Interstitial inflammation has emerged as a key event in the development of acute renal failure. To gain better insight into the nature of these inflammatory processes the interplay between tubular epithelial cells, endothelial cells, and neutrophils (PMN) was investigated. A co-culture transmigration model was developed, composed of human dermal microvascular endothelial (HDMEC) and human renal proximal tubular cells (HK-2) cultured on opposite sides of Transwell® growth supports. Correct formation of an endo-epithelial bilayer was verified by light and electron microscopy. The model was used to study the effects of endotoxin, TNF, and alpha-MSH by measuring PMN migration and cytokine release. To distinguish between individual roles of microvascular endothelial and epithelial cells in transmigration processes, migration of PMN was investigated separately in HK-2 and HDMEC-monolayers. Sequential migration of PMN through endothelium and epithelium could be observed and was significantly increased after pro-inflammatory stimulation with either TNF- or endotoxin (3.5 ± 0.58 and 2.76 ± 0.64 fold vs. control, respectively). Co-incubation with alpha-MSH inhibited the transmigration of PMN through the bilayer after pro-inflammatory stimulation with LPS but not after TNF. The bilayers produced significant amounts of IL-8 and IL-6 mostly released from the epithelial cells. Alpha-MSH decreased LPS induced IL-6 secretion by 30 % but had no effect on IL-8 secretion. We established a transmigration model showing sequential migration of PMN across microvascular endothelial and renal tubular epithelial cells stimulated by TNF- and endotoxin. Anti-inflammatory effects of alpha-MSH in this bilayer model is demonstrated by inhibition on PMN transmigration and IL-6 secretion.