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*
Division of Nephrology, University of Washington, Seattle,
Washington
Scios, Inc., Sunnyvale, California
Division of Nephrology, University of Erlangen, Erlangen,
Germany
Department of Clinical Pathology, University of Vienna, Vienna,
Austria
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Division of Nephrology, University of Florida, Gainesville,
Florida.
Correspondence to Dr. Duk-Hee Kang, Division of Nephrology, Baylor College of Medicine, One Baylor Plaza, Alkek N520, Houston, TX 77030. Phone: 713-798-5835; Fax: 713-798-5010; E-mail: dkang{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) inhibited VEGF mRNA expression and protein secretion
by cultured tubular epithelial cells of the medullary thick ascending limb,
under both normoxic and hypoxic conditions. Impaired angiogenesis
characterizes the remnant kidney model and is correlated with progression. The
impaired angiogenesis may be mediated by alterations in the renal expression
of TSP-1 and VEGF, with the latter being regulated by macrophage-associated
cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is increasing evidence, however, that the microvasculature may play a critically important role in progressive renal disease. Bohle et al. (1) observed a remarkable loss of peritubular capillaries in interstitial fibrosis in human disease, and a loss of peritubular capillaries was also observed in several experimental models of interstitial fibrosis (2,3). A loss of capillaries would result in impaired delivery of oxygen and nutrients to the tubules and interstitial cells, producing chronic ischemia. A particularly vulnerable area would be the outer medulla, which normally exists in a borderline hypoxic environment because of countercurrent circulation and the high metabolic demands of the tubular epithelial cells in the thick ascending limb (4). Hypoxia has been demonstrated to induce tubular cell and fibroblast proliferation, matrix synthesis, cytokine release, and upregulation of tubular cell Fas expression (5,6,7). Fibrotic scarring resulting from ischemia would exacerbate hypoxia by increasing the oxygen diffusion gradient; therefore, chronic hypoxia may have a crucial role in progressive renal disease.
Yamanaka and co-workers (8,9) also observed a loss of glomerular endothelial cells in two experimental models of progressive glomerulosclerosis. In both the remnant kidney (RK) model and anti-glomerular basement membrane (anti-GBM) disease, an ineffectual glomerular endothelial proliferative response was demonstrated in association with progressive endothelial cell loss. Those authors postulated that glomerular endothelial loss results in denudation of the GBM, with activation of the coagulation system, capillary collapse, and subsequent glomerulosclerosis (8,9).
The pathogenesis of the progressive capillary loss in chronic renal disease has not been determined. Although there are likely to be specific factors that mediate endothelial cell death, such as oxidants and angiotensin II (10,11), there may also be ineffectual repair (2,3,8,9). This contrasts with acute glomerular injury, in which complete capillary repair is common (12,13). In this study, we examined potential mechanisms for the inadequate repair of the endothelium in the RK model, to investigate the role of the renal microvasculature in progressive renal disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preoperative and postoperative 24-h urinary protein excretion rates were measured using the sulfosalicylic acid method, and blood urea nitrogen (BUN) levels were determined colorimetrically with a commercial kit (Sigma Diagnostics, St. Louis, MO). Systolic arterial BP was monitored with a tail cuff sphygmomanometer, using an automated system with a photoelectric sensor (IITC; Life Sciences, Woodland Hills, CA), which has been demonstrated to be closely correlated with intra-arterial measurement of BP (15).
Renal Morphologic and Immunohistochemical Analyses
Tissue for light microscopy and immunoperoxidase staining was fixed in
methyl Carnoy's solution and embedded in paraffin. Four-micrometer sections
were stained with the periodic acid-Schiff re-agent and counterstained with
hematoxylin. Indirect immunoperoxidase staining of 4-µm sections was
performed as described previously
(16), with the following
specific monoclonal and polyclonal antibodies: endothelial cells were detected
with the mouse monoclonal antibody JG-12, directed against a 70-kD cell
membrane antigen present on rat endothelial cells
(16), and the monoclonal
anti-endothelial cell antibody RECA-1 (a gift of Adrian Duijvestijn;
University of Limberg, The Netherlands)
(17); vascular endothelial
growth factor (VEGF) with a rabbit polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA); thrombospondin-1 (TSP-1) with the mouse
monoclonal antibody A6.1 (Neomarkers, Fremont, CA); and monocytes/macrophages
with the mouse monoclonal antibody ED-1 (Serotec, Indianapolis, IN). Control
experiments included omission of the primary antibody and substitution of the
primary antibody with preimmune rabbit or mouse serum.
