Prevention of neutrophil extravasation by hepatocyte growth factor leads to attenuations of tubular apoptosis and renal dysfunction in mouse ischemic kidneys

Abstract

Ischemia and reperfusion (I/R) injuries occur in numerous organs under pathophysiological conditions. In this process, neutrophils play important roles in eliciting parenchymal injuries. Using a murine model of renal I/R, we show that hepatocyte growth factor (HGF) is a natural ligand that inhibits endothelial injuries and neutrophil extravasation. In mice after renal I/R, plasma HGF levels increased, along with c-Met/HGF receptor phosphorylation in the vascular endothelium. However, this c-Met activation was transient, associated with a decrease in endogenous HGF level, and followed by neutrophil infiltration and renal dysfunction. Suppression of endothelial c-Met phosphorylation by anti-HGF IgG led to rapid progressions of neutrophil extravasation, tubular apoptosis, and renal dysfunction. Inversely, enhancement of the c-Met activation by exogenous HGF blocked endothelial/tubular apoptotic injuries and acute renal failure. In this process, HGF prevented endothelial nuclear factor kappaB activation and inhibited induction of an adhesion molecule (ICAM-1), resulting in attenuated vascular edema and neutrophil infiltration. Thus, we conclude that 1) the HGF/c-Met system of endothelial cells confers an initial barrier to block neutrophil infiltration, and 2) transient and insufficient HGF production allows manifestation of postischemic renal failure. Our study provides a rationale for why HGF supplementation elicits therapeutic effects in ischemic kidneys.

Figure 1

Changes in HGF levels during progression of neutrophil infiltration and tubular apoptosis in mice after renal I/R. A: Changes in the number of neutrophils (identified as Gr1-positive interstitial cells per ×200 field), noted in interstitial spaces of I/R-undergone kidneys (original magnification, ×330). The means of infiltrated neutrophils in each time point were calculated based on individual values (n = 6), which were determined on at least 20 fields per mouse. B: Alterations of tubular epithelial cell apoptosis (as determined by TUNEL staining) in ischemic kidneys (×330). C: Changes of plasma and renal HGF levels (as determined by a rodent HGF-specific ELISA system) after the release from warm ischemia. Statistical differences: *P < 0.05, **P < 0.01 versus pre-ischemia group (ie, 0-hour). For abbreviations, see text.

Figure 2

Acceleration of renal injuries and dysfunction in ischemic mouse kidneys by an anti-HGF IgG. A: Suppression of c-Met protein expression and phosphorylation by an antibody specific for rodent HGF. Left: Renal tissues were collected 1 hour after the I/R treatment, and then renal extract was immunoprecipitated with an anti-c-Met IgG and probed on PVDF membranes with an anti-phosphospecific c-Met IgG, as reported previously. Right: The band density was quantified using a densitometric analysis, with aid of computer software (NIH image). Data are expressed as mean ± SD (n = 4). **P < 0.01 versus normal IgG. B: Effects of the HGF-neutralizing treatment on BUN and plasma creatinine (Cr) levels. Mice were treated with anti-HGF IgG immediately after the surgical treatment of renal I/R and killed 8-hour postischemia. Data are shown as mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus normal IgG group. C: Aggravating effects of anti-HGF IgG on tubular apoptosis in the renal I/R-undergone mice (8 hours postischemia). Representative findings of the apoptotic tubules in TUNEL-stained sections (×160). D: Acceleration of interstitial neutrophil infiltrations in the kidneys at 8 hours after HGF neutralization, as evidenced by counting Gr1-positive interstitial cells per ×200-field.

Figure 3

c-Met localization in mouse ischemic kidneys. A: Western blotting analysis for c-Met expression levels in kidneys after I/R. β-actin was detected as an internal control. Representative data were shown, and the density was quantified using a densitometric analysis, with aid of computer software (NIH image). B: Immunohistochemical findings of phosphorylated c-Met/HGF receptor in vascular endothelial cells in mice before and after renal I/R (original magnification: ×360). Renal tissues were fixed in 70% ethanol and then subjected to a process for paraffin embedding. Anti-phosphorylated c-Met IgG (pMet-1234/1235) was applied on the dewaxed sections, followed by an ABC immuno-peroxidase method. Insets: Normal rabbit IgG was applied on renal tissues (1 and 18 hours postischemia) as a negative control. Arrowheads: Positive signals in tubules.

