Sulodexide decreases albuminuria and regulates matrix protein accumulation in C57BL/6 mice with streptozotocin-induced type I diabetic nephropathy

Abstract

Objective: Sulodexide is a mixture of glycosaminoglycans that may reduce proteinuria in diabetic nephropathy (DN), but its mechanism of action and effect on renal histology is not known. We investigated the effect of sulodexide on disease manifestations in a murine model of type I DN.

Methods: Male C57BL/6 mice were rendered diabetic with streptozotocin. After the onset of proteinuria, mice were randomized to receive sulodexide (1 mg/kg/day) or saline for up to 12 weeks and renal function, histology and fibrosis were examined. The effect of sulodexide on fibrogenesis in murine mesangial cells (MMC) was also investigated.

Results: Mice with DN showed progressive albuminuria and renal deterioration over time, accompanied by mesangial expansion, PKC and ERK activation, increased renal expression of TGF-β1, fibronectin and collagen type I, III and IV, but decreased glomerular perlecan expression. Sulodexide treatment significantly reduced albuminuria, improved renal function, increased glomerular perlecan expression and reduced collagen type I and IV expression and ERK activation. Intra-glomerular PKC-α activation was not affected by sulodexide treatment whereas glomerular expression of fibronectin and collagen type III was increased. MMC stimulated with 30 mM D-glucose showed increased PKC and ERK mediated fibronectin and collagen type III synthesis. Sulodexide alone significantly increased fibronectin and collagen type III synthesis in a dose-dependent manner in MMC and this increase was further enhanced in the presence of 30 mM D-glucose. Sulodexide showed a dose-dependent inhibition of 30 mM D-glucose-induced PKC-βII and ERK phosphorylation, but had no effect on PKC-α or PKC-βI phosphorylation.

Conclusions: Our data demonstrated that while sulodexide treatment reduced proteinuria and improved renal function, it had differential effects on signaling pathways and matrix protein synthesis in the kidney of C57BL/6 mice with DN.

Figure 1. The effect of sulodexide on blood glucose, body weight, and kidney weight-to-body weight ratio in control and DN C57BL/6 mice.

(A) Blood glucose level, (B) body weight and (C) kidney weight-to-body weight ratio in control and DN mice treated with saline or sulodexide are shown. Results are expressed as mean+SD of data obtained from 6 mice per group. DN, diabetic nephropathy. *P<0.001, with vs without DN for the same time-point.

Figure 2. The effect of sulodexide on albuminuria and renal function in control and DN C57BL/6 mice.

(A) Urine albumin-to-creatinine (ACR) ratio, (B) serum creatinine level and (C) serum urea level in control and DN mice treated with saline or sulodexide are shown. Results are expressed as mean+SD of data obtained from 6 mice per group. Insert in (A) shows the effect of sulodexide on ACR in mice with microalbuminuria (white circle) and macroalbuminuria (black circle) with time. *P<0.01, **P<0.001, with vs without DN for the same time-point, ^# P<0.01, DN baseline vs sulodexide-treated DN mice, ^§ P<0.001, DN baseline vs saline-treated DN mice, ^‡ P<0.01, saline vs sulodexide treatment for the same time-point.

Figure 3. The effect of sulodexide treatment on renal histology in control and DN C57BL/6 mice.

(A) Representative images of PAS stained renal specimens obtained from control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Mesangial expansion, thickening of the Bowman’s capsule, and increased matrix accumulation are observed in DN mice and these changes are reduced in mice treated with sulodexide, to levels similar to those observed in non-diabetic mice. Image-based computer assisted analysis was performed to semi-quantify (B) glomerular sclerotic index as determined by the extent of PAS-positive staining and (C) surface area of the glomerular tuft. (D) Representative images of renal sections stained for collagen deposition using Masson’s trichrome staining (depicted by the blue color). Asterisks denote tubular atrophy. Image-based computer assisted analysis was performed to semi-quantify (E) the amount of collagen deposition in the glomeruli and (F) tubulo-interstitial changes in control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ***P<0.001, DN mice vs non-diabetic mice for the same treatment at the same time-point, ^### P<0.001, DN baseline vs non-diabetic baseline, ^§ P<0.05, ^§§ P<0.01, ^§§§ P<0.001, compared to baseline for the same group, ^‡‡‡ P<0.001, saline vs sulodexide for the same time-point in DN mice. Original magnification x1000 for panels (A) and (D).

Figure 4. The effect of sulodexide on perlecan expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of perlecan core protein in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of perlecan expression in snap frozen renal tissue from control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. (C) Image-based computer assisted analysis was performed to semi-quantify the amount of perlecan in the glomeruli of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§§§ P<0.001, compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 5. The effect of sulodexide on heparanase expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of heparanase in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of heparanase protein expression in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Image-based computer assisted analysis was performed to semi-quantify the amount of heparanase in the (C) glomeruli and (D) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§ P<0.05, ^§§ P<0.01, ^§§§ P<0.001, compared to baseline for the same group, ^## P<0.01, ^### P<0.001, DN baseline vs non-diabetic baseline, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡‡ P<0.01, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 6. The effect of sulodexide on phosphorylated PKC-α expression in the kidneys of control and DN C57BL/6 mice.

