Attenuation of Glycerol-Induced Acute Kidney Injury by Previous Partial Hepatectomy: Role of Hepatocyte Growth Factor/c-met Axis in Tubular Protection

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Original Paper

Nephron Exp Nephrol 2007;107:e95–e106 DOI: 10.1159/000109828

Attenuation of Glycerol-Induced Acute Kidney Injury by Previous Partial Hepatectomy:Role of Hepatocyte Growth Factor/c-met Axis in Tubular Protection

Eduardo Homsi a Patricia Janino a Subrata K. Biswas a Shinya Mizuno b

Toshikazu Nakamura b Jose B. Lopes de Faria a

a Division of Nephrology, Department of Medicine, School of Medical Sciences, State University ofCampinas, São Paulo , Brazil; b Division of Molecular Regenerative Medicine, Department of Biochemistryand Molecular Biology, Osaka University Graduate School of Medicine, Osaka , Japan

infiltration. The regenerative response was less intense 24 and 72 h after glycerol administration in this group. The anti-HGF treatment disclosed an important role of HGF in the re-duction of tubular injury, particularly apoptosis. Overexpres-sion of heme oxygenase-1 was observed in Gly-AKI+HPTX rats, but was not associated with HPTX-induced renal pro-tection. Conclusion: We conclude that Gly-AKI+HPTX rats have a reduced susceptibility to renal injury instead of an increased regenerative response and that endogenous HGF overexpression is responsible for suppression of tubular apoptosis. Copyright © 2007 S. Karger AG, Basel

Introduction

Acute kidney injury secondary to glycerol-induced rhabdomyolysis is characterized by intense cortical acute tubular necrosis, preglomerular vasoconstriction, tu-bular apoptosis in the renal medulla, and mononuclear leukocyte infiltration [1, 2] . The mechanisms of renalaggression involve both ischemia due to shift of intravas-cular fluid to necrotic muscle and myoglobin nephrotox-icity [3, 4] . Myoglobin reabsorbed at proximal tubules in-

Key Words

Acute kidney injury � Acute renal failure � Hepatocyte growth factor � Hepatocyte growth factor receptor c-met � Apoptosis � Inflammation � Heme oxygenase

Abstract

Background/Aims: Previous partial hepatectomy (HPTX) can attenuate glycerol-induced acute kidney injury (Gly-AKI). The aim of this study was to explore the pathophysi-ological mechanisms and the role of hepatocyte growthfactor (HGF) in kidney protection. Methods: Rats were sub-jected to HPTX 24 h before glycerol administration. Renal function, acute tubular necrosis, apoptosis, leukocyte infil-tration, and the expression of HGF, c-met, monocyte che-moattractant protein-1, interleukin-1 � , and heme oxygen-ase-1 were evaluated 24 h after glycerol injection. The regenerative response was analyzed from 6 to 72 h after glycerol injection (BrdU incorporation). In a separate series of experiments, Gly-AKI+HPTX rats were treated with anti-HGF antibody. Results: Gly-AKI+HPTX rats showed an in-creased expression of renal HGF and c-met as well as an im-proved creatinine clearance and reduced acute tubular necrosis and apoptosis, cytokine expression, and leukocyte

Received: November 6, 2006 Accepted: June 29, 2007 Published online: October 16, 2007

Eduardo Homsi Av. Conselheiro Rodrigues Alves 775, Ap. 251 Vila Mariana 04014-012 São Paulo (Brazil) Tel. +55 115 575 5357, Fax +55 115 084 8221, E-Mail [email protected]

© 2007 S. Karger AG, Basel1660–2129/07/1073–0095$23.50/0

Accessible online at:www.karger.com/nee

http://dx.doi.org/10.1159%2F000109828

Homsi /Janino /Biswas /Mizuno /Nakamura /Lopes de Faria

Nephron Exp Nephrol 2007;107:e95–e106e96

duces heme-iron-mediated oxidative stress that is pri-mordial in the pathogenesis of injuries in this model [5–7] .

Hepatocyte growth factor (HGF) was originally dis-covered as a potent mitogen for mature hepatocytes in a primary culture system [8, 9] . Subsequently, it has been shown [10–12] that HGF exerts mitogenic responses and participates in regeneration and protection of various or-gans after injuries. In the renal tissue, HGF plays an im-portant role in tubular regeneration [10, 11, 13, 14] and in the antiapoptotic response of the tubular epithelial cells [15, 16] . The HGF receptor c-met is a tyrosine kinase type receptor and is a protooncogene product expressed in the epithelial cellular membrane. After glycerol-induced acute kidney injury (Gly-AKI), the renal c-met mRNA expression increases markedly after 12 and 24 h, whereas the renal HGF mRNA expression remains unaffected and the renal HGF protein concentration decreases [17, 18] . However, the plasma level of HGF increases in Gly-AKI [18, 19] , implicating that HGF synthesis in distant organs may exert a reparative role in Gly-AKI. The ad-ministration of exogenous human recombinant HGF during the initial phase of Gly-AKI reduces the mortality and accelerates functional recovery and tubular regen-eration in rats [18, 20] .

