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Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Correspondence to Dr. Youhua Liu, Department of Pathology, University of Pittsburgh School of Medicine, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: 412-648-1916; E-mail: liuy{at}msx.upmc.edu
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Abstract |
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Introduction |
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Hepatocyte growth factor (HGF) was originally characterized as a potent mitogen for hepatocytes (7,8). Subsequent studies have indicated that HGF exerts mitogenic, motogenic, tubulogenic, and anti-apoptotic activities in a wide spectrum of target cells, including renal tubular epithelial cells (9–11). The pleiotropic properties of HGF render it specifically suited for promoting tissue repair and regeneration after injury (12–14). Previous studies in our laboratory have demonstrated that HGF is rapidly upregulated in blood circulation and in multiple organs after acute renal injury induced by folic acid, whereas its specific receptor, c-met, is exclusively induced in the kidneys (15). This observation establishes that the HGF/c-met signaling system targets its action to damaged organ by site-specific upregulation of its receptor while maximally mobilizing its ligand (15). Moreover, induction of c-met receptor by other growth factors, such as epidermal growth factor and insulin-like growth factors-I in renal tubular epithelial cells raises an intriguing possibility that the HGF/c-met system could represent a shared and converging pathway that leads to acceleration of renal regeneration after injury.
Because HGF protein is rapidly cleared by the liver in vivo, it has a very short half-life in blood circulation (16). Maintenance of a sustained level of exogenous HGF protein in the circulation by injections, even when given at very short intervals, has proved to be challenging. One strategy to circumvent this problem is to develop an effective gene delivery approach (17,18) that allows sustainable expression of HGF protein in vivo. We recently described a highly efficient approach to introduce and express exogenous HGF gene by systemic administration of naked plasmid vector (19). This gene delivery strategy results in remarkable level of exogenous HGF protein in the circulation as well as in the kidneys of normal adult animals in a controllable fashion (19). Hence, this means of gene transfer may provide an efficient way to evaluate the therapeutic efficacy of HGF for acute renal failure.
In this study, we show that administration of HGF expression plasmid by intravenous injection significantly ameliorates acute renal injury induced by folic acid. Our results suggest that exogenous HGF not only promotes renal tubular repair after injury but also functions as a cytoprotective agent to minimize the initial injury, to protect tubular epithelial cells against both apoptotic and necrotic death, and to preserve normal kidney structure and functions.
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Materials and Methods |
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Plasmid Injection
The recombinant human HGF expression plasmid (pCMV-HGF) that contains full-length human HGF cDNA driven under a human cytomegalovirus promoter was cloned as described elsewhere (20). The empty expression plasmid vector pcDNA3 was purchased from Invitrogen (San Diego, CA). Plasmid DNA was administered into mice by a hydrodynamic-based gene transfer technique via rapid injection of a large volume of DNA solution through the tail vein, as described elsewhere (21). Briefly, 20 µg of plasmid DNA was diluted in 1.6 ml of saline and injected via the tail vein into the circulation within 5 to 10 s. Mice from control group were injected with 20 µg of empty plasmid vector pcDNA3 in 1.6 ml of saline in an identical fashion. Mice were divided into five groups: (1) normal control group, (2) folic acid/control group in which mice were preadministered empty pcDNA3 plasmid 24 h before folic acid injection, (3) folic acid/preadministration of pCMV-HGF plasmid group in which mice were preadministered pCMV-HGF plasmid 24 h before folic acid injection, (4) folic acid/pCMV-HGF plasmid group in which mice were simultaneously injected with pCMV-HGF plasmid and folic acid, and (5) folic acid/HGF group in which mice were simultaneously injected with folic acid and recombinant human HGF protein (Genentech, Inc., South San Francisco, CA) at 200 µg/kg body wt. Six mice from each group at each time point were killed at 1, 2, 3, and 6 d postinjection of folic acid, respectively, except the normal control group, in which six mice were only killed at day 1. Serum was collected at the time of death (day 1, 2, 3, and 6) as well as at day 5. One part of the kidney was processed for tissue histology. Another part was snap-frozen in Tissue-Tek O.C.T. compound for subsequent cryosectioning. The remaining kidney was immediately frozen in liquid nitrogen and stored at -80°C for tissue lysate preparation.
