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Am J Physiol Renal Physiol 294: F928-F936, 2008. First published February 13, 2008; doi:10.1152/ajprenal.00596.2007
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Renal ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, a novel VEGF inhibitor

David P. Basile,1 Katherine Fredrich,2 Bhadrani Chelladurai,1 Ellen C. Leonard,1 and Alan R. Parrish3

1Department of Cellular and Integrative Physiology, Indiana University, Indianapolis, Indiana; 2Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin; and 3Department of Systems Biology and Translational Medicine, Texas A&M University, College Station, Texas

Submitted 18 December 2007 ; accepted in final form 6 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reductions in vascular density occur following acute ischemia-reperfusion (I/R) injury that may predispose the development of chronic kidney disease. The mechanisms mediating vascular loss are not clear but may relate to the lack of effective vascular repair responses. To determine the regulation of the VEGF/VEGFR pathway following I/R injury, male Sprague-Dawley rats were subjected to bilateral renal ischemia (45 min) and allowed to recover for 1, 3, 7, and 35 days. VEGF mRNA expression was repressed by greater than 50% of control values up to 3 days postischemia, while VEGF protein was repressed for up to 7 days postischemia. The renal mRNA expression of receptors was not altered postischemia; however, VEGFR1 (flt-1) protein was transiently reduced in kidney while soluble flt-1 was elevated in plasma at 7 days following injury. Microarray analysis of angiogenesis-related genes identified the enhanced expression of a number of genes, among these was ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif-1), a secreted VEGF inhibitor. The altered expression of ADAMTS-1 was confirmed using RT-PCR and Western blot analysis; immunofluorescence localized its expression to proximal tubules following I/R injury. Other genes identified using microarray included aminopeptidase N, Smad-1, and Id-3 and their localization was also examined using immunohistochemistry. In summary, the data indicate no clear pattern of anti-angiogenic gene expression following renal I/R injury. However, the studies do suggest an overall inhibition of the VEGF pathway during the early injury and repair phase of renal ischemia that may contribute to an overall reduction in renal microvascular density.

microarray; rarefaction

ISCHEMIA-REPERFUSION (I/R) injury in rodents results in acute kidney injury (AKI) characterized by a loss of glomerular filtration rate and overt damage in the proximal tubule epithelium. I/R injury results in immediate increases in renal vascular resistance and persistent reductions in renal blood flow in the postreperfusion period. These alterations in renal vascular resistance contribute to the overall severity of AKI and are mediated either directly via injury to the renal microvasculature or secondary to alterations in tubular metabolism or inflammation that may impinge on renal blood flow (1, 9, 34). The resolution of vascular resistance following I/R is an important component in the recovery from AKI and is critical to the reestablishment of renal function and the recovery process.

Despite the resolution of blood flow during recovery from I/R, there remains a persistent rarefaction of renal blood vessels following the initial resolution of AKI that does not appear to be reversible. While the mechanism and time course of capillary loss following I/R are not known, reductions in capillary density are clearly established within 4 wk and show no evidence of resolution thereafter (4). This reduction in renal microvessels predisposes hypoxia and is hypothesized to contribute toward the subsequent development of interstitial fibrosis following recovery from I/R injury (2, 3). In this regard, I/R-induced AKI is similar to other models of progressive renal disease in which vascular rarefaction precedes development of interstitial scar formation (13, 14, 27, 33).

The loss of the renal vasculature appears in stark contrast to the regenerative potential of the renal tubular system following I/R. As a possible explanation for the lack of vascular repair, we hypothesized that renal I/R results in a net shift of expressed factors in favor of anti-angiogenesis vs. angiogenesis/vascular stabilization (2). For example, we already identified potential inhibitors of angiogenesis that are stimulated in the setting of I/R, such as TGF-β, PAI-I, MMPs, and angiostatin (6, 7, 32).

