Heparanase Levels Are Elevated in the Urine and Plasma of Type 2 Diabetes Patients and Associate with Blood Glucose Levels

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans. Utilizing an ELISA method capable of detection and quantification of heparanase, we examined heparanase levels in the plasma and urine of a cohort of 29 patients diagnosed with type 2 diabetes mellitus (T2DM), 14 T2DM patients who underwent kidney transplantation, and 47 healthy volunteers. We provide evidence that heparanase levels in the urine of T2DM patients are markedly elevated compared to healthy controls (1162±181 vs. 156±29.6 pg/ml for T2DM and healthy controls, respectively), increase that is statistically highly significant (P<0.0001). Notably, heparanase levels were appreciably decreased in the urine of T2DM patients who underwent kidney transplantation, albeit remained still higher than healthy individuals (P<0.0001). Increased heparanase levels were also found in the plasma of T2DM patients. Importantly, urine heparanase was associated with elevated blood glucose levels, implying that glucose mediates heparanase upregulation and secretion into the urine and blood. Utilizing an in vitro system, we show that insulin stimulates heparanase secretion by kidney 293 cells, and even higher secretion is observed when insulin is added to cells maintained under high glucose conditions. These results provide evidence for a significant involvement of heparanase in diabetic complications

Clinical Significance of Urine Heparanase in Bladder Cancer Progression1

Heparanase is an endo-β-glucuronidase capable of cleaving heparan sulfate (HS), an activity implicated in tumor metastasis. Heparanase expression is upregulated in primary human tumors, correlating with reduced post operative survival and elevated microvessel density. An ELISA method was used to quantify heparanase in urine from 282 individuals. Urine was collected from healthy volunteers (n = 41), patients diagnosed with noncancerous pathologic disorders (n = 90), and bladder cancer patients (n = 92). Fifty-nine bladder carcinoma patients after transurethral resection (TUR) with no evidence of disease (NED) were also included. Heparanase levels were significantly elevated in urine from bladder cancer patients compared with healthy controls (P < .001) and with noncancerous urinary disorders (P < .05). Heparanase elevation strongly correlated with tumor grade (P < .001) and stage (P = .027). An optimal cutoff value of 154 pg/ml was determined. Of 199 individuals enrolled (59 patients after TUR and 24 patients with recurring disease were excluded), 65 had heparanase levels above 154 pg/ml. Only 3 of 65 (4.6%) were healthy individuals. In contrast, 52.3% (34 of 65) of individuals with heparanase levels above 154 pg/ml were bladder cancer patients. The results indicate that urine heparanase levels are elevated during bladder cancer progression, suggesting that the ELISA method may be applied for bladder cancer diagnosis

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

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

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