Hepatocyte Growth Factor Receptor in Acute Tubular Necrosis

J Am Soc Nephrol 12: 531–540, 2001 Hepatocyte Growth Factor Receptor in Acute Tubular Necrosis

RALPH RABKIN,* FERNANDO FERVENZA,* TANNY TSAO,* RICHARD SIBLEY,† MICHAEL FRIEDLAENDER,* FAY HSU,* CHARLES LASSMAN,† MICHAEL HAUSMANN,* PHIL HUIE,† and RALPH H. SCHWALL‡ *Research Service Veterans Affairs Palo Alto Health Care System and Departments of *Medicine and †Pathology, Stanford University, Palo Alto, California, and ‡Department of Molecular Oncology, Genentech Inc., South San Francisco, California.

Abstract. In acute tubular necrosis, there are early transient as early as 12 h after injury. Histologic analyses demonstrated increases in circulating and local bioactive hepatocyte growth that the increase in c-MET immunoreactivity was most marked factor (HGF) levels and renal HGF receptor (c-MET) gene in the most severely damaged nephron segments in the outer expression. It has therefore been suggested that endogenous medulla. In injured proximal tubules, the receptor was redis- HGF may play a role in initiating renal repair. To test this tributed from an apical location to an intracellular location. hypothesis, changes in the levels, activity, and anatomic dis- DNA synthesis was increased in the injured kidneys, especially tribution of c-MET protein were characterized in relation to the in the outer medulla, where the increase in c-MET protein onset and localization of DNA synthesis in kidneys of rats with levels was most prominent. The increase in DNA synthesis was ischemia-induced acute tubular necrosis. Whole-kidney first detected 12 h after the initial increase in activated c-MET c-MET protein levels were significantly increased in the in- levels. It is concluded that the early increases in the levels of jured kidneys 12 h after injury and rose to a maximum after c-MET protein and activated receptor support the hypothesis 1 d, exceeding the control values by sevenfold. Eight days after that HGF participates in the initiation of renal regeneration. In injury, c-MET levels, although decreasing, were still elevated addition, the persistent elevation of c-Met protein levels sug- above control values. An increase in the levels of activated gests that prolonged and even late treatment with HGF may be c-MET, i.e., tyrosine-phosphorylated c-MET, was also evident of therapeutic value

Recovery of normal renal function after acute tubular necrosis released into the circulation or locally stored bound to the (ATN) requires regeneration of damaged tubular epithelium, a extracellular matrix (7). In the rat kidney, HGF is produced in process in which growth factors play an important role. Several the peritubular interstitium, most likely by endothelial cells and studies have examined the ability of selected growth factors to macrophages (9). In addition to stimulating cell growth and modulate the course of experimental ATN in rats. Earlier migration in cell culture, it stimulates the formation of tubular studies indicated that epidermal growth factor attenuates the structures and cellular differentiation (7,8). HGF is secreted as severity and duration of acute renal failure (1), and we and a biologically inactive precursor that is activated by serine other investigators recently demonstrated that insulin-like protease cleavage. These proteases are induced in injured tis- growth factor I treatment is also effective (2,3). Hepatocyte sues and serve to increase the formation of the mature active growth factor (HGF) is another growth factor that may play a form (10). Active HGF acts on target cells through a cell role in renal regeneration after injury, and its administration membrane tyrosine kinase receptor known as c-MET. Binding does have a salutary effect on the course of experimental acute of HGF to c-MET causes receptor homodimerization, auto- renal failure, stimulating DNA synthesis and accelerating re- phosphorylation, and activation of tyrosine kinase activity, covery (4–6). HGF is a potent mitogen not only for hepato- which in turn activates complex downstream signaling cas- cytes but also for endothelial cells and several types of epithe- cades, including the phosphatidylinositol-3-kinase and mito- lial cells, including renal tubular cells (7,8). This growth factor gen-activated protein kinase pathways (7,8). is produced throughout the body in cells of mesenchymal Support for a role for endogenous HGF in regeneration after origin and is secreted in an immature inactive form that is ATN comes from studies that demonstrated early transient increases in HGF levels in injured kidneys. HGF gene expression is simultaneously increased in uninjured tissues such as Received July 20, 1999. Accepted June 16, 2000. liver, lung, and spleen (4,11,12), and these tissues then release Correspondence to Dr. Ralph Rabkin, VAPAHCS (111R), 3801 Miranda the growth factor into the circulation. HGF bioactivity in Avenue, Palo Alto, CA 94304. Phone: 650-858-3985; Fax: 650-849-0213; E-mail: [email protected] kidney and plasma is increased, reaching a peak 12 h after 1046-6673/1203-0531 injury and returning to baseline levels after 2 to 3 d, indicating Journal of the American Society of Nephrology that HGF functions in both an endocrine manner and a local Copyright © 2001 by the American Society of Nephrology paracrine/autocrine manner (4,13). Kidney c-MET mRNA lev- 532 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 531–540, 2001