To examine whether there was any evidence of endothelial cell proliferation, double-immunostaining was performed with an anti-endothelial cell antibody (JG-12, and IgG antibody) and an antibody to the proliferating cell nuclear antigen (PCNA) (19A2, an IgM monoclonal antibody; Coulter, Hialeah, FL). The anti-PCNA antibody and JG-12 were incubated simultaneously overnight at 4°C, followed sequentially by incubation with biotinylated rabbit anti-mouse IgM serum, incubation with peroxidase-conjugated avidin D (Vector, Burlingame, CA) color development with diaminobenzidine (DAB) and nickel chloride, and incubation in 3% H2O2 for 8 min, to eliminate any remaining peroxidase activity. Subsequently, sections were incubated with biotinylated horse anti-mouse IgG for 30 min at room temperature, followed by peroxidase-conjugated avidin D and DAB (16).
To examine the relationship between VEGF expression and macrophage infiltration, double-immunostaining with the anti-VEGF antibody (a rabbit polyclonal antibody) and an antibody to a cell-specific marker for macrophages (ED-1, a mouse monoclonal IgG1 antibody) was performed using an indirect immunoperoxidase technique. Samples were incubated with ED-1 overnight at 4°C, followed sequentially by biotinylated rabbit anti-mouse IgG1 (Zymed, San Francisco, CA) and peroxidase-conjugated avidin D, with color development using DAB and nickel chloride. After incubation in 3% H2O2 for 8 min, the anti-VEGF antibody was applied for 2 h at room temperature, followed by biotinylated rat anti-rabbit IgG antibody (Vector) for 30 min and peroxidase-conjugated avidin D and DAB without nickel chloride. Control experiments included omission of either the primary or secondary antibody.
Quantification of Morphologic Data
The number of glomerular capillary loops per glomerular cross-section, as
identified by positive JG-12 staining, was counted by a single observer in all
glomeruli of tissue sections, at x 400 magnification. Glomerular
capillary density was also measured and defined as the number of capillaries
per glomerular cross-sectional area (per 0.01 mm2); the latter was
determined by morphometric measurements using computer image analysis (Optimas
6.2; Media Cybernetics, Silver Springs, MD). Peritubular capillary densities
were quantified in two ways. The area of peritubular capillary staining by
JG-12 was expressed as the percent positive area per 100 cortical tubules
using computer image analysis, to account for changes in tubular size and to
exclude the effects of tubular dilation and/or atrophy on peritubular
capillary numbers. The other index, i.e., peritubular capillary
rarefaction index, was determined by counting the numbers of squares in 10
x 10 grids that did not contain JG-12-positive peritubular capillary
staining, in at least 20 nonoverlapping sequential fields, at x100
magnification. The minimal possible capillary rarefaction index is 0,
i.e., every square in the grid contains a JG-12-positive peritubular
capillary, whereas the maximal score is 100, i.e., JG-12-positive
peritubular capillaries are absent from every square in the grid
(18). Changes in capillary
density were confirmed by staining tissue sections with RECA-1, which is an
antibody to a different endothelial cell antigen. The mean numbers of
proliferating endothelial cells (JG-12-and PCNA-positive cells) in glomeruli
and peritubular areas in each biopsy were calculated in a blinded manner, as
the mean numbers of positive cells in individual glomeruli and
0.25-mm2 grids, respectively, at x200 magnification.
