Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells

Background Both interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a lpha) are pleiotropic cytokines that have been implicated in the developmen t of glomerular and tubular injury in various forms of immune-mediated rena l disease, including glomerulonephritis. Although TNF-alpha has been shown to stimulate IL-6 production in renal cells in culture, the signaling mecha nisms that regulate IL-6 production are not fully understood. The aim of th is study was to examine the role of the p38 and extracellular signal-regula ted kinase (ERK) mitogen-activated protein kinase (MAPK) pathways in regula ting TNF alpha-mediated IL-6 production from both primary human mesangial c ells (HMCs) and human proximal tubular (HPT) cells. Methods. Primary mesangial and proximal tubular cells were prepared from ne phrectomized human kidney tissue. Cells were treated for 24 hours with TNF- alpha in the presence and absence of the specific p38 and ERK12 MAPK inhibi tors SB203580 and PD98059, respectively, either alone or in combination. IL -6 levels in the cell culture media were measured by enzyme-linked immunoso rbent assay. MAPK activation was demonstrated by immunoblot for the active kinase (tyrosine/threonine phosphorylated) in whole cell extracts using pho spho-specific antibodies. p38 MAPK activity in HPT cells was measured using an in vitro immunokinase assay using ATF2 as the substrate. Results. TNF-alpha (0.1 to 100 ng/ml) stimulated a dose-dependent increase in IL-6 production in both renal cell types. The activation of the p38 and the ERK1,2 MAPKs occurred following TNF-alpha stimulation. The role of thes e activations in IL-6 production was confirmed by the ability of both inhib itors SB203580 (1 to 30 mu M) and PD98059 (0.01 to 10 mu M) to inhibit basa l and TNF-alpha-stimulated IL-6 production in both cell types. The addition of both inhibitors in combination caused greater decreases in IL-6 product ion compared with either inhibitor alone. Pretreatment with SB203580 (10 mu M) had no effect on basal or TNF-alpha-stimulated phosphorylation of p38 M APK but completely abolished TNF-alpha-stimulated p38 MAPK activity. PD9805 9 decreased both basal and TNF-alpha-stimulated phosphorylation of ERK1,2. Conclusions. This study provides evidence that both the p38 and ERK MAPK pa thways are important for the regulation of the production of IL-6 from the proximal tubular and glomerular mesangial regions of the nephron. In respon se to TNF-alpha, the activation of both pathways leads to IL-G production. These findings could aid in an understanding of the cellular mechanisms tha t regulate IL-6 production and could provide insights into possible pharmac ological strategies in inflammatory renal disease.