Determination of HGF Levels by Enzyme-Linked Immunosorbent Assay
For measurement of tissue HGF level, kidneys from mice were homogenized in the HGF extraction buffer that contained 20 mM Tris-HCl (pH 7.5), 2 M NaCl, 0.1% Tween-80, 1 mM ethylenediaminetetraacetate, and 1 mM phenylmethylsulfonyl fluoride, as described elsewhere (19). After centrifugation at 19,000 x g for 20 min at 4°C, the supernatant was recovered for determination of HGF by use of an enzyme-linked immunosorbent assay (ELISA) method. This assay uses a sandwich method that consists of three steps of antigen-antibody reactions. Briefly, the 96-well microtiter plates (Nunc-Immuno Module, Fisher Scientific, Pittsburgh, PA) were incubated with 50 µl of uncoupled monoclonal anti-HGF antibody (H14) per well diluted in 50 mM Tris HCl (pH 8.0) at a final concentration of 1.5 µg/ml at room temperature for 16 h. H14 anti-human HGF antibody was prepared by use of a standard protocol of hybridoma technology, as described elsewhere (19). This antibody could detect human HGF protein but not rodent HGF in an ELISA assay and in immunohistochemical staining (19). The plates were washed three times with phosphate-buffered saline (PBS) that contained 0.05% Tween 20 (pH 7.4) and were blocked with 200 µl of blocker solution (PBS with 1% bovine serum albumin [BSA]) at room temperature for 2 h. The plates were extensively washed five times with PBS containing 0.05% Tween 20 and kept at 4°C. Fifty-microliter aliquots of standard human HGF solution or tissue samples were added to the wells and incubated for 2 h at room temperature. After extensive washing, a 100-µl aliquot of biotinylated goat anti-human HGF polyclonal antibody (purchased from R & D Systems, Minneapolis, MN) at a dilution of 1:2000 was added, and the plates were incubated for another 2 h. After being washed, they were then incubated with 100 µl of horseradish peroxidase–conjugated streptavidin (Zymed Laboratories, South San Francisco, CA) at a dilution of 1:20,000 and subsequently with enzyme substrate solution that contained 0.1 mg/ml of tetramethylbenzidine and 0.006% H2O2 in 0.1 M sodium citrate (pH 6.0). The plates were allowed to stand for 30 min at room temperature, and the reaction was stopped by addition of 50 µl 4N H2SO4. Absorbance was read at 405 nm by an automatic Emax Precision Microplate Reader (Molecular Devices Co., Sunnyvale, CA). Samples were diluted if necessary to give an optical density that was within the linear portion of the standard curve. Total protein levels were determined by use of a bicinconinic-acid protein assay kit (Sigma) with BSA as a standard. The concentration of HGF in kidneys was expressed as nanograms per milligram total protein.
Western Immunoblot Analysis
For analysis of human HGF transgene expression, total kidney extracts as described above for ELISA were used for Western immunoblotting. For detection of other proteins, kidney tissues were homogenized by a polytron homogenizer (Brinkmann) in RIPA lysis buffer (1% NP40, 0.1% sodium dodecyl sulfate [SDS], 100 µg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml antipain, and 2 µg/ml leupeptin in PBS) on ice, and the supernatants were collected after centrifugation at 13,000 x g at 4°C for 20 min. Protein concentration was determined by use of a bicinconinic-acid protein assay kit (Sigma), and tissue lysates were mixed with an equal amount of 2x SDS loading buffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, and 0.2% bromophenol blue), as described elsewhere (15). Samples were heated at 100°C for 5 to 10 min before loading and separated on precast 10% or 15% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins were electrotransferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL) in transfer buffer that contained 48 mM Tris-HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C for 1 h. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% Carnation nonfat milk in TBS buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20). The membranes were then incubated for 16 h at 4°C with various primary antibodies in blocking buffer that contained 5% milk. Monoclonal anti-human HGF antibody (H14) was generated in our laboratory as described above. The phosphospecific Akt antibody as well as the Akt antibody that detects total Akt (phosphorylation state–independent) was obtained from New England BioLabs (Beverly, MA). The antibodies against proliferating cell nuclear antigen (PCNA), Bcl-xL, Bax, and ß-actin were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). After extensive washing three times, the membranes were then incubated with horseradish peroxidase–conjugated secondary antibody (Bio-Rad) for 1 h at room temperature in 1% nonfat milk. The signals were visualized by the enhanced chemiluminescence system (Amersham). Quantitation was performed by measuring the intensity of the hybridization signals with the use of NIH Image analysis software.
