Suramin Attenuates Renal Fibrosis Figure 7. Effect of suramin on phosphorylation of STAT3 and ERK1/2 in rat remnant kidney. (A) Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against phospho-STAT3 (p-STAT3), STAT3, phospho-ERK1/2 (p-ERK1/2), ERK1/2. Expression levels of p- STAT3 (B), STAT3 (C), p-ERK1/2 (D), ERK1/2 (E) were quantified by densitometry and normalized with GAPDH. 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.g007 In the present study, we observed increased expression of MCP-1 and RANTES in the remnant kidney, and suramin treatment significantly reduced their expression. Furthermore, suramin inhibited renal ablation-induced phosphorylation of NF-kB, a key transcriptional factor involved in the expression of multiple proinflammatory chemokines and cytokines, including MCP-1 and RANTES. In addition, STAT3, another trancriptional factor, is also highly phosphorylated in the remnant kidney, and its phosphorylation is inhibited by suramin. This suggests that the production of cytokines and chemokines in the damaged kidneys can be transcriptionally regulated by both NF-kappaB and STAT3. In support of this hypothesis, we have recently demonstrated that the inhibition of STAT3 by S3I-201 suppressed the expression of TNF-alpha, IL-1beta, and ICAM-1 without affecting that of MCP-1 in the mouse kidney injured by ureteral obstruction. Since NF-kappaB is well known to play an important role in regulating MCP-1 expression [50], these findings suggest that NF-kappaB and STAT3 can operate in concert to regulate expression of cytokines and chemokines in injured kidneys. It should be emphasized that NF-kappaB and STAT3 may regulate expression not only of cytokines/chemokines, leading to inflammatory responses, but also other genes involving renal fibrogenesis. Recently, we reported that STAT3 activity is required for the expression of TGF-beta1 and TGF-beta1 receptor II [27]. NF-kappaB has also been shown to be able to interact with AP-1 to regulate the expression of collagen, fibronectin and TGF-beta1, enhancing ECM accumulation [51]. Notably, AP-1 transcriptional factor is subjected to regulation by the ERK1/2 signaling pathway, and suramin treatment also blocked ERK1/2 phosphorylation in the remnant kidney. Therefore, it is likely that suramin inhibits the inflammatory response in the fibrotic kidney through multiple signaling pathways and transcrip- tional factors. It is well known that suramin exhibits its pharmacological effects by interfering with the interaction of multiple growth factors/ cytokines and their receptors [5,6,7,8].. Previous studies have also shown that suramin is a potent antagonist of PDGF, EGF and TGF-beta receptors whereas activation of these three receptors is PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36194