els are also transiently increased after ischemia- and folic rRNA in samples of total RNA subjected to electrophoresis through a acid-induced injuries (13–15), and whole-kidney c-MET pro- denaturing agarose gel containing 1.7% formaldehyde. Protection tein levels are elevated 12 and 24 h after folic acid adminis- assays were repeated at least once. tration (13). Taken together, the early increases in circulating HGF levels and kidney c-MET expression suggest that this Preparation of Riboprobes ligand-receptor pair may play a role in promoting regeneration The c-MET template was kindly provided by Dr. Paul Godowski of injured kidneys. To more firmly establish whether HGF and (Genentech Inc.). This cDNA fragment was prepared by first perform- c-MET do indeed participate in the regenerative process, we ing reverse transcription of total rat liver mRNA using two primers with homology to both the human and mouse c-MET mRNA se- have examined the response of the receptor in greater detail. quences, i.e., 5'-CGTAGGATCCATATGTGGCTGGGACTT-3' and We report that, in rats with ischemic ATN, both the level and 5'-GCATGGATCCACCTTTAACTGCTTCAGG-3' (17,18). After state of activation of the c-MET receptor protein increase early PCR amplification, the resulting 221-bp cDNA fragment was inserted after injury, before the onset of DNA synthesis. Furthermore, into a pBluescript II SK(ϩ) plasmid. The plasmid was linearized with the increase in c-MET levels is most prominent in the more SstII, and the T7 promoter was used for transcription of the riboprobe. severely injured nephron segments, where maximal DNA synthesis is observed. Therefore, our data, although not providing Preparation of Crude Kidney Membranes direct evidence for a role for HGF in renal regeneration, do For measurement of c-MET levels in kidney plasma membranes, support the hypothesis that HGF participates in the early re- crude membranes were isolated, as described previously, from control generative process. Finally, we also report that c-MET protein and ATN kidneys 2 and 8 d after surgery (16). In brief, kidney levels remain elevated at a time when the elevated circulating homogenates were prepared in 8% (wt/wt) sucrose and subjected to HGF levels are known to have returned to baseline values differential centrifugation. Crude membranes were collected after ϫ (4,13), suggesting that prolonged and even delayed treatment centrifugation at 47,000 g, washed, resuspended in calcium-free Ϫ with HGF may be of therapeutic value in acute renal failure. Krebs-Ringer Hepes buffer (pH 7.4), and stored at 80°C. Western Immunoblotting Materials and Methods c-MET and phosphorylated c-MET were detected according to Animals and Tissue Collection previously described methods. In brief, frozen kidney was powdered Male Sprague-Dawley rats, weighing approximately 225 g and under liquid nitrogen and homogenized in 5 volumes of ice-cold RIPA allowed free access to food and water, were anesthetized with intra- lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% peritoneally administered xylazine (10 mg/kg) and ketamine (75 mg/ Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 ␮g/ml leupep- kg). Body temperature was maintained at 37°C. The kidneys were tin, 5 ␮g/ml aprotinin, 1 ␮g/ml pepstatin, 1 mM sodium vanadate, and exposed via a flank incision (2). In one-half of the rats, the renal 1 mM NaF. The homogenate was incubated at 4°C with shaking for pedicle was clamped for 35 min (ATN group); the other rats served as 1 h and then clarified by centrifugation at 40,000 ϫ g for 30 min. sham-operated control animals. After recovery from the procedure, Plasma membranes were solubilized in the same buffer. For detection the ATN rats were allowed ad libitum dietary intake and the control of c-MET, the supernate was added to sodium dodecyl sulfate (SDS) animals were pair-fed. Twelve hours or 1, 2, or 8 d after surgery, the sample buffer with 2-mercaptoethanol, and the solution was heated for rats were anesthetized, blood was collected, and the kidneys were 5 min at 95°C. For detection of c-MET phosphorylation, 1 mg of rapidly excised and frozen in liquid nitrogen. Other rats were given tissue extract was incubated overnight at 4°C with anti-murine c-MET 100 mg/kg bromodeoxyuridine (BrdU) 2 h before euthanasia, at which antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and precipi- time the kidneys were perfusion-fixed, via the abdominal aorta, with tated with protein A-agarose. The immunoprecipitates were washed cold 4% paraformaldehyde for 10 min. Kidney slices and small bowel several times and resuspended in SDS sample buffer with 2-mercap- samples (positive control samples) were then stored overnight in cold toethanol and were then heated at 95°C for 5 min. The solubilized 4% paraformaldehyde and embedded in paraffin blocks, from which proteins (50-␮g aliquots) were separated by SDS-polyacrylamide gel ␮ 5- m sections were cut and processed for immunohistochemical electrophoresis (7.5% running gel) and transferred to nitrocellulose analysis. membranes, which were blocked with 5% nonfat dry milk for detection of c-MET or 1% bovine serum albumin in Tris-buffered saline Solution Hybridization/RNAse Protection Assays with 0.05% Tween for detection of tyrosine phosphorylation. The ␮ Total RNA was extracted according to the method of Chomczyn- membranes were then incubated overnight at 4°C with 1 g/ml ski, and mRNA amounts were quantified by means of a solution anti-murine c-MET or anti-phosphotyrosine antibody (Santa Cruz hybridization/ribonuclease protection assay, as described previously Biotechnology), washed, and incubated for1hatroom temperature (16). In brief, total RNA (20 ␮g) was hybridized overnight at 42°C with horseradish peroxidase-conjugated secondary antibody; proteins with approximately 200,000 cpm of [␣-32P]CTP-labeled antisense were observed by using the enhanced chemiluminescence method c-MET RNA probe in 75% formamide/400 mM NaCl. After hybrid- (Amersham, Arlington Heights, IL) and were quantified by ization, the mixture was added to RNAse digestion buffer containing densitometry.