Frozen Section and HGF Staining
Cryosections were prepared at 5 µm thick in a cryostat by routine procedures and fixed in cold methanol for 10 min at -20°C. Immunostaining for human HGF protein was performed by use of the Vector M.O.M. immunodetection kit, according to the protocol specified by the manufacturer (Vector Laboratories, Burlingame, CA). Briefly, crysections were incubated with M.O.M. blocking reagent for 1 h at room temperature to block endogenous mouse immunoglobulins in the tissue and to reduce background staining. After being washed with PBS, sections were incubated with primary anti-HGF monoclonal antibody (H14) at a 1:500 dilution in PBS that contained 1% BSA overnight at 4°C. Sections were then incubated for 1 h with secondary anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a dilution of 1:200 in PBS that contained 1% BSA, before being washed extensively with PBS. Slides were mounted with antifade mounting media (Vector Laboratories) and examined on Nikon (Melville, NY) Eclipse E600 Epi-fluorescence microscope equipped with a digital camera. As a negative control, the primary antibody was omitted, and no staining occurred.
Serum Creatinine Determination
Serum was collected from animals at different time points as indicated. Serum creatinine level was determined by use of a creatinine assay kit, according to the protocols specified by the manufacturer (Sigma Chemical Co.). The level of creatinine was expressed as milligrams per 100 ml (dl).
Transferase-Mediated dUTP Nick-End Labeling Staining
In situ detection of DNA fragmentation was performed on kidney cryosections by use of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining with Apoptosis Detection System (Promega, Madison, WI). Briefly, cryosections were washed by immersion into PBS and then treated with protease K at 20 µg/ml in TE buffer (100 mM Tris-HCl [pH 8.0] and 50 mM ethylenediaminetetraacetate) for 10 min. After pre-equilibration in 100 µl buffer that contained 200 mM potassium cacodylate, 25 mM Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg/ml BSA, and 2.5 mM cobalt chloride, strands of DNA were end-labeled by incubation at 37°C for 1 h in 50 µM fluorescein–12-dUTP, 100 µM dATP, 10 mM Tris-HCl (pH 7.6), 1 mM EDTA, and terminal deoxynucleotidyl transferase. The reaction was stopped by adding 2x sodium chloride/sodium citrate hybridization buffer for 15 min. The slides were stained by immersion in 1 µg/ml of propidium iodide (Sigma) in PBS. After being washed, the slides were mounted and observed on a Nikon Eclipse E600 Epifluorescence microscope.
Histology and Immunohistochemical Staining
Paraffin-embedded kidney sections from the mice were prepared by a routine procedure (22) to be 4 µm thick. Sections were stained with hematoxylin/eosin for general histology. Immunohistochemical staining was performed by use of the Vector M.O.M. immunodetection kit, as described above (Vector Laboratories). The primary antibodies used were anti-PCNA and anti-Bcl-xL (Santa Cruz Biochemical). As a negative control, the primary antibody was replaced with nonimmune normal IgG, and no staining occurred.
Statistical Analyses
Animals were randomly assigned to control and treatment groups. Statistical analyses of the data were performed by the t test with the use of SigmaStat software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.
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150 ng of exogenous HGF per mg of total tissue protein in the kidneys at day 1 after a single injection of 20 µg of pCMV-HGF per mouse (Figure 1). Under the same conditions, exogenous HGF was undetectable in the kidneys at any given time points when mice were injected with empty pcDNA3 plasmid in an identical manner (Figure 1). The expression of exogenous HGF transgene in the kidneys was sustained at least 3 d. Although the renal human HGF level tended to decline thereafter, a significant level of exogenous HGF (100 ng/mg protein) was still detected in the kidneys at day 6 after the delivery of human HGF gene (Figure 1). In contrast, intravenous injection of purified recombinant human HGF protein at 200 µg/kg of body wt only resulted in moderate level of HGF protein in the kidneys (Figure 1). The level of human HGF protein after direct injection only reached to 25% of that achieved by gene transfer approach, which suggests that systemic administration of naked plasmid vector provides a highly efficient way to deliver exogenous HGF protein to the kidneys in vivo.