After partial hepatectomy, there is a marked increase in plasma HGF [21] and HGF mRNA expression in sev-eral organs, the most intense HGF mRNA expression be-ing observed in the kidneys 6 h after partial hepatectomy [22] . The elevated systemic level of endogenous HGF may affect the outcome of distant organ injuries early after partial hepatectomy. In a previous study [23] , we have shown that Gly-AKI in prehepatectomized rats had an attenuated course, with rapid recovery of the renal func-tion and less tubular injury. In the present study, the ob-jectives are to explore the pathophysiological mecha-nisms involved in Gly-AKI protection in prehepatecto-mized rats and to evaluate the role of the endogenous HGF/c-met axis in this response.

Materials and Methods

Animal Treatments Wistar rats (weighing 200–250 g) were bred in the animal fa-

cility at our institution. The experiments were performed in ac-cordance with the guidelines established by the Brazilian College for Animal Experimentation. Gly-AKI was induced after over-night fast by a single intramuscular injection of 50% glycerol(6 ml/kg in the first series and 5 ml/kg in the second series of ex-periments) divided into both lower hind limbs. Partial (70%) hep-

atectomy was performed according to the method described by Higgins and Handerson [24] 24 h before glycerol injection in all experiments. Control rats received intramuscular saline injec-tions and underwent sham hepatectomy. The first series of ex-periments comprised four groups: control (sham hepatectomy and saline injection), control+hptx (70% hepatectomy and saline injection), Gly-AKI (sham hepatectomy and glycerol injection), and Gly-AKI+hptx (70% hepatectomy and glycerol injection). The aims were to assess the effect of partial hepatectomy on the renal expression of HGF and its receptor c-met and to evaluate the effect of hepatectomy on Gly-AKI outcome, regarding renal func-tion, tubular injury, renal inflammation, and tubular regenera-tion. In a separate series of experiments, the role of HGF in the renal response to partial hepatectomy was evaluated through neutralization of endogenous HGF, using rabbit antirat HGF an-tibody, as previously described [25] . The rats were divided into five groups: control, control+hptx (for HO-1 expression), Gly-AKI, Gly-AKI+hptx+normal rabbit IgG, and Gly-AKI+hptx+anti-HGF IgG. The anti-HGF antibody or normal IgG was adminis-tered at a dose of 4 mg/kg, divided in four intraperitoneal injec-tions every 12 h, initiating 1 h after hepatectomy, based on a previous method [26] . All data in this series were collected 24 h after glycerol injection (12 h after the last anti-HGF injection).

Renal Function Studies The rats were expanded with water (10% of body weight) by

gavage, and after 2 h spontaneously voided urine was collected. At the end of the urine collection, blood was drawn through car-diac puncture. Serum and urinary creatinine levels were mea-sured by an automated analyzer (Cobas Mira; Roche, Basel, Swit-zerland). The creatinine clearance was calculated and normalized to 100 g body weight.

Histological Analysis Acute Tubular Necrosis. The kidneys were harvested, fixed in

10% formalin, and embedded in paraffin (the same fixative was used in all histological studies, unless otherwise specified). Lon-gitudinal cuts (4 � m) of the midportion of the kidneys were stained with hematoxylin and eosin. The number of totally ne-crotic and desquamated tubules (absence of nuclei) was quanti-fied in 15 random cortical high-power fields (HPF; ! 400) for each rat.

Terminal Deoxynucleotidyl Transferase (TdT) Mediated 2-De-oxyuridine 5 � -Triphosphate (dUTP) Nick End Labeling (TUNEL) Assay. In situ detection of DNA fragmentation characteristic of apoptotic cells was performed using the TUNEL method. Briefly, longitudinal sections of the embedded kidneys were dewaxed, re-hydrated, and endogenous peroxidase blocked in 3% hydrogen peroxide. The tissue was incubated with Proteinase K (Boehrin-ger Mannheim, Indianapolis, Ind., USA) 20 mg/ml for 15 min at room temperature and then rinsed in TdT buffer (Amersham Pharmacia Biotech, Piscataway, N.J., USA; 30 m M Tris, 140 m M sodium cacodylate, and 1 m M cobalt chloride, pH 7.2) and incu-bated with TdT 1: 50 and biotinylated dUTP (Invitrogen, Carls-bad, Calif., USA) 1: 50 in TdT buffer for 60 min at room tempera-ture. Labeled nuclei were detected with Vectastain ABC (Vector Laboratories, Burlingame, Calif., USA) incubation for 30 min and developed by 3,3 � -diaminobenzidine tetrahydrochloride (Dako North America, Carpinteria, Calif., USA) as a substrate chromo-gen solution. The sections were counterstained with hematoxylin.