RNAse A (100 mg/ml) and RNAse T1 (2000 U/ml) and was incubated at 30°C for 1 h. Proteinase K (200 mg/ml) was then added, and Immunohistochemical Analyses incubation was continued at 37°C for 30 min. Protected hybrids were c-MET and BrdU were detected with appropriate antisera by means then precipitated with ethanol and size-separated on a 5% polyacryl- of the avidin-biotin immunoperoxidase procedure. For localization of amide/8 M urea denaturing gel. Autoradiographs were obtained, and c-MET, two different, affinity-purified, rabbit IgG polyclonal anti- protected bands were measured by densitometry. Sample loading was bodies were used at dilutions of 1:400. One antibody was directed controlled by measuring the level of ethidium bromide-stained 18S against the last 21 amino acids of the carboxyl terminus of the mouse J Am Soc Nephrol 12: 531–540, 2001 HGF Receptor in ATN 533 c-MET protein (Santa Cruz Biotechnology). The other antibody was Results directed against the extracellular domain of human c-MET and was Serum Creatinine Levels prepared by stimulating antibodies against a recombinant c-MET-IgG In rats subjected to bilateral renovascular occlusion, serum fusion protein (19). For detection of c-Met, deparaffinized kidney creatinine levels were elevated at 12 h and returned to control sections were incubated first with 3% hydrogen peroxide for 30 min values by day 8 (Table 1). (to block endogenous peroxidase) and then with 10% nonimmune goat serum for1hatroom temperature (to prevent nonspecific antibody binding), followed by overnight incubation at 4°C with the Kidney c-MET mRNA Levels Are Increased after murine or human anti-c-MET antibody. After a wash with phosphate- Ischemic Injury buffered saline, sections were incubated with biotinylated goat anti- As shown in Figure 1, there were significant increases in rabbit IgG (dilution, 1:400; Vector Laboratories) for 30 min, followed c-MET mRNA levels 6 and 24 h after injury. This finding is by incubation with horseradish peroxidase-conjugated streptavidin for consistent with earlier reports (4,13,15). 30 min at room temperature. The secondary antibody was observed after the addition of 3,3'-diaminobenzidine in 0.02% urea/hydrogen Kidney c-MET Protein Levels Are Increased after peroxide. Kidneys from at least three control rats and three ATN rats Injury were examined at each time point of the study. Nonspecific signals were excluded by incubating sections with nonimmune rabbit serum To determine whether the increase in c-MET gene expres- or with anti-c-MET antibody that had been preabsorbed with the sion is followed by an increase in cellular receptor protein c-MET-IgG fusion protein or with a tumor necrosis factor receptor- levels, we measured whole-kidney c-MET protein levels using IgG fusion protein (20). Sections were counterstained with Gill’s Western immunoblotting. The results are presented in Figures hematoxylin. For detection of BrdU, the sections were incubated for 2 and 3A. As early as 12 h after injury, there was a significant 25 min at 37°C in phosphate-buffered saline with 0.1% trypsin and increase in c-MET protein levels, which reached a maximum at 0.1% CaCl2 and were then incubated for 45 min at 70°C with 95% 24 h. At that time, the c-MET levels in the ATN rat kidneys formamide in 0.15 trisodium citrate. After the sections were rinsed, were 7 times greater than those in the sham-operated control they were incubated for1hatroom temperature with mouse mono- kidneys. The c-MET levels then decreased but were still sig- clonal anti-BrdU antibody diluted 1:50 in 1% fetal bovine serum nificantly higher than control values after 8 d. Because kidney (Dako Products, Carpenteria, CA). The secondary antibody was bio- c-MET levels reflect protein levels in whole cells and because tinylated goat anti-mouse IgG (Vector) and was detected using the peroxidase reaction, as described above. the immunohistochemical studies described below demonstrated redistribution of the receptors after injury, we also measured plasma membrane c-MET levels on days 2 and 8. DNA Synthesis Like the cell levels, plasma membrane c-MET levels increased DNA synthesis was determined from the incorporation of BrdU after injury (Figure 4). into DNA, as measured by immunohistochemical analyses, and was quantitated by counting the number of labeled tubular cell nuclei in six randomly selected, high-power (ϫ400) fields in each kidney Onset of Increased DNA Synthesis Occurs after the cortex. Increase in c-MET Protein Levels In contrast to the early increase in c-Met protein levels, Biochemical Measurements DNA synthesis in the ATN kidneys at 12 h after injury did not Serum creatinine levels were determined in a Beckman creatinine differ from that observed in the control kidneys (Figure 3B). analyzer (Beckman Instruments, Fullerton, CA). Tissue protein con- By 24 h, there was a marked (18-fold) increase in DNA centrations were determined using the Bio-Rad method (Bio-Rad synthesis, which was sustained at 48 h and returned to baseline Laboratories, Richmond, CA). values by day 8.

