control kidneys (sham alone or sham plus suramin treatment); and its expression was elevated in the remnant kidney. Like MCP-1, RANTES was also predominantly expressed in the renal tubules (Fig. 5A). Suramin treatment reduced its expression (Fig. 5B). These results, in conjunction with our recent observations that suramin inhibits expression of multiple cytokines including TGF- beta1, TNF-alpha, Interleukin-1, ICAM-1 [12] in the model of UUO, suggest that suramin has a strong inhibitory effect on inflammatory responses in the renal kidney. Suramin reduces NF-kappaB activation in the rat remnant kidney NF-kappaB is a ubiquitous eukaryotic transcription factor that regulates gene expression of cytokines and enzymes involved in controlling inflammatory responses [35]. Phosphorylation of the p65 unit of NF-kappaB initiates its activation and plays an important role in regulating the specificity of the NF-kappaB-dependent gene expression [36]. Suramin may regulate expression of multiple cytokines and chemokines, through activation of NF-kappaB. To test Suramin Attenuates Renal Fibrosis this hypothesis, we determined the phosphorylation of p65 NF- kappaB in rat remnant kidney treated with/without suramin by Western blot analysis. Figure 6 shows that the phosphorylated NF- kappaB p65 was not detectable in the kidney of sham-operated rats, but significantly increased in the remnant kidney. Suramin treatment decreased its phosphorylation. Expression of total p65 NF-kappaB was not affected by renal ablation and suramin. These data suggest that suramin is effective in inhibiting NF-kappaB activation in the remnant kidney, providing the molecular basis for decreased expression of chemokines/cytokines in the injured kidney treated with suramin after urethral obstruction and 5/6 renal ablation. Suramin abolishes phosphorylation of STAT3 and ERK1/2 renal ablation In addition to NF-kappaB, STAT3 and ERK1/2 pathways also play an important role in modulating inflammatory responses [37,38] and renal fibroblast proliferation. We thus further examined the effect of suramin on STAT3 and ERK1/2 phosphorylation in the remnant kidney. As shown in Fig. 7, increased STAT3 and ERK1/2 phosphorylation (activation) was Figure 4. Effect of suramin on MCP-1 expression in rat remnant kidney. (A) Photomicrographs (4006) illustrating MCP-1-stained sections of kidney tissue on week 4 after various treatments as indicated. (B) MCP-1 staining graphic presentation of quantitative data. Data are represented as the mean 6 S.E.M (n = 6). Means with different superscript letters are significantly different from one another (P,0.05). doi:10.1371/journal.pone.0036194.g004 Figure 5. Effect of suramin on RANTES expression in rat remnant kidney. (A) Photomicrographs (4006) illustrating RANTES-stained sections of kidney tissue on week 4 after various treatments as indicated. (B) RANTES staining graphic presentation of quantitative data. Data are represented as the mean 6 S.E.M (n = 6). Means with different superscript letters are significantly different from one another (P,0.05). doi:10.1371/journal.pone.0036194.g005 PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e36194