Nevertheless, the hypothesis is counterintuitive since hypoxia following I/R may be expected to stimulate the expression of proangiogenic molecules, particularly vascular endothelial cell growth factor (VEGF), in a HIF1{alpha}-dependent fashion (26). Because renal I/R results in persistent hypoxia and damage to the vascular system, we therefore sought to investigate the VEGF system in both the early injury and regenerative phases of this model as a possible explanation for the lack of vascular repair. Initially, an a priori approach was used that employed standard methodologies to examine the expression of the VEGF pathway during recovery from renal I/R injury. As a complement to these studies, array-based approaches were also utilized to glean additional information regarding the expression of angiogenesis-related genes within the context of renal I/R. These studies led to the identification of an additional candidate gene called ADAMTS-1, a known inhibitor of VEGF activity, which is induced in the setting of renal I/R injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and surgical procedures. Male Sprague-Dawley (Harlan, Indianapolis, IN) rats weighing 250–300 g were housed with a 12:12-h light-dark cycle. Animals were fed standard laboratory chow (Harlan, Teklad, Indianapolis, IN) with 0.8% sodium content; food and water were available ad libitum. Care of the rats before and during the experimental procedures was conducted in accordance with the policies of National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All protocols had received prior approval by the Indiana University Institutional Animal Care and Use Committee.

Rats were subjected to a standard renal I/R to induce AKI after 45 min of bilateral renal artery clamping; sham-surgeries were performed in a similar fashion except that the renal pedicle was visualized but not touched (7, 20). Blood samples were obtained at 24 h and at the time of animal death to evaluate the degree of AKI by serum creatinine determination using standard methodologies (7, 20).

Initial studies (group I) were conducted to obtain tissues for analysis of mRNA and protein expression using real-time PCR and Western blot analysis. In these studies, after removal of the clamps, rats were allowed to recover for 1, 3, 7, and 35 days postischemia or sham-surgery. At the time of death (between 0900 and 1200), animals were anesthetized with ketamine/xylazine/acepromazine (2.0/0.6/0.3 mg/kg). The kidneys were removed and cut bilaterally; one-half of each kidney was snap-frozen in liquid nitrogen and stored at –70°C for subsequent biochemical analysis. In addition to these animals, a separate group of animals was treated identically and tissues were used to obtain RNA for use in microarray analysis (group II). Additional surgeries were performed on rats that were used to obtain tissues for immunofluorescent analysis. In these studies, injury levels were determined at 24 h following I/R by evaluation of serum creatinine and were similar to values we reported previously (3, 32). For these experiments, kidneys were fixed by immersion in 2% paraformaldehyde, processed with 20% sucrose in PBS, and prepared for cryostat sectioning.

RNA isolation and real-time PCR. Total RNA was isolated from whole kidney using a Qiagen total RNA isolation kit according to the manufacturer's recommendations. Real-time PCR was carried out as described previously (5); briefly, FAM-labeled primers (Lux primers, Invitrogen, Carlsbad, CA) specific for each of the genes of interest are shown in Table 1. Real-time PCR reactions were carried out on an ABI Prism 7900HT (ABI, Applied Biosytems, Foster City, CA) using a TaqMan One-Step RT-PCR Master Mix Reagents Kit (ABI) according to the manufacturer's recommendation. Real-time reactions were carried out simultaneously for 18S RNA in parallel wells. Standard curves (0.01–2 ng total RNA) were generated using pooled RNA samples from control rats for both 18S RNA and the mRNA for the genes of interest to determine the relative fold-change between sham-operated and postischemic kidney. Values for specific genes were normalized to values obtained for 18S RNA in each sample and data were expressed relative to normalized values for sham-operated controls.