The percentage areas of the cortex and outer medulla that were positive for VEGF were measured by computer image analysis (Optimas 6.2) (16). In each biopsy, the negative background staining was calibrated to zero, and the area of positive staining above the background level in each field was measured. Each measurement was derived from computer analysis of the integrated logarithm of the inverse gray value, which is proportional to the total amount of absorbing material in the light path. This system enables the percentage area of positive staining in each biopsy to be accurately quantified. Glomerular and periglomerular TSP-1 expression was graded by counting the percentage of positive glomeruli or glomeruli surrounded by TSP-1 staining, respectively. Tubulointerstitial TSP-1 expression was quantified by counting the number of tubules exhibiting positive TSP-1 staining in 10 sequentially selected, 1-mm2 grids, at x100 magnification. The mean numbers of macrophages (ED-1-positive cells) in glomeruli and the interstitial area in each biopsy were calculated, in a blinded manner, by averaging the total number of positive cells in each glomerulus or in 30 sequentially selected 0.25-mm2 grids, respectively, at x200 magnification.
The percentage of glomeruli exhibiting focal or global glomerulosclerosis was determined by evaluation of all glomeruli present in the biopsy. Glomerulosclerosis was defined as segmental increases in the glomerular matrix, segmental collapse, obliteration of capillary lumina, and accumulation of hyaline, often with synechial attachment to Bowman's capsule. Tubulointerstitial injury was defined as inflammatory cell infiltration, tubular dilation and/or atrophy, or interstitial fibrosis. Injuries were graded semiquantitatively by a blinded observer, who examined at least 40 cortical fields (magnification, x100) in periodic acid-Schiff-stained biopsies. Only cortical tubules were included in the following scoring system (16): 0, normal; 1, involvement of <10% of the cortex; 2, involvement of 10 to 25% of the cortex; 3, involvement of 26 to 50% of the cortex; 4, involvement of 51 to 75% of the cortex; 5, extensive damage involving >75% of the cortex.
Western Blot Analyses
Isolation of whole protein from the RK was performed by tissue
homogenization in Tris-glycine buffer with proteinase inhibitors (Complete;
Roche, Indianapolis, IN). After determination of the protein concentration
using the Bio-Rad protein assay (Bio-Rad, Richmond, CA), protein samples (30
µg) were mixed with reducing buffer, boiled, resolved on 7.5% sodium
dodecyl sulfate (SDS)-polyacrylamide gels, and transferred to nitrocellulose
membranes by electroblotting. Membranes were blocked with 5% (wt/vol) nonfat
milk powder in Tris-buffered saline for 30 min at room temperature. An
affinity-purified rabbit polyclonal antibody to human VEGF (Santa Cruz
Biotechnology), which recognizes all four isoforms of human VEGF and is
reported to cross-react with rat VEGF, was used. After incubation of the
membrane with alkaline phosphatase-conjugated mouse anti-rabbit antibody, the
bands were observed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium tablets (Sigma Chemical Co., St. Louis, MO). Positive
immunoreactive bands were quantified by densitometry.
In Vitro Effects of Macrophage-Derived Cytokines on Tubular VEGF
Expression
Murine thick ascending limb (mTAL) cells were obtained by microdissection
of normal mouse (C57BL/6J) kidneys. Cells were identified as pure mTAL cells
by their uniform cobblestone appearance when grown to confluence and by their
uniform positive staining for Tamm-Horsfall protein,
Na+/K+-ATPase, and inducible nitric oxide synthase. For
passage of mTAL cells, confluent cells were washed with Hanks' balanced salt
solution (HBSS) (Irvine Scientific, Santa Ana, CA) and then incubated for 5
min at 37°C with 1 ml of HBSS containing 0.05% trypsin (Irvine Scientific)
and 0.01% ethylenediaminetetraacetate (Irvine Scientific), until the cells
were detached. Free cells were suspended in mTAL medium containing 14% NuSerum
(Becton-Dickson, Franklin Lakes, NJ), hydrocortisone (40 ng/ml), thyroxine (2
ng/ml; Sigma), penicillin, and streptomycin (Life Technologies, Grand Island,
NY), centrifuged at 1200 rpm for 5 min, and seeded. All data presented are
from experiments performed with cells from passages 8 to 18.