HGF in Hepatectomy-Induced Gly-AKI Protection

Nephron Exp Nephrol 2007;107:e95–e106 e97

Positive cells were counted in twelve random high-power tubu-lointerstitial fields in the cortex and in twelve in the medulla (both ! 400).

Immunohistochemistry Assessment of Cellular Proliferation by 5-Bromo-2 � -Deoxiuri-

dine (BrdU) Labeling. BrdU 100 mg/kg (Calbiochem, San Diego, Calif., USA) was injected intraperitoneally 1 h before sacrifice. Longitudinal sections of the midkidney were dewaxed and rehy-drated, followed by antigen retrieval and blockade of nonspecific binding and endogenous peroxidases. Tissue sections were incu-bated in a 1: 50 dilution of 1% bovine serum albumin and mono-clonal anti-BrdU antibody (Dako, Glostrup, Denmark) for 1.5 h at room temperature and biotinylated secondary anti-mouse IgG antibody (Dako) 1: 200 for 1 h. ABC reagent (Dako) was applied to slides for 30 min, followed by diaminobenzidine tetrahydro-chloride (Dako). Hematoxylin counterstaining and dehydration were done, and the slides were mounted with Entellan � (Merck, Darmstadt, Germany). The BrdU-positive cells were scored in twelve randomly selected high-power tubulointerstitial fields in the cortex and in twelve in the medulla (both ! 400).

Macrophages (Anti-ED-1), T Lymphocytes (Anti-MAS010P), In-terleukin-1 � (IL-1 � ), and Monocyte Chemoattractant Protein-1 (MCP-1). Slides were dewaxed, and endogenous peroxidase was blocked in 3% H 2 O 2. Antigen retrieval was accomplished by ex-posing sections to microwaves in 10 m M citrate buffer (pH 6.0). The slides were incubated for 1 h with primary anti-ED-1 antibody 1: 100 (AbD Serotec, Oxford, UK) and anti-IL-1 � antibody 1: 100 (polyclonal rabbit antirat; Santa Cruz Biotechnology, Santa Cruz, Calif., USA ) or overnight at 4 ° C with anti-MAS010 antibody 1: 50 (monoclonal mouse antirat T lymphocytes; Accurate Chemical and Scientific, Westbury, N.Y., USA) and anti-MCP-1 goat antirat anti-body 1: 800 (Santa Cruz Biotechnology) and subsequently with spe-cific biotinylated secondary antibodies 1: 400 for 1 h. The reaction product was detected with an ABC kit (Dako) and color developed with diaminobenzidine tetrahydrochloride (Dako). ED-1- and MAS010P-positive cells were counted in twelve randomly selected cortical and ten medullar tubulointerstitial HPFs ( ! 400). For anal-ysis of IL-1 � and MCP-1 expression, 15 random cortical and 10 medullar HPFs ( ! 400) were captured by means of a computerized video microscope. Using the KS 300 Imaging System (Carl Zeiss Microimaging, Jena, Germany), the HPFs were grid lined (10 ! 10) and the number of stained grid fields was counted in each HPF. The results are expressed as percentage of positive grid fields.

c-Met. Kidneys were perfused in vivo via the abdominal aorta with cold 4% paraformaldehyde solution for 10 min. After perfu-sion, the kidneys were fixed overnight at 4 ° C in 4% paraformal-dehyde and embedded in paraffin blocks. The slides were de-waxed and endogenous peroxidase blocked in 3% H 2 O 2 for 30 min. Nonspecific bindings were blocked in 10% goat serum for1 h. The slides were then incubated with primary antibody (poly-clonal rabbit antirat; Santa Cruz Biotechnology) 1: 400 overnight at 4 ° C. Subsequently, the slides were incubated with biotin-con-jugated secondary antibody (goat antirabbit; Santa Cruz Biotech-nology) 1: 400 for 30 min. The reaction product was detected with an ABC Kit (Dako), and the slides were developed with diamino-benzidine tetrahydrochloride (Dako). Using the same imaging procedure as described above; ten cortical and ten medullar HPFs were grid lined (10 ! 10), and the percentage of positive grid fields/HPF was counted.

Western Immunoblotting for HGF, Heme Oxygenase-1(HO-1), and MCP-1 Renal cortical fragments were homogenized at 4 ° C in 30 m M

Tris HCl (pH 7.5), 10 m M EGTA, 5 m M EDTA, 1 m M dithiothrei-tol, 250 m M sucrose, and 40 � l/ml protease inhibitor cocktail (Complete Mini; Roche). The homogenized solution was centri-fuged at 11,000 g at 4 ° C for 10 min. A sample of the supernatant was used for protein quantification using the Bradford protein assay (Bio-Rad Laboratories, Hercules, Calif., USA).

