1 Department of Pediatrics, Chaim Sheba Medical Center, Tel Hashomer, 2 Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, 3 Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute and 4 Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Correspondence and offprint requests to: Dr Ilan Tsarfaty, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 69978. Email: ilants{at}post.tau.ac.il
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Abstract |
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Methods. We examined the in situ distribution patterns of the Tyr1356-phosphorylated form of Met with a novel monoclonal antibody following renal injury and renal hypertrophy in rats. Sections of the kidney following either sham operation, transient ischaemia of one kidney or unilateral nephrectomy were analysed using indirect immunofluorescence staining and confocal laser scanning microscopy analysis of total Met protein levels and Tyr1356-phosphorylated Met (Met and pMet, respectively).
Results. At 6 h post-treatment, pMet increases in ischaemic kidneys compared with sham-operated kidneys, and these changes become substantial after 48 h in both medulla and cortex of ischaemic kidneys (P < 0.001). We also show significant up-regulation of Met predominantly in the medulla of ischaemic kidneys, 48 h following injury (P < 0.009). Inter-estingly, the stimulus for hypertrophy in the remnant kidney after uninephrectomy and the contra-lateral kidney during ischaemia is not accom-panied by significant up-regulation of Met or pMet staining compared with sham operation at both time points.
Conclusions. We demonstrate in this work, for the first time, in situ detection of tyrosine kinase growth factor receptor docking site activation during pathological processes in the kidney. Using this methodology, we show a significant increase in Met docking site activity in both renal medulla and cortex solely following stimulation by ischaemia and repair.
Keywords: HGF/SF; ischaemia; Met; signal transduction; tyrosine kinase docking site
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Introduction |
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Important roles for Met and HGF/SF have been suggested in both kidney development and regeneration following injury [1]. We have demonstrated previously that co-expression of HGF/SF and Met in murine 3T3 fibroblasts results in the expression of several epithelial markers including tight junction proteins, desmosome-like cell interactions and lumen formation [6,7]. Moreover, Met was highly expressed in kidney cells undergoing mesenchymalepithelial conversion and in tubular structures [7]. In addition, MetHGF/SF induces tubulogenesis of primary renal proximal epithelial cells [8]. In animal models of toxic or ischaemic acute renal failure, MetHGF/SF act in a renotropic and nephroprotective manner [1]. During experimental acute renal failure, elevated HGF/SF mRNA levels were found in kidney and liver, but Met mRNA was up-regulated selectively at the site of greatest tubular injury or hypertrophy [9]. An increase of HGF/SF protein and mRNA levels was also detected in rats after unilateral nephrectomy (UNX) and CCl4 treatment, with concomitant internalization of Met [10]. In addition, expression of Met in the kidney increased after UNX, ischaemia or folic acid treatment [11]. Thus, while elevated mRNA and protein levels of Met have been reported after kidney injury, no data exist regarding Met activation and specific involvement of the receptors docking site. The active state of the Drosophila epidermal growth factor (EGF) receptor signalling pathway was monitored by examination of the in situ distribution of the active phosphorylated form of mitogen-activated protein kinase, which is downstream of the receptors docking site, with a specific monoclonal antibody [12]. To localize and measure activation of Met in situ, we have developed a novel monoclonal antibody that recognizes Met docking site phosphorylated Tyr1356 (anti-pMet). Using Met and pMet antibodies in conjunction with confocal laser scanning microscopy (CLSM) analysis, we demonstrate differential up-regulation of Met docking site activity and Met expression following several types of renal injury.
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Subjects and methods |
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Kidney sections
Kidneys from five animals of each group were dissected to obtain cortex and medulla. To ensure that no medulla was included in cortex samples, these samples contained only outer cortex by removal of juxtamedullary cortex. Kidney sections were all examined microscopically [haematoxylin and eosin (H&E) staining].