When the mTAL cells had grown to 70% confluence in multiwell plates (Becton
Dickinson), the 14% NuSerum-containing medium was removed and the cells were
washed three times with HBSS. The culture medium was changed to serum-free
mTAL medium for 24 h before each experiment. After synchronization of cell
growth for 24 h, mTAL cells were washed three times with HBSS and exposed to
interleukin-1ß (IL-1ß) (0.1 to 100 ng/ml), IL-6 (0.1 to 100 ng/ml),
or tumor necrosis factor- (TNF-) (0.1 to 100 ng/ml) (all from
Sigma) for 3 to 24 h. All experiments were also conducted under hypoxic
conditions. To achieve hypoxia, mTAL cells, with or without cytokines, were
incubated in a GasPak anaerobic culture pouch (BBL Microbiology Systems,
Kansas City, MO), using hydrogen and a palladium catalyst to remove all traces
of oxygen. At the end of a 24-h exposure to hypoxia, the partial pressure of
oxygen in the culture medium was measured using a pH/blood gas analyzer;
values were in the range of 25 to 30 mmHg and 120 to 130 mmHg for hypoxic and
normoxic conditions, respectively.
To quantify the level of VEGF secretion under the different conditions, VEGF protein was measured in mTAL cell culture supernatants using a commercial mouse enzyme-linked immunosorbent assay kit for VEGF (R & D Systems, Minneapolis, MN), which is sensitive to levels of 3 pg/ml. The interassay coefficient of variation was <7% and the intra-assay coefficient of variation was <5% among the standards. All data for VEGF production determined in enzyme-linked immunosorbent assays were expressed as picograms per 105 cells.
Cell viability under the experimental conditions was examined by using lactate dehydrogenase (LDH) assays. After exposure of cells to various cytokines under normoxic or hypoxic conditions, supernatants were collected and filtered (to remove any dead and detached cells), and the LDH activity was measured using an enzymatic method based on the oxidation of NADH to NAD during the reduction of pyruvate, which is catalyzed by LDH (Sigma). LDH release was calculated as a percentage of LDH amounts in the supernatant, compared with total LDH amounts in the lysed mTAL cells plus the supernatant.
RNA Isolation and Reverse Transcription-PCR of VEGF mRNA
After incubation of cells with various proinflammatory cytokines
(IL-1ß, IL-6, or TNF-, 10 ng/ml) under normoxic or hypoxic
conditions, total RNA was prepared from the mTAL cell monolayers using the
RNeasy96 total RNA isolation protocol (Qiagen, Valencia, CA). Total RNA was
similarly isolated from cortical and medullary tissue samples from both
sham-operated kidneys and RK at 8 wk (n = 4 each).
Total RNA was analyzed by quantitative real-time PCR (19), using an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). This system is based on the ability of the 5'-nuclease activity of Taq polymerase to cleave a nonextendable, dual-labeled, fluorogenic hybridization probe during the extension phase of PCR. The probe is labeled with a reporter fluorescent dye 6-carboxyfluorescein) at the 5' end and a quencher fluorescent dye (6-carboxytetramethyl-rhodamine) at the 3' end. When the probe is intact, reporter emission is quenched by the physical proximity of the reporter and quencher fluorescence dyes. However, during the extension phase of PCR, the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye from the probe, with a concomitant increase in reporter fluorescence.
The following sequence-specific primers and probes for mouse VEGF and 18S rRNA were designed using Primer Express software (PE Applied Biosystems): for VEGF: forward, 5'-GGAGCAGAAAGCCCATGAAGT-3'; reverse, 5'-GTCTCAATCGGACGGCAATAG-3'; probe, 5'-6-carboxyfluorescein-TGAAGTTCATGGACGTCTACCAGCGCA-6-carboxytetramethyl-rhodamine-3'; for 18S rRNA: forward, 5'-CGGCTACCACATCCAAGGAA-3'; reverse, 5'-GCTGGAATTACCGCGGCT-3'; probe, 5'-6-carboxyfluorescein-TGCTGGCACCAGACTTGCCCTC-6-carboxytetramethyl-rhodamine-3'.
