Department of Pathology, Nippon Medical School, Tokyo, Japan
Correspondence to Dr. Akira Shimizu, Department of Pathology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Phone: +81-3-3822-2131 (ext. 5261); Fax: +81-3-5685-3067; E-mail: ashimizu{at}nms.ac.jp
Vascular endothelial growth factor (VEGF) is essential formaintenance of the glomerular capillary network. The presentstudy investigated the effects of VEGF in rats with progressivecrescentic glomerulonephritis (GN). Necrotizing and crescenticGN was induced in rats by injection of anti-rat glomerular basementmembrane (GBM) antibody. The alterations of glomerular capillariesand glomerular VEGF expression were assessed. In addition, theeffects of continuous VEGF165 administration (10 µg/100g per d) on glomerular capillaries, glomerular inflammation,and the course of crescentic GN were examined. The appropriatetiming of VEGF administration in progressive GN also was evaluated.In anti-GBM GN, necrotizing and crescentic glomerular lesionsoccurred by day 7, and newly formed necrotizing lesions reoccurredby week 3. Expression of VEGF was markedly reduced in necrotizingand crescentic lesions. Capillary repair was impaired aftercapillary destruction in necrotizing and crescentic glomeruli,which rapidly progressed to sclerotic glomeruli with chronicrenal failure. In contrast, in the rats that received VEGF165administration from day 7, the necrotizing and crescentic lesionsrecovered and renal function significantly improved in week4. This was evident by proliferating endothelial cells and glomerularcapillary repair. In addition, VEGF administration decreasedintercellular adhesion molecule-1 and monocyte chemoattractantprotein-1 expression in glomeruli (particularly on endothelialcells), reduced glomerular infiltrating CD8-postive and ED-1–positivecells, and inhibited the newly formed necrotizing lesions. VEGFadministration was apparently effective against both the inflammatoryand necrotizing glomerular lesions. These results suggest thatVEGF administration resolves glomerular inflammation and acceleratesglomerular recovery in the progressive necrotizing and crescenticGN. The therapeutic application of VEGF may be clinically usefulfor severe GN accompanied by extensive glomerular inflammationand endothelial injury.
Glomerular capillary and endothelial injury plays an importantrole in the pathogenesis of renal diseases and is viewed asa crucial factor in disease progression (1–4). Recentstudies of experimental glomerulonephritis (GN) have indicatedthat impairment of the capillary repair process in glomerulardamage may be associated with the continuation of mesangialproliferation and the accumulation of mesangial matrix, whichresults in the development of glomerular sclerosis and renaldysfunction (5–7). However, in recovery models of GN,complete capillary repair in damaged glomeruli can lead to fullrecovery of the glomerular architecture with resolution of themesangial proliferation (8–10). These findings demonstratethat capillary repair is a crucial event to allow for glomerularhealing as well as recovery from mesangial proliferative GNaccompanied by destruction of the glomerular capillary network.One of the most severe types of GN accompanied by destructionof the glomerular capillary network is diffuse necrotizing andcrescentic GN. Progression to crescentic GN is determined bythe severity of the injury to the glomerular capillary walls(11,12). Our recent study demonstrates that the progressionof glomerular sclerosis with renal dysfunction in necrotizingand crescentic GN is associated with destruction of the capillarynetwork in the necrotizing lesions and subsequently the impairmentof the glomerular capillary repair process (5). In addition,capillary regression caused by endothelial cell apoptosis contributesto the development of glomerular sclerosis. These results suggestthat the development of necrotizing and crescentic GN and renaldysfunction may be prevented by the therapy for stimulationof angiogenesis.
Several studies have indicated that the most important angiogenicfactor in glomeruli is vascular endothelial growth factor (VEGF)(13–15). VEGF has been known as a proliferative and survivalfactor for endothelial cells. It plays a highly dynamic rolein the regulation of angiogenesis through specific receptorson endothelial cells (16,17). The systemic administration ofVEGF can mediate glomerular endothelial cell proliferation inseveral experimental models of renal diseases, including thromboticmicroangiopathy, renal ablation, and mesangial proliferativeGN (6,18–20). In the present study, the diffuse necrotizingand crescentic GN was induced in Wistar-Kyoto (WKY) rats byinjection of anti-rat glomerular basement membrane (GBM) antibody.We determined the serial changes in VEGF production in the glomeruliand the alterations of glomerular capillaries. In addition,we examined the potential beneficial effects of systemic VEGF165administration in the development of glomerular inflammationand in the progression of crescentic GN.