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  		Table 1. Primers used for real-time PCR analysis

 
Construction of rat angiogenesis oligonucleotide arrays. A pathway-specific microarray employing oligonucleotides (70-mers) spotted onto glass slides, as described previously by Wang et al. (38), was synthesized to evaluate potential alterations in angiogenesis-related genes. This array comprised 132 genes selected based on their putative involvement in regulating vascular and/or endothelial cell homeostasis. Positive control 70-mer oligonucleotides corresponding to genes of wide tissue expression such as β-actin and GAPDH as well as positive controls with relevance to the model of renal I/R injury were also included. Negative control spots consisted of 50% water/50% DMSO. To minimize cross-hybridization, the TIGR rat gene index containing a nonredundant set of expressed mRNA sequences was used as the complete genome source (http//:www.tigr.org/tdb/tfi/rgi) for selecting 70-mer oligonucleotide sequences using the PICK70 program (http://ftp.roedu.net/mirrors/sf.net/a/ar/arrayoligosel//). Oligonucleotides (unmodified, Operon, Germantown, MD) were diluted with 50% DMSO and spotted with a minimum of six replications per slide using a four-pin arrayer (Affymetrix, Santa Clara, CA) on microglass slides (Corning Glass Works, Corning, NY) that had been coated with poly-L-lysine. Printed slides were UV crosslinked, blocked with succinic anhydride, and stored in the dark at room temperature.

Microarray hybridization and data analysis. Total RNA (0.1 µg) was reverse transcribed and labeled with Cy3 or Cy5 using the Ovation amionallyl RNA amplification and labeling system (Nugen) according to the manufacturer's instructions and slides were hybridized using standard conditions. Slides were dried and coated with DyeSaver and scanned in a GenePix Personal 4100A Scanner (Axon Instruments). Raw values of fluorescent intensity in each spot were obtained from microarray images using GenePix 4.1 software. These raw data were categorized, selected, and adjusted to yield log-transformed, normalized ratios following the systematic method that was described previously (5). Log-transformed ratios were obtained from average signal intensities among replicates for each unique element on the array. Hybridizations were carried out using kidney RNA derived from one sham and one postischemic rat; ratios for each unique oligonucleotide element were then averaged across four hybridizations, representing four unique animals pairs, at each time point postsurgery (i.e., 1 day, 7 days). Mathematical manipulation and averaging of microarray data were facilitated using Stata Intercooled 8.2 (Stata, College Station, TX). Determination of differential expression was based on 95% C.I. of all ratios. Raw data from GenePix 4.1 from the eight hybridizations were uploaded to the Gene Expression Ominbus (GEO) website http://www.ncbi.nlm.nih.gov/geo/; the series accession number is GSE7040.

Angiogenesis PCR array. Rat Angiogenesis RT2 Profiler PCR Array and RT2 Real-Time SyBR Green/ROX PCR Mix were purchased from SuperArray Bioscience (Frederick, MD). PCR was performed on ABI Prism 7900 HT (Applied Biosystems), according to the manufacturer's instructions. Samples from three sham and three postischemic kidneys were compared at both 1 and 3 days postsurgery. For data analysis, the {Delta}{Delta}Ct method was used with the aid of a Microsoft excel spreadsheet containing algorithms provided by the manufacturer. Fold-changes were then calculated and expressed as log-normalized ratios of values from postischemic/sham-operated tissues.

Immunohistochemistry and Western blotting. For Western blot analysis, frozen kidney samples were homogenized in 50 mM Tris·HCl, pH 8.0, containing 10% DMSO and 2 mM PMSF using a Teflon and glass homogenizer. The homogenate was cleared of large tissue fragments by centrifugation for 5 min at 3,000 g. Protein content was determined using a Bio-Rad protein assay. One-hundred micrograms of total protein were loaded onto either 7.5% (Flt, Flk, ADAMTS-1, aminopeptidase N) or 10% (VEGF) precast polyacrylamide gels (Bio-Rad) and proteins were separated at 100 V for between 90 and 120 min, depending on the size of the particular protein of interest. Protein samples were then transferred onto nitrocellulose sheets (Bio-Rad) at 20 V for 12 h at 4°C. After being blocked, membranes were incubated overnight with primary antibody (1 µg/ml VEGF, Santa Cruz Biotechnology; 1 µg/ml Flt; 1 µg/ml FLK, Santa Cruz Biotechnology; 0.2 µg/ml aminopeptidase N, R&D Systems; 0.2 µg/ml ADAMTS1, Sigma). Blots were then incubated by a species-appropriate horseradish peroxidase-labeled secondary antibody and developed using chemiluminescent substrate (Pierce, Supersignal). Chemiluminescent signals were visualized and quantified using a FujiFilm LAS1000 image acquisition system and analysis software. For quantification, signals for bands of interest were subtracted from local background; data are expressed for each individual sample relative to the mean signal derived from the sham-operated control group for each antibody.

