Suramin: a potential therapy for diabetic nephropathy.

OBJECTIVE: To determine whether delayed administration of a single dose of suramin, a drug that has been used extensively in humans to treat trypanosomiasis, attenuates renal injury in a leptin receptor deficient C57BLKS/J db/db type 2 diabetic nephropathy (T2DN) mouse model. RESEARCH DESIGN AND METHODS: Groups of female non-diabetic (control) db/m and diabetic db/db mice of 8 and 16 weeks of age, respectively, were treated with suramin (10 mg/kg) or saline i.v. All animals were euthanized one week later. Measurements in mice 1 week following treatment included the following: body weight; blood glucose; urinary protein excretion; pathological lesions in glomeruli and proximal tubules; changes in protein expression of pro-inflammatory transcription factor nuclear factor κB (NF-κB) and intracellular adhesion molecule-1 (ICAM-1), profibrotic transforming growth factor-β1 (TGF-β1), phospho-SMAD-3 and alpha-smooth muscle actin (α-SMA); and immunohistochemical analysis of leukocyte infiltration and collagen 1A2 (COL1A2) deposition. RESULTS: Immunoblot analysis revealed increased NF-κB, ICAM-1, TGF-β1, phospho-SMAD-3, and α-SMA proteins in both 9 and 17 week db/db mice as compared to db/m control mice. Immunohistochemical analysis revealed moderate leukocyte infiltration and collagen 1A2 (COL1A2) deposition in 9 week db/db mice that was increased in the 17 week db/db mice. Importantly, suramin significantly decreased expression of all these markers in 9 week db/db mice and partially decreased in 17 week db/db mice without altering body weight, blood glucose or urinary protein excretion. There was no difference in creatinine clearance between 9 week db/m and db/db mice ± suramin. Importantly, in the 17 week db/db mice suramin intervention reversed the impaired creatinine clearance and overt histological damage. CONCLUSIONS: Delayed administration of a single dose of suramin in a model of T2DN attenuated inflammation and fibrosis as well as improved renal function, supporting the use of suramin in T2DN

Effect of delayed administration of suramin on deposition of renal fibrogenic material COL1A2 in 9/17 week db/db mice.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073655#pone-0073655-g001" target="_blank">Figure 1</a>. Representative photomicrographs of COL1A2 deposition in kidney sections from 9/17 week non-diabetic and diabetic mice ± suramin intervention, respectively. All fields were chosen from the cortical regions of the kidney sections. Original magnification, 200 X. <b>A</b>. Quantitative analysis of renal COL1A2 assessed by brown staining in the epithelial cells lining the proximal tubules (arrows) in a total of 25 fields in the cortical region of kidney sections. Data are expressed as mean ± SE (n = 4). * Significantly different from respective non-diabetic <i>db/m</i> mice (<i>p</i>≤0.05). # Significantly different from vehicle-treated diabetic <i>db/db</i> mice (<i>p</i>≤0.05). @ Significantly different from vehicle-treated 9 week diabetic <i>db/db</i> mice (<i>p</i>≤0.05).</p

Effect of delayed administration of suramin on urinary protein excretion and creatinine clearance (CrCl) in 9/17 week db/db mice.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073655#pone-0073655-g001" target="_blank">Figure 1</a>. Urine was collected on ice 24 h before euthanization and (<b>A</b>) Urinary protein excretion normalized to urinary creatinine; (<b>B</b>) Creatinine clearance; in 9 and 17 week non-diabetic and diabetic mice ± suramin intervention, respectively, were measured. Data are expressed as mean ± SE (n = 3–6). * Significantly different from suramin-treated diabetic <i>db/db</i> mice. (<i>p</i>≤0.05).</p

Effect of delayed administration of suramin on body weights and serum glucose levels in 9/17 week db/db mice.