Fifty micrograms of total protein in 5% glycerol, 0.03% bro-mophenol blue, and 10 m M dithiothreitol was loaded in 10% so-dium dodecyl sulfate-polyacrylamide gel (for HGF) or 12.5% gel (for HO-1 and MCP-1) and eletrophoresed in Laemmli solution. Molecular weight marker (Rainbow; Amersham Pharmacia Bio-tech) was used as standard. Proteins were transferred to nitrocel-lulose membranes (Bio-Rad Laboratories) in transfer buffer (50 m M Tris-HCl, pH 7.0, 380 m M glycine, 0.1% sodium dodecyl sul-fate, and 20% methanol). Nonspecific binding was blocked by in-cubating the membranes for 1 h with 5% nonfat milk at room temperature. Primary antibodies were: goat antirat HGF (Santa Cruz Biotechnology), mouse antirat HO-1 (StressGen Biochemi-cals, Victoria, B.C., Canada), and goat antirat MCP-1 (Santa Cruz Biotechnology). The membranes were incubated at 4 ° C overnight in 5% nonfat milk in phosphate-buffered saline/Tween 20 at a di-lution of 1: 500 (for HGF and HO-1) and of 1: 200 (for MCP-1). Af-ter washing with phosphate-buffered saline/Tween 20, horserad-ish peroxidase conjugated secondary antibodies diluted 1:20,000 (for HGF), 1: 2,500 (for HO-1) and 1: 40,000 (for MCP-1) were in-cubated for 1 h at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence method (Su-perSignal ECL-HRP Substrate System; Pierce, Rockford, Ill., USA). Immunoblot for � -actin was used for load control. Quan-titation of the bands was done by optical densitometry using Im-ageJ software, version 1.37 (National Institutes of Health, Bethes-da, Md., USA).

Plasma HGF Measurement The HGF concentrations of the plasma samples collected 24 h

after glycerol injection were determined using an enzyme immu-noassay kit specific for rat HGF (Institute of Immunology, Tokyo, Japan).

Statistics Data are expressed as mean values 8 SEM. Analysis of vari-

ance and the Student-Newman-Keuls test were used to test para-metric values and the Mann-Witney or the Kruskal-Wallis/Dunn test for nonparametric values. p ! 0.05 was considered statisti-cally significant.

Results

Functional and Morphological Effects of Previous Partial Hepatectomy in Gly-AKI Outcomes Prehepatectomized rats showed milder Gly-AKI. The

renal function estimated by creatinine clearance was more preserved in this group than in the sham-hepatec-tomized Gly-AKI rats ( fig. 1 ).



The histological evaluation showed remarkable acute cortical tubular necrosis 24 h after glycerol administra-tion that was reduced by previous partial hepatectomy ( fig. 2 a). The TUNEL assay showed a significant increase in tubular epithelial apoptotic cells, mainly in the renal medulla, 6 h after glycerol injection. In prehepatecto-mized Gly-AKI rats, apoptosis was prevented, and the number of TUNEL-positive cells was similar to that of the control group ( fig. 2 b).

HGF/c-met Expression after Partial Hepatectomy and Gly-AKI The expression of HGF protein in the renal cortex was

evaluated by Western blot. The expression of HGF was scant in controls and moderate in prehepatectomized controls and in the Gly-AKI rats at 24 h. There was a clear increase in the expression of the precursor (90 kDa) and active form (50 kDa) of renal cortical HGF in prehepatec-tomized Gly-AKI rats ( fig. 3 a, b). The plasma level of HGF was increased 24 h after glycerol injection, but there was not difference between Gly-AKI and Gly-AKI+hptx rats ( fig. 3 c).

The expression of the HGF receptor c-met was evalu-ated by immunohistochemistry. The c-met expression in renal cortex and medulla was limited in control (i.e., sham-hptx/sham-Gly) rats, but was significantly in-creased after partial hepatectomy (control+hptx group)

and 24 h after glycerol injection (Gly-AKI group). The most extensive cortical expression was observed, when both stimuli were present (Gly-AKI+hptx rats; fig. 4 ).

Time Course of Tubular Regeneration We evaluated the tubular cell BrdU incorporation as a

marker of mitosis at different intervals after glycerol in-jection. A strong proliferative activity was seen only 24 h after the glycerol injection, initially present more in the renal medulla and later in the cortex. Prehepatectomized

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Fig. 1. Evaluation of the renal function 24 h after glycerol injec-tion. The creatinine clearance was significantly reduced in the Gly-AKI group as compared with control and prehepatectomized control (control+hptx) groups. The prehepatectomized Gly-AKI group (Gly-AKI+hptx) showed a creatinine clearance higher than the Gly-AKI group but lower than the controls. n = 7–10 rats/group. * p ! 0.01 versus the other groups.