Figure 4

Inhibitory effect of HGF on neutrophil adhesion and ICAM-1/nuclear NF-κB location in vitro. A: HGF-mediated suppressions of adhesions between HUVEC and peripheral blood-derived neutrophils. HUVECs were incubated for 3 hours with TNF-α (5 ng/ml). HGF (0–30 ng/ml) or vehicle (BSA) was pulsed into the HUVEC culture 30 minutes before adding TNF-α. After washing the cells with medium, neutrophils were transferred onto the HUVEC cell layer. The co-culture was kept for an additional 30 minutes, and then neutrophil adhesion was quantified using a MPO assay. B: Immunoblot analysis of ICAM-1 in whole-HUVEC cell lysates (top), which were harvested 3 hours after adding TNF-α (5 ng/ml), HGF (30 ng/ml) plus TNF-α, or HGF. The remaining HUVECs were fixed on a glass disk with 70% ethanol and then stained using an anti-ICAM-1 IgG (bottom). C: Inhibitory effects of HGF on TNF-α-induced NF-κB activation. Top: Immunoblot analysis of NF-κB (p65) in the nuclear extraction of HUVEC, incubated for 3 hours. Middle: Changes in nuclear location of NF-κB, as revealed by immunocytochemistry. The positive percentage of NF-κB-stained nucleoli were counted in >1000 cells per well (n = 6). Bottom: DNA-binding ELISA for detecting activated NF-κB in the whole-cell lysates of HUVECs. The culture condition was the same as in Figure 4A. Data were shown as means ± SD (n = 6). **P < 0.01 versus BSA; ^#P < 0.05 versus TNF-α alone.

Figure 5

Changes in plasma human HGF levels during administrations of rh-HGF to mice after renal ischemia. A: An experimental protocol of rh-HGF supplement therapy in the mouse model of postischemic acute renal failure. rh-HGF (or saline) was subcutaneously injected into mice immediately after renal I/R, and s.c. injections were repeated at an interval of 6 hours. The mice were killed at 3 and 18 hours to determine the effect of HGF on postischemic renal damages. B: Time course changes in plasma human HGF levels at 1, 3, and 18 hours after start of rh-HGF supplement therapy. The plasma was obtained from postorbital veins, and human HGF levels were determined using a human HGF-specific ELISA kit. S, saline; H, rh-HGF; n.d., not detectable.

Figure 6

HGF-induced suppressions of endothelial nuclear NF-κB and ICAM-1 expressions, related to attenuated vascular injuries in mice. A: Immunohistochemical detections of phosphorylated c-Met and nuclear NF-κB in renal vessels of the ischemic mice. The kidneys were collected at 3 hours after renal I/R and then subjected to immunohistochemistry for phosphorylated c-Met as well as for NF-κB (brown signals) and CD-31 (red signals), respectively. Arrowheads, vascular endothelium. #, tubular epithelial areas. Top inset: The time-matched renal tissue was immunostained with normal rabbit IgG and anti-CD31 mouse IgG (ie, negative control). Bottom inset: Expanded finding of the NF-κB-positive nuclear staining. B: Changes in endothelial ICAM-1 scores in kidneys, as determined by histological scores. Renal tissues were sampled before or 3 hours after renal I/R and then subjected to immunohistochemical procedures. Typical findings of endothelial ICAM-1 expression were shown. Arrowheads, endothelial surface. C: Suppression of endothelial apoptosis by rh-HGF in renal tissues, obtained at 3 hours after renal I/R. The kidneys were fixed in cold 70% ethanol, sectioned, and immunostained using an anti-mouse CD31 IgG. The CD31-stained tissue sections were refixed in 10% formalin and then subjected to TUNEL staining, as reported. D: Changes in peri-vascular edematous lesions (H&E staining). Arrowheads, edematous lesions. #, inflammatory cell accumulation. All data are shown as means ± SD (n = 6). In these experiments, statistical differences are expressed as *P < 0.05; **P < 0.01.

Figure 7

Preventive effects of rh-HGF supplement therapy toward neutrophil infiltration, tubular apoptosis, and renal dysfunction in mice that have undergone renal I/R. A: Changes in neutrophil accumulations in interstitial spaces of ischemic kidneys, treated with or without rh-HGF therapy (Gr1 staining, ×300). Renal tissues were collected at 3 and 18 hour after the renal I/R challenge and then subjected to immunohistochemical procedures. Furthermore, granulocyte-specific MPO activities were measured in a part of samples used in immunohistochemistry. In this assay, the saline control level (3 hours postischemia) was defined as 100%. B: Decrease in tubular apoptosis levels and of BUN levels in the mouse model of renal I/R injury by rh-HGF treatment. Data are shown as means ± SD (n = 6). *P < 0.05; **P < 0.01 compared with the saline-injected control group.