MC were e posed to patho physiologic Hcy concentration that has been pre viously shown to modulate MC behaviour. The results choose size revealed that several cytokines were sig nificantly affected by this manoeuvre, including TIMP 1, MIP 2, interferon gamma and fractalkine. MIP 2 influ ences leukocyte migration and has been shown to mediate inflammatory infiltration in glomerular disease. Accordingly, we chose to e plore the influence of Hcy on MIP 2 and to relate the observations to leukocyte interac tion with glomerular MC in an in vitro assay system. Homocysteine induces MIP 2 e pression and increases MIP 2 protein Initially we determined the influence of variable Hcy con centrations on MIP 2 e pression by qRT PCR. The results indicated a significant impact on e pression at 50 and 100 M.

Another sulphur containing amino acid, that is structurally similar to DL Hcy did not influence e pression. Hence changes in MIP 2 e pression can be attributed to an effect specific to Hcy, rather than to structural similari ties with L Cys. Subsequently, the e pression of MIP 2 induced by Hcy in MC was quantified by western blot analysis. In line with the e pression data, Hcy significantly increased MIP 2 protein levels in MC. Of note, MIP 2 e pression increased 2. 5 fold at 50 MHcy, com pared to e pression at 100 M L Cys. MIP 2 lev els did not increase further when Hcy concentration was increased to 100 M. Homocysteine induced MIP 2 requires p38MAPK and PI3kinase but not P42 44 MAPK Signaling MIP 2 induction has been reported to be MAPK and PI 3 Kinase dependent.

Hence, we investigated role of MAPK and PI 3 Kinase in MIP 2 e pression induced by Hcy. Hcy induced MIP 2 was significantly attenuated by a PI 3 Kinase inhibitor and by an inhibitor of a p38MAPK. In contrast, use of a p42 44 MAPK inhibitor did not significantly alter Hcy induced MIP 2. Immunohistochemistry was employed as another analyt ical tool to e amine the effect of Hcy on mesangial MIP 2. Cells were e posed to Hcy, in the absence and presence of inhibitors to p38MAPK and PI3 Kinase. MIP 2 e pression in medium supplemented with FBS and L Cys represented control condi tions. As revealed in figure 2, panel C, the e pression of MIP 2 was increased by Hcy compared to control. Hcy induced of MIP 2 was abolished by LY294002 and SB203580.

These results suggest that Hcy induced e pression of MIP 2 in MC was mediated by p38MAPK and PI 3 K signalling pathways and are consist ent with the results derived from Western blotting analy sis. Hcy activates p85 PI 3 Kinase and p38MAPK in mesangial cells In an effort to corroborate the observations related to blunting of the effect of Hcy on MIP 2 by inhibitors of PI3 Kinase and p38MAPK, western blotting analyses was employed to determine levels of activated p38MAPK and PI3 Kinase in Drug_discovery MC e posed to ele vated levels of e tracellular Hcy. Hcy induced time dependent increases in p38 MAPK phosphorylation between 10 and 30 minutes.