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Complications

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

  1. Rachel Goldberg1,
  2. Ariel M. Rubinstein1,
  3. Natali Gil1,
  4. Esther Hermano1,
  5. Jin-Ping Li2,
  6. Johan van der Vlag3,
  7. Ruth Atzmon1,
  8. Amichay Meirovitz1 and
  9. Michael Elkin1⇑
  1. 1Sharett Institute, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
  2. 2Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
  3. 3Nephrology Research Laboratory, Department of Nephrology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands
  1. Corresponding author: Michael Elkin, melkin{at}hadassah.org.il.
  1. R.G. and A.M.R. contributed equally to this work.

Diabetes 2014 Dec; 63(12): 4302-4313. https://doi.org/10.2337/db14-0001
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    Figure 1

    Induction of TNF-α in the kidneys of diabetic mice correlates with the overexpression of heparanase. Kidney tissue was harvested from nondiabetic and diabetic WT and Hpse-KO mice 10 weeks after the induction of diabetes by STZ, lysed, and analyzed for TNF-α (A) and heparanase (Hpse) (B) expression by qRT-PCR. At least three mice per condition were analyzed. Error bars represent ±SE. C and D: The distribution and number of TNF-α+ cells in the tubulointerstitial and glomerular compartments of nondiabetic and diabetic mice were determined using immunofluorescent staining with anti–TNF-α (red) antibody. C, top: TNF-α+ cells in the cortical interstitial compartment were quantified per microscopic field (0.07 mm2), based on six sections from four independent mice in each group, under ×400 magnification. C, bottom: The number of TNF-α+ 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. D: Representative immunofluorescent (top) and light microscopy (bottom) images are shown, ×400 magnification. Glomeruli are delineated by dashed lines. D, diabetic; KO, knockout; ND, nondiabetic.

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    Figure 2

    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.

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    Figure 3

    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.

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    Figure 4

    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).

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    Figure 5

    A model of a heparanase-driven sequence of events powering chronic inflammation and kidney injury in DN. Increased glucose levels induce heparanase gene expression in diabetic kidney (A) (acting via an early growth response 1 [EGR1]-dependent mechanism, among other pathways; B). C: The overexpressed 65-kDa heparanase proenzyme (latent HPSE) is processed into its enzymatically active (8- + 50-kDa) form (active HPSE) by CatL, which is supplied by tubular cells activated by DM components. D: Active heparanase sustains macrophages stimulation by DM, resulting in increased production of kidney-damaging cytokines (i.e., TNF-α; E), thus fostering DN development and progression (F).

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Role of Heparanase-Driven Inflammatory Cascade in Pathogenesis of Diabetic Nephropathy
Rachel Goldberg, Ariel M. Rubinstein, Natali Gil, Esther Hermano, Jin-Ping Li, Johan van der Vlag, Ruth Atzmon, Amichay Meirovitz, Michael Elkin
Diabetes Dec 2014, 63 (12) 4302-4313; DOI: 10.2337/db14-0001

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Role of Heparanase-Driven Inflammatory Cascade in Pathogenesis of Diabetic Nephropathy
Rachel Goldberg, Ariel M. Rubinstein, Natali Gil, Esther Hermano, Jin-Ping Li, Johan van der Vlag, Ruth Atzmon, Amichay Meirovitz, Michael Elkin
Diabetes Dec 2014, 63 (12) 4302-4313; DOI: 10.2337/db14-0001
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