Effects of Suramin on Polycystic Kidney Disease in a Mouse Model of Polycystin-1 Deficiency

Abstract

The aberrant activation of the purinergic signaling pathway has been shown to promote cyst growth and fluid secretion in autosomal dominant polycystic kidney disease (ADPKD). Suramin is an anti-parasitic drug that has strong anti-purinergic properties. Whether suramin could have a therapeutic effect on ADPKD has not been fully investigated. We examined the effect of suramin on cyst progression in a Pkd1 microRNAs transgenic mouse model that presented stable Pkd1 knockdown and moderate disease progression. The Pkd1-deficient mice were treated with suramin (60 mg/kg) by intraperitoneal injection twice a week from postnatal days 35 to 90. Kidney-to-body weight ratios, cyst indices, and blood urea nitrogen (BUN) levels were measured. Cell proliferation and macrophage infiltration were determined by immunohistochemistry. The suramin-treated group had significantly lower renal cyst densities, cell proliferation, and macrophage infiltration compared with saline-treated controls. Suramin significantly inhibited ERK phosphorylation and the expression of Il1b, Il6, Nlrp3, Tgfb, Fn1, P2rx7, and P2ry2 mRNAs in the kidneys. However, BUN levels remained high despite the reduction in cyst growth. Furthermore, plasma cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels were significantly higher in the suramin-treated group compared with the control group. Periodic acid-Schiff staining revealed degenerative changes and epithelial cell vacuolation in the non-cystic renal tubules, which indicated phospholipidosis following suramin treatment. These results suggest that suramin may reduce renal cyst growth and inflammation, but the associated tubular cell injuries could limit its therapeutic potential. Other purinergic receptor antagonists with less nephrotoxicity may deserve further investigation for the treatment of ADPKD.

Keywords: polycystic kidney disease; purinergic signaling; suramin.

Figure 1

The expression of P2X7 receptors in wild-type and Pkd1-miR Tg mice. (A) Representative images of kidney sections showing diffuse immunostaining for P2X7 receptors in renal tubules in wild-type mice (n = 5). (B) Strong staining for P2X7 receptors is noted in the cyst-lining epithelial cells in the Pkd1 miR Tg mice at 30 days of postnatal age (n = 5). (C) Diffuse staining for P2X7 receptors in cyst walls and non-cystic tubules in the Pkd1-miR Tg mice at 60 days of age (n = 5). (D) Comparison of the renal mRNA expression of P2rx7 and other pro-inflammatory and pro-fibrotic genes in the kidneys of wild-type and Pkd1 miR Tg mice according to different postnatal ages (n = 10, 9, 5, 7, respectively). Data are mean ± SEM. Scale bar, 50 µm.

Figure 2

Suramin treatment reduces cyst burden in Pkd1 miR Tg mice. (A) Schematic illustration of the experimental groups and the time frame of suramin administration. (B) Representative images of whole kidney sections (H&E stain) in the suramin-treated Pkd1-miR Tg mice compared to the vehicle-treated controls. (C) Kidney-to-body weight ratios, (D) cystic indices, and (E) BUN levels in different experimental groups as indicated (n = 6, 8, 22, and 20 per group, respectively). Data are mean ± SEM. S, suramin; V, vehicle. Scale bar, 1 mm.

Figure 3

Suramin treatment inhibits the proliferation of cyst-lining epithelial cells in Pkd1 miR Tg mice. (A) Immunostaining for BrdU incorporation in kidney sections from Pkd1-miR Tg mice in vehicle- and suramin-treated groups. Arrows indicate BrdU-positive cyst lining epithelial cells. Scale bar, 50 µm. (B) Quantitative image analysis showed a significant reduction (p < 0.01) in the number of BrdU-positive cells in the suramin-treated Pkd1-miR Tg mice (n = 20) compared with the vehicle-treated mutant mice (n = 21) using an unpaired, two-tailed t-test. Data are mean ± SEM.

Figure 4

Suramin treatment inhibited renal inflammation and macrophage infiltration. (A) Immunostaining of kidney sections for F4/80 in different treatment groups as indicated. Quantitative image analysis showed a significant decrease in the percentage of F4/80 positive cells in the suramin-treated Pkd1-miR Tg mice compared with the vehicle-treated controls. Scale bar, 50 µm. (B) Suramin treatment reduced the mRNA expression of pro-inflammatory genes and purinergic receptor genes in the kidneys, as determined by qRT-PCR (n = 6, 8, 22, and 20 per group, respectively). Data are mean ± SEM.

Figure 5

Effect of suramin treatment on renal fibrosis in Pkd1-miR Tg mice. (A) Comparison of the renal Fn1, Col1a2, and Tgfb1 mRNA expression in different study groups as indicated. The internal control gene used for normalization was rodent 18S ribosomal RNA. (B) Immunohistochemistry staining for fibronectin in the kidneys of wild-type and Pkd1-miR Tg mice. Quantitative analysis showed increased fibronectin deposition in the cystic kidneys of Pkd1-miR Tg mice, but no significant changes were noted after treatment with suramin. Scale bar, 50 µm. Data are mean ± SEM. n = 6, 8, 22, and 20 per group, respectively.

Figure 6

Suramin reduces the phosphorylation of ERK1/2 in Pkd1-miR Tg mice. Kidney lysates from Pkd1-miR Tg mice and wild-type mice with or without suramin treatment were analyzed by Western blotting analysis. (A) Representative immunoblots of phosphorylated ERK1/2, total ERK1/2, and GAPDH. (B) The densitometric analysis of p-ERK1/2 normalized to total ERK1/2 protein expression. GAPDH was used for loading control. Data are mean ± SEM (n = 6, 8, 22, and 20 per group respectively).

Figure 7

Suramin induces cytoplasmic vacuolation and kidney injuries in non-cystic tubules. (A) Plasma levels of kidney injury markers (KIM-1, NGAL, cystatin C) were increased in the suramin-treated Pkd1-miR Tg mice compared to the vehicle-treated mutant mice. Data are mean ± SEM (n = 6, 8, 22, and 20 per group respectively). (B) Representative periodic acid-Schiff (PAS) staining in the kidney sections from the vehicle- and suramin-treated Pkd1-miR Tg mice (n = 3 per group). Arrow indicates the cytoplasmic vacuolation and degeneration of proximal tubules. Scale bar, 50 µm.