Role of Heparanase-Driven Inflammatory Cascade in Pathogenesis of Diabetic Nephropathy

Heparanase sensitizes macrophages to activation by DM components in vitro. A: Mouse peritoneal macrophages were plated in medium containing low glucose (1.0 g/L) and either remained untreated (Cont) or incubated (2 h, 37°C) with the following: active recombinant heparanase (Hpse; 0.8 μg/mL); high glucose (hg; 4.5 g/L); Hpse + hg; AGE (60 μg/mL) + hg; Hpse + AGE + hg; IFN-γ (100 units) + AGE + hg; Hpse + IFN-γ + AGE + hg; albumin (BSA, 30 μg/mL) + IFN-γ + AGE + hg; Hpse + BSA + IFN-γ + AGE + hg; and iHpa. TNF-α secretion was evaluated by ELISA of the conditioned medium. Note that active heparanase enzyme, purified as described by Blich et al. (52), markedly enhanced the production of TNF-α by macrophages activated by various combinations of DM components. B: Induction of NF-κB signaling in macrophages activated by the combination of kidney DM components (glucose 4.5 g/L; AGE 60 μg/mL; albumin 30 μg/mL) in the absence and the presence of heparanase (Hpse; 0.8 μg/mL) was assessed by immunofluorescent analysis using anti-phospho-p65 (yellow) antibodies. Macrophage nuclei were counterstained with DRAQ5 (red). Bi: Resting macrophages (Mϕ). Bii: Mϕ activated by DM. Biii: Mϕ activated by DM in the presence of Hpse. Biv: Mϕ activated by LPS (100 ng/mL) served as a positive control. C: Quantification of the effect of heparanase on the nuclear localization of phospho-p65 in macrophages activated by DM. The nuclear accumulation of p65 in ≥100 cells/condition was analyzed. The percentage of cells with nuclear p65 is indicated. Error bars represent SE. *P < 0.04, **P < 0.02.

A and B: Increased number of TNF-α–expressing macrophages in diabetic kidneys of WT mice compared with Hpse-KO mice. A: Diabetes was induced by STZ, as described in Research Design and Methods. On experimental week 16, nondiabetic (ND) and diabetic (D) WT and Hpse-KO (KO) mice were killed, and their kidney tissue was processed for double-immunofluorescent analysis with anti-F4/80 (yellow) and anti–TNF-α (red) antibodies. Cell nuclei were counterstained with DRAQ5 (blue). Representative images are shown. F4/80⁺TNF-α⁺ double-positive cells in the tubulointerstitial (white arrows) and glomerular (green arrowheads) compartments of diabetic kidneys in WT mice are indicated. Glomeruli are delineated by dashed lines. Scale bars, 50 μm. B, top: F4/80⁺TNF-α⁺ double-immunostained cells in the tubulointerstitial compartment were counted per microscopic field (0.07 mm²), based on six sections from four independent mice of each group, under ×400 magnification. B, bottom: The number of double-immunostained glomerular cells per glomerular cross-section (GCS) was counted in ≥40 glomeruli per animal. Data are the mean ± SE, n = 4 mice per condition. *P < 0.01, **P < 0.001. C: Macrophages from WT mice express higher levels of TNF-α compared with Hpse-KO mice. Peritoneal macrophages derived from WT and Hpse-KO mice were either untreated or incubated (2 h, 37°C) with DM components (120 μg/mL AGE and BSA, 100 units IFN-γ). TNF-α expression was evaluated by qRT-PCR. **P < 0.001, ***P < 0.008.

Induction of CatL under diabetic conditions in vivo and in vitro. A–D: CatL protein levels in kidneys from nondiabetic and diabetic mice. Kidneys were harvested from STZ-treated type 1 diabetic mice (A and C) and db/db type 2 diabetic mice (B and D) and were analyzed for CatL protein levels by immunoblotting showing 43-kDa procathepsin L (Pro) and 25-kDa (active) forms of the enzyme (A and B) and immunostaining (C and D). C and D, top: Note the CatL staining (brown) in the tubular compartment of diabetic kidney (original magnification ×200). C and D, bottom: Specimens were scored according to CatL immunoreactive intensity (0–3) and distribution (0–4). The intensity was scored as follows: 0, no staining; 1, weak staining; 2, medium staining; 3, strong staining. The percentage of positive cells was scored as “0” (<5%), “1” (5–25%), “2” (25–50%), “3” (50–75%), and “4” (>75%). The data shown are the mean ± SE of staining scores, n = 4 mice per condition. **P < 0.001. E and F: Induction of CatL in proximal tubuli cell line HK-2 by DM components. HK-2 cells were either untreated (Cont) or incubated (24 h, 37°C) with DM components (4.5 g/L glucose, 120 μg/mL AGE and BSA, 100 units IFN-γ) and processed for Western blot analysis (E) and immunofluorescent staining (F). F, right panel: Staining intensity was quantified using Zen software (Carl Zeiss) per microscopic field (0.012 mm²), based on 40 fields per condition. Data shown are the mean intensity ± SE. *P = 0.006. G: Activation of latent heparanase by DM-stimulated HK-2 cells. Heparanase enzymatic activity was examined using sulfate-labeled ECM as the substrate (32,38,39) in lysates of HK-2 cells, either untreated (HK2) or stimulated by DM components (HK2 + DM), as described above, in the absence or presence of 65-kDa heparanase precursor (65 Hpse) purified as shown previously (32). Note the increased enzymatic activity upon incubation of 65 Hpse with DM-stimulated HK-2 cells (HK2 + DM + 65 Hpse).