<p>Non-diabetic <i>db/m</i> heterozygous mice of 8/16 weeks and diabetic <i>db/db</i> homozygous mice of 8/16 weeks, respectively, were treated with either saline vehicle or 10 mg suramin/kg (i.v., dissolved in saline). Mice in all groups were terminated a week later. (<b>A</b>) Body weights; and (<b>B</b>) Serum glucose levels; in 9 and 17 week non-diabetic and diabetic mice ± suramin intervention, respectively, were measured. Data are expressed as mean ± SE (n = 6–10). <b>*</b> Significantly different from respective non-diabetic <i>db/m</i> mice. (<i>p</i>≤0.05). <b>#</b> Significantly different from 9 week diabetic <i>db/db</i> mice. (<i>p</i>≤0.05).</p

Effect of delayed administration of suramin on renal pro-fibrotic markers in 9/17 week db/db mice.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073655#pone-0073655-g001" target="_blank">Figure 1</a>. Representative Western blots showing profibrotic markers: Renal TGF-β₁, renal phospho-Smad-3 and α-SMA in kidneys of 9/17 week <i>db/db</i> mice ± suramin intervention. (<b>A</b>) Densitometric analysis of renal TGF-β₁; (<b>B</b>) Renal phospho-Smad-3; and (<b>C</b>) Renal α-SMA. Data were normalized by GAPDH which served as internal control. Data are expressed as mean ± SE (n = 4). <b>*</b> Significantly different from respective non-diabetic <i>db/m</i> mice (<i>p</i>≤0.05). # Significantly different from vehicle-treated diabetic <i>db/db</i> mice (<i>p</i>≤0.05). @ Significantly different from vehicle-treated 9 week diabetic <i>db/db</i> mice (<i>p</i>≤0.05).</p

Effect of delayed administration of suramin on renal inflammation in 9/17 week db/db mice.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073655#pone-0073655-g001" target="_blank">Figure 1</a>. Representative Western blots showing inflammatory markers: phospho-p65 and ICAM-1 in kidneys of 9/17 week <i>db/db</i> mice ± suramin intervention. (<b>A</b>) Densitometric analysis of renal phospho-p65; and (<b>B</b>) renal ICAM-1. Data were normalized by GAPDH which served as internal control. Data are expressed as mean ± SE (n = 4). <b>*</b> Significantly different from respective non-diabetic <i>db/m</i> mice (<i>p</i>≤0.05). # Significantly different from vehicle-treated diabetic <i>db/db</i> mice (<i>p</i>≤0.05). @ Significantly different from vehicle-treated 9 week diabetic <i>db/db</i> mice (<i>p</i>≤0.05).</p

Effect of delayed administration of suramin on renal injury in 17 week db/db mice.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073655#pone-0073655-g001" target="_blank">Figure 1</a>. Representative photomicrographs of PAS-stained kidney sections from 17 week non-diabetic and diabetic mice ± suramin intervention, respectively. (<b>A</b>). Quantitation of glomeruli with more than 50% of their volume occupied by mesangial matrix in 17 week non-diabetic and diabetic mice ± suramin intervention. (<b>B</b>) Quantitation of proximal tubules with vacuolization in 17 week non-diabetic and diabetic mice ± suramin intervention. Data are expressed as mean ± SE (n = 6). <b>*</b> Significantly different from respective non-diabetic <i>db/m</i> mice. (<i>p</i>≤0.05). <b>#</b> Significantly different from vehicle-treated diabetic <i>db/db</i> mice. (<i>p</i>≤0.05). <b>PT</b>, Proximal tubule. All fields were chosen from the cortical regions of the kidney sections. Original magnification, 200 X. It should be noted that we could not detect any glomeruli with mesangial matrix expansion or proximal tubules with vacuolization in the vehicle-treated non-diabetic <i>db/m</i> control mice kidneys.</p

Effect of delayed administration of suramin on renal leukocyte infiltration in 9/17 week db/db mice.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073655#pone-0073655-g001" target="_blank">Figure 1</a>. Representative photomicrographs of neutrophil and monocyte staining assessed by formation of stable pinkish-red colored (arrows) complex of free naphthol and diazonium salts following incubation of kidney sections from 9/17 week non-diabetic and diabetic mice ± suramin intervention, respectively. All fields were chosen from the cortical regions of the kidney sections. Original magnification, 200 X. <b>A</b>. Quantitative analysis of renal leukocyte infiltration assessed by number of pink colored dots in a total of 25 fields in the cortical region of kidney sections. Data are expressed as mean ± SE (n = 4). <b>*</b> Significantly different from respective non-diabetic <i>db/m</i> mice (<i>p</i>≤0.05). @ Significantly different from vehicle-treated 9 week diabetic <i>db/db</i> mice (<i>p</i>≤0.05).</p