Anti-GBM GN Model in WKY Rats
The Ethics Review Committee for Animal Experimentation of NipponMedical School approved the animal experiments described inthe present study. Inbred male WKY rats (Charles River Japan,Kanagawa, Japan) that weighed 100 g were used for all experiments.Anti-GBM GN was induced by injection of rabbit anti-rat GBMantibody at a dose of 50 µg IgG/100 g body wt on day 0(5,21). In the present study, we performed three experiments.In experiment 1, for assessing the alterations of VEGF expressionand the glomerular capillary network in the development of anti-GBMGN, five rats were killed on day 7 and 2, 3, 4, 6, and 8 wkafter the administration of anti-GBM antibody. In experiment2, for investigating the beneficial effects of exogenous VEGF,rats were treated with recombinant human VEGF165 (Life TechnologiesBRL, Tokyo, Japan) dissolved in saline (VEGF-treated group)or saline alone (control group) injections. Rats were administeredVEGF165 at a dose of 10 µg/100 g body wt per d or salineusing an intraperitoneal micro-osmotic pump (Alzet osmotic pump;Alza, Mountain View, CA) starting at day 7 and ending at day28 (3 wk). In each group, five rats underwent biopsy or werekilled on day 7 (before VEGF administration) and 2, 3, 4, 6,and 8 wk after the disease induction. In experiment 3, for examiningthe appropriate timing of VEGF administration in anti-GBM GN,rats in the various phases of GN were administered VEGF165 (10µg/100 g body wt per d) or vehicle using the same method.VEGF165 was initiated on day 2 (glomerular inflammation phase),day 7 (necrotizing glomerular phase), week 2 (proliferativeglomerular phase), and week 4 (sclerotic glomerular phase) andcontinued for 1 wk duration, respectively. In each group, fiverats underwent biopsy or were killed on, before, and 1 wk aftertreatment with VEGF165. For estimating renal function, urineand blood samples were collected for measurement of urinaryprotein, plasma creatinine, and blood urea nitrogen using anautoanalyzer (SRL, Tokyo, Japan).
Histopathologic and Immunohistochemical Examination
After removal of the kidney, renal tissues were fixed in 20%buffered formalin and embedded in paraffin for light microscopicexamination. Tissues were stained with hematoxylin and eosin,periodic acid-Schiff, and periodic acid-methenamine Silver forhistopathologic examination.
The following primary antibodies were used for immunohistochemistry.(1) Polyclonal rabbit anti-rat thrombomodulin (TM) antibody(provided by Dr. David Stern, Columbia University, New York,NY), which has been used as a marker for endothelial cells (5,6,9,10).Biotinylated anti-rat TM antibody was prepared using a biotinlabeling kit (Boehringer Mannheim, Mannheim, Germany) (5). (2)Monoclonal mouse anti-rat endothelial cell antigen-1 (RECA-1)antibody (Serotec, Oxford, UK), which has also been used asa marker for endothelial cells. (3) Monoclonal mouse anti-proliferatingcell nuclear antigen (PCNA) antibody (PC10; DAKO, Glostrup,Denmark), which is a marker for cellular proliferation. (4)Polyclonal rabbit anti-VEGF antibody (6), which can detect VEGF-producingcells. Biotinylated anti-VEGF antibody was prepared using abiotin labeling kit (Boehringer Mannheim). (5) Monoclonal mouseanti–flk-1 antibody (A-3; Santa Cruz Biotechnology, SantaCruz, CA), which can detect cells that express VEGF receptor-2(VEGFR-2). (6) Monoclonal mouse anti-rat CD8 antibody (Nichirei,Tokyo, Japan). (7) Monoclonal mouse anti-rat ED-1 antibody (BMA,Nagoya, Japan), which can detect infiltrating macrophages. (8)Monoclonal mouse anti-intercellular adhesion molecule-1 (anti-ICAM-1)antibody (G-5; Santa Cruz Biotechnology). (9) Polyclonal goatanti-monocyte chemoattractant protein-1 (anti–MCP-1) antibody(R-17; Santa Cruz Biotechnology). (9) Polyclonal goat anti-typeIV collagen antibody (Southern Biotechnology Associates, Birmingham,AL), which is used for evaluation of mesangial matrix accumulationand glomerular sclerosis. For immunohistochemistry for TM, PCNA,CD8, ED-1, and type IV collagen, 20%-buffered, formalin-fixed,paraffin-embedded tissue sections were used and the specimenswere stained by the standard avidin-biotin-peroxidase complextechnique. For TM, VEGF, flk-1, ED-1, and type IV collagen,tissue sections were incubated with 0.1% pepsin for 60 min,0.4% pepsin for 20 min, 0.1% proteinase for 5 min, 0.1% pepsinfor 45 min, 0.1% pepsin for 30 min, and 0.1% proteinase for5 min, respectively, before incubation with the primary antibody.For optimizing the detection of PCNA and CD8, sections weremicrowaved for 10 min in 0.01 M sodium citrate (pH 6.0) andin 4% urea, respectively, after dewaxing. Proliferating endothelialcells were identified after double immunohistochemistry stainingwith PCNA and TM using the color modification method of 3,3'-diaminobendizine(DAB) precipitation by nickel chloride, which changes DAB colorfrom brown to black (5,6,10). Sections were incubated with PCNAfollowed by a peroxidase-conjugated goat anti-mouse IgG andH2O2, nickel chloride–containing DAB. Sections were thenincubated with biotinylated anti-rat TM antibody and an avidin-biotinperoxidase complex followed by H2O2 containing DAB. For detectingICAM-1 and MCP-1 expression in glomeruli, 4-µm frozensections were stained by the standard indirect technique andwere observed with a fluorescence microscope. For detectingICAM-1 and MCP-1 expression on endothelial cells, double immunohistochemistrystaining with RECA-1 and ICAM-1 or MCP-1 was performed. Four-micrometerfrozen sections were stained with anti–RECA-1 antibody(mouse IgG1) and followed by FITC-labeled goat anti-mouse IgG1antibody (ZYMED, San Francisco, CA). Sections were then incubatedwith anti–ICAM-1 (mouse IgG2a) or anti–MCP-1 (goatIgG) and followed by Texas-red conjugated goat anti-mouse IgG2aor rabbit anti-goat IgG antibodies (Biomeda, Foster City, CA).Specimens were examined under a confocal laser scanning microscope(CLSM, TCS-SP; Leica Lasertechnik, Heidelberg, Germany) basedon an upright microscope (DMRB; Leica Lasertechnik) equippedwith a krypton/argon laser. For all biopsies, negative controlswere used in which the primary antibody was substituted withequivalent concentrations of an irrelevant antibody or normalrabbit IgG (DAKO). All control sections were negative.