Fig. 2. Histological findings in Gly-AKI and the effects of previ-ous partial hepatectomy (hptx). a Light microscopic (hematoxi-lin-eosin) evaluation showed evident cortical acute tubular ne-crosis at 24 h in the Gly-AKI group that was significantly reduced in prehepatectomized rats (Gly-AKI+hptx). n = 10/group. * p ! 0.001. b TUNEL staining showed that tubular apoptotic cells pre-dominated in the renal medulla and were significantly increased 6 h after glycerol injection both in cortex and medulla (Gly-AKI group). Previous hepatectomy (Gly-AKI+hptx) prevented the in-crease in apoptosis after glycerol injecton. n = 5–7 rats/group. * p ! 0.001 versus the other groups – medulla; * * p ! 0.05 versus the other groups – cortex.



rats showed a mitotic index comparable to that of sham-hepatectomized rats, when the mitotic activity was low (controls and rats 12 h after glycerol injection). During the peak of tubular proliferation, the prehepatectomized Gly-AKI rats showed less mitosis than the sham-hepatec-tomized animals ( fig. 5 ).

Effect of Partial Hepatectomy on Renal Inflammation in the Gly-AKI Rats The leukocyte adherence or infiltration in the renal pa-

renchyma as well as the expression of MCP-1 and IL-1 � were evaluated by immunohistochemistry. The macro-phage infiltration was intense 24 h after the glycerol injec-tion, particularly in the renal medulla. T lymphocyte counts were also significantly increased in deep renal cor-tex and outer medulla of the Gly-AKI rats. The expression of MCP-1 and IL-1 � was observed preferentially in the renal cortex tubules and was enhanced 24 h after the glyc-

erol injection. Previous partial hepatectomy reduced cel-lular infiltration and cytokine expression ( table 1 ).

Participation of HGF in the Renal Protection Observed in Prehepatectomized Rats The concomitant increase in ligand HGF and its re-

ceptor, c-met, prompted us to hypothesize that the HGF/c-met axis may play a key role in the hepatectomy-in-duced renal protection in our Gly-AKI model. To test our hypothesis, we injected anti-HGF neutralizing antibody immediately after 70% partial hepatectomy (i.e., every12 h, four times, i.p.), as previously reported [24] . Dur-ing this experimental period, the Gly-AKI rats showed a significant increase in the number of necrotic and apo-ptotic tubular cells, while previous hepatectomy strongly suppressed the necrotic and apoptotic changes ( fig. 6 a, b). Of note, anti-HGF IgG diminished the hepatectomy-mediated tubular protection. Especially, the number of

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Fig. 3. HGF expression 24 h after glycerol injection. a Western blot of renal cortical homogenates showing the expression of the HGF. There was a moderate increase in the expression of HGF in pre-hepatectomized controls (control+hptx) and Gly-AKI rats. The expression was intense in the prehepatectomized Gly-AKI (Gly-AKI+hptx) group, showing both the precursor (90 kDa) and ac-tive (50 kDa) forms. The right lane was loaded with control re-

combinant human HGF (4 ng diluted in running buffer). Each lane represents a distinct animal. b Optic densitometry analysis, confirming the increased normalized renal HGF expression in the Gly-AKI+hptx group. * p ! 0.01 versus the other groups. c Plasma HGF level, showing a similar increase in Gly-AKI and Gly-AKI+hptx groups in comparison with the controls. * * p ! 0.05 versus controls.



TUNEL-positive tubular epithelial cells was increased in anti-HGF IgG-treated AKI rats to a fourfold level of nor-mal IgG-treated rats, thus suggesting an important role of endogenous HGF for the prevention of tubular apop-tosis (and possibly necrosis, in part). In this process, anti-HGF IgG did not alter the hepatectomy-mediated im-provement in the renal function, as estimated by creati-nine clearance ( fig. 6 c). A possible explanation for the difference between histological and functional param-eters is that the prehepatectomized rats may have an at-tenuated preglomerular vasoconstrictive response to tu-bular injury. An increased afferent arteriolar resistance is the major determinant of a reduced glomerular filtration rate in Gly-AKI rats [1] . Alternatively, the histological changes seen in anti-HGF IgG-treated rats might be re-flected in renal dysfunction only later in the course of Gly-AKI. Further follow-up studies would be necessary to address this notion.