Immunohistochemical localization was carried out on cryostat sections (10 µm) that were stored frozen and desiccated following sectioning. Sections were blocked with PBS supplemented with 10% goat serum (or rabbit serum, as required) for 1 h and incubated for 12–18 h at 4°C with primary antibodies as follows: 2 µg/ml VEGF, 2 µg/ml aminopeptidase N, 10 µg/ml ADAMTS1. Following several rinses in cold PBS, tissues were incubated for 1 h in species-specific secondary antibody labeled either with FITC or Cy3. After being rinsed, slides were coverslipped in a solution of 50 glycerol/20% Mowiol (Calbiochem) and allowed to dry overnight. Tissues were visualized by epifluorescence microscopy using a Nikon Optiphot-2 upright microscope equipped with 100-W mercury lamp, a Spot digital camera, and image acquisition software (version 3.4.5; Diagnostic Instruments).

Some localization was carried out on paraffin-embedded sections. For these studies, the Smad-1 antibody was a generous gift from Dr. M. Landstrom, Ludwig Cancer Research Center (1:500). These antibodies were developed by the indirect biotin-streptavidin-horseradish peroxidase technique using aminoethyl carbazole (AEC) as a substrate (Zymed) and visulalized using standard light microscopy.

Cell culture studies. HK-2 cells, a proximal tubular cell line derived from normal human kidney, were purchased from ATCC and cultured in keratinocyte serum-free medium (GIBCO) supplemented with 5 ng/ml recombinant epidermal growth factor and 0.05 mg/ml bovine pituitary extract. Cells were passed (1:5) every 4 days and used between passages 3 and 6. The supernatant was removed and cell pellet was washed with cold PBS and then lysed in buffer (10 mM Tris·HCl, pH 7.6, 1% SDS; 1 mM PMSF; 1 mM leupeptin; 1 mM orthovandate) and boiled for 5 min. The homogenates were spun at 18,000 g for 10 min and the supernatant was collected.

Statistical analyses. Statistical analysis of spotted microarray data is described above. Data from RT-PCR arrays were analyzed similarly and also utilized an evaluation of significance from Student's t-test with an adjustment for multiple comparisons using a false discovery rate (FDR) correction described by Benjamini and Hochber (8). Data from Western blot and independent real-time PCR analyses were carried out using Student's t-test and P level was adjusted using Bonferroni's correction for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Renal expression of the VEGF-VEGFR system following I/R. VEGF expression is stimulated by hypoxia in several in vitro and in vivo settings, an effect that is thought to be stimulated by the activation of HIF1{alpha} (26). VEGF-1 mRNA was measured by reverse-transcriptase real-time PCR in kidneys of rats following sham surgery or at different times following I/R injury. The expression of VEGF mRNA was not increased at any time points following I/R but rather was significantly depressed at early time points following I/R (1–3 days) relative to levels obtained in shams (Fig. 1). This lack of response was observed despite the fact that heme oxygenase-1 (HO-1), a gene reported to be regulated by HIF1{alpha}, was enhanced by four- to sixfold between days 1–7 in response to I/R (Fig. 1).