For electron microscopic examination, the kidney tissue wasfixed in 2.5% glutaraldehyde solution in phosphate buffer (pH7.4) and postfixed with 1% osmium tetroxide, dehydrated, andembedded in Epok 812. Ultrathin sections were stained with uranylacetate and lead citrate and then examined with an electronmicroscope (model H7100, Hitachi Corp., Tokyo, Japan).
Isolation of Glomeruli and Western Blot Analysis for VEGF165, ICAM-1, and MCP-1
For examining the production of VEGF, ICAM-1, and MCP-1 in glomerulibefore and after disease induction, Western blotting was performedusing polyclonal rabbit anti-VEGF antibody (147; Santa CruzBiotechnology), monoclonal mouse anti–ICAM-1 antibody(G-5; Santa Cruz Biotechnology), or monoclonal mouse anti–MCP-1antibody (MB10; IBL, Gunma, Japan), respectively. For this purpose,glomeruli were isolated using a standard three-stage sievingmethod (6). Isolated glomeruli were homogenized in lysis buffer.After centrifugation at 15,000 x g for 30 min at 4°C, thesupernatant was collected and used for analysis. Samples thatcontained 10 µg of protein per lane were separated on10% acrylamide gel by SDS-PAGE. After electrophoresis, the separatedprotein was transferred to a Hybond-P nitrocellulose membrane(Amersham Life Science, Buckinghamshire, UK) and incubated withanti-VEGF antibody (1:2000), anti–ICAM-1 antibody (1:1000),or anti–MCP-1 antibody (1:1500). Bound antibody was detectedwith peroxidase-conjugated anti-rabbit IgG antibody (1:1000)or peroxidase-conjugated anti-mouse IgG antibody (1:5000), respectively,with the enhanced chemiluminescence detection system (ECL Westernblotting detection regents; Amersham). Membranes were washedand then exposed to film. For confirming equal loading of eachprotein, the membrane was stripped and reblotted with anti–-actinantibody (Sigma, St. Louis, MO). Densitometric analysis of thebands was performed with NIH Image software.
Quantification of Histopathologic Findings
In each kidney sample, >30 cross-sections of glomeruli wereexamined sequentially for the following parameters: (1) Glomerularendothelial cells: The mean number of nuclei of TM-positivecells per glomerular cross-section; (2) proliferating endothelialcells: The mean number of both PCNA- and TM-positive cells perglomerular cross-section; (3) glomerular capillaries: The meannumber of glomerular capillary lumina surround by TM-positivecells per glomerular cross-section; (4) infiltrating CD8+ cells:The mean number of CD8-positive cells per glomerular cross-section;(5) infiltrating macrophages: The mean number of ED-1–positivecells per glomerular cross-section; (6) necrotizing glomeruli:The mean percentage of glomeruli with necrotic lesions in periodicacid-methenamine Silver–stained sections; (7) glomerularsclerosis: The mean semiquantitative staining score of typeIV collagen per glomerular cross-section (6) (scores 0 to 4:score 0, no localized increase of staining; score 1, up to 25%of the glomerular tuft showing focally increased staining; score2, 25 to 50% of the glomerular tuft demonstrating a focallystrong staining; score 3, 50 to 75% of the glomerular tuft stainedstrongly in a focal manner; score 4, >75% of the glomerulartuft stained strongly). Glomerular cross-sections that containedonly a small portion of the glomerular tuft were excluded fromthe analysis. All histopathologic evaluations were performedby investigators who were blinded to the treatment modality(saline versus VEGF). These results were expressed as the mean± SD, and statistical analysis was performed using thet test.
Glomerular VEGF and Impaired Capillary Repair in Anti-GBM GN
A severe necrotizing and crescentic GN was produced in WKY ratsby a single injection of anti-rat GBM antibody on day 0. Manyleukocytes infiltrated the glomeruli, and, subsequently, severenecrotizing lesions occurred with cellular crescents by day7 (Figure 1A). Newly formed necrotizing glomerular lesions occurredrepeatedly by week 3 (Figure 1B), and these injured areas progressedto global sclerosis of the glomeruli between week 4 and week8 (Figure 1, C and D). In necrotizing lesions, TM-positive endothelialcells disappeared concordant with destruction of the glomerularcapillary network (Figure 1E).
Figure 1. Necrotizing and crescentic glomeruli progress to global sclerotic glomeruli without capillary regeneration in anti–glomerular basement membrane (anti-GBM) glomerulonephritis (GN) on day 7 (A and E), week 2 (B and F), week 4 (C and G), and week 8 (D and H). (A through D) Severe necrotizing glomeruli with cellular crescents are noted on day 7 and gradually progress to global sclerotic glomeruli with fibrous crescent by week 8. Newly formed necrotizing glomerular lesions, which are characterized by fibrinoid necrotizing lesions, are detected on day 7 and week 2. (E through H) During the progression of necrotizing and crescent lesion to global sclerosis, thrombomodulin (TM)-positive endothelial cells and TM-positive glomerular capillaries gradually reduce in damaged glomeruli. Magnification, x600 (periodic acid-methenamine Silver [PAM] stain in A through D; TM stain in E through H).