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90-kD band under nonreducing conditions. The relative abundance of exogenous HGF protein was largely consistent at days 1, 2, 3, and 6, respectively, after HGF plasmid injection (Figure 1B). Direct injection of recombinant HGF protein also yielded a weak band in the Western blot of whole-kidney lysates at day 1, which disappeared beyond day 3 after initial injection (Figure 1B). Immunofluorescence staining revealed that HGF transgene expression was largely limited to renal glomeruli in the kidneys (Figure 1, C and D). More than 90% of glomeruli were positively stained for human HGF protein, whereas tubular epithelium was essentially negative for human HGF transgene expression. As expected, no staining was observed in the kidneys from mice that received the empty vector pcDNA3 injection (Figure 1C).
Expression of Exogenous HGF Gene Prevents Acute Renal Injury in Mice
Administration of a high dose of folic acid in mice induced severe acute renal failure characterized by a rapid decline of renal function, tubular epithelial cell death, and morphologic abnormalities. Figure 2A shows the levels of serum creatinine, an indicative marker for kidney dysfunctions, at different time points after mice were preadministered either empty pcDNA3 or pCMV-HGF plasmid 24 h before folic acid injection. Mice that received empty pcDNA3 vector displayed a rapid and marked increase in serum creatinine as early as 1 d after injection of folic acid. Serum creatinine remained significantly elevated at least to 3 d and declined toward the baseline level thereafter (Figure 2A). This dynamic pattern of serum creatinine levels manifests the course of altered kidney functions typically seen in this model of acute renal failure (15,23). In contrast, single preadministration of pCMV-HGF plasmid dramatically prevented folic acid–caused renal dysfunctions. Serum creatinine remained relatively constant at the baseline levels during the entire experimental scheme (Figure 2A), which indicates that preadministration of exogenous HGF gene essentially precludes acute renal failure induced by folic acid.
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HGF Gene Expression Preserves the Integrity of Kidney Morphology
Concomitant with rapid decline of renal functions, kidney morphology underwent dramatic changes during folic acid–induced acute renal injury. Figure 3A shows a representative micrograph of the kidney morphology typically seen at day 1 after injection of folic acid, which exhibited tubular cast formation, calcification and dilation, loss of brush borders, tubular epithelial cell necrosis and apoptosis, and interstitial infiltration. Preadministration of HGF plasmid largely prevented the morphologic injuries after acute renal failure caused by folic acid (Figure 3B). The morphology of these kidneys was almost indistinguishable from that of normal control one, which suggests that HGF gene therapy dramatically preserves the integrity of normal kidney morphology. Simultaneous injection of HGF plasmid also significantly ameliorated morphologic injury (Figure 3C). However, direct injection of recombinant HGF protein exhibited a much lower level of protection of renal structure against acute damage caused by folic acid (Figure 3D).
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Discussion |
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Compared with infusion of purified recombinant protein, HGF gene delivery via the naked plasmid vector results in a much more effective therapy for amelioration of acute renal failure. This is mostly likely due to the nature of persistent expression of HGF achieved by administration of the naked plasmid vector. In agreement with this notion, renal exogenous HGF protein levels remained elevated for at least 6 d after a single injection of HGF plasmid vector (Figure 1). Because of its rapid clearance by the liver in vivo, delivery of recombinant HGF protein by injection only produces transient elevation of HGF in blood circulation, and that may well explain the relative inefficiency of a single injection of recombinant HGF protein in this study. It is conceivable that multiple repeated injections of HGF protein at very short intervals are required for the effective prevention of acute renal failure (13). Alternatively, the difference in the levels of exogenous HGF protein in the kidneys after intravenous administration of the HGF gene versus recombinant protein may play a critical role in determining the therapeutic outcome. The systemic administration of naked plasmid has been shown to be an efficient way to introduce and express the HGF gene in vivo (19); the level of exogenous HGF protein in the kidneys is much higher after injection with HGF plasmid than that with recombinant protein (Figure 1). The inverse correlation between the level of HGF in the kidneys and renal dysfunction likely highlights a dose-dependent action of exogenous HGF for the amelioration of acute renal failure in vivo.
