Determination of heparanase levels in urine (A, B) and plasma (C, D) of individuals from the study groups.

<p>Shown are average (±SE; A, C) and median (B, D) values quantified by an ELISA method, as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017312#s2" target="_blank">Materials and Methods</a>’.</p

Demographic description of enrolled individuals.

<p>Demographic description of enrolled individuals.</p

Immunofluorescent staining. Heparanase transfected 293 cells were left untreated as control (Con) or stimulated with insulin (250 and 50 pM) for 2 h.

<p>Cells were then fixed, stained with monoclonal anti-heparanase antibody (upper panel, green) and examined by confocal microscopy. Merged images with nuclear counterstaining (red) are shown in the lower panels. Note more diffused heparanase-positive vesicles in response to insulin stimulation.</p

Insulin cooperates with glucose to stimulate secretion of enzymatically active heparanase.

<p><b>A</b>. Immunoblotting. Heparanase-transfected 293 cells were cultured under normal (0.45%) or high (3%) glucose conditions in serum free medium for 20 h. Cells were left untreated (0) or incubated with the indicated concentration of insulin for 2 h. Cell conditioned medium (1 ml) was then collected, and TCA-precipitates were subjected to immunoblotting applying anti-heparanase (upper panel) and anti-cathepsin D (second panel) antibodies. Densitometry analysis of the active 50 kDa heparanase is shown in the lower panel. <b>B</b>. Heparanase enzymatic activity. Corresponding medium samples of untreated cells (control; ♦) or cells treated with insulin (50 pM) under low (▪) or high (▴) glucose were applied onto 35 mm dishes coated with ³⁵S-labeled ECM for 20 h. The medium was then collected and sulfate labeled HS degradation fragments were analyzed by gel filtration, as described under "<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017312#s2" target="_blank">Materials and Methods</a>". <b>C.</b> Kinetics. Heparanase transfected 293 cells were grown under serum-free conditions and left untreated (Con) or stimulated with insulin under low (0.45%; Ins) or high (3%; Ins+glu) glucose levels. At the time indicated, conditioned medium was collected and subjected to immunoblotting applying anti-heparanase antibody.</p

Heparanase levels in the urine and plasma of healthy volunteers (control), type 2 diabetic patients (T2DM) and T2DM patients who underwent kidney transplantation.

<p>Heparanase levels in the urine and plasma of healthy volunteers (control), type 2 diabetic patients (T2DM) and T2DM patients who underwent kidney transplantation.</p

Association between blood glucose and heparanase levels.

<p><b>A, B</b>. Urine and plasma heparanase levels presented according to gender. Shown are average (±SE) values of heparanase levels in urine (A) and plasma (B) quantified by ELISA in males (M) and females (F) of healthy volunteers (control), T2DM patients and T2DM patients who underwent kidney transplantation. <b>C, D</b>. Association between urine and plasma heparanase and blood glucose levels. Heparanase levels in plasma and urine were correlated with blood glucose levels using the non-parametric Spearman's rank test. Heparanase levels in the urine were found to correlate with blood glucose (<b>C</b>; r = 0.52, p = 0.0001); blood glucose was also associated with plasma heparanase (<b>D</b>; r = 0.38, p = 0.003).</p