Antibodies
The following antibodies were used: rabbit polyclonal SP260 anti-murine Met peptide antibody (1:500 and 1:50 dilutions for western blot and immunofluorescence, respectively), rabbit polyclonal C28 anti-human Met peptide antibody [1:50 dilution for immunofluorescence (IF)] (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-phosphorylated docking site of Met at Tyr1356 (Tyr1354 in mouse and Tyr1356 in humans; anti-pMet antibody). A phosphotyrosine peptide corresponding to the human Met autophosphorylation site [Val His Val Asn Ala Thr Tyr (PO3H) Val Asn Val Lys Cys] was synthesized and analysed by mass spectrometry to verify the presence of the phosphorylated residue. The phosphorylated peptide was linked to keyhole limpet haemocyanin (KLH) to increase its immunogenicity and injected into mice. Antibody specificity was verified initially by recognition of the phosphorylated peptide and not the unphosphorylated peptide. Further conformation was achieved by enzyme-linked immunosorbent assay (ELISA) and western blot analysis using MadineDarby canine kidney (MDCK) cells treated with HGF/SF compared with untreated cells. The pMet antibody (from hybridoma supernatant) was used in 1:20 and 1:50 dilutions for western blot and immunofluorescence, respectively. Anti-phospho-Tyr monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY) was used for western blot in 1:1333 dilution.
Generation of Met mutants
Seven different expression vectors were constructed by introducing point mutations substituting the three major tyrosines in the Metmu (Y1347, Y1354 and Y1363) docking site to phenylalanine. The constructs were co-transfected into NIH-3T3 cells along with a plasmid coding for human HGF/SF as previously described [13]. The different transfectants were designated according to the mutation introduced: the wild-type clone was designated YYY and the mutated Met clones: FFY, FYY, FYF, YFY, YFF, YYF and FFF.
Immunoprecipitation and western blot analysis of Met and pMet tyrosine in MDCK and mouse kidney
For in vitro analysis, near-confluent MDCK cells were either treated or not with human recombinant purified HGF/SF (50 ng/ml) for 10 min at room temperature. HGF/SF was purified and assayed from the conditioned medium of HMH cells (NIH-3T3 cells transfected with Methu and HGF/SFhu) according to Rong et al. [14].
For in vivo analysis, mice were injected with saline or 130 ng/g purified HGF/SF and, after 10 min, were rapidly sacrificed and kidneys were harvested and frozen in liquid nitrogen. Kidneys were homogenized in 1 ml of lysis buffer (20 mM TrisHCl pH 7.8, 100 mM NaCl, 50 mM NaF, 1% NP-40, 0.1% SDS, 2 mM EDTA, 10% glycerol) with a protease inhibitor cocktail (Boehringer Mannheim, Germany) and 1 mM sodium orthovanadate using an electric homo-genizer (ULTRA-TURRAX, IKA Labortechnik, Staufen, Germany). MDCK cells were collected, washed twice with cold phosphate-buffered saline (PBS) and lysed in 1 ml of lysis buffer as described above. Cell and tissue lysates were clarified by centrifugation, and 1 mg of cell lysate protein was immunoprecipitated with SP260 anti-Met antibody. The immunoprecipitates were subjected to western blot analysis using (i) SP260 anti-Met antibody, (ii) 4G10 anti-phospho-Tyr antibody or (iii) anti-pMet antibody.
Visualization was achieved using horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody or HRP-conjugated protein A (1:5000) (Amersham, Arlington Heights, IL), the enhanced chemiluminescence reaction and exposure to X-ray film (Fuji, Japan).
Immunofluorescence staining and confocal microscopy
Paraffin blocks from kidney grafts were serially sectioned at 56 µm. Tissue samples were stained with H&E to identify morphological structures. Staining and antibody specificity were determined as previously described [15]. In brief, serial sections were deparaffinized, and blocked [5% bovine serum albumin (BSA), 10% normal donkey serum in PBS] for 10 min. The sections were incubated with primary antibody for 1 h at room temperature. Following three washes in PBS, sections were labelled with either donkey anti-rabbit or anti-mouse antibody conjugated to either fluorescein isothiocyanate (FITC), cyanine (Cy5) or rhodamine (Jackson Immuno Research Laboratories, West Grove, PA) diluted 1:50 for 1 h at room temperature. Subsequently, slides were washed three times in PBS and mounted with coverslips using GelMount (Biomeda, Foster City, CA).