HGF transgene expression is predominantly localized in the glomeruli of the kidneys, which is probably due to the characteristic nature of the hydrodynamic and anatomical flow of the injected plasmid DNA solution in this gene delivery procedure (19,21). It is reasonable to speculate that glomerular structure is well suited to access and trap the naked plasmid vector in blood circulation after systemic administration via the tail vein. However, because HGF is a secreted protein, its presence at a particular location may not necessarily indicate the site of in situ gene expression. Thus, the exact cellular origin of HGF transgene expression in the kidneys remains to be determined, perhaps by detecting its mRNA by use of more sophisticated method such as in situ hybridization. Of interest, despite its glomeruli-specific localization of human HGF protein after gene transfer, renal tubular epithelial cells at the site of injury in this model readily responded to exogenous HGF to initiate various cellular actions. Such spatial dissociation between exogenous HGF production and its targets essentially mimics the paracrine and endocrine mechanisms of endogenous HGF action in vivo. Tubular epithelial cells do not express endogenous HGF under any circumstances, yet they are the targets of HGF produced and secreted from not only the kidney but also distal intact organs such as the lung and liver after acute renal injury (15). It is not entirely understood how exogenous HGF produced in glomeruli precisely targets its action to the damaged tubules in acute renal failure. However, previous studies from our laboratory have suggested that local upregulation of c-met receptor in the injured tubules probably plays a crucial role in conferring the specificity of HGF action (15).
Although acute renal failure induced by an ischemic or nephrotoxic insult is classically designated as acute tubular necrosis, emerging evidence suggests that apoptotic cell death may play a significant role in the pathogenesis of most forms of acute renal failure (24,29). One of the interesting findings in this study is that preconditioning the kidneys with overexpression of exogenous HGF gene largely protects tubular epithelial cells from cell death and thereby markedly prevents acute renal injury in the first place in vivo. Regardless of the mechanisms, these results underscore that HGF is a very potent survival factor capable of protecting cells against both apoptosis and necrosis in vivo. This notion is consistent with reports elsewhere that have shown an early induction of endogenous HGF after injury in vivo as well as its strong cell survival-promoting ability in vitro (30–34). Therefore, it is quite possible that the mechanistic role of HGF in ameliorating acute renal failure is predominately mediated by acting as a cytoprotective agent to minimize the initial injury and cell death in the first place, rather than by solely promoting reparative regeneration after injury, as previously emphasized.
Renal tubular cells that are lethally injured after folic acid injection can die by two distinct mechanisms: necrosis or apoptosis. Apoptosis is considered to be an active cell suicide process that involves signal transduction cascades (35,36). Although the degradative process of necrosis is accompanied by mitochondrial swelling and early loss of plasma membrane integrity that leads to the release of injurious substances from the cytosol and triggers inflammatory reaction in the surrounding tissue, apoptosis is involved by a progressive breakdown of the cell into membrane-bound particles, termed apoptotic bodies, that are rapidly phagocytosed by macrophages and neighboring healthy epithelial cells (24). Although it is well documented that HGF protects a wide variety of cells against apoptosis in vitro (30,33), no convincing evidence has demonstrated that HGF can protect cells against necrosis. On the contrary, we reported elsewhere that HGF effectively protects cultured renal tubular cells from apoptosis induced by a low dose of cisplatin but not necrosis caused by a high dose of the same agent (30). Hence, the exhibition of efficient prevention of both apoptosis and necrosis in vivo after HGF gene transfer is quite surprising. This discrepancy of the outcomes between in vitro and in vivo studies is not completely understood at this stage. It should be noted that apoptosis and necrosis could share some pathways in response to death stimuli, despite their divergence in the mechanism of cell death. The cell fate is often determined by the severity of the injury, with extremely severe insults causing necrosis and milder insults of the same type inducing apoptosis (37–39). Thus, it is possible that, after HGF gene transfer, cell survival signaling in the kidney is so high that it protects renal tubular cells not only from apoptosis but also from necrosis, the form of cell death in response to the most severe death challenges.
