Positive staining in nuclei-free areas of the mesangium, was assessed in 30 randomly selected glomeruli and scored in a blinded manner on a scale of 0 to 4, where 0 = 0? , 1 = .5?5 , 2 = .25?0 , 3 = .50?5 , 4 = .75 deposition. The scores reflected variations in the extent rather than intensity of staining, and the reproducibility of this scoring system has been documented [22]. The `sclerotic index’ referred to the mean score. Collagen deposition was assessed with Masson’s Title Loaded From File trichrome staining of 30 glomeruli, scored in a blinded manner using the aforementioned system and expressed as an arbitrary unit (AU). Tubulo-interstitial changes such as collagen deposition, tubular dilation and/or atrophy, and inflammatory cell infiltration were assessed in 10 non-overlapping areas free of glomeruli, and graded on a scale 1?, where 0 = normal, 0.5 = small focal areas of damage, 1 = ,10 , 2 = 10?5 , 3 = 26?5 and 4 = .75 damage in the renal cortex [22] and expressed as mean tubulointerstitial score for each group.Cytochemical StainingDetection of phosphorylated ERK and PKC-a, Title Loaded From File TGF-b1, fibronectin, and collagen type I, III and IV in paraffin-embedded kidney sections (5 mm) from non-diabetic or DN mice treated with saline or sulodexide was performed with specific antibodies (dilution 1:50), followed by peroxidase-anti-peroxidase staining, counterstained with hematoxylin and examined with an Axioskop 2 plus microscope as previously described [23,24]. Staining in the capillary loops, mesangium and tubulo-interstitium was semiquantitatively assessed in at least 30 glomeruli and tubules per mouse kidney, and the extent of staining graded as follows: 0 = 0?5 staining, 1 = .5?5 staining, 2 = .25?0 staining, 3 = .50?5 staining, 4 = .75 staining [22].Figure 2. The effect of sulodexide on albuminuria and renal function in control and DN C57BL/6 mice. (A) Urine albumin-tocreatinine (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 1655472 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 timepoint, #P,0.01, DN baseline vs sulodexide-treated DN mice, 1P,0.001, DN baseline vs saline-treated DN mice, {P,0.01, saline vs sulodexide treatment for the same time-point. doi:10.1371/journal.pone.0054501.gImmunohistochemical Analysis of Perlecan ExpressionSnap frozen renal sections (8 mm) were blocked with 3 BSA in PBS, incubated with rabbit anti-mouse perlecan antibody followed by the appropriate secondary antibody in a darkened humidified chamber. All incubation periods were 1 h at 37uC and sections were washed thrice with PBS between steps. Sections were mounted with fluorescent mountant and epifluorescence viewedSulodexide and Diabetic NephropathySulodexide and Diabetic NephropathyFigure 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.Positive staining in nuclei-free areas of the mesangium, was assessed in 30 randomly selected glomeruli and scored in a blinded manner on a scale of 0 to 4, where 0 = 0? , 1 = .5?5 , 2 = .25?0 , 3 = .50?5 , 4 = .75 deposition. The scores reflected variations in the extent rather than intensity of staining, and the reproducibility of this scoring system has been documented [22]. The `sclerotic index’ referred to the mean score. Collagen deposition was assessed with Masson’s trichrome staining of 30 glomeruli, scored in a blinded manner using the aforementioned system and expressed as an arbitrary unit (AU). Tubulo-interstitial changes such as collagen deposition, tubular dilation and/or atrophy, and inflammatory cell infiltration were assessed in 10 non-overlapping areas free of glomeruli, and graded on a scale 1?, where 0 = normal, 0.5 = small focal areas of damage, 1 = ,10 , 2 = 10?5 , 3 = 26?5 and 4 = .75 damage in the renal cortex [22] and expressed as mean tubulointerstitial score for each group.Cytochemical StainingDetection of phosphorylated ERK and PKC-a, TGF-b1, fibronectin, and collagen type I, III and IV in paraffin-embedded kidney sections (5 mm) from non-diabetic or DN mice treated with saline or sulodexide was performed with specific antibodies (dilution 1:50), followed by peroxidase-anti-peroxidase staining, counterstained with hematoxylin and examined with an Axioskop 2 plus microscope as previously described [23,24]. Staining in the capillary loops, mesangium and tubulo-interstitium was semiquantitatively assessed in at least 30 glomeruli and tubules per mouse kidney, and the extent of staining graded as follows: 0 = 0?5 staining, 1 = .5?5 staining, 2 = .25?0 staining, 3 = .50?5 staining, 4 = .75 staining [22].Figure 2. The effect of sulodexide on albuminuria and renal function in control and DN C57BL/6 mice. (A) Urine albumin-tocreatinine (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 1655472 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 timepoint, #P,0.01, DN baseline vs sulodexide-treated DN mice, 1P,0.001, DN baseline vs saline-treated DN mice, {P,0.01, saline vs sulodexide treatment for the same time-point. doi:10.1371/journal.pone.0054501.gImmunohistochemical Analysis of Perlecan ExpressionSnap frozen renal sections (8 mm) were blocked with 3 BSA in PBS, incubated with rabbit anti-mouse perlecan antibody followed by the appropriate secondary antibody in a darkened humidified chamber. All incubation periods were 1 h at 37uC and sections were washed thrice with PBS between steps. Sections were mounted with fluorescent mountant and epifluorescence viewedSulodexide and Diabetic NephropathySulodexide and Diabetic NephropathyFigure 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.
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