Figure 1
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  		Fig. 1. Real-time PCR results evaluating mRNA levels of VEGF pathway-related genes following recovery from renal ischemia-reperfusion (I/R) injury. VEGF, VEGF-R1, VEGF-R2, and heme oxygenase-1 (HO-1) were assayed by real-time PCR in whole kidney RNA samples from sham-operated controls and from animals following I/R injury at 1, 3, 7, and 35 days postischemia. Values were normalized to the level of 28S RNA and expressed relative to normalized values of sham-operated controls. Means ± SE of n = 5 animals per time point. *P < 0.05 by Student's t-test with Bonferonni correction for multiple comparisons relative to expression in sham-operated controls.

 
Figure 2 and Table 2 show data from representative Western blots of kidneys during recovery from AKI demonstrating an inhibition of VEGF expression at 1, 3, and 7 days postischemia (Fig. 2A); VEGF levels recovered at later time points and were indistinguishable from sham-operated controls at 35 days postischemia (Fig. 2B). Immunofluorescence histochemistry demonstrated that VEGF was most prominent in tubular epithelium of sham-operated control rats (Fig. 3A). The signal was absent after substituting the primary antibody to a nonimmune IgG (Fig. 3D) and reduced after preincubating the antibody with 1 µg/ml recombinant VEGF (Fig. 3C). Relative to the signal observed in sham-operated controls, immunofluorescent signals were difficult to detect in proximal tubules of kidneys derived from postischemic rats (Fig. 3B).


Figure 2
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  		Fig. 2. Western blot of VEGF and VEGF-R1. A: immunoreactive bands for VEGF and VEGF-R1 (flt-1) and VEGF-R2 (Flk-1) in extracts derived from sham-operated controls and from rats at 1, 3, and 7 days postischemia. B: bands from a separate set of gels using tissues derived from sham and post-I/R rats at 35 days postsurgery for VEGF and VEGF-R1. See Table 2 for summary.

 

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  		Table 2. Protein expression levels of VEGF and VEGF-R1 by Western blot densitometry

 

Figure 3
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  		Fig. 3. Immunofluorescent analysis of VEGF expression in kidney following I/R injury. Shown are photomicrographs of sections through renal cortex rat kidney following either sham surgery (A, C, D) or 3 days following recovery from I/R (B). Note presence of signal in proximal tubules (arrow) in sections of kidney from sham-operated rat incubated with VEGF antibody. The signal is reduced in sections from sham-operated controls when coincubated with 10 µg/ml recombinant VEGF (C) or when primary antibody is eliminated (D). The signal was consistently reduced following I/R injury (B). Micrographs are illustrative of tissues from 4 animals per group. Bar is 50 µm.

 
VEGF receptors, VEGF-R1 and VEGF-R2 (Flt-1 FLK-1, respectively), were unaffected by I/R at the level of RNA when compared with shams (Fig. 1). The only observed alteration in VEGF-R protein expression was a transient reduction in VEGFR1 (Flt1) protein at 7 days (Fig. 2A, Table 2) that returned to control values by 35 days postischemia (Fig. 2, B and C). I/R injury had no demonstrable effect on VEGF-R2 (Flk-1) protein levels at the time points evaluated (24 h through 7 days; Fig. 2). These data indicate that the VEGF system is not enhanced in the early phase of I/R injury but rather indicate moderate repression in the early postischemic state.

Soluble VEGF-R (s-flt) expression following I/R injury. A soluble form of the VEGF-R1 referred to as s-Flt is reported to have VEGF inhibitory properties. As shown in Fig. 4, a truncated version of VEGF-R1 was present in plasma at ~100 kDa, relative to the full-length form (~160–180 kDa) observed in kidney tissue extract. This immunoreactive band is consistent with the reported size of s-Flt and was substantially elevated at 7 days postischemia (>5-fold, P < 0.05, by Student's t-test).


Figure 4
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  		Fig. 4. Western blot analysis of flt-1/s-flt in serum of postischemic rats following I/R. Shown are Western blots using an antibody for flt-1 protein using extracts derived from kidney tissue or 10 µl rat plasma as labeled. Note the full-length flt-1 receptor present in kidney tissue ~160 kDa while there was a significant band in plasma at ~100 kDa corresponding to the reported sizes for s-flt. The s-flt bland in 7 day postischemic plasma was >5-fold that of sham-operated plasma as determined by densitometry (P < 0.05).