The signaling transduction events that lead to the protection of renal tubular epithelial cells from cell death in vivo are not entirely clear. Using cultured renal tubular cells as a model system, we previously identified a dual mechanism leading to HGF protection against apoptosis (32). HGF phosphorylates and activates Akt kinase in a phosphoinositide 3-kinase–dependent manner, which in turn phosphorylates the proapoptotic Bad protein and leads to its cytosolic sequestration and inactivation (32). Meanwhile, HGF also induces prosurvival Bcl-xL protein expression in a delayed response fashion (32,33). Not surprisingly, we found that this dual mechanism may also well explain for HGF protection against cell death in vivo. Systemic administration of HGF plasmid vector induces activation of Akt kinase and preserves prosurvival Bcl-xL protein expression in the kidneys after folic acid injection (Figures 6 and 7). Expression of Bcl-xL protein is specifically localized in tubular epithelia, where the injuries take place (Figure 7). It is worthwhile to note that the activation of Akt kinase in vivo may be underestimated at day 1 after injury in this study, given the fact that HGF-induced Akt activation is an extremely rapid event that starts as early as 5 min in vitro (32). Of interest, despite similar levels of Akt activation and Bcl-xL induction after either preadministration or simultaneous administration of HGF gene, dramatic difference in renal protection is found between these two groups. Because preadministration presumably results in early activation of survival signaling in the kidneys, this observation likely suggests that not only the level but also that timing of survival signaling activation play an important role in determining cell death/survival. Taken together, the concomitant activation of multiple survival signaling, such as Akt and Bcl-xL, likely provides a molecular clue for understanding the remarkable ability of HGF to protect cells from death in vivo.
This study may have important implications in designing therapy for clinical acute renal failure. Although the role of growth factors in ameliorating acute renal failure has been increasingly recognized (4–6), studies elsewhere have emphasized their roles in promoting cell proliferation and accelerating tubular regeneration and recovery after injury. Little is known about their roles in the protection of tubules from cell death and the prevention of acute renal injury in the first place. The results presented in this study strongly suggest that the beneficial role of HGF may largely be mediated through protection of cells from initial injury and cell death. Thus, usage of growth factors at 1 or 2 d after injurious insult may exhibit much fewer therapeutic benefits. Consistent with this view, a recent clinical trial of insulin-like growth factor-I in established acute renal failure failed to demonstrate any therapeutic efficacy (40). Hence, early administration of growth factors such as HGF before or immediately after the insult may result in a better therapeutic outcome for acute renal failure in patients. Undoubtedly, the efficacy of HGF gene therapy via naked plasmid vector, as shown in this study, will provide a novel avenue for exploring a therapeutic strategy for clinical acute renal failure.
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, pcDNA3 group; 
, preadministration of pCMV-HGF group;
, purified rhHGF protein group. Data are presented as mean ± SEM from six animals per group at each time point. (B) Western blot demonstrates human HGF protein abundance in the kidneys at different time points after injection of plasmid vector or HGF protein, respectively. Whole-kidney extracts (made from the pool of kidneys from six animals per group) were separated on a sodium dodecyl sulfate–polyacrylamide gel and immunoblotted with a specific monoclonal antibody against human HGF. Purified recombinant human HGF (purified HGF) (5 ng) was also loaded in the adjacent lane, to confirm the correct size of hybridized signal and to estimate the amounts of HGF protein in each sample. The same blot was stripped and reprobed with actin to confirm equal loading. The asterisk (*) denotes the group that was preadministered with pCMV-HGF plasmid 24 h prior to folic acid injection. (C and D) Immunofluorescence staining shows the localization of human HGF protein in the kidneys after the injection of plasmid vector. Positive staining for human HGF protein was largely limited to renal glomeruli at day 1 after intravenous injection of pCMV-HGF plasmid (D). No HGF staining was observed at day 1 in the kidneys that received pcDNA3 plasmid (C). Scale bar, 40 µm.
, pcDNA3.
