Specific endothelial heparin-binding EGF-like growth factor deletion ameliorates renal injury induced by chronic angiotensin II infusion

Abstract

Transactivation of EGF receptor (EGFR) by angiotensin II (Ang II) plays important roles in the initiation and progression of chronic kidney diseases. Studies suggest that heparin-binding EGF-like factor (HB-EGF) may be a critical mediator in this process, but its role in vivo has not been investigated. In the current study, we found that in response to Ang II infusion, kidneys from endothelial HB-EGF deletion mice had significantly reduced EGFR activation compared with controls. Meanwhile, deletion of endothelial HB-EGF expression decreased Ang II infusion related renal injury, as demonstrated by 1) less albuminuria; 2) less glomerulosclerosis; 3) preserved endothelial integrity and decreased podocyte injury, as shown by greater glomerular tuft area and WT1-positive cells, and fewer apoptotic cells measured by cleaved caspase 3 staining; 4) reduced inflammation in the perivascular area and interstitium measured by F4/80 and CD3 immunostaining; and 5) reduced renal fibrosis. In conclusion, our results suggest that shedding of HB-EGF from endothelium plays an important role in Ang II-induced renal injury by linking Ang II-AT1R with EGFR transactivation. Inhibition of HB-EGF shedding could be a potential therapeutic strategy for chronic kidney disease.

Keywords: angiotensin II; chronic kidney disease; heparin-binding epidermal growth factor-like factor.

Fig. 1.

Induction and confirmation of heparin-binding (HB) epidermal growth factor (EGF)-like factor deletion. A: X-gal staining (blue) showed that HB-EGF gene was expressed and had been deleted in the endothelium of glomerular and peritubular capillaries in HB^endo−/− mouse kidneys 2 wk after tamoxifen (TAM) injection. The kidney from HB^lox/lox mice was used as a negative control. Counterstain: sirius red. B: experimental protocol. HB-EGF deletion was induced by TAM injection at the age of 8 wk. Uninephrectomy (UNx) was performed in all mice, and minipumps infusing angiotensin II (Ang II; n = 7) or normal saline (NS; n = 6) were implanted subcutaneously and replaced after 4 wk. Measurements and tissue sampling were performed before and after Ang II or normal saline infusion.

Fig. 2.

EGFR activation and a disintegrin and metalloprotease 17 (ADAM17) augmentation by chronic Ang II infusion. A: compared with saline-infused mice, Ang II infusion increased kidney phospho-EGFR (p-EGFR) levels in both HB^lox/lox and HB^endo−/− mice, with the latter having much lower levels of p-EGFR. The total amount of EGFR was almost identical in all groups. Levels of ADAM17 were also increased in Ang II-infused kidneys compared with saline-infused groups. B and C: densitometric analysis of the Western blot bands in A expressed as p-EGFR/EGFR and ADAM17/β-actin. *P < 0.05 compared with normal saline infusions; ^P < 0.05 compared with Ang II-infused mice with endothelial HB-EGF deletion. Values represent the mean and SE of at least 3 independent experiments (n = 6–7 in each group). D: 8 wk after chronic Ang II infusion, increased p-EGFR immunostaining was seen in glomeruli, proximal tubules, perivascular infiltrating cells, and smooth muscle cells in the large blood vessels in the control HB^lox/lox mice, with significantly decreased expression in HB^endo−/− mouse kidneys. No p-EGFR immunostaining was detected in saline-infused groups.

Fig. 3.

Characterization of physical parameters of Ang II- or saline-infused mice. A: compared with saline-infused mice, systolic blood pressure (SBP) was increased in Ang II-infused mice, with no significant differences between HB^lox/lox and HB^endo−/− mice. B: telemetry studies were performed on Ang II-infused mice (n = 4 in each group). There were no significance differences between Ang II-infused HB^lox/lox and HB^endo−/− mice. In both groups, increased SBP levels were detected 2 days after Ang II infusion peaked around 4 days and remained elevated until the end of the experiment. C: heart rate profiles recorded by telemetry. There is an 8–10% increase in heart rate after Ang II infusion, with comparable levels in both groups (P > 0.05). D: mice body weight (BW) was slightly decreased after 4-wk Ang II infusion, whereas no significant differences were detected compared with saline-infused mice. E: at the end of the study, kidney and heart were weighed. No significant differences were seen in kidney weight (KW)/BW ratios in either Ang II- or saline-infused mice. F: compared with Ang II-infused HB^lox/lox mice, HB^endo−/− mice with endothelial HB-EGF deletion significantly reduced cardiac hypertrophy, indicated by lower heart weight (HW)/BW ratios. All the results shown are means ± SE of 6–7 mice in each group. *P < 0.05.

