Figure 9. Effect of suramin on phosphorylation of Smad-3 phosphorylation in rat remnant kidney. Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against phospho-Smad (p-Smad), and Smad (A). Expression levels of p-Smad was quantified by densitometry and normalized with Smad(B). 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.g009 to suppression of macrophage infiltration, inhibition of chemokine expression and inactivation of transcriptional factors. As renal fibrogenesis is associated with the increased production of numerous cytokines/growth factors and subsequent activation of their receptors and signaling pathways, suramin with its ability to inhibit the interaction of multiple cytokine/growth factor with their receptors may offer improved therapeutic benefits in kidney fibrotic diseases. Materials and Methods Chemicals and Antibodies Antibodies to p-STAT3, STAT3, p-ERK1/2, ERK1/2, p- EGFR, p-PDGFR-beta, PDGFR-beta, p-p65NF-kappaB, p65NF- kappaB were purchased from Cell Signaling Technology (Dan- vers, MA). Antibodies to GAPDH, EGFR, MCP-1, and RANTES were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ED-1 antibody was purchased from Serotec (Oxford, UK). Suramin and all other chemicals were from Sigma (St. Louis, MO). References 1. Anderson S, Rennke HG, Brenner BM (1986) Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. J Clin Invest 77: 1993–2000. 2. Fujihara CK, Malheiros DM, Zatz R, Noronha IL (1998) Mycophenolate mofetil attenuates renal injury in the rat remnant kidney. Kidney Int 54: 1510–1519. 3. Hou FF, Zhang X, Zhang GH, Xie D, Chen PY, et al. (2006) Efficacy and safety of benazepril for advanced chronic renal insufficiency. N Engl J Med 354: 131–140. Suramin Attenuates Renal Fibrosis Remnant Kidney Models and Suramin Treatment The remnant kidney model was created in male Sprague- Dawley rats that weighed 180 to 200 g (Charles River Labora- tories, Wilmington, MA). Five-sixths of normal renal mass was surgically ablated according to our previous protocols [12]. Briefly, 20 animals underwent subtotal nephrectomy involving right subcapsular nephrectomy and infarction of approximately two- thirds of the left kidney by ligation of the posterior and one or two anterior extra renal branches of the renal artery. In addition, 12 rats underwent a sham operation (laparotomy and manipulation of the renal pedicles). On the second week after surgery, rats were randomly divided into suramin treatment and non-treatment groups. Suramin was given at 10 mg/kg once per week for two weeks. 24-hour urine samples were collected in metabolic cages starting at day 1 and weekly for the determination of urinary levels of protein. 28 days after surgery, all animals were sacrificed and the kidneys were then collected for further analysis. Immunoblot Analysis Immunoblot analysis of tissue samples was conducted as described previously [53]. The densitometry analysis of immuno- blot results was conducted by using NIH Image software (National Institutes of Health, Bethesda, MD). Immunohistochemical Staining Immunohistochemical staining was performed according to the procedure described in our previous studies [53]. ED1, MCP-1, and RANTES staining were semi quantified by a computer-aided morphometric analysis (MetaMorph; Universal Imaging Co., Downingtown, PA). Briefly, a grid containing 117 (13X9) sampling points was superimposed on images of cortical high-power field (6400). The number of grid points overlying positive area (except tubular lumen and glomeruli) were counted and expressed as a percentage of all sampling points, as described previously [27]. For each kidney, 10 randomly selected, non-overlapping fields were analyzed in a blinded manner. Statistical Analysis All the experiments were conducted at least three times. Data depicted in graphs represent the means 6 SEM for each group. Inter-group comparisons were made using one-way analysis of variance (ANOVA). Multiple means were compared using Tukey’s test. The differences between two groups were determined by Student t-test. Statistical significant difference between mean values was marked in each graph. P,0.05 is considered significant. Author Contributions Conceived and designed the experiments: SZ NL. Performed the experiments: NL SH ET RG. Analyzed the data: NL SZ. Contributed reagents/materials/analysis tools: RG. Wrote the paper: NL GB SZ. 4. Lewis EJ, Hunsicker LG, Bain RP, Rohde RD (1993) The effect of angiotensin- converting-enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N Engl J Med 329: 1456–1462. 5. Coffey RJ, Jr., Leof EB, Shipley GD, Moses HL (1987) Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J Cell Physiol 132: 143–148. 6. Kloen P, Jennings CL, Gebhardt MC, Springfield DS, Mankin HJ (1994) Suramin inhibits growth and transforming growth factor-beta 1 (TGF-beta 1) binding in osteosarcoma cell lines. Eur J Cancer 30A: 678–682. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e36194