5188 ± 234 vs.3876 ± 99 pg/ml) and TNF-α (141 ± 30 vs.45 ± 3 pg/ml). In addition, Stx2+SubAB increased TNF-α level (85 ± 13 vs. 45 ± 3). The toxins did not have anyeffect on IL-6 and TNF-α release by HK-2, but SubAB and Stx2+SubAB decreased IL-8levels in these cells. Finally, differences in the pro-inflammatory mediators releasewere observed in the HGEC and HK-2 coculture compartments compared to HGEC andHK-2 monocultures (n=1).ConclusionsThese results show thatendothelium-epithelium communication modulates the inflammatory responsescaused by Stx2 and SubAB on renal cells and may contribute to the early eventsof ARF.