Limited Effect of HGF on Inflammatory Events during Progression of Gly-AKI in Prehepatectomized Rats There was a significant increase in the number of ED1-

positive cells 24 h after the glycerol injection, indicating infiltration of macrophages/monocytes in the intersti-

tium of the damaged renal tissues. Of interest, previous hepatectomy suppressed the macrophage infiltration, while anti-HGF IgG treatment did not change the mac-rophage infiltration caused by the glycerol injection in prehepatectomized rats ( fig. 7 a). Consistent with the changes in the infiltration of ED1-positive cells, renal MCP-1 expression was evident in the Gly-AKI rats and was reduced to a level similar to that of controls in Gly-AKI rats that had undergone previous hepatectomy ( fig. 7 b, c). In this process, anti-HGF IgG did not modify the reduction of the MCP-1 expression induced by hepa-tectomy, suggesting that endogenous HGF may not be re-lated to the hepatectomy-mediated suppression of macro-phage infiltration, at least during the early stage of AKI.

Role of HO-1 in the Renal Protection Afforded by Partial Hepatectomy In a previous study [20] , it was demonstrated that HGF

administration could increase the renal expression ofHO-1 in rats. As HO-1 is a protective enzyme in Gly-AKI [27] , we evaluated the HO-1 expression by Western blot-ting in our study groups. The HO-1 protein expression was increased in the renal cortex 24 h after the glycerol injec-tion. In prehepatectomized Gly-AKI rats, the expression

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Fig. 4. Immunostaining for c-met in the renal pa-renchyma. a Representative slide of a cortical HPF ( ! 400) image of immunostaining for c-met in a control rat and a Gly-AKI rat. b Quantitative analysis of c-met expression at 24 h. Images ( ! 400) were grid lined 10 ! 10 using the KS 300 Imaging System. The percentage of positive grid fields was counted in ten random cortical and ten random medullary fields. The c-met positivity was significantly increased in both compartments in prehepatectomized controls (control+hptx), Gly-AKI rats, or prehepatectomized Gly-AKI rats (Gly-AKI+hptx) as compared with the controls.n = 6–11 rats/group. * p ! 0.001 versus control cortex; * * p ! 0.05 versus control medulla.



was still higher than in the controls, but was at a level in-ferior to that in the sham-hepatectomized Gly-AKI rats. The anti-HGF treatment did not change evidently the ex-pression seen in prehepatectomized Gly-AKI rats ( fig. 8 ).

Discussion

This study confirms our previous finding that partial hepatectomy attenuates Gly-AKI [23] . Prehepatecto-mized Gly-AKI rats showed a more preserved renal func-tion and less tubular injury and interstitial inflammation

when compared with sham-hepatectomized Gly-AKI rats. We observed an increased renal HGF and tubular c-met protein expression after partial hepatectomy, even in control rats. Prehepatectomized Gly-AKI rats showed a greater renal expression of HGF/c-met proteins. The HGF plasma level was increased in Gly-AKI, but the level was similar, regardless the rats being submitted to prior par-tial hepatectomy or not. This finding suggests that the renal synthesis of HGF in the prehepatectomized Gly-AKI rats was responsible for its increased expression in the renal cortex of this group at 24 h. The HGF plays an essential role in renal tubular repair following acute kid-

Table 1. Renal inflammation 24 h after glycerol injection

Control Control + hptx Gly-AKI Gly-AKI + hptx

Macrophages/!400 HPF, cortex 4.581.4 4.883.0 18.983.1b 14.283.4b

Macrophages/!400 HPF, medulla 4.080.6 2.681.3 107.1824.2a 26.489.9T lymphocytes/!400 HPF, cortex 9.681.9 15.783.7 22.182.9b 19.683.2T lymphocytes/!400 HPF, medulla 13.482.6 11.883.2 19.983.5 12.682.6MCP-1-positive cortical grid fields, % 9.683.8 10.185.5 33.885.4b 17.488.6MCP-1-positive medullar grid fields, % 0.880.5 6.583.8 8.284.6 9.785.4IL-1�-positive cortical grid fields, % 1.581.0 1.981.5 40.583.5a 15.685.2b

IL-1�-positive medullar grid fields, % 15.187.1 12.686.1 9.085.4 16.887.3

Leukocytes were counted in 12 cortical and 10 medullar HPF. MCP-1 and IL-1� grid fields: images were captured by videomicroscopy and grid lined 10 ! 10; KS 300 Imaging System.

The percentages of positive grid fields/HPF were counted in 15 cortical and 10 medullar HPF/rat.a p < 0.05 vs. other groups; b p < 0.05 vs. controls; n = 6–8 rats/group.