Statistical Analyses Phosphorylated c-MET Levels Are Increased after For comparison between groups, data were analyzed with the Renal Injury unpaired t test. P values of Ͻ0.05 were considered significant. Results As depicted in Figure 5, there were distinct increases in are expressed as mean Ϯ SEM. phosphorylated c-Met levels at 12 and 24 h after surgery in the

Table 1. Serum Creatinine Levelsa

Serum Creatinine Levels (mg/dl) Group 0.5 d 1 d 2 d 8 d

Sham 0.6 Ϯ 0.03 0.6 Ϯ 0.03 0.5 Ϯ 0.04 0.5 Ϯ 0.05 ATN 2.0 Ϯ 0.19b 2.2 Ϯ 0.43c 2.2 Ϯ 0.52b 0.6 Ϯ 0.07

a n ϭ 3 to 8 rats/group per time point. ATN, acute tubular necrosis. b P Ͻ 0.01. c P Ͻ 0.05. 534 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 531–540, 2001

Figure 1. c-MET mRNA levels in kidneys of rats 6 or 24 h after a sham operation (control animals) or bilateral ischemic renal injury (acute tubular necrosis [ATN]). The mRNA levels were measured using a solution hybridization/nuclease protection assay. The 18S rRNA levels in the same total RNA samples were assessed by ethidium bromide staining after electrophoretic separation in a denaturing agarose gel.