The podocytes expressed VEGF in normal glomeruli before diseaseinduction (data not shown). However, the expression of VEGFin podocytes was markedly decreased or lost around necrotizinglesions or within cellular crescents (Figure 2A). In addition,the expression of VEGF was not upregulated in segmental necrotizingand crescentic lesions, although VEGF expression was detectedin podocytes or cells in mesangial areas (probably activatedmesangial cells and infiltrating macrophages) in remaining histologicwell-preserved glomerular segments (Figure 2A). In accordancewith the expansion of the injured glomerular areas by week 8,remaining podocytes and cells in mesangial areas with VEGF expressiongradually reduced (Figure 2B). In parallel with the decreasein the relative area of VEGF expression in glomeruli, the proteinlevel of VEGF165 gradually decreased in isolated glomeruli byweek 8 (Figure 3).
Figure 2. Vascular endothelial growth factor (VEGF) expression (A and B), VEGF receptor-2 (VEGFR-2; flk-1) expression (C), and rare endothelial cell proliferation (D) in anti-GBM GN in week 2 (A, C, and D) and in week 4 (B). (A) In damaged glomeruli in week 2, remaining podocytes and cells in mesangial areas express VEGF. However, the expression of VEGF is diminished in segmental necrotizing (*) and crescentic lesions. (B) In week 4, injured areas progress to global sclerosis, and the expression of VEGF is markedly reduced in damaged glomeruli. (C) In damaged glomeruli, VEGFR-2 (flk-1) is expressed on endothelial cells in remaining glomerular capillaries. However, positive staining for VEGFR-2 is lost in the necrotizing and proliferative lesions in accordance with capillary destruction. (D) Both TM- and proliferating cell nuclear antigen (PCNA)-positive proliferating endothelial cells () are noted in the mildly damaged lesions. However, no proliferating endothelial cells are present in severely damaged necrotizing lesions (*). Magnification, x600 (VEGF stain in A and B; flk-1 stain in C; double stain with PCNA [black] and TM [brown] in D).
Figure 3. Decrease of glomerular VEGF protein during the progression of crescentic GN. (A) Western blot analysis shows that bands of molecular weight corresponding to the VEGF165 are detected in the lane of recombinant human VEGF165 (lane rh) and in the lane of protein lysate extracted from isolated glomeruli on day 0 (lane 0), day 7 (lane 1w), week 2 (lane 2w), week 3 (lane 3w), week 4 (lane 4w), week 6 (lane 6w), and week 8 (lane 8w) after disease induction. -Actin protein is detected almost equally in each lane. (B) Densitometric analysis of A shows that VEGF165 protein levels of isolated glomeruli gradually decreased during the progression of crescentic GN by week 8. Each bar represents the levels of VEGF165 protein normalized to levels of -actin protein and is shown as a percentage of the day 0 (lane 0) value.
VEGFR-2 (flk-1) was expressed on glomerular endothelial cellsin the remaining capillaries in damaged glomeruli (Figure 2C).However, in accordance with the decreased expression of glomerularVEGF, rare proliferating endothelial cells were present alongwith impaired capillary regeneration in necrotizing lesions(Figure 2D). In damaged glomeruli, capillary repair was rarelyseen between week 2 and week 4, and the TM-positive capillarylumina gradually reduced during the development of glomerularsclerosis by week 8 (Figure 1, E through H).
VEGF165 Accelerates Glomerular Capillary Repair in Anti-GBM GN
We next examined the effects of VEGF165-induced angiogenesison the course of anti-GBM GN. Systemic administration of VEGF165significantly enhanced endothelial cell proliferation and glomerularcapillary repair by week 4. Numerous proliferating endothelialcells (both PCNA- and TM-positive cells) were found within necrotizinglesions (Figure 4, A and B), and regenerating capillary networksdeveloped near the crescentic or the adhesive lesions (Figure 4, C and D).The regenerating capillaries had morphologicallyactivated endothelial cells (Figure 4, E and F). The numberof proliferating endothelial cells was increased significantlyby week 2 (Figure 5A), which was followed by a rapid recoveryin the number of total TM-positive glomerular endothelial cellsby week 4 (Figure 5B). In parallel with capillary regeneration,the number of glomerular capillary lumina per glomerular cross-sectionalso increased (Figure 5C). Even in rats that were treated withVEGF165 continuously by week 4, VEGF-mediated endothelial cellproliferation could be inhibited before occurrence of endothelialcell overproliferation (Figure 5). VEGF165, which was administeredat a dose of 10 µg/100 g body wt per d, did not mediateendothelial cell proliferation in normal organs, including liver,lungs, heart, and the digestive tract (data not shown).
Figure 4. Glomerular capillary regeneration and proliferating endothelial cells in the VEGF-treated group. Regenerating capillaries with both PCNA- and TM-positive cells are detected within the proliferative lesions in week 2 (A) and near the adhesive lesions in week 4 (B; double stain with PCNA [black] and TM [brown]). Glomerular capillary network develops near the cellular crescent in week 2 (C) and near the adhesive lesions in week 4 (D). (E) The developing capillary (*) is seen in the damaged area in week 2. (F) Activated endothelial cells (*), characterized by swelling of nuclei and loss of fenestration, are noted in regenerating capillaries in week 2. Magnifications: x600 in A and B; x800 in C and D (PAM stain); x1000 in E; x3000 in F.
Figure 5. The number of both PCNA- and TM-positive cells (A), the number of TM-positive endothelial cells (B), and the number of TM-positive capillary lumina (C) per glomerular cross-section in rats that were treated with VEGF (•) or vehicle (). Values are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. During the treatment with VEGF between week 2 (1 wk after the starting of VEGF) and week 4, the number of both PCNA- and TM-positive proliferating cells, TM-positive endothelial cells, and TM-positive capillary lumina per glomerular cross-sections increase with the regeneration and reconstruction of the glomerular capillary network.
