Hased from Santa Cruz Biotechnology Inc. (Genetimes Technology International Holding Ltd, Hong Kong). Phosphorylated PKC-bII and ERK were purchased from Cell Signaling Technology (Gene Company, Hong Kong). Fibronectin antibody was purchased from 317318-84-6 supplier Sigma-Aldrich Chemical Company (Tin Hang Technology Ltd, Hong Kong). QuantiChrom albumin, creatinine and urea assay kits were purchased from BioAssay Systems (California, USA). Accu-Chek Advantage II Glucostix test strips and AccuChek Advantage blood glucose meter were purchased from Roche Diagnostics (DKSH Hong Kong Ltd, Hong Kong). Sulodexide (Vessel Due F) was purchased from Alfa Wassermann (Guangzhou, China).Animal StudiesMale C57BL/6 mice at 6? weeks of age were purchased from the Laboratory Animal Unit (University of Hong Kong, Hong Kong) and received standard chow and water ad libitum. After one week acclimatizing to their surroundings, mice were GNF-7 fasted for 6 h prior to intra-peritoneal injection of streptozotocin (STZ, 50 mg/ kg) in 10 mM citrate buffer, pH 4.5, administered on five consecutive days. Diabetes mellitus was confirmed by tail vein blood sampling of glucose concentration, measured with AccuChek Advantage II Glucostix test strips. Spot urine was tested weekly for albuminuria with QuantiChrom albumin assay kit until sacrifice. Mice with elevated blood glucose levels (.10 mM) and albuminuria (.100 mg/dl) on two separate occasions two days apart (defined as `baseline’ in the animal studies) were randomizedFigure 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. doi:10.1371/journal.pone.0054501.gSulodexide and Diabetic Nephropathyto receive treatment with either saline (vehicle control) or sulodexide (1 mg/kg/day) by oral gavage for 2, 4, 8 or 12 weeks (6 mice per time-point for 1655472 each group). After 2, 4, 8 and 12 weeks of treatment, mice were sacrificed, blood samples were obtained by cardiac puncture and the kidneys harvested, decapsulated and weighed. The left kidney was cut perpendicular to the long-axis and one half of the kidney was snap frozen in OCT followed by immersion in liquid nitrogen, while the second half was fixed in 10 neutral-buffered formalin followed by paraffin embedding. Renal cortical tissue from the right kidney was separated from the medulla and frozen at 280uC until mRNA isolation. Six diabetic mice that had just developed proteinuria were also sacrificed to obtain baseline values for clinical, histological and morphometrical parameters. Negative control groups included non-diabetic male C57BL/6 mice treated with either saline or sulodexide for 12 weeks. Serum creatinine and urea levels were measured using QuantiChrom creatinine and urea assay kits respectively.Histological Assessment of the KidneyParaffin-embedded kidney sections (5 mm) were stained with periodic acid-Schiff (PAS) and Masson’s trichrome for histologic and morphometric analysis. Thirty cross-sectional profiles of PASstained glomeruli were captured for each mouse. The glomerular tuft area was determined using Axiovision 4.3 software (Zeiss, Hong Kong). Assessment of mesangial matrix accumulation, denoted by PAS-.Hased from Santa Cruz Biotechnology Inc. (Genetimes Technology International Holding Ltd, Hong Kong). Phosphorylated PKC-bII and ERK were purchased from Cell Signaling Technology (Gene Company, Hong Kong). Fibronectin antibody was purchased from Sigma-Aldrich Chemical Company (Tin Hang Technology Ltd, Hong Kong). QuantiChrom albumin, creatinine and urea assay kits were purchased from BioAssay Systems (California, USA). Accu-Chek Advantage II Glucostix test strips and AccuChek Advantage blood glucose meter were purchased from Roche Diagnostics (DKSH Hong Kong Ltd, Hong Kong). Sulodexide (Vessel Due F) was purchased from Alfa Wassermann (Guangzhou, China).Animal StudiesMale C57BL/6 mice at 6? weeks of age were purchased from the Laboratory Animal Unit (University of Hong Kong, Hong Kong) and received standard chow and water ad libitum. After one week acclimatizing to their surroundings, mice were fasted for 6 h prior to intra-peritoneal injection of streptozotocin (STZ, 50 mg/ kg) in 10 mM citrate buffer, pH 4.5, administered on five consecutive days. Diabetes mellitus was confirmed by tail vein blood sampling of glucose concentration, measured with AccuChek Advantage II Glucostix test strips. Spot urine was tested weekly for albuminuria with QuantiChrom albumin assay kit until sacrifice. Mice with elevated blood glucose levels (.10 mM) and albuminuria (.100 mg/dl) on two separate occasions two days apart (defined as `baseline’ in the animal studies) were randomizedFigure 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. doi:10.1371/journal.pone.0054501.gSulodexide and Diabetic Nephropathyto receive treatment with either saline (vehicle control) or sulodexide (1 mg/kg/day) by oral gavage for 2, 4, 8 or 12 weeks (6 mice per time-point for 1655472 each group). After 2, 4, 8 and 12 weeks of treatment, mice were sacrificed, blood samples were obtained by cardiac puncture and the kidneys harvested, decapsulated and weighed. The left kidney was cut perpendicular to the long-axis and one half of the kidney was snap frozen in OCT followed by immersion in liquid nitrogen, while the second half was fixed in 10 neutral-buffered formalin followed by paraffin embedding. Renal cortical tissue from the right kidney was separated from the medulla and frozen at 280uC until mRNA isolation. Six diabetic mice that had just developed proteinuria were also sacrificed to obtain baseline values for clinical, histological and morphometrical parameters. Negative control groups included non-diabetic male C57BL/6 mice treated with either saline or sulodexide for 12 weeks. Serum creatinine and urea levels were measured using QuantiChrom creatinine and urea assay kits respectively.Histological Assessment of the KidneyParaffin-embedded kidney sections (5 mm) were stained with periodic acid-Schiff (PAS) and Masson’s trichrome for histologic and morphometric analysis. Thirty cross-sectional profiles of PASstained glomeruli were captured for each mouse. The glomerular tuft area was determined using Axiovision 4.3 software (Zeiss, Hong Kong). Assessment of mesangial matrix accumulation, denoted by PAS-.