Figure 2. Western immunoblot analysis of kidney c-MET protein after ischemic ATN or a sham operation (Control). Solubilized kidney homogenates were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, under reducing conditions, and transferred to nitrocellulose membranes. After incubation with anti-c-MET antibody, the 145-kD signal was detected by enhanced Figure 3. (A) Time course of changes in c-MET protein levels in ATN chemiluminescence. kidneys and sham-operated control kidneys. Autolumigraphs were quantitated by densitometry, and results were expressed relative to those for the paired control animals (assigned a value of 1). (B) Time course of DNA synthesis. DNA synthesis was estimated from the injured kidneys, compared with control values. Phosphorylated number of cortical tubular cell nuclei that were positive for bromode- receptors were barely detectable in the control animals and oxyuridine (BrdU) in each high-power field (hpf) (ϫ400). Results are were thus not quantifiable by densitometry. Because the recep- mean Ϯ SEM for three or four animals/group per time point. tor protein levels were greater in the ATN samples (Figure 2), the increase in phosphorylation in this group could simply reflect an increase in the number of receptors in the basal state increase in phosphorylated receptor levels may have occurred of autophosphorylation. Alternatively, the increased phosphor- even earlier. ylation could reflect a true increase in the state of receptor tyrosine phosphorylation, or a combination of both of these c-MET Protein Levels Are Increased in the Injured possibilities. Whichever is the case, it is likely that the in- Regions of the Kidney, and c-MET Protein Is creased levels of phosphorylated c-MET promote the action of Redistributed within Damaged Proximal Tubule Cells HGF. Because this increase in the levels of phosphorylated Low-power views of control kidney sections demonstrated receptors preceded the onset of DNA synthesis in the injured low-level c-MET immunoreactivity in all regions of the kid- kidneys (Figure 3B), this finding lends support to the hypoth- ney, but especially evident in the outer medulla (Figure 6A). esis that endogenous HGF participates in initiating the regen- The ATN kidneys exhibited a general increase in c-MET eration of injured kidneys. Because the earliest time point immunostaining, which was strongest in the outer medulla (the examined was 12 h after the insult, it is possible that an region of the kidney sustaining the most severe injury) (Figure J Am Soc Nephrol 12: 531–540, 2001 HGF Receptor in ATN 535

ischemic damage. These findings are presented in Figure 7B for kidneys obtained 24 h after injury. Similar patterns were observed after 12 and 48 h. Figure 7B demonstrates that, in the injured cortex, some proximal tubules exhibited the same apical polar c-MET distribution as observed in control kidneys. In other tubules, the c-MET signal was present in a punctate pattern in the cell cytosol. This appearance is consistent with the presence of receptors in endocytotic vesicles and may reflect endocytosis of the receptor after it is activated by HGF (21). The more severely injured cells were diffusely stained, Figure 4. Western immunoblot analysis of kidney plasma membrane and the detached cells were intensely stained. These latter c-Met protein. Plasma membranes were prepared from rats 2 and 8 d changes likely reflect the loss of polarity that follows cellular after ischemic injury or a sham operation and were processed as injury (22). Despite this receptor redistribution, it is important described for Figure 2 (n ϭ 3 rats/group per time point). to note that the immunoblot analysis demonstrated overall increases in whole-cell and plasma membrane receptor protein levels (Figure 4). Examination of the outer stripe of the outer zone of the medulla of control kidneys revealed diffuse c-MET staining in all nephron segments (Figure 7C). Interestingly, in contrast to the cortical proximal tubules, outer medullary proximal tubular cells lacked brush border c-MET immunostaining and c-MET appeared to be localized intracellularly. This variance in im- Figure 5. Tyrosine phosphorylation of c-MET in kidneys of ATN and munostaining likely reflects differences in the structure and control rats 12 and 24 h after injury. Solubilized kidney proteins from function of the different segments of the proximal tubules. The three rats/group per time point were immunoprecipitated with anti-c- ATN kidneys demonstrated an increase in immunoreactivity in MET antibody, separated by SDS-polyacrylamide gel electrophoresis, the outer stripe of the medulla, and this was most intense in the and blotted with an anti-phosphotyrosine antibody. The phosphory- injured nephrons (presumably, the straight portion of the prox- lated 145-kD protein was detected by enhanced chemiluminescence. imal tubules) (Figure 7D). Both detached and attached cells were heavily stained. We suggest that the increase in c-MET staining in the detached cells reflects an unsuccessful protec- 6B). It is noteworthy that BrdU immunoreactivity (a measure tive response to injury that occurs before detachment and of DNA synthesis) was maximally increased in this same cellular necrosis. In the inner stripe of the outer zone of the region of the injured kidneys (Figure 6E). These findings medulla, c-MET expression was evident in all nephron seg- provide further support for a role for HGF in renal regenera- ments (Figure 8A). In the injured kidneys, the tubular cells tion. To exclude the possibility that this increase in c-MET appeared swollen and were diffusely immunostained (Figure immunostaining might reflect nonspecific binding of the anti- 8B). The papillae of the control kidneys demonstrated c-MET c-MET antibody to damaged cells, the effect of using antigen- staining distributed throughout the collecting ducts (Figure preabsorbed antibody was tested (20). Preabsorption with a 8C). The localization of the signal was poorly defined, but c-MET-IgG fusion protein largely blocked the c-MET signal staining appeared more prominent in the apical membrane. (Figure 6C). In contrast, preincubation of antibody with a Staining was also evident in interstitial cells and thin-walled tumor necrosis factor receptor-IgG fusion protein failed to structures consistent in appearance with the thin limbs of Henle block the signal (data not shown). We also observed that two and the vasa recta. There did not seem to be any major different c-MET-specific antibodies, i.e., anti-murine and anti- difference in c-MET immunostaining between control and human c-MET antibodies, yielded essentially identical results ATN kidneys (Figure 8D). Eight days after the original isch- and no signal was obtained with nonimmune rabbit serum (data emic insult, the intensity of immunostaining in the ATN kid- not shown). neys decreased (data not shown), which is consistent with the At higher magnification, an interesting pattern of c-MET Western immunoblots presented earlier. Many but not all of the distribution in the cortex was apparent. In control kidneys, the proximal tubules had regained the apical localization of c-MET c-MET signal was strongest in the distal tubules and collecting staining at that time. Light staining of the cellular interstitial ducts. In those nephron segments, staining was localized infiltrate was present at that time. To rule out the possibility mostly in the apical and basolateral membranes (Figure 7A). that the altered c-MET immunostaining in the ATN kidneys The proximal tubular cells also stained for c-MET. There the might be caused by nonspecific binding of the anti-c-MET signal was clearly present in the brush border membrane. In antibody to damaged cells, the effects of using antigen-preab- occasional proximal tubules, there were infrequent areas of sorbed antibody or omitting this antibody altogether were cytoplasmic staining. Focal staining of glomerular epithelial tested (20). Preabsorption with a c-MET-IgG fusion protein cells was also evident. In the injured cortex, a range of patchy largely but not completely blocked the c-MET signal (Figure changes was evident, reflecting the variable severity of the 9A), whereas in the absence of the antibody there was no 536 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 531–540, 2001

