In this report, we describe that NF-B is spontaneously activated in isolated, normal glomeruli. Ex vivo incubation of isolated rat glomeruli triggered expression of a NF-B-dependent gene, monocyte chemoattractant protein-1 (MCP-1), in parallel with down-regulation of IB and IB proteins and activation of p65 NF-B subunit. The induction of MCP-1 was also observed in mesangial cells co-incubated with isolated glomeruli or exposed to medium conditioned by isolated glomeruli (GCM), which was abrogated by inhibition of NF-B. The activation of NF-B by glomerulus-derived factors was confirmed using reporter mesangial cells that produce secreted alkaline phosphatase (SEAP) under the control of the B enhancer element. When the reporter cells were adoptively transferred into normal glomeruli, expression of SEAP mRNA and activity of SEAP were also up-regulated in the explanted glomeruli. The molecular weight of factors responsible for activation of NF-B was > 50 kD, and TNF- was identified as one of glomerulus-derived activators. To examine upstream events involved, we focused on mitogen-activated protein (MAP) kinases that are spontaneously activated in explanted glomeruli. Selective suppression of extracellular signal-regulated kinase (ERK) or p38 MAP kinase significantly attenuated activation of NF-B in mesangial cells triggered by co-culture with isolated glomeruli. Interestingly, the suppressive effects by MAP kinase inhibitors were not observed in mesangial cells treated with GCM. These data suggested that NF-B was spontaneously activated in explanted glomeruli via autocrine/paracrine factors including TNF- and that the production of NF-B activators by glomeruli was, at least in part, through MAP kinase pathways.