Primers were used at a concentration of 200 nM and probes at a concentration of 100 nM in each reaction. Multiscribe reverse transcriptase and AmpliTaq Gold polymerase (PE Applied Biosystems) were used for all reverse transcription (RT)-PCR assays. Relative quantitation of 18S rRNA and VEGF mRNA was performed using the comparative threshold cycle number for each sample, fitted to a five-point standard curve (ABI Prism 7700 User Bulletin 2; PE Applied Biosystems). Expression levels were normalized to 18S rRNA levels and related to relevant control values.
Northern Blot Analysis of VEGF mRNA
Total RNA was also analyzed by Northern blot analysis. After
spectrophotometric determination of RNA purity and concentration, 20-µg RNA
samples were denatured and subjected to electrophoresis through 1% agarose
gels containing 2.2 M formaldehyde. RNA was then transferred to nylon membrane
filters (Hybond-N; Amersham, Piscataway, NJ) by capillary action and was
cross-linked using a Stratalinker (Stratagene, La Jolla, CA), followed by
prehybridization for 30 min at 37°C with ExpressHyb hybridization solution
(Clontech, Palo Alto, CA). The probe for VEGF (kindly provided by Dr. Mark E.
Cooper, University of Melbourne, Victoria, Australia) was labeled with
[32P]dCTP by random-primed DNA synthesis. Hybridization was
performed at 37°C for 1 h, and then filters were washed three times for 20
min each [first wash in 2x SSC/0.1% SDS at 65°C, second wash in
1x SSC/0.1% SDS at 65°C, and third wash in 0.1x SSC/0.1% SDS
at room temperature] before exposure to x-ray film (Kodak). The relative
autoradiographic intensities of the Northern blots were determined by scanning
densitometry. All results were corrected for differences in RNA loading by
rehybridization with an oligonucleotide probe for 18S rRNA.
Statistical Analyses
All data are presented as mean ± SD. Differences in the various
parameters between the sham and RK groups were evaluated by unpaired
comparisons for nonparametric data. Differences in parameters at each time
point after RK surgery were compared by paired t test. The
relationships between variables were assessed by Pearson correlation analysis.
Significance was defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Changes in the Renal Microvasculature
The number of JG-12-positive glomerular capillary loops per glomerular
cross-section revealed no significant changes during the first 4 wk after
renal ablation, although there was a trend toward an increase in capillary
number, compared with the baseline value
(Figure 1A). However, at 8 wk
after renal ablation, the number of glomerular capillary loops per glomerular
cross-section was significantly reduced
(Figure 1, A, C, and D).
Glomerular capillary density expressed as the number of capillary loops per
0.01 mm2 of glomerular cross-sectional area was more markedly
decreased at week 8, compared with sham-operated rats (14.2 ± 5.2
versus 30.1 ± 8.4 loops/0.01 mm2, RK
versus sham, P < 0.05). Peritubular capillary loss,
expressed as an increased capillary rarefaction index (i.e.,
percentage area with no capillaries) and decreased capillary density
(i.e., amount of capillary staining/100 tubules), was observed
earlier than the changes in glomerular capillaries. Focal peritubular
capillary loss was present by 2 wk after renal ablation
(Figure 1B) and further
increased with time Figure 1, B, E, and
F). At 8 wk, the peritubular capillary rarefaction index was
markedly increased up to 22.5% and this increase was associated with a
significant decrease in peritubular capillary density (10.2 ± 1.8
versus 0.78 ± 0.23%, baseline versus 8 wk, P
< 0.01), indicating significant capillary loss with considerable areas of
renal tissue devoid of capillaries.
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PCNA and JG-12 double-staining documented peritubular capillary endothelial cell proliferation beginning at 1 wk, followed by glomerular endothelial cell proliferation at 2 wk after the 5/6 nephrectomy (Figure 2). Both glomerular and peritubular capillary endothelial cell proliferation then subsided and eventually decreased to levels below those observed for the control group, at weeks 8 and 4, respectively (Figure 2).
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Similar changes in the microvasculature were noted with an antibody to a different endothelial cell antigen (RECA-1), suggesting that the findings observed were likely attributable to true changes in the microvasculature, as opposed to alterations in endothelial antigen expression.