Figure 6. Immunohistochemical localization of the hepatocyte growth factor receptor (c-MET) and BrdU. (A, B, and C) c-MET immunostaining in kidneys from sham-operated control rats (A) and ATN rats 24 h after an ischemic insult (B and C). Note the dramatic increase in c-MET immunostaining in the injured kidney, especially the outer medulla (B). In contrast, when an ATN kidney was probed with anti-c-MET antibody that had been preabsorbed with the receptor protein used to stimulate antibody formation, the c-MET signal was essentially abolished (C). This establishes the specificity of the immunostaining. (D and E) BrdU immunostaining in kidneys from sham-operated control rats (D) and ATN rats 48 h after injury (E). Note the pronounced immunostaining for BrdU in the outer medulla, where c-MET immunostaining is most marked. Magnification, ϫ1.2.

immunostaining at all (Figure 9B). Therefore, there seems to threefold higher than those in control animals. This increase in be a low level of nonspecific immunostaining, which cannot whole-kidney c-MET protein levels was accompanied by a account for the changes in c-MET expression observed in the similar increase in kidney plasma membrane c-MET levels, injured kidneys. It is also possible that the low level of immu- which would favor an increase in tissue sensitivity to HGF. It nostaining observed reflects incomplete preabsorption of the is important to note that the increase in kidney c-MET protein anti-c-MET antibody. levels preceded the increase in DNA synthesis, which was first evident 24 h after injury. Furthermore, the greatest increases in Discussion c-MET and DNA synthesis occurred in the same region of the Administration of recombinant HGF to rodents with acute kidney, namely the outer medulla, where injury was most renal failure accelerates recovery, and it has been suggested marked. Because circulating and local kidney HGF levels are that endogenous HGF may play a role in the natural sponta- also elevated as early as 2 to 6 h after renal injury (4,13), this neous recovery process. The aim of these experiments was to sequence of events supports the hypothesis that HGF plays a test this hypothesis. To this end, we examined the changes in role in initiating renal regeneration, and it is consistent with the the expression and activity of the HGF receptor, c-MET, in the recent findings of Liu et al. (13) in folic acid-induced acute kidneys of rats with ischemia-induced ATN. We observed that, renal failure. Additional support is provided by our finding that as early as 12 h after the ischemic insult, there was a fourfold c-MET tyrosine phosphorylation, a process that reflects recep- increase in c-MET protein levels. This was preceded by an tor activation, is increased within 12 h after injury. Whether the increase in c-MET gene expression that was evident at 6 h after increased tyrosine phosphorylation reflects an increase in the ischemia. c-MET protein levels increased to a maximum after number of receptors in the basal state of phosphorylation 24 h, at which time the levels in the ATN kidneys exceeded the and/or an absolute increase in the state of receptor phosphor- values for the sham-operated control kidneys by sevenfold. ylation is unclear, because the number of receptors was in- Levels then decreased, but even after 8 d the levels were still creased in the injured kidneys. In any case, because tyrosine J Am Soc Nephrol 12: 531–540, 2001 HGF Receptor in ATN 537