It is interesting that the cellular crescents, which developedaround necrotizing lesions by day 7 (Figure 6A), were transientin nature. Recovery of necrotizing lesions correlated with thegradual subsidence of extraglomerular cellular crescents betweenweek 2 and week 4 (Figure 6, B through D). Although small adhesivelesions were evident in damaged glomeruli, even in the VEGF-treatedgroup, the capillary structure developed around and/or withinthe adhesive lesions (Figures 4D and 6D).
Figure 6. VEGF administration prevents glomerular inflammation and accelerates glomerular repair. (A) In week 1 (before VEGF administration), the necrotizing lesions with cellular crescents occur with glomerular inflammation. (B) In week 2 (1 wk after the starting of VEGF), glomerular inflammation resolves and small cellular crescents remain. In week 3 (C) and week 4 (D), necrotizing lesions recover with small adhesive lesions. Newly formed fibrinoid necrotizing glomerular lesions are markedly reduced during VEGF treatment. Magnification, x200 (PAM stain).
VEGF165 Resolves Glomerular Inflammation in Anti-GBM GN
We examined the effect of systemic administration of VEGF165on glomerular inflammation leading to necrotizing and crescenticglomerular injury, because newly formed fibrinoid necrotizinglesions were markedly reduced after VEGF administration (Figure 6, B through D).This model is characterized by the accumulationand activation of CD8-positive lymphocytes and ED-1–positivemacrophages in glomeruli, through MCP-1 and ICAM-1 pathways,which are crucial for the initiation and subsequent progressionof anti-GBM GN (22–25). ICAM-1 and MCP-1 were expressedat detectable levels on day 7 and significantly in week 2 indamaged glomeruli (Figure 7, A and C), especially on glomerularendothelial cells, glomerular epithelial cells, and cells increscent in the vehicle-infused control group (Figure 7, E and G).In contrast, VEGF165 administration strongly suppressedthe upregulation of ICAM-1 and MCP-1 in damaged glomeruli (Figure 7, B and D),particularly on glomerular endothelial cells (Figure 7, F and H).Western blotting analysis also showed that proteinlevels of ICAM-1 and MCP-1 in isolated glomeruli increased noticeablyin week 1 and markedly in week 2 after disease induction inthe control group (Figure 8). VEGF administration strongly suppressedthe upregulation of ICAM-1 and MCP-1 protein levels in week2. Many CD8-positive cells and ED-1–positive macrophagesinfiltrated the glomeruli on day 7. In the vehicle-infused controlgroup, infiltration of these cells continued together with theoccurrence of newly formed necrotizing lesions by week 3 (Figures 9, A and C, and 10). In contrast, the glomerular CD8-positivecells and ED-1–positive cells were rapidly reduced afteradministration of VEGF (Figure 10, A and B), and a few of thesecells were seen in damaged glomeruli in the VEGF-treated group(Figure 9, B and D). After the resolution of the cell infiltration,newly formed necrotizing lesions in glomeruli were markedlydiminished during VEGF treatment (Figure 10C). In parallel withthe resolution of the glomerular inflammation and the recoveryfrom necrotizing and crescentic GN, VEGF-treated rats showedsignificant improvement in the degree of glomerular sclerosis,renal function, and proteinuria in week 4 compared with thevehicle-treated control rats (Figure 11).
Figure 7. Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) expression in glomeruli and ICAM-1 and MCP-1 expression on glomerular endothelial cells (double stain with rat endothelial cell antigen-1 (RECA-1) [green] and ICAM-1 [red] or MCP-1 [red]). In the vehicle-infused control group (A, C, E, and G) in week 2, ICAM-1 (A and E) and MCP-1 (C and G) are expressed strongly in glomeruli, especially on endothelial cells, epithelial cells, and cells in crescent (E and G). However, in the VEGF-treated group (B, D, F, and H) in week 2, the expressions of ICAM-1 (B) and MCP-1 (D) are weak. Importantly, double immunostaining of RECA-1 and ICAM-1 (F) or MCP-1 (H) show that VEGF administration suppresses strongly the upregulation of ICAM-1 and MCP-1 on glomerular endothelial cells. Magnification, x400 (ICAM-1 stain in A and B; MCP-1 stain in C and D).
Figure 8. Western blotting of ICAM-1 and MCP-1 protein in isolated glomeruli in vehicle- or VEGF-treated rats. -Actin protein is detected almost equally in each lane. ICAM-1 and MCP-1 protein are noted weakly in the lane of protein lysate extracted from isolated glomeruli on day 0 (lane 0). Protein levels of ICAM-1 and MCP-1 in glomeruli increase in week 1 (lane 1w) and week 2 (lane VEGF [–] 2w) after disease induction. However, VEGF administration suppresses strongly the upregulation of ICAM-1 and MCP-1 protein levels in week 2 (lane VEGF [+] 2w). (B) Densitometric analysis of A. Each bar represents the levels of ICAM-1 and MCP-1 protein normalized to levels of -actin protein and is shown as a percentage of the VEGF (–) 2w value.