Renal VEGF Expression
Figure 3 presents the
changes in renal VEGF expression detected by immunohistochemical staining. In
sham-operated rats, there was constitutive expression of VEGF in tubules
within the outer medulla and the medullary rays
(Figure 3A), similar to that
observed in normal kidneys
(20,21).
No significant change in the percent positive area of renal VEGF expression
(which reflects primarily tubular expression) was noted during the first 2 wk
after renal ablation. However, a decrease in VEGF immunostaining became
evident 4 wk after the 5/6 nephrectomy, with marked change by week 8
(Figure 3, B and C).
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In sham-operated rats, VEGF was constitutively expressed in podocytes of the glomeruli (Figure 4A), as in normal kidneys (20,21). In the RK model, glomerular VEGF immunostaining in podocytes demonstrated a mild increase at weeks 1 and 2. However, VEGF staining in podocytes was markedly decreased by 8 wk (Figure 4, B and C), especially in glomeruli with macrophage infiltration (Figure 4B). Figure 5 presents the results of RT-PCR and Western blot analyses of RK tissue (8 wk) for VEGF, revealing the downregulation of VEGF mRNA and protein in both the cortex and medulla in this model of progressive renal disease (Figure 5).
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Renal TSP-1 Expression
De novo expression of TSP-1 in glomerular and tubular areas was
evident 1 and 2 wk after renal ablation, respectively. TSP-1 was first
observed in peritubular and periglomerular areas and in platelets and platelet
aggregates in glomeruli (Figure 6, A and
B). TSP-1 expression was increased in interstitial areas and
glomeruli at 1 and 2 wk, respectively, and the increased expression was
sustained at 8 wk (Figure 6, C to
F).
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Macrophage Infiltration and Renal Expression of VEGF and TSP-1
There was a significant increase in the number of ED-1-positive cells
within both the glomeruli and the tubulointerstitium of the RK group. The
increase in the number of ED-1-positive cells began at 1 wk and inexorably
increased thereafter (Table
2).
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Double-immunostaining with ED-1 and anti-VEGF revealed that the glomeruli with the most marked macrophage infiltration exhibited more dramatic reductions in VEGF staining in podocytes (Figure 4B). Conversely, glomerular VEGF expression was preserved in glomeruli without significant macrophage infiltration (Figure 4C). Interestingly, tubulointerstitial macrophage infiltration was also spatially and quantitatively associated with a significant decrease in VEGF immunostaining in the cortex and the outer and inner medulla (Figure 7, A through C). For individual animals, a significant inverse correlation between the number of tubulointerstitial macrophages and the percent positive area of VEGF immunostaining was observed (Figure 7C). Similarly, there was a positive correlation between TSP-1 expression and the number of glomerular and interstitial macrophages (Figure 7, D and E). The number of ED-1-positive cells in the tubulointerstitium was also correlated with the loss of peritubular capillaries, as quantified by the peritubular capillary rarefaction index (Figure 7F).
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Relationship between Capillary Loss and Changes in VEGF and TSP-1
Expression
There was a significant inverse correlation between tubular TSP-1
expression and peritubular capillary density (r2 = -0.69,
P < 0.05), as well as between glomerular TSP-1 expression and
glomerular capillary loop number per glomerular cross-section
(r2 = -0.55, P < 0.05). Tubular VEGF
expression was also correlated with peritubular capillary density
(r2 = 0.66, P < 0.01).
Relationship between Changes in the Renal Microvasculature and Renal
Structure and Function
The glomerular capillary number per glomerular cross-section and the
peritubular capillary density demonstrated significant inverse correlations
with 24-h urinary protein excretion (r2 = -0.63 and -0.57,
respectively; P < 0.05). A weak but significant correlation
between the peritubular capillary rarefaction index and BUN levels was also
observed from 2 wk after renal mass reduction (r2 = 0.46,
P < 0.05). Glomerular capillary numbers were correlated with the
percentage of glomeruli demonstrating sclerosis (r2 =
-0.55, P < 0.05). Interstitial fibrosis scores were also inversely
correlated with the peritubular capillary density (r2 =
-0.80, P < 0.05).
