Background: The beta-D-endoglycosidase heparanase has been proposed as an important contributor to loss of glomerular charge in proteinuria. Expression of heparanase was, therefore, determined in acute puromycin aminonucleoside (PAN) nephrosis.
Methods: A rabbit polyclonal antibody was produced against a 17-amino acid peptide derived from the predicted amino acid sequence of heparanase. The antibody was validated by Western blot. Immunohistochemical staining and Western blotting were used to localize heparanase protein in normal kidneys and kidneys from rats with PAN nephrosis. Northern blot analysis was used to determine mRNA expression.
Results: Immunohistochemical staining showed that heparanase protein was localized to tubular cells in the distal convoluted tubules, thick ascending limb of the loop of Henle, and transitional cell epithelium in normal kidney. Minimal expression was noted in normal glomeruli. Western blot analysis of protein from isolated normal glomeruli showed minimal expression of the 65 kD proheparanase protein. A marked increase in the staining for heparanase was found at day 5 of the PAN nephrosis model, at approximately the time of onset of proteinuria, and at day 14. Expression was predominantly seen in podocytes. At day 5, only the 65 kD proheparanase species was identified, but at day 14, mature 58 kD heparanase also was present. Northern blot analysis of sieved glomeruli at day 14 confirmed an increase in heparanase mRNA. The human podocyte cell line 56/10A1 also produced both proheparanase and mature heparanase, suggesting that podocytes can activate heparanase without other cell types.
Conclusion: The previously mentioned data confirm that the novel beta-D-endoglycosidase heparanase is up-regulated and activated in glomeruli from rats with proteinuria. Heparanase may be involved, therefore, in the loss of glomerular charge seen in proteinuria. Moreover, the presence of heparanase in normal tubules suggests that it may also be involved in cell migration or turnover.