Figure 7. Immunohistochemical localization of c-MET in the kidney cortex and outer medulla of sham-operated control rats and ATN rats 24 h after an ischemic episode. Magnification, ϫ220. (A) Control kidney cortex. Proximal tubules show distinct brush border membrane staining (arrows). Distal tubules are heavily stained (*). (B) ATN kidney cortex. Several proximal tubules exhibit the normal pattern of immunoreactivity, with prominent brush border membrane staining (closed arrows). Other proximal tubules, while maintaining an apical signal, exhibit a punctate pattern of immunoreactivity in the cytosol (open arrows). Still other proximal tubular cells have lost the apical signal and exhibit a more diffuse pattern of staining (arrowheads). Detached cells in the tubular lumen are heavily stained. There is intense staining of the distal tubules (*). (C and D) Outer stripe of the outer zone of the medulla of control (C) and ATN (D) kidneys. In the control kidney, there is diffuse immunostaining of the proximal and distal nephron segments. Unlike the cortical proximal tubules, the outer medullary proximal tubule segments lack brush border membrane immunoreactivity. In the injured kidney, there is increased immunoreactivity in the damaged tubules and numerous sloughed cells. phosphorylation of c-MET serves to activate signal transduc- contrast, Tsarfaty et al. (26) reported that c-MET is localized to tion, the increase in phosphorylation supports a role for HGF in the apical pole of cells lining human breast tissue mammary renal regeneration. ducts. These varying descriptions of the polar distribution of c-MET immunostaining in normal rat kidneys was strongest c-MET may reflect tissue-specific cellular differences in the in the cortical distal tubules and collecting ducts and medullary localization of the receptor, as well as perhaps technical collecting ducts. In these nephron segments, c-MET appeared differences. to be localized to the apical and basolateral membranes. Im- We observed striking changes in the cellular distribution and munostaining in the proximal tubular cells was weak and intensity of c-MET immunostaining in acutely injured rat kid- localized mainly to the brush border membrane. Liu et al. (23) neys, which was not readily evident when the antibody was also described prominent staining of distal tubular cells, with preabsorbed with the antigen. In the outer medulla, which was weak staining of proximal tubules, but did not comment on the the most prominent site of ischemia-induced ATN and subse- subcellular receptor distribution. Because the kidneys were not quent DNA synthesis, there was a marked increase in c-MET perfusion-fixed in that study, the apical location of c-MET may levels, especially in the more severely damaged cells. Both have been obscured. Those authors did note that early diabetes detached and attached cells were heavily immunostained. Be- is associated with an increase in c-MET protein levels, and cause increases in c-MET mRNA and protein levels were they proposed that the HGF/c-MET system may play a role in detected as early as 6 and 12 h after injury, respectively, and diabetic renal hypertrophy, as has been suggested for compen- because cellular necrosis and detachment evolve during a 12- satory renal growth (9). Crepaldi et al. (24) observed that to 24-h period (27), we suggest that immunostaining of the c-MET is localized exclusively to the basolateral pole of cul- detached cells reflects a failed protective response that oc- tured MDCK cells (a dog kidney cell line with distal nephron curred before detachment. However, it might be argued that the features). This finding may well reflect a cell line phenomenon. increase in c-MET levels in the injured cells might in some In the same study, those authors localized c-MET to the baso- way contribute to their injury. Arguing against this are reports lateral pole of the epithelium lining the human colon. Simi- that early treatment with HGF ameliorates the severity of larly, Nusrat et al. (25) localized c-MET to the basolateral injuries (4–6). membrane of native crypt epithelial cells of human intestine. In In the cortex, there were patchy changes in the proximal 538 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 531–540, 2001

Figure 8. Immunohistochemical localization of c-MET in the inner stripe of the outer zone of the medulla and papilla of sham-operated control and ATN rats 24 h after an ischemic episode. Magnification, ϫ220. Inner stripe of the outer zone of the medulla of control (A) and ATN (B) kidneys. The tubular cells of the injured kidney appear swollen and are diffusely stained. Papillae of control (C) and ATN (D) kidneys.

