Figure 6. Effect of suramin on NF-kappaB activation in rat remnant kidney. Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against phospho-p65 NF-kappaB, p65NF-kappaB. Expression levels of p-p65 NF-kappaB(B) were quanti- fied by densitometry and normalized with p65 NF-kappaB. 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.g006 detected in rat remnant kidney and suramin treatment abolished this response. Remnant kidney injury also increased expression levels of total STAT3 and ERK1/2, but their expression was not affected by suramin. This data indicates that suramin may also suppress renal inflammation and fibroblast activation by interfer- ing with these two signaling pathways. Suramin abrogates phosphorylation of EGFR and PDGFR in rat remnant kidney Activation of intracellular signaling pathways is usually secondary to the activation of membrane receptors. EGFR and PDGFR have been shown to mediate activation of all three signaling pathways described above [36,39], and they play a central role in the activation and proliferation of renal fibroblasts leading to renal fibrogenesis in a variety of pathological settings [40,41,42,43]. We examined the effect of suramin on the activation of these two receptors. PDGFR receptor and its active form (p-PDGFR) were not detectable in the normal kidney; however, renal ablation induced their expression (Fig. 8). In contrast, a small amount of total EGFR and its active form (p-EGFR) was detectable in the normal kidney, and its expression was increased in the remnant kidney (Fig. 8). Although administration of suramin totally blocked renal ablation- induced phosphorylation of PDGFR and EGFR, expression of total PDGFR and EGFR were not affected by this treatment (Fig. 8). This data supports the role of suramin as a potent inhibitor for multiple growth factor receptors and also suggests the importance of PDGFR and EGFR in mediating the anti-fibrotic action of suramin. Suramin inhibits Smad-3 phosphorylation in the remnant kidney TGF-betasignaling is associated with almost all forms of kidney disease characterized by renal fibrosis and inflammation, and Suramin Attenuates Renal Fibrosis smad-3 is a critical component of the intracellular pathway that transmits TGF-beta signals from the cell surface into the nucleus [44,45]. To determine whether suramin would have an inhibitory effect on the TGF-beta signaling pathway, we examined the effect of suramin on Smad-3 phosphorylation in the remnant kidney. Fig. 9 shows that renal ablation induced Smad-3 phosphorylation, which was suppressed by suramin administration. Phosphorylated smad-3 was not detectable in the sham–operated kidney with/ without administration of suramin. Total Smad-3 expression was the same in each group. Thus, suramin substantially blocked TGF- beta signaling, which may mediate the antifibrotic effects of this compound in the remnant kidney. Discussion Renal fibrosis is the final common pathway for most forms of progressive renal disease and involves glomerular and vascular sclerosis, and/or interstitial fibrosis. Most renal disorders (whether glomerular or tubulointerstitial, congenital or acquired) lead to renal fibrosis. Thus, there is a great interest in identifying and developing drugs to prevent or reverse fibrosis. Recently, we demonstrated that suramin inhibits activation and proliferation of renal interstitial fibroblasts and attenuates the development of renal interstitial fibrosis when given either immediately after injury or 3 days after initiation of injury in UUO model [12,13], suggesting it may be a potential drug for treatment of fibrotic disease. However, it remains unclear whether suramin also improves glomerular and vascular lesions as these pathological changes are not typical in UUO model. As the remnant kidney is characterized by the slow development of glomerulosclerosis, vascular sclerosis, tubulointer- stitial fibrosis and renal inflammation [14,15], mimicking human disease, we further tested the efficacy of suramin in this model. Our results revealed that suramin administration is effective in preventing the development of glomerular and vascular sclerosis and attenuating inflammatory response. To our knowledge, we are the first to demonstrate that suramin exhibits a renoprotective effect in a model of hypertenisve glomerulosclerosis. However, the mechanims by which suramin protects against development of glomerulosclerosis are not fully understood. Although blood pressure is an important factor associated with this process [46], we recently observed renoprotec- tion with suramin in the remnant kidney that was independent of effects on systemic hypertension [12]. This indicates that suramin may attenuate sclerosis through nonhemodynamic factors. Among numerous nonhemodynamic factors, macrophages have been shown to play an important pathogenic role in the progression of glomerulosclerosis. For example, glomerular infiltration of mac- rophages was observed within 1 to 2 wk of renal ablation, before the development of renal scarring [47]. Glomerular macrophage depletion by X-irradiation has been shown to ameliorate the progression of the glomerular injury in the remnant kidney [15]. Further, macrophage-conditioned medium has been reported to promote prosclerotic responses in cultured rat mesangial cells [48]. In the current study, we examined the effect of suramin on the macrophage infiltration in the kidney and found that suramin treatment significantly suppresed macrophage infiltration as revealed by both histochemistry and immunoblot analysis. This suggests that a reduction of macrophage by suramin may thus have contributed to amelioration of sclerosis. Suramin-mediated suppression of macrophage infiltration may be through inhibition of chemokine expression. Previous studies have indicated that production of MCP-1 and RANTES is able to build up the chemokine gradient that serves as a chemotactic signal to attract macrophages to the injured site in the kidney [49]. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e36194