Evidence that Macrophage-Derived Cytokines Decrease VEGF Expression
in mTAL Cells
The observation that there was a spatial and quantitative correlation of
macrophages with sites of decreased VEGF expression suggested that macrophages
could be releasing factors that might modulate VEGF expression. We therefore
conducted studies to determine whether several of the more important
macrophage proinflammatory cytokines could regulate VEGF expression in renal
tubular cells. mTAL cells were exposed to IL-1ß, IL-6, or TNF-
(range, 0.1 to 100 ng/ml). Exposure of mTAL cells to IL-1ß or IL-6 for 24
h significantly inhibited VEGF protein secretion
(Figure 8A). TNF-
decreased VEGF secretion only at the highest dose (100 ng/ml) tested. At the
concentrations used, there was no evidence for cytotoxicity, as documented by
LDH release. We further investigated the effects of these cytokines in
altering VEGF expression in response to hypoxia. Hypoxia increased VEGF
protein secretion by 2.4-fold, compared with normoxic control animals
(P < 0.05). Similar to their effects on normoxic cells, all three
cytokines also inhibited VEGF secretion
(Figure 8B). The percentage
inhibition of VEGF protein secretion by IL-1ß was comparable under
normoxic and hypoxic conditions. However, IL-6 and TNF- induced more
profound decreases in VEGF protein secretion under hypoxic conditions.
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IL-1ß, IL-6, and TNF- also reduced VEGF mRNA expression in mTAL
cells, as measured in real-time RT-PCR and Northern blot analyses. Both types
of analyses documented significant reductions in VEGF mRNA expression in mTAL
cells in response to IL-1ß, IL-6, and TNF-, under both normoxic
and hypoxic conditions (Figure
9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our first finding was that an initial angiogenic response follows subtotal renal ablation, as documented by an increased level of proliferation of peritubular and glomerular endothelial cells by weeks 1 and 2, respectively. Yamanaka and co-workers (8) also reported early proliferation of glomerular endothelial cells, and other groups reported proliferation of mesangial cells, tubular cells, and fibroblasts (22,23). Morphometric studies also documented early increases in both the length and number of glomerular capillaries (24,25), suggesting angiogenesis. The mechanism of this increased endothelial cell proliferation is unknown but may be related to mesangial cell production of growth factors such as platelet-derived growth factor and transforming growth factor-ß (TGF-ß) (26,27), to platelet-associated factors (27), or to shear stress-induced activation of endothelial cells, with the release of TGF-ß (26). Although TGF-ß and other factors may well contribute to the early angiogenic response (28), we also observed that there was an increase in podocyte expression of the potent angiogenic factor VEGF at weeks 1 and 2 after renal ablation. The observation that VEGF expression increases in podocytes in association with endothelial cell proliferation suggests that VEGF may have a role in this process, perhaps via diffusion across the GBM because of the strong affinity of VEGF for heparan sulfate (29).
Unfortunately, the initial proliferative response by the glomerular and peritubular capillary endothelium was not sustained and there was progressive capillary loss that was significant as early as week 4 for the peritubular capillaries and by week 8 for the glomerular capillaries. Although factors mediating endothelial cell death, such as oxidants, angiotensin II, and Fas (10,11), are likely to be important in the progressive endothelial cell loss, the observation in this study that endothelial cell proliferation subsided to levels significantly below those observed for both sham-operated and normal rats demonstrates that an impaired angiogenic response is also contributory. These data are consistent with recent findings that impaired angiogenesis and/or endothelial cell proliferation occurs in other models of progressive renal disease, such as observed with aging or anti-GBM disease (2,3,9). Kitamura et al. (8) also reported impaired glomerular endothelial proliferation late in the RK model. The inability to stimulate endothelial repair in the setting of endothelial cell loss would be expected to amplify the microvascular disease and contribute to progression.