Fig. 4.

Endothelial HB-EGF deletion slowed down renal injury induced by chronic Ang II infusion. A: compared with saline-infused mice, albuminuria, as indicated by increased urinary albumin/creatinine ratios (ACR), was present after 2 wk of Ang II infusion and increased continuously with prolonged Ang II infusion. Compared with HB^lox/lox controls, HB^endo−/− mice had significantly lower ACR. (*P < 0.05 or #P < 0.001 compared with normal saline-infused mice; ^P < 0.05 or ΨP < 0.001 compared with Ang II-infused HB^endo−/− mice). B: periodic acid-Schiff (PAS)-stained kidney sections from Ang II- or saline-infused mice. In Ang II-infused mouse kidneys, tubular casts and glomerulosclerosis were detected in HB^lox/lox controls, whereas only mesangial expansion was seen in HB^endo−/− mice. No obvious renal morphological changes were observed in the kidneys of saline-infused mice. C: glomerular sclerosis shown by PAS staining in B was assessed by glomerular score (0–4) and shown as means ± SE of 6–7 samples in each group. D: renal fibrosis shown by anti-α-smooth muscle actin-Cy3 (α-SMA-Cy3) immunofluorescence staining. Original magnification ×400. All images represent the results of 6–7 mice in each group.

Fig. 5.

Glomerular injury and apoptosis in the kidneys of saline- or Ang II-infused mice. A, top: glomerular capillary density shown by Von Willebrand factor (vWF) immunostaining; middle: podocytes shown by WT1 staining; bottom: apoptosis shown by cleaved caspase-3 (CC3) immunostaining. B: representative images of glomerular capillary tuft quantification by converting color image to binary image using ImageJ software, as described in the materials and methods. C: quantification of glomerular capillary density through vWF staining in A. *P < 0.05 compared with all of the other groups. D: quantification of WT1-positive cells. *P < 0.05 compared with all of the other groups. E: quantification of CC3-positive cells. *P < 0.05 compared with normal saline-infused groups; ^P < 0.05 compared with Ang II-infused HB^endo−/− mouse kidneys. HPF, high-power field.

Fig. 6.

Representative images of VEGF-A staining in kidney sections from saline- or Ang II-infused mice. No obvious VEGF-A staining was detected in saline-infused kidneys in either group. In the kidneys from Ang II-infused mice, strong VEGF-A staining was seen in the glomeruli and vasculature in HB^lox/lox control, which was decreased dramatically in the HB^endo−/− kidneys.

Fig. 7.

Endothelial HB-EGF deletion reduced Ang II-induced renal inflammation. A: F4/80 staining indicated macrophage infiltration in the HB^lox/lox control kidneys after Ang II infusion, whereas only sparse F4/80-positive cells were detected in the HB^endo−/− kidneys. B: compared with Ang II-infused kidneys from HB^endo−/− mice, Ang II-infused HB^lox/lox kidneys showed increased interstitial staining of iNOS, an M1 marker, and weak positivity for arginase I, an M2 marker. C: infiltration of CD3-positive lymphocytes was seen in the periglomerular and perivascular regions in Ang II-infused HB^lox/lox control kidneys but was rarely detected in Ang II-infused HB^endo−/− kidneys. D: some of the infiltrating CD3-positive lymphocytes also expressed p-EGFR. E: more CD4- vs. CD8-positive cells were detected in the perivascular infiltration areas in both Ang II-infused groups, with HB^lox/lox control kidneys having significantly higher numbers of both CD4- and CD8-positive infiltrated cells.

Fig. 8.

Endothelial HB-EGF deletion lessened Ang II-induced inflammation cytokine production. A and B: increased proinflammatory cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1) were detected by ELISA in serum of Ang II-infused HB^lox/lox control mice, and their levels were significantly reduced in Ang II-infused HB^endo−/− mice. *P < 0.05 compared with all other groups. Values represent the mean SE of 6–7 mice in each group. C: both IL-6 and MCP-1 immunoreactivity were strongly detected in smooth muscle cells and perivascular infiltrating cells, with IL-6 also expressed in tubular epithelial cells, after Ang II infusion in the HB^lox/lox mice, which was markedly reduced in HB^endo−/− mice. D: strongtransforming growth factor-β (TGF-β) immunostaining was seen in the glomeruli, interstitium, and perivascular infiltrating cells after Ang II infusion in HB^lox/lox kidneys but was markedly reduced in HB^endo−/− kidneys.