 
Identification of genes with potential vasculotrophic activity using targeted arrays. While VEGF is a prominent vasculotrophic factor, there are a multitude of additional factors with potential influence on vascular stability that have not been formally investigated in renal I/R injury. Therefore, to complement studies on VEGF expression, we generated a spotted oligonucleotide microarray containing up to 132 genes relevant to angiogensis. Positive control gene spots (e.g., β-actin, GAPDH) were easily detectable (not shown) and additional control genes with well-established alterations in expression following I/R injury were also included to verify the utility of this microarray format. The expression of fibronectin, osteopontin, and TIMP-1 was significantly enhanced relative to the sham-operated control while the expression of EGF was repressed (Table 3). A fifth positive control, TGF-β1, had low signal intensity and was not shown to be differentially expressed on these arrays.


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  		Table 3. Summary of genes identified as significantly altered in postischemic rat kidney using angiogenesis-targeted microarray format

 
Only 62 of the 132 experimental gene spots (~47%) showed average signal intensities greater than two times that observed in negative control blank spots. Table 2 lists genes identified as differentially expressed at 1 day and 7 days postischemia as determined by their distribution of normalized log2 ratios. In addition to the positive control genes, only 2 of the 62 (experimental) genes were considered differentially expressed at 1 day postischemia; these were ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif) and aminopeptidase N (Anpep). Two additional genes were identified at 7 days postischemia; these were Id3 (inhibitor of DNA binding-3) and Smad 1.

Because the spotted array format may lack sensitivity, studies were also conducted using a more sensitive commercial PCR-based array system. The system includes 84 angiogenesis genes of which 60 genes were considered detectable (~71%). Of the 84 genes, 44 of these genes were also common to the small spotted array format; 19 were detectable in both systems, while 16 were detectable by PCR but not by spotted array. Of the remaining nine genes, seven were detectable by spotted array only and two were not detectable by either format.

Comparisons were carried out on RNA from sham-operated control and postischemic rat kidneys at 1 day and 3 days post-I/R injury. Data shown in Table 4 are log-transformed expression ratios from genes considered differentially expressed at 3 days following injury. Model relevant genes such as EGF and fibronectin demonstrated appropriate and predicted changes in response to ischemia, and alterations in CCl2, CxCl1, MMP2, and Mdk are consistent with previous reports in this model (22, 29, 31, 39). None of the 16 genes that were detectable by PCR format, but not in the spotted array, were altered by I/R injury. Similar to results obtained using spotted arrays, aminopeptidase N (Anpep) and Id3 were detectable in this format and found to be differentially expressed. Other genes identified using spotted arrays (ADAMTS-1 and Smad-1) were not present in the PCR format. A reduction in VEGF-A expression could also be appreciated with the PCR format, but not in the spotted array format. When comparisons were made at 1 day postischemia, similar trends were observed; however, due to larger variances, only the differential expression of EGF and Anpep was statistically significant (not shown).


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  		Table 4. Summary of genes identified as significantly different using RT2 profiler PCR array at 3 days post-I/R injury

 
The expression of genes identified using spotted arrays was subjected to more detailed characterization. ADAMTS-1 is a secreted metalloproteinase with VEGF inhibitory activity. Its increased expression following I/R injury was verified by real-time PCR (Fig. 5) and Western blot analysis (Fig. 6A). ADAMTS-1-like immunoreactivity was elevated in proximal tubules following I/R relative to the signal observed in tubules of sham-operated rats for at least 7 days postischemia (Fig. 6, C vs. D). The potential expression of ADAMTS-1 in the tubular epithelium is also suggested by its identification in extracts of HK-1 human cultured proximal tubule cell line by Western blot (Fig. 6B). However, ADAMTS-1 expression was not altered in HK-1 cells subjected to simulated hypoxia-reoxygenation (data not shown).