Representative images of (A) phosphorylated PKC-α in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Image-based computer assisted analysis was performed to semi-quantify the amount of phosphorylated PKC-α in the (B) glomeruli and (C) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§§§ P<0.001, compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 7. The effect of sulodexide on phosphorylated ERK expression in the kidneys of control and DN C57BL/6 mice.

Representative images of (A) phosphorylated ERK in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Image-based computer assisted analysis was performed to semi-quantify the amount of phosphorylated ERK in the (B) glomeruli and (C) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§§§ P<0.001, compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡ P<0.05, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 8. The effect of sulodexide on TGF-β1 gene and protein expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of TGF-β1 in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of TGF-β1 protein expression in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Image-based computer assisted analysis was performed to semi-quantify the amount of TGF-β1 in the (C) glomeruli and (D) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§ P<0.05, ^§§ P<0.01, ^§§§ P<0.001, compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, *P<0.05, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 9. The effect of sulodexide on collagen type I gene and protein expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of collagen type I (Coll I) in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of collagen type I expression in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Image-based computer assisted analysis was performed to semi-quantify the amount of collagen type I in the (C) glomeruli and (D) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§ P<0.05, ^§§ P<0.01, ^§§§ P<0.001, compared to baseline for the same group, ^# P<0.05, ^### P<0.001, DN baseline vs non-diabetic baseline, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡ P<0.05, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 10. The effect of sulodexide on collagen type IV gene and protein expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of collagen type IV (Coll IV) in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of collagen type IV expression in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Image-based computer assisted analysis was performed to semi-quantify the amount of collagen type IV in the (C) glomeruli and (D) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§ P<0.05, ^§§§ P<0.001, compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, **P<0.01, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 11. The effect of sulodexide on collagen type III gene and protein expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of collagen type III (Coll III) in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of collagen type III expression in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Boxed areas are enlarged to compare glomerular expression of collagen type III (depicted by arrows). Image-based computer assisted analysis was performed to semi-quantify the amount of collagen type III in the (C) glomeruli and (D) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§ P<0.05, ^§§§ P<0.001 compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, *P<0.05, **P<0.01, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡‡ P<0.01, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 12. The effect of sulodexide on fibronectin gene and protein expression in renal tissue in control and DN C57BL/6 mice.

(A) Gene expression of fibronectin (FN) in control and DN mice treated with saline or sulodexide as determined by real-time PCR. (B) Representative images of fibronectin expression in control and DN mice at baseline and after 12 weeks treatment with saline or sulodexide are shown. Original magnification x1000. Boxed areas are enlarged to compare glomerular expression of fibronectin (depicted by arrows). Image-based computer assisted analysis was performed to semi-quantify the amount of fibronectin in the (C) glomeruli and (D) tubulo-interstitium of control and DN mice. Results are expressed as mean+SD of data obtained from 6 mice per group. ^§ P<0.05, ^§§ P<0.01, ^§§§ P<0.001 compared to baseline for the same group, ^### P<0.001, DN baseline vs non-diabetic baseline, **P<0.01, ***P<0.001, DN mice vs non-diabetic mice for the same treatment, ^‡ P<0.05, ^‡‡ P<0.01, ^‡‡‡ P<0.001, saline vs sulodexide treatment for the same time-point in DN mice.

Figure 13. The effect of Gö6976, PD98059 and sulodexide on fibronectin and collagen type III synthesis and phosphorylation of signaling pathways in murine mesangial cells.

(A) Western blot analysis showing the effect of 5 mM D-glucose, 30 mM D-glucose and 30 mM mannitol in the presence or absence Gö6976 or PD98059 on fibronectin and collagen type III synthesis in murine mesangial cells after 24 h incubation (upper panel). The intensity of the bands were analyzed by densitometric scan using ImageJ (NIH), normalized to β-actin and expressed as arbitrary densitometric units (DU) (lower panels). ***P<0.001, 5 mM D-glucose vs 30 mM D-glucose, ^### P<0.001, 30 mM D-glucose vs 30 mM mannitol, ^§§ P<0.01 or ^§§§ P<0.001, with vs without inhibitor for the same stimulation. (B) Western blot analysis showing the effect of 5 mM D-glucose, 30 mM D-glucose and 30 mM mannitol in the presence or absence of sulodexide on fibronectin and collagen type III synthesis in murine mesangial cells after 24 h incubation. (C) Western blot analysis showing the effect of 5 mM D-glucose, 30 mM D-glucose and 30 mM mannitol in the presence or absence of sulodexide on ERK PKC-α, PKC-βI or PKC-βII phosphorylation in murine mesangial cells after 24 h incubation.

Figure 14. Schematic diagram summarizing the pathways through which glomerulosclerosis and tubulo-interstitial fibrosis are induced in C57BL/6 mice following streptozotocin administration and the differential effect of sulodexide on fibrotic processes.