Figure 9. Glomerular infiltrating CD8-positive cells (A and B) and ED-1–positive macrophages (C and D) in the vehicle-treated (A and C) or VEGF-treated (B and D) groups in week 2. Many CD8-positive cells and ED-1–positive macrophages are found in glomeruli in vehicle-treated rats. However, the glomerular CD8-positive cells and ED-1–positive cells are reduced rapidly after VEGF administration, and a few of these cells are seen in glomeruli in the VEGF-treated group. Magnification, x600 (CD8 stain in A and B; ED-1 stain in C and D).
Figure 10. The number of infiltrating CD8-positive cells (A) and ED-1–positive macrophages (B) in glomeruli and the percentage of necrotizing glomeruli (C) in VEGF-treated (•) or vehicle-treated () groups. Values are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. Glomerular CD8-positive cells, glomerular ED-1–positive cells, and necrotizing glomeruli are diminished rapidly after administration of VEGF, and lower levels continue by week 4.
Figure 11. Glomerular sclerosis (A through C), proteinuria (D), blood urea nitrogen (E), and serum creatinine (F) levels in VEGF- or vehicle-treated groups by week 4. In week 4, glomerular sclerosis with type IV collagen deposition develops in vehicle-infused control groups (A), but minimal sclerosis is detected in adhesion area in the VEGF-treated group (B). (C through F) Values are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. VEGF administration substantially inhibits glomerular sclerosis, reduces proteinuria, and prevents the loss of renal function. Magnification, x200 in A and B (type IV collagen stain).
After Stopping of VEGF165 Treatment in Anti-GBM GN
After stopping treatment with VEGF, marked cellular infiltrationrecurred with the formation of severe necrotizing lesions andcrescent by week 6 (Figure 12, A through D). Necrotizing andcrescentic glomeruli progressed to sclerotic glomeruli withproteinuria and renal dysfunction by week 8 (Figure 12, E through H).However, the progression of glomerular sclerosis and renaldysfunction in the VEGF-treated group were delayed by VEGF165treatment for 3 wk, and the degree of glomerular sclerosis,proteinuria, and renal function was better in the VEGF-treatedgroup than in the vehicle-infused control group in week 8.
Figure 12. Necrotizing and crescentic glomerular lesion in week 6 (A); the number of infiltrating CD8-positive cells (B) and ED-1–positive macrophages (C) in glomeruli; the percentage of necrotizing glomeruli (D); the degree of glomerular sclerosis (E); and proteinuria (F), blood urea nitrogen (G), and serum creatinine (H) levels after stopping of VEGF treatment. After stopping of VEGF165 treatment, necrotizing and crescentic glomerular lesions recur with marked cellular infiltration by week 6. Although there are less progression of glomerular sclerosis and renal dysfunction in the VEGF-treated group than the in control group in week 8, glomerular sclerosis, proteinuria, and renal dysfunction rapidly develop after stopping of VEGF165 administration.
Timing of VEGF165 Treatment in Anti-GBM GN
We examined the appropriate timing of VEGF treatment in progressiveGN (Figure 13). In rats with glomerular inflammation, administrationof VEGF from day 2 (glomerular inflammation phase) preventednecrotizing and crescentic glomerular lesions, proteinuria,and renal dysfunction by day 9. When VEGF was started on day7 (necrotizing glomerular phase), both proteinuria and renaldysfunction were almost completely recovered by week 2. Ratsthat received VEGF from week 2 (proliferative glomerular phase)showed partial but significant recovery of proteinuria and renaldysfunction by week 3. In the rats that were treated with VEGFfrom week 4 (sclerotic glomerular phase), administered VEGFcould not enhance the angiogenic capillary repair in scleroticglomerular lesions, but VEGF tended to inhibit the progressionof renal dysfunction and proteinuria by week 5.
Figure 13. Various timing of VEGF (•) or vehicle () treatment and the morphologic alterations (A through D) or changes of renal functions (E and F). Systemic VEGF administration was initiated on day 2 (graph: 2d to 9d), day 7 (graph: 1w to 2w), week 2 (graph: 2w to 3w), and week 4 (graph: 4w to 5w) and continued for 1 wk. Values are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. In the rats that were treated with VEGF from the early phase of GN, necrotizing and crescentic lesions are prevented (A, control on day 9; B, VEGF-treated group on day 9), and urinary protein and blood urea nitrogen levels seem to improve 1 wk after VEGF treatment. In the rats that were treated with VEGF from the late phase of GN (after development of glomerular sclerosis), VEGF does not mediate enough angiogenic capillary repair in sclerotic lesions (C, control in week 5; D, VEGF-treated group in week 5). However, VEGF tends to inhibit the development of renal dysfunction and proteinuria. Magnification, x200 in A through D (PAM stain).
We have demonstrated in the present study that the progressionof necrotizing and crescentic GN is accompanied by a decreaseof VEGF expression in damaged glomeruli and impaired angiogeniccapillary repair after the destruction of the glomerular capillarynetwork. Systemic administration of VEGF165 successfully inducedglomerular healing, including glomerular capillary repair andresolution of extraglomerular crescent. In addition, VEGF165administration led to a decrease in ICAM-1 and MCP-1 expressionin glomeruli, reduced glomerular infiltrating CD8+ cells andmacrophages, and inhibited acute inflammatory necrotizing andcrescentic glomerular destruction. Importantly, VEGF significantlyameliorated renal damage when administration was initiated afterappearance of necrotizing and crescentic lesions. In addition,VEGF tended to prevent progression of renal dysfunction whenadministration was started after development of glomerular sclerosis.These findings suggest a possible effect on established humanrenal diseases, and the therapeutic application of VEGF maybe clinically useful for progressive GN accompanied by severeendothelial injury, capillary destruction, and glomerular inflammation.