In this study, we investigated whether the impaired angiogenic response was attributable to loss of an angiogenic factor, increased expression of an antiangiogenic factor, or a combination of both. One of the most important angiogenic factors is VEGF, and this factor is constitutively expressed in podocytes and in tubular epithelial cells of the outer medulla and the medullary rays (20). In addition to being an angiogenic factor, VEGF is an endothelial cell survival factor under a variety of conditions (30,31). We observed a loss of VEGF in both podocytes and the tubules of the outer medulla in association with reduced glomerular and peritubular capillary densities and the development of glomerulosclerosis and interstitial fibrosis. These data suggest that a loss of VEGF could be responsible for the impaired angiogenesis and may contribute to the progressive renal scarring. We have noted a similar loss of VEGF in podocytes and tubules in aging-associated glomerulosclerosis and interstitial fibrosis in rats (32). In addition, a loss of VEGF has been observed in podocytes in glomerulosclerosis (33) and in the outer medullary tubules in chronic interstitial disease in human patients (21), suggesting that downregulation of VEGF expression may be important in human renal disease.
The impaired angiogenic response could also be attributable to increased expression of antiangiogenic factors. TSP-1 is an attractive candidate because it has many actions that oppose those of VEGF, in that it inhibits both basic fibroblast growth factor- and VEGF-mediated endothelial cell proliferation (34) and independently induces endothelial cell apoptosis (35). TSP-1 is also expressed in the interstitium in both experimental (36,37) and human (38) interstitial fibrosis and is a strong predictor of the subsequent development of interstitial fibrosis (37). Although TSP-1 may contribute to the development of fibrosis because of its ability to activate TGF-ß (39), our observation that TSP-1 levels were increased in glomeruli and the interstitium and were correlated with the loss of glomerular and peritubular capillaries suggests that TSP-1 may promote renal scarring via effects on the endothelium.
We also addressed potential mechanisms underlying the downregulation of
VEGF expression. An important finding was the observation that the loss of
VEGF expression by podocytes and tubular cells was strongly correlated, both
spatially and quantitatively, with macrophage infiltration. It is well
documented that macrophages may have proinflammatory or reparative phenotypes
and can express both proangiogenic and antiangiogenic factors
(40). Macrophages may
therefore be regarded as a "double-edged sword," because they may
be important in some angiogenic responses, such as those in wounds or tumors
(40), but they also play an
important role in renal damage and progressive scarring
(41). Although some of the
tubular loss of VEGF in our study could reflect tubular damage, this cannot
account for the diffuse loss of VEGF from the outer medullary tubules. We
therefore explored the possibility that key macrophage-derived cytokines,
including IL-1ß, IL-6, and TNF-, may affect VEGF expression by
tubular cells. Interestingly, IL-1ß, IL-6, and TNF- (albeit at a
higher concentration) were all able to significantly inhibit the secretion of
VEGF protein by mTAL cells in vitro and to suppress VEGF mRNA levels.
The inhibitory effects of these cytokines on VEGF expression were also
demonstrated under hypoxic conditions similar to those normally present in the
outer medulla. These observations are the first to demonstrate that
macrophage-associated cytokines can negatively modulate the constitutive
production of VEGF by distal tubular epithelial cells. Inhibition of VEGF
would be expected to negatively affect the ability of the microvasculature to
respond to injury and thus represents another mechanism by which macrophages
can contribute to progressive renal disease.
Proteinuria, which is well recognized as being an important risk factor for progression, was also correlated with capillary loss. Given the strong association of proteinuria with interstitial macrophage infiltration (42), it seems possible that proteinuria may indirectly contribute to microvascular loss, via stimulation of local macrophage accumulation.
In conclusion, the RK model is associated with a brief but unsustained
angiogenic response, followed by progressive glomerular and peritubular
capillary loss and renal scarring. The loss of capillaries is correlated with
a loss of VEGF and an increase in TSP-1 levels in the kidney, conditions that
favor endothelial cell loss and impaired angiogenesis. The observation that
sites of macrophage accumulation were correlated with the loss of VEGF
suggests that macrophages may play a role in the downregulation of VEGF. This
proposal was supported by in vitro data demonstrating that
macrophage-derived cytokines (IL-1ß, IL-6, and TNF-) significantly
suppressed VEGF expression in mTAL cells. Therefore, progressive renal disease
in this model is associated with impaired angiogenesis and capillary loss,
suggesting a critical role for microvascular disease in either causing or
contributing to the development of end-stage renal disease.
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