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Fig. 5. Effect of previous partial hepatectomy on tubular regen-eration after Gly-AKI. The immunohistochemistry for BrdU ( ! 400 HPF) showed a low proliferative activity in controls and early after glycerol injection. There was marked increase in tubu-

lar proliferation at 24 h in the renal medulla ( a ) and at 72 h in the renal cortex ( b ). Prehepatectomized rats (hptx) showed a lower proliferative activity during the course of Gly-AKI. n = 6–10 rats/group. * p ! 0.01 versus related Gly-AKI+hptx.



ney injury, but there is also evidence that the HGF can reduce the initial tubular injury [10, 11, 28] . Interestingly, the prehepatectomized rats never showed an increased tubular regeneration after Gly-AKI. Therefore, the pres-ervation of renal function and architecture in the pre-hepatectomized Gly-AKI rats was a consequence of the increased resistance to Gly-AKI (including apoptosis and

necrosis). In order to ascertain the role of endogenous HGF overexpression in the renal resistance to Gly-AKI, we treated a group of prehepatectomized rats with neu-tralizing anti-HGF antibody. The anti-HGF treatment showed that endogenous HGF played an important role in the prevention of tubular injury, particularly tubular cell apoptosis, in prehepatectomized rats. The stimula-tion of the HGF/c-met system before induction of Gly-AKI showed the preponderance of the tubular survival effect over the proregenerative action of HGF.

Several studies utilizing different methods of treatment with HGF, either in vivo or in renal epithelial cell culture, consistently showed the antiapoptotic effect of HGF after tubular injuries [15, 16, 29–33] . Most in vivo studies also showed that HGF treatment reduced the magnitude of acute tubular necrosis 1 or 2 days after AKI induction, but the mitotic indexes were increased in the HGF-treated an-imals [10, 11] ; accelerated repair instead of reduced necro-sis could be responsible for this finding [31–34] . de Souza Durão et al. [35] showed direct evidence that HGF can re-duce apoptosis and necrosis in Madin-Darby canine kid-ney cells subjected to hypoxia. The signal transduction events that lead HGF to protect cells from apoptosis are not entirely clear. Liu [36] showed that HGF phosphorylates and activates Akt kinase in a phosphoinositide-3-kinase-dependent manner. Activated Akt kinase can phosphory-late and inactivate proapoptotic Bad protein, but also can phosphorylate and activate I � B kinase [37] . The I � B kinase complex phosphorylates I � B- � , leading to nuclear trans-location of NF- � B (nuclear factor-kappa B) p65 subunit, and thereby stimulates the NF- � B activity. Activated NF- � B may regulate positively the transcription of antiapop-totic proteins like Bcl-xl [38] . Mice treated with HGF-en-

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Fig. 6. Effect of treatment with anti-HGF neutralizing antibody on kidney injury 24 h after glycerol injection in prehepatecto-mized (hptx) rats. a Prehepatectomized Gly-AKI rats (Gly-AKI+hptx+IgG) showed less acute tubular necrosis per ! 400 HPF than the sham-hepatectomyzed Gly-AKI rats, and an in-crease in acute tubular necrosis was observed when anti-HGF an-tibody (Gly-AKI+hptx+anti-HGF) was administered, but it was not significant. b TUNEL-positive (apoptotic) cells in the renal medulla per ! 400 HPF were increased 24 h after glycerol injec-tion (Gly-AKI group). Prehepatectomy (Gly-AKI+hptx+IgG) pre-vented this increase, and anti-HGF treatment (Gly-AKI+hptx+anti-HGF) restored it. c The reduction in the creatinine clearance observed in the Gly-AKI rats was prevented by previous partial hepatectomy (Gly-AKI+hptx+IgG), and this protection was not modified by anti-HGF treatment (Gly-AKI+hptx+anti-HGF). n = 6–9 rats/group. * p ! 0.01 versus the other groups; * * p ! 0.01 ver-sus control and Gly-ARF+hptx+IgG rats.



coded plasmid showed increased prosurvival Bcl-xl pro-tein expression and reduced renal apoptosis in response to folic-acid-induced acute kidney injury [32] . Considering the tubular c-met/HGF receptor overexpression in Gly-AKI rats, we predict that an increased level of endogenous HGF can abrogate tubular apoptosis also in Gly-AKI.

There is accumulating evidence that an inflammatory reaction is involved in the pathogenesis of acute renal fail-ure [39] . We observed an increased renal tubular expres-sion of MCP-1 and IL-1 � cytokines as well as an increased infiltration of mononuclear leukocytes 24 h after Gly-AKI. In the prehepatectomyzed Gly-AKI rats, the inflam-matory response was significantly milder. Several studies showed the anti-inflammatory properties of HGF in renal injuries, including reductions in MCP-1, intercellular ad-hesion molecule-1, E-selectin expression, and leukocyte infiltration [30, 40–43] . The present findings using an AKI model appear reciprocal to these previous reports, once inflammatory events in prehepatectomized rats (such as macrophage infiltration and MCP-1 induction) were not restored, when HGF was blocked. This reaction seems to be specific for prehepatectomized rats, since we have recently observed exacerbation of the inflammatory response in nonhepatectomized Gly-AKI rats treated with the same anti-HGF antibody [unpubl. data]. The discrep-ant response to HGF blockade in these models suggests that anti-inflammatory or antioxidative mechanisms in-dependent of HGF, e.g., an increased unconjugated biliru-bin level, may be relevant in prehepatectomized rats. This point must be further explored in the future.