In neonatal rodents, VEGF and its receptors play a criticalrole in glomerular capillary formation and endothelial celldifferentiation in developing glomeruli, and VEGF has been knownto be an essential molecule for kidney development, especiallyglomerulogenesis (13–15). In the recovery models of GN,glomerular capillary repair with endothelial cell proliferationis associated with upregulated expression of VEGF and its receptor,VEGFR-2 (8). In addition, a recent report using radiolabeledVEGF indicated that administered 125I-VEGF accumulates in thekidney even in normal rats and that the major VEGF binding sitesin kidney are in the glomeruli (26). In diseased glomeruli withupregulation of VEGFR-2, such as in the case of diabetic nephropathy,the binding of administered 125I-VEGF in glomeruli is increased(26). In support of this evidence, systemic administration ofVEGF121 or VEGF165 stimulates endothelial cell proliferationin several experimental models of renal diseases (6,18–20).Our previous studies demonstrated that glomerular capillaryrepair occurs through capillary regeneration from the remainingendothelial cells in the lesions, as well as from new capillarygrowth in the glomerular vascular pole (6,10). Recently, severalinvestigators have established the presence of bone marrow–derivedendothelial progenitor cells in the adult circulation and demonstratedthat these cells contribute to glomerular healing, includingglomerular capillary repair in experimental and human renaldiseases (27–29). VEGF augments circulating endothelialprogenitor cells and also induces engraftment of these cellsinto vasculature (30,31). In the present study, systemic administrationof VEGF165 can stimulate angiogenic capillary repair in damagedglomeruli and improves renal function in necrotizing and crescenticGN. Further studies are ongoing to identify whether bone marrow–derivedendothelial progenitor cells are essentially involved in capillaryrepair in this model and whether the number of these cells isincreased by VEGF administration.
Importantly, VEGF mediates not only recovery of intraglomerularnecrotizing lesions but also the resolution of extraglomerularcellular crescents. Glomerular crescents occupy the urinaryspace by covering and compressing the glomerular tuft, and crescentformation contributes directly to renal dysfunction throughthe reduction of glomerular filtration and urinary flow (11,12).The development of necrotizing lesions with capillary rupturemay be essential in the process of crescent formation (11,12).In the present study, cellular crescent resolved in parallelwith the recovery of necrotizing lesions after VEGF administration.Our results suggest that the acceleration of capillary repairby VEGF may reduce capillary wall injury and rupture leadingto crescent formation, through the recovery of intraglomerularnecrotizing lesions.
Infiltration by CD8-positive cells and macrophages in glomerulithrough chemokines and adhesion molecules is a crucial eventfor the initiation and subsequent progression of anti-GBM GN(22,23). A key role for ICAM-1 and MCP-1 in leukocyte infiltrationin this model has been indicated from studies using neutralizingantibodies against these chemokine and adhesion molecules (23–25).VEGF is known as a proinflammatory cytokine (17,32), can actas a chemoattractant for leukocytes, and can also stimulatethe expression of adhesion molecules including ICAM-1 and chemokinesincluding MCP-1 (32). However, several studies have shown thatsystemic administration of VEGF does not affect the leukocyteaccumulation in kidneys in several models of renal diseases(6,18–20). Recent in vivo studies have also demonstratedthat VEGF may induce arterial nitric oxide, prostacyclin, Bcl-2,and heme oxygenase-1 in endothelial cells and can act as a cytoprotectivefactor for endothelial cells and as a vascular protective factorin the adult vasculature and in disease (33–37). In thesestudies, VEGF can induce enhancement of endothelial functionsthat mediate the inhibition of vascular smooth muscle cell proliferation,prevention of endothelial cell apoptosis, prolongation of endothelialcell survival, suppression of coagulation system, and inhibitionof inflammation. In addition, a recent study demonstrated thatthe administration of a single bolus dose of VEGF attenuatesinflammation-induced leukocyte–endothelium interactionat both microvascular and macrovascular levels (38). In thepresent study, the expression of ICAM-1 and MCP-1 in glomeruli,particularly on the glomerular endothelial cells, which increasedin the anti-GBM GN, was strongly suppressed by VEGF165 administration.This resulted in the resolution of glomerular inflammation andreduced the leukocyte-mediated necrotizing glomerular injury.These findings suggest that systemic VEGF165 administrationcan induce the resolution of glomerular inflammation in anti-GBMGN through the stabilization of glomerular endothelial cells.It also strongly supports the idea that VEGF could be a cytoprotectivefactor for endothelial cells and vascular protective factorfor glomerular capillaries in anti-GBM GN.
During VEGF administration for 3 wk, we expected that a similarmechanism of accommodation in organ transplantation could developand the intensity of anti-GBM GN, after stopping treatment withVEGF, could be reduced. However, after stopping treatment withVEGF, marked necrotizing and crescentic GN recurred. Our findingssuggest that although the beneficial effect of VEGF cannot continuefor a long period in this model after stopping of VEGF165 treatment,the resolution of glomerular inflammation in anti-GBM GN isdependent on VEGF165 administration.
