Involvement of human heparanase in the pathogenesis of diabetic nephropathy.

Background: Decreased heparan sulfate proteoglycan content of the glomerular basement membrane has been described in proteinuric patients with diabetic nephropathy. Heparanase is an endo-b-Dglucuronidase that cleaves negatively charged heparan sulfate side chains in the basement membrane and extracellular matrix. Objectives: To investigate whether urine from type I diabetic patients differs in heparanase activity from control subjects and whether resident glomerular cells could be the source of urinary heparanase. Methods: Using soluble S-HSPG and sulfate-labeled extracellular matrix we assessed heparanase activity in human glomerular epithelial cells, rat mesangial cells, and urine from 73 type I diabetic patients. Heparanase activity resulted in the conversion of a high molecular weight sulfate-labeled HSPG into heparan sulfate degradation fragments as determined by gel filtration analysis. Results: High heparanase activity was found in lysates of both epithelial and mesangial cells. Immunohistochemical staining localized the heparanase protein to both glomeruli capillaries and tubular epithelium. Heparanase activity was detected in the urine of 16% and 25% of the normoalbuminuric and microalbuminuric diabetic patients, respectively. Urine from 40 healthy individuals did not possess detectable heparanase. Urinary heparanase activity was associated with worse glycemic control. Conclusion: We suggest that heparanase enzyme participates in the turnover of glomerular HSPG. Hyperglycemia enhances heparanase activity and/or secretion in some diabetic patients, resulting in the loss of albumin permselective properties of the GBM. IMAJ 2002;4:996±1002 Diabetic nephropathy is the most important single disorder leading to renal failure in the western world. Nephropathy develops in approximately 30% of patients with insulin-dependent diabetes mellitus. Microalbuminuria, occurring 10±15 years following the diagnosis of IDDM, is at present the earliest clinical marker identifying patients at risk to develop nephropathy [1]. The inability to recognize the subset of patients destined to develop diabetic nephropathy prior to the occurrence of microalbuminuria prevents early intervention which may modify the progressive nature of the disease. Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the cell surface and extracellular matrix [2]. The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The negatively charged nature of the molecule is due to Nand Olinked sulfate moieties [2]. The formation of a nearly albumin-free urine follows restriction to passage of proteins by the glomerular capillary wall, coupled with proximal tubular reabsorption of small amounts of filtered protein [3]. The permselective properties of the glomelular capillary wall represent the summation of size and charge limitation to ultrafiltration imposed by a fenestrated endothelium, the glomerular basement membrane and by epithelial podocyte foot processes with their interconnecting slit diaphgragms [4]. Alterations in size and charge-dependent selectivity of the glomerular capillary wall have been described in proteinuric patients with both diabetic and non-diabetic glomerulopathies [5,6]. These alterations can be attributed to structural modifications in anionic HSPG molecules, mainly agrin and perlecan, situated in the GBM [7]. Indeed, alterations in glomerular HSPG content and structure were reported in diabetic patients and are associated with its increased permeability to albumin [8,9]. Moreover, as suggested by the Steno hypothesis, altered HSPG metabolism is the underlying cause for both renal and extrarenal diabetic complications [10]. Heparanase is an endo-b-D-glucuronidase that cleaves HS at specific interchain sites [11]. Heparanase activity was found to correlate with the metastatic potential of cancer cells [11±13] and with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses [14]. Degradation of HS by heparanase results in the release of heparin-binding growth factors, enzymes and plasma proteins that are sequestered by HS in basement membranes and cell surfaces [11]. Partial sequencing of heparanase purified from human placenta, platelets and hepatoma cells, followed by screening of expressed sequence tag (EST) databases led to the Original Articles Dedicated to the memory of Prof. Amiram Eldor whose inspiration, wisdom, and encouragement contributed to the accomplishment of this study and the heparanase research project in general. HSPG = heparan sulfate proteoglycan GBM = glomerular basement membrane IDDM = insulin-dependent diabetes mellitus HS = heparan sulfate 996 A. Katz et al. IMAJ . Vol 4 . November 2002 cloning of a cDNA and gene encoding the heparanase protein [13,15]. Only one sequence was identified, consistent with the notion that this is the dominant endoglucuronidase in mammalian tissues [11±13,15]. Expression of the cloned cDNA in insect and mammalian cells yielded 65 and 50 kDa latent and highly active heparanase, respectively [11±13,15]. At present, there is no information regarding enzymatic activity of heparanase in the kidney, both in health and disease. Removal of HSPG by in situ enzymatic digestion with bacterial heparinase was shown to cause an increased permeability of the GBM to ferritin, and I-labelled albumin ± both negatively charged molecules [16]. The present study examined whether urine from type I diabetic patients differs in heparanase activity from control subjects and whether resident glomerular cells could be the source of urinary heparanase. Materials and Methods