Tigated. Specific inhibitors to PKC (Go6976, 10 mM) and ERK (PD98059, 50 mM) were used ?to determine whether fibronectin and collagen type III synthesis was mediated through PKC or ERK phosphorylation.normalized to total ERK, PKC-a, PKC-bI and PKC-bII respectively.Statistical AnalysesResults are expressed as mean+SD. Statistical analysis was performed using GraphPad Prism version 5.0 for Windows, (GraphPad Software, San Diego, CA, USA). Differences were assessed by ANOVA followed by Bonferroni’s multiple comparison post-test. Two-tailed P,0.05 was considered statistically significant.Western Blot AnalysisWhole cell lysates of MMC were obtained by solubilizing cells cultured under control or experimental conditions in 20 mM A196 web sodium acetate (pH 6.0) containing 4 M urea and 1 Triton X100 (200 ml). Aliquots of each cell lysate (10 mg total protein content determined with a modified Lowry assay) were denatured in sample buffer at 95uC for 5 min and subjected to SDS-PAGE. Samples were electrophoresed on 8 acrylamide gels to investigate fibronectin and collagen type I and III synthesis, and on 12 acrylamide gels to investigate ERK, PKC-a, PKC-bI and PKCbII phosphorylation [24]. Proteins were transferred onto nitrocellulose membranes using a mini-gel transfer system at 100 V for 1 h at 4uC. Equal loading of proteins was confirmed by staining the membranes with Ponceau S solution. Membranes were immunoblotted with primary antibodies to fibronectin, collagen type III, b-actin, total and phosphorylated (phospho) ERK, PKCa, PKC-bI and PKC-bII, followed by the relevant horseradish peroxidase-conjugated secondary antibodies as previously described [25]. Bands were visualized by ECL and the band intensity semi-quantitated by densitometry using ImageJ (NIH) software, normalized to their respective house-keeping protein and expressed as arbitrary densitometric unit (DU). Fibronectin and collagen type III were normalized to b-actin, and phospho-ERK, phospho-PKC-a, phospho-PKC-bI and phospho-PKC-bII MedChemExpress LY2409021 wereResultsPersistent proteinuria accompanied by blood glucose level above 30 mM was observed in 50 of male C57BL/6 mice 4? weeks 1655472 after intra-peritoneal STZ administration. These mice were then randomized to either saline or sulodexide treatment for periods up to 12 weeks, with non-diabetic mice under same treatments as negative controls.Sulodexide Reduces Albuminuria and Renal Function Deterioration in DN MiceThere was no difference in the survival of saline- or sulodexidetreated DN or non-diabetic mice (data not shown). Elevated blood glucose level remained stable over time in sulodexide-treated DN mice, and was comparable to saline-treated controls (Figure 1A). Sulodexide did not affect the blood glucose level in non-diabetic mice. Mice with DN failed to gain weight, and their weight was 49.80 that of their non-diabetic counterpart after 12 weeks (22.4363.32 and 44.6865.91 g respectively, P,0.001) (Figure 1B). The kidney weight-to-body weight ratio was significantly higher in DN mice compared with non-diabetic mice (1.1160.26 vs 0.4760.04 after 12 weeks, P,0.001) (Figure 1C). SulodexideSulodexide and Diabetic Nephropathytreatment did not affect body weight or kidney weight-to-body weight ratio (Figure 1B and C). Urine albumin-to-creatinine ratio (ACR) increased over time in DN mice (105.30651.47 vs 19.42612.65 g/mmol, 12 weeks vs baseline, P,0.001), which was markedly reduced with sulodexide treatment (Figure 2A). After 12 weeks of sulodexide treatmen.Tigated. Specific inhibitors to PKC (Go6976, 10 mM) and ERK (PD98059, 50 mM) were used ?to determine whether fibronectin and collagen type III synthesis was mediated through PKC or ERK phosphorylation.normalized to total ERK, PKC-a, PKC-bI and PKC-bII respectively.Statistical AnalysesResults are expressed as mean+SD. Statistical analysis was performed using GraphPad Prism version 5.0 for Windows, (GraphPad Software, San Diego, CA, USA). Differences were assessed by ANOVA followed by Bonferroni’s multiple comparison post-test. Two-tailed P,0.05 was considered statistically significant.Western Blot AnalysisWhole cell lysates of MMC were obtained by solubilizing cells cultured under control or experimental conditions in 20 mM sodium acetate (pH 6.0) containing 4 M urea and 1 Triton X100 (200 ml). Aliquots of each cell lysate (10 mg total protein content determined with a modified Lowry assay) were denatured in sample buffer at 95uC for 5 min and subjected to SDS-PAGE. Samples were electrophoresed on 8 acrylamide gels to investigate fibronectin and collagen type I and III synthesis, and on 12 acrylamide gels to investigate ERK, PKC-a, PKC-bI and PKCbII phosphorylation [24]. Proteins were transferred onto nitrocellulose membranes using a mini-gel transfer system at 100 V for 1 h at 4uC. Equal loading of proteins was confirmed by staining the membranes with Ponceau S solution. Membranes were immunoblotted with primary antibodies to fibronectin, collagen type III, b-actin, total and phosphorylated (phospho) ERK, PKCa, PKC-bI and PKC-bII, followed by the relevant horseradish peroxidase-conjugated secondary antibodies as previously described [25]. Bands were visualized by ECL and the band intensity semi-quantitated by densitometry using ImageJ (NIH) software, normalized to their respective house-keeping protein and expressed as arbitrary densitometric unit (DU). Fibronectin and collagen type III were normalized to b-actin, and phospho-ERK, phospho-PKC-a, phospho-PKC-bI and phospho-PKC-bII wereResultsPersistent proteinuria accompanied by blood glucose level above 30 mM was observed in 50 of male C57BL/6 mice 4? weeks 1655472 after intra-peritoneal STZ administration. These mice were then randomized to either saline or sulodexide treatment for periods up to 12 weeks, with non-diabetic mice under same treatments as negative controls.Sulodexide Reduces Albuminuria and Renal Function Deterioration in DN MiceThere was no difference in the survival of saline- or sulodexidetreated DN or non-diabetic mice (data not shown). Elevated blood glucose level remained stable over time in sulodexide-treated DN mice, and was comparable to saline-treated controls (Figure 1A). Sulodexide did not affect the blood glucose level in non-diabetic mice. Mice with DN failed to gain weight, and their weight was 49.80 that of their non-diabetic counterpart after 12 weeks (22.4363.32 and 44.6865.91 g respectively, P,0.001) (Figure 1B). The kidney weight-to-body weight ratio was significantly higher in DN mice compared with non-diabetic mice (1.1160.26 vs 0.4760.04 after 12 weeks, P,0.001) (Figure 1C). SulodexideSulodexide and Diabetic Nephropathytreatment did not affect body weight or kidney weight-to-body weight ratio (Figure 1B and C). Urine albumin-to-creatinine ratio (ACR) increased over time in DN mice (105.30651.47 vs 19.42612.65 g/mmol, 12 weeks vs baseline, P,0.001), which was markedly reduced with sulodexide treatment (Figure 2A). After 12 weeks of sulodexide treatmen.