Podocytes express VEGF in normal glomeruli. In GN, both residentglomerular cells, including podocytes, endothelial cells, activatedmesangial cells, and infiltrating leukocytes, can release glomerularVEGF (6,8,18,19). Our results in the present study showed thatthe expression of VEGF on podocytes was markedly reduced innecrotizing and crescentic lesions. In addition, VEGF expressionwas not upregulated in both resident glomerular cells and infiltratingleukocytes during the progression of crescentic GN. A possibleexplanation for the loss of VEGF expression in damaged glomerulicould be through the loss of or severe injury to glomerularpodocytes and mesangial and endothelial cells by necrotizingglomerular destruction and crescent formation. In addition,a variety of cytokines and growth factors are involved in theinitiation and progression of anti-GBM GN (11,18,25), and severalof these, such as, IL-1, IL-6, and TNF-, downregulate VEGF production(18). In the present study, the mechanisms of this decreaseof VEGF production in glomeruli was not fully investigated,but it may seem that impaired capillary repair and capillaryregression in this model is paralleled by a progressive lossin glomerular VEGF expression. We therefore presume that impairedglomerular capillary regeneration and capillary regression leadingto the progression of anti-GBM GN and irreversible glomerularscarring may be associated with VEGF depletion.
Recently, the therapeutic effects of angiogenic growth factorshave been investigated clinically and in animal models. Theadministration of VEGF, basic fibroblast growth factor (FGF-2),hepatocyte growth factor (HGF), or vectors encoding these proteinsresults in the improvement of hemodynamics and increased capillarydensity in ischemic tissues (39,40). In renal disease, theseangiogenic growth factors are known to mediate endothelial cellproliferation in glomeruli (6,8,13–15,18–20,41,42).However, intravenous injection of FGF-2 results in glomerularpodocyte injury, promoting glomerular sclerosis (43). Althoughsystemic administration of HGF can stimulate endothelial cellproliferation and capillary repair in glomeruli (42), our preliminarystudy using anti-GBM GN showed no or minimal effects of administeredHGF on resolution of glomerular inflammation (A.S. and T.M.,personal communication). On the basis of this background, weexamined the beneficial effects of VEGF165 and conclude thatsystemic administration of VEGF165 resolves glomerular inflammationand accelerates glomerular repair in the progressive necrotizingand crescentic GN. Systemic administration of VEGF165 may betherapeutically effective in GN accompanied by extensive endothelialdamage and severe glomerular inflammation.
Acknowledgments
We express special thanks to Dr. David Stern (Columbia University,New York, NY) and Dr. Yukio Yuzawa (School of Medicine, NagoyaUniversity, Nagoya, Japan) for providing the anti-TM antibody;Dr. Yasuhiro Natori and Dr. Naoyuki Nakao (Research Institute,International Medical Center of Japan, Tokyo, Japan) for providingthe anti-GBM antibody; Dr. Clive Patience (Immerge Biotherapeutics,Cambridge, MA) and John M. Lavelle (Transplantation BiologyResearch Center, Massachusetts General Hospital, Boston, MA)for excellent advice and critical review of the manuscript.We are also grateful to Dr. Toshiyuki Ishiwata (Department ofPathology, Nippon Medical School) for advice and Mr. TakashiArai, Ms. Mitsue Kataoka, Ms. Arimi Ishikawa, and Ms. NaomiTamura for expert technical assistance.
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Received for publication January 19, 2004.
Accepted for publication July 14, 2004.
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Top
Abstract
Introduction
100 g were used for all experiments. Anti-GBM GN was induced by injection of rabbit anti-rat GBM antibody at a dose of 50 µg IgG/100 g body wt on day 0 (5,21). In the present study, we performed three experiments. In experiment 1, for assessing the alterations of VEGF expression and the glomerular capillary network in the development of anti-GBM GN, five rats were killed on day 7 and 2, 3, 4, 6, and 8 wk after the administration of anti-GBM antibody. In experiment 2, for investigating the beneficial effects of exogenous VEGF, rats were treated with recombinant human VEGF165 (Life Technologies BRL, Tokyo, Japan) dissolved in saline (VEGF-treated group) or saline alone (control group) injections. Rats were administered VEGF165 at a dose of 10 µg/100 g body wt per d or saline using an intraperitoneal micro-osmotic pump (Alzet osmotic pump; Alza, Mountain View, CA) starting at day 7 and ending at day 28 (3 wk). In each group, five rats underwent biopsy or were killed on day 7 (before VEGF administration) and 2, 3, 4, 6, and 8 wk after the disease induction. In experiment 3, for examining the appropriate timing of VEGF administration in anti-GBM GN, rats in the various phases of GN were administered VEGF165 (10 µg/100 g body wt per d) or vehicle using the same method. VEGF165 was initiated on day 2 (glomerular inflammation phase), day 7 (necrotizing glomerular phase), week 2 (proliferative glomerular phase), and week 4 (sclerotic glomerular phase) and continued for 1 wk duration, respectively. In each group, five rats underwent biopsy or were killed on, before, and 1 wk after treatment with VEGF165. For estimating renal function, urine and blood samples were collected for measurement of urinary protein, plasma creatinine, and blood urea nitrogen using an autoanalyzer (SRL, Tokyo, Japan). 
-actin antibody (Sigma, St. Louis, MO). Densitometric analysis of the bands was performed with NIH Image software. 
). 
, IL-6, and TNF-
) are noted in the mildly damaged lesions. However, no proliferating endothelial cells are present in severely damaged necrotizing lesions (*). Magnification, x600 (VEGF stain in A and B; flk-1 stain in C; double stain with PCNA [black] and TM [brown] in D).
). Values are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. During the treatment with VEGF between week 2 (1 wk after the starting of VEGF) and week 4, the number of both PCNA- and TM-positive proliferating cells, TM-positive endothelial cells, and TM-positive capillary lumina per glomerular cross-sections increase with the regeneration and reconstruction of the glomerular capillary network.