Figure 9. (A) Photomicrograph of the cortex of 24-h post-ATN kidneys probed with anti-c-Met antibody that had been preabsorbed with the receptor protein used to stimulate antibody formation. (B) Photomicrograph of the outer stripe of the outer zone of the medulla probed with secondary antibody only; the anti-c-MET antibody was omitted. The photomicrographs correspond to the regions shown in Figure 7, B and D, respectively. Magnification, ϫ220. tubules. Some tubules maintained the same apical polar distri- normally observed, and it is conceivable that elevated tubular bution of c-MET as observed in control kidneys, whereas other fluid HGF levels could serve to activate the apical receptors, tubules exhibited a punctate pattern of staining in the cell causing receptor-mediated endocytosis (28). The activated re- cytosol. In more severely injured proximal tubular cells, there ceptors could then activate other signaling proteins and so was a loss of the apical distribution of c-MET, with staining in induce a regenerative response. Also relevant is the report by the cytosol; cells that had detached from the basement mem- Tajima et al. (21), who noted a decrease in plasma membrane brane were intensely and diffusely stained. These latter HGF receptor binding in the remaining kidney after unine- changes likely reflect the general loss of polarity that follows phrectomy and in regenerating liver. They attributed this de- ischemic tubular cell injury (22). The situation in the modestly crease in specific binding to either increased receptor occu- injured cells is less clear. In these cells, the punctate intracel- pancy or increased internalization in response to elevated lular distribution of the receptor may reflect increased receptor- circulating HGF levels. Taking that report together with all of mediated endocytosis in response to elevated HGF levels (21). our findings, we propose that tubular cells respond to injury by Indeed, in human patients with ATN, there is a marked in- increasing c-MET expression, a response that is followed by crease in urinary HGF levels above the low levels that are increased receptor activation, receptor internalization, signal J Am Soc Nephrol 12: 531–540, 2001 HGF Receptor in ATN 539 transduction, and subsequent promotion of a regenerative re- 9. Nagaike M, Hirao S, Tajima H, Noji S, Taniguchi S, Matsumoto sponse. Furthermore, when the injury is severe and cell polarity K, Nakamura T: Renotropic functions of hepatocyte growth is lost, it seems that the receptors become distributed through- factor in renal regeneration after unilateral nephrectomy. J Biol out the cell. Examination of the kidneys 8 d after injury Chem 266: 22781–22784, 1991 revealed that many but not all of the proximal tubules demon- 10. Miyazawa K, Shimomura T, Naka D, Kitamura N: Proteolytic activation of hepatocyte growth factor in response to tissue strated normalization of receptor distribution. injury. J Biol Chem 269: 8966–8970, 1994 In conclusion, we have demonstrated that, after an ischemic 11. Kono S, Nagaike M, Matsumoto K, Nakamura T: Marked in- renal insult, there are early increases in c-MET protein levels duction of hepatocyte growth factor mRNA in intact kidney and and activity that precede the onset of DNA synthesis. Further- spleen in response to injury of distant organs. Biochem Biophys more, the greatest increases in receptor protein expression and Res Commun 186: 991–998, 1992 DNA synthesis both occur in the most severely injured nephron 12. Yanagita K, Nagaike M, Ishibashi H, Yoshiyuki N, Matsumoto segments in the outer medulla. Taken together, this sequence of K, Nakamura T: Lung may have an endocrine function producing events and their anatomic colocalization support the hypothesis hepatocyte growth factor in response to injury of distal organs. that HGF and c-MET participate in promoting recovery from Biochem Biophys Res Commun 182: 802–809, 1992 injury. Finally, it is important to note that the c-MET levels 13. Liu Y, Tolbert EM, Lin L, Thursby MA, Sun AM, Nakamura T, remain elevated as much as 8 d after the ischemic insult. This Dworkin LD: Up-regulation of hepatocyte growth factor receptor: An amplification and targeting mechanism for hepatocyte finding, together with the report that serum HGF levels are growth factor action in acute renal failure. Kidney Int 55: 442– elevated only transiently after acute experimental renal injury, 453, 1999 suggests that prolonged and even late treatment with HGF may 14. Ishibashi K, Sasaki S, Sakamoto A, Hoshino Y, Nakamura T, be of therapeutic benefit. 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