Heme oxygenase (HO) is the enzyme implicated in the degradation of heme. The inducible form HO-1 is in-duced by heme, oxidants, and cytokines [44] ; therefore, HO-1 is intensely expressed in Gly-AKI [27] . HO breaks the heme ring and produces biliverdin and carbon mon-oxide, and iron is released and incorporated to ferritin. Subsequently, biliverdin is reduced to bilirubin by bili-verdin reductase. Besides the removal of heme that is po-tentially oxidant and toxic, HO-1 products, such as bili-rubin, carbon monoxide, and ferritin, are cytoprotec-tants with recognizable antioxidant, anti-inflammatory, antiapoptotic, and vasodilatory properties [45–47] . Nath et al. [48] , using HO-1-deficient mice, convincingly showed the importance of HO-1 in limiting kidney in-jury and mortality in Gly-AKI and hemoglobin-induced AKI. Nagano et al. [20] showed that intravenous admin-istration of HGF before Gly-AKI enhanced HO-1 mRNA expression which could participate in the renal protective effect of HGF. In our study, the HO-1 protein expression was clearly increased in the renal cortex 24 h after glyc-

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Fig. 7. Macrophage infiltration and MCP-1 expression 24 h after glycerol injection in rats. a Macrophage infiltration was observed in the renal parenchyma 24 h after glycerol injection (Gly-AKI), and it was reduced in prehepatectomized rats (Gly-AKI+hptx+IgG) as well as in the prehepatectomized rats treated with anti-HGF antibody (Gly-AKI+hptx+anti-HGF). * p ! 0.01 versus the other groups. b The immunostaining for MCP-1 showed in-creased protein expression in Gly-AKI that was prevented by pre-hepatectomy (Gly-AKI+hptx+IgG), even when anti-HGF was ad-ministered to the rats (Gly-AKI+hptx+anti-HGF). * p ! 0.01 ver-sus the other groups. c The same findings were confirmed on renal MCP-1 Western blotting. C = Controls; G = Gly-AKI; HG = Gly-AKI+hptx. n = 6–9 rats/group. Macrophages counted in 12 cortical plus 10 medullar ! 400 HPF. The percentage of positive grid fields was counted in 15 random cortical fields in 10 ! 10 grid-lined HPF, using the KS 300 Imaging System.



erol injection. Prehepatectomized rats also showed an in-creased expression of HO-1, although to a lower level than sham-hepatectomized rats. This finding excludes the hypothesis that the HGF-mediated increase in renal HO-1 was responsible for the renal resistance to Gly-AKI in prehepatectomized rats.

HGF may be an important organotropic ligand for min-imizing hepatic and renal injuries. In this study, we found that previous hepatic injury (i.e., 70% hepatectomy) led to renal protection during progression of Gly-AKI in rats, as-sociated with an increase in the HGF levels. Inversely, per-sistent renal diseases lead to attenuation of virus-induced hepatitis, possibly via an increase in the HGF levels [49] . These results strengthened the hypothesis that endoge-nous HGF/c-met may confer an intrinsic repair system for attenuating parenchymal damage in several organs.

In summary, previous partial hepatectomy preserved the renal function and reduced tubular injury, namely necrosis and apoptosis, as well as reduced the renal in-flammatory response to Gly-AKI. The prehepatecto-mized Gly-AKI rats showed a remarkable expression of renal HGF/c-met proteins, and this system was respon-sible for the reduction in tubular injury, mainly tubular apoptosis. Based on all available data, we would like to

emphasize that induction (or enhancement) of the endog-enous HGF/c-met axis in the kidney is in part responsible for hepatectomy-mediated renal protection, as noted in the case of Gly-AKI. Our model may provide an experi-mental clue to elucidate how HGF produces tubular pro-tective outcomes during the progression of AKI.

Acknowledgment

Financial support was provided by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil.

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Fig. 8. HO-1 expression 24 h after glycerol. a Western blot of renal cortical homoge-nates, showing the expression of HO-1. b Optic densitometry analysis of normal-ized HO-1 expression. The expression of HO-1 was increased in Gly-AKI as com-pared with the other groups. In prehepa-tectomized rats treated with normal IgG (Gly-AKI+hptx+IgG) or anti-HGF IgG (Gly-AKI+hptx+anti-HGF), the expres-sion was higher than in controls but lower than in Gly-AKI. * p ! 0.01 versus the other groups; * * p ! 0.01 versus controls.

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Documents

Attenuation of Glycerol-Induced Acute Kidney Injury by Previous Partial Hepatectomy: Role of Hepatocyte Growth Factor/c-met Axis in Tubular Protection