Figure 5
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  		Fig. 5. Real-time PCR analysis of mRNA levels of genes identified by microarray. Levels of both ADAMTS-1 and aminopeptidase N (Anpep) were assayed in whole kidney RNA samples by real-time PCR following sham surgery or following recovery from I/R at 1, 3, and 7 days postsurgery. Values were normalized to the level of 28S RNA and expressed relative to normalized values of sham-operated controls. Means ± SE of n = 5 animals per time point. *P < 0.05 by Student's t-test with Bonnferonni correction for repeated measures relative to sham-operated control.

 

Figure 6
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  		Fig. 6. Characterization of ADAMTS-1 protein following renal I/R. A: immunoreactive bands corresponding to ADAMTS-1 protein (~110 kDa) in extracts derived from sham-operated controls and from rats at 1, 3, and 7 days postischemia. The intensity of ADAMTS-1 was significantly increased by 1.9 ± 0.3, 1.5 ± 0.2, and 1.4 ± 0.2 relative to values obtained in sham-operated controls at 1, 3, and 7 days postischemia (P < 0.05 by Student's t-test, n = 5 per group). B: immunoreactive band of the same size obtained from extracts HK-1 proximal tubule cells. C: immunofluorescence through renal outer medulla of kidney sections stained using an antibody against ADAMTS-1. There was little evidence of positive signal in kidneys from sham-operated controls; however, there was increased signal observed in the tubules of postischemic animals at 3 days postischemia (B–D). Arrow indicates appearance of regenerating proximal tubule. E: kidney from 3 days postischemia in the absence of primary antibody. Data are representative of a minimum of 4 animals per group. Bar is 50 µm.

 
The other three identified genes were also further investigated. The repression of aminopeptidase N mRNA was verified by real-time PCR analysis (Fig. 5) and also by Western blots (Fig. 7C). Using immunofluorescence histochemistry, strong aminopeptidase N-immunoreactive signal was found in sham-operated controls (Fig. 7A) and was primarily localized in brush-border membrane (Fig. 7 inset, arrows). The signal corresponding to aminopeptidase N signal was substantially reduced in the brush-border membrane following I/R (Fig. 7B). Smad-1 signal was enhanced in postischemic animals (at 7 days postinjury) in tubular epithelium (not shown) while commercially available antibodies against Id-3 were not useful in identifying the localization of this protein in the rat kidney using immunohistochemistry (not shown).


Figure 7
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  		Fig. 7. Characterization of aminopeptidase N protein following renal I/R. Aminopeptidase N immunofluorescent signals were evaluated in cryostat sections and are shown in sham-operated rat kidney cortex (A) with signal clearly present in proximal tubule brush border (see inset); reduced signals were clearly evident in cortex at 3 days of recovery from I/R injury (B). Magnification is shown. C: representative Western blot experiments demonstrating a reduction in aminopeptidase N in response to renal I/R injury. By densitometry, the intensity of aminopeptidase N signal was decreased by 0.92 ± 0.03, 0.46 ± 0.11, 0.22 ± 0.04 relative to values obtained in sham-operated controls at 1, 3, and 7 days postischemia, respectively (not significant at day 1; P < 0.05 at days 3 and 7 by Student's t-test, n = 5 per group).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Tubular regeneration is an important element of the initial recovery from AKI induced by I/R; however, the structural recovery of the vasculature is impaired and this may be critical in the compromise of long-term function following I/R (1). Although both structural and functional alterations in microvascular cells have been described within hours of I/R injury, the mechanisms of cellular loss and time course of vascular rarefaction have not been delineated (1). It is suggested that reduced vascular trophic support postischemia may either promote vessel loss and/or compromise vascular repair or regeneration following injury.

The current study was conducted to characterize additional potential vasculotrophic/angiogenic factors in the kidney in the model of rat I/R. The expression of VEGF or VEGF receptors has previously been investigated at relatively early time points following I/R injury. Reports by Kanellis et al. and Vannay et al. (11, 12