Immunostained sections were analysed using a 410 Zeiss (Obercocen, Germany) CLSM with the following configuration: 25 mW krypton/argon (488, 568 nm) and HeNe (633 nm) laser lines. When comparing fluorescence intensities, identical CLSM parameters (e.g. scanning line and laser light) were used. The brightness and contrast used for CLSM analysis were the same for all the images. All of the fluorescent signals acquired were above the autofluorescent background as measured from a control slide stained with the secondary antibody without a primary antibody. Comparison analysis was performed to determine the autofluorescent signal in the region analysed. All signal analysed was above the background signal. To compare the relative levels of protein expression, we used the percentage positive area (PPA) image analysis procedure [15]. In brief, PPA was calculated as the ratio between the positive stained area and the total cellular area. The positive stained area was determined by measuring the fluorescent intensity of the image, which is above the positive cut-off intensity. Positive cut-off intensities were determined based on the fluorescence intensities histogram for each antibody staining. Total cellular area was determined by measuring the fluorescent intensity above the surrounding background of the image, and depicts the cellular autofluorescence. The PPA calculation allows for the quantitative comparison of protein expression in the tissue sections. The PPA data shown represent the calculated average of at least four different CLSM fields. Images were printed using a Codonics 1600 dye sublimation colour printer (Codonics, Middleburg Heights, OH).
Statistical analysis
Statistical analysis of the PPA data was performed using the Students t-test in Microsoft Excel (Microsoft, Redmond, WA), and interaction between groups was examined using two-way ANOVA. For multiple comparisons between treatment groups, we used Statistica Software (StatSoft, Tulsa, OK) and the Tukey parameter test.
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Results |
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Met and pMet staining in sections of kidney 6 h following treatment
Figure 3 demonstrates Met and pMet staining in the renal cortex (Figure 3A and B) and medulla (Figure 3C and D) 6 h after anaesthesia (control group), surgical stress (sham operation), renal injury (ischaemic kidney) or a stimulus for hypertrophy (contralateral to the ischaemic kidney and UNX). Quantification of Met and pMet staining was performed using double immunofluorescence staining with anti-Met antibody (green staining) and anti-pMet antibody (red staining) by CLSM and PPA analysis (see Subjects and methods). Co-localization is an important analysis option, which is enhanced in CLSM investigations due to its focal and specific multiple label detection abilities. A common method of co-localization is merging of green and red images, to produce one image in which a yellow colour appears where co-localization exists. Thus, yellow represents co-localization of both Met and pMet. As shown, cortical Met staining was similar for all groups analysed (Figure 3A and B). In addition, medullary Met staining in the ischaemic kidneys was identical to that found in the sham-operated animals and was not significantly higher than in the control group (Figure 3C and D).
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Met and pMet staining in sections of kidney 48 h following treatment
Figure 4 demonstrates Met and pMet staining in cortical and medullary (Figure 4A and B, and C and D, respectively) kidney sections of the various treatment groups. For cortical Met, staining increased in ischaemic kidneys 48 h after renal injury compared with the control group (P = 0.04), but was not significantly increased compared with sham-operated animals or the other groups (Figure 4A and B). A more predominant and significant increase in Met staining was observed in the medulla of ischaemic kidneys (P = 0.001 compared with the control and UNX groups; P = 0.009 compared with sham operation; P = 0.003 compared with contralateral ischaemic kidney) (Figure 4C and D). Thus, a significant change in Met staining was observed in the renal medulla 48 h after ischaemia.
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Consistent with our findings for the earlier time point, after 48 h, neither a stimulus for hypertrophy in the remaining kidney following UNX and the contralateral kidney during ischaemia, nor the surgical stress of the sham-operated animals induced a significant elevation in cortical or medullary Met staining compared with the control group. For pMet, staining increased in both cortex and medulla of the UNX animals 48 h following treatment, compared with the control group (P = 0.012 and P = 0.039, respectively) (Figure 4A and B, and C and D, respectively). Nevertheless, these staining levels were similar to those found in the sham-operated animals and therefore are not substantial.
Met and pMet staining in the kidney: comparison between 6 and 48 h
Once it was established that Met and pMet are differentially up-regulated in kidney sections of the various groups, at specific time points after treatment, we examined sections of the kidneys to determine whether staining levels changed in each group with time.
Indeed, we found that cortical Met staining in the ischaemic kidney, UNX and sham operation groups at 48 h was significantly elevated (P = 0.05) compared with the respective values measured in each group 6 h following treatment (data not shown). A significant increase in cortex and medullary Met staining between these time points was found only in the ischaemic kidneys (P = 0.03 and P = 0.004, respectively) (Figure 5A).
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Discussion |
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Over the past few years, a role has been suggested for MetHGF/SF as mediators of compensatory renal cell growth and repair after injury [1]. In vitro, HGF/SF induced mitogenic and morphogenetic responses in renal epithelial cells. In animal models of acute renal failure, HGF/SF has been shown to act in a renotropic manner [1]. In order to elucidate the possible site and mode of action of HGF/SF following renal injury or renal hypertrophy, several groups previously have studied the expression of its high-affinity receptor, Met, in these situations [9,11,17,18] (Table 1). Nevertheless, to date, levels of Met activity have not been determined during kidney regeneration in vivo. In an attempt to elucidate the role of this receptor tyrosine kinase further in both reparative hyperplasia and renal tubular hypertrophy, Met docking site phosphorylation concomitant with total Met levels were examined in situ. Analysis was performed in the cortex and medulla of normal and sham-operated rat kidneys as well as models of tubular hyperplasia (unilateral renal ischaemia/reflow) and tubular hypertrophy (UNX). Our results demonstrate a remarkable increase in pMet and Met staining following kidney ischaemia and hyperplastic stimuli. Similar up-regulation was not observed in the contralateral, undamaged kidney, the remaining kidney after UNX or in sham-operated kidneys. Thus, Met expression and docking site activity were selectively up-regulated in the injured ischaemic kidneys, but not in kidneys undergoing hypertrophy or surgical stress. The most dramatic changes of receptor expression and docking site activation were detected 48 h after ischaemic insult. The increase in Met levels and docking site activity noted in the remnant kidney after 48 h was non-specific, as the surgical stress of the sham-operated animals resulted in similar levels.
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The most likely explanation for the discrepancies found in both the inductive stimuli and Met expression patterns is a different temporal response rate at the gene, protein and phosphorylated protein levels. It is possible that a stronger stimulus, i.e. cell damage or necrosis rather than glomerular hyperfiltration, might be needed for significant up-regulation of Met protein levels. Indeed, Met protein levels were also shown to be increased following renal tubular injury by folic acid [17]. Prat et al. [19] have previously demonstrated high levels of Met transcripts in various adult organs, including the kidney, but Met protein was barely detectable. They suggested that inefficient translation or instability of the Met protein would perturb its levels [19]. Moreover, the expression of met mRNA and even protein does not necessarily indicate activation of Met signalling pathways. An additional explanation for the discrepancies between our findings and previous reports is the possibility that phosphorylation of tyrosine residues other than Tyr1354, which we did not measure and which might have a different temporal expression profile (e.g. Tyr1347 and Tyr1363), plays a role in signal transduction leading to cell proliferation after ischaemic injury.
Our results illustrate how monitoring of in situ Met docking site activity assists in identifying a functional increase before receptor abundance increases, and in determining whether it is selectively up-regulated in different regions of the kidney. In situ Met activation was confined primarily to the tubular epithelial cells, and a significant increase in tyrosine phosphorylation of the Met docking site in both cortical and medullary tubular cells was observed 48 h after the ischaemic insult with the advent of tissue regeneration and proliferation. It is of note that cortical up-regulation occurred in the absence of the juxta-medullary cortex, which was dissected away to ensure that no medulla was included in pMet determination. Moreover, in the ischaemic kidneys, we observed a trend towards higher levels of Met docking site activity, but not protein levels, already at 6 h following renal injury. At 48 h, we could demonstrate widespread Met activation in both cortical and medullary tubular cells, while only the renal medulla exhibited a significant increase in Met peptide levels. The significance of Met protein induction and docking site activation in kidney ischaemia is emphasized by the fact that protein synthesis shutdown occurs following acute tubular necrosis. This elevation, accompanied by increased expression of integrins, laminins and fibronectin [20], is most probably associated with restoration of cell polarization following the ischaemic injury.
We suggest that HGF/SF activates Met and induces alterations in Met docking site activity, which regulates the extent and site of cell proliferation as well as tissue regeneration following injury. These results support the hypothesis that Met signal transduction plays a prominent role in kidney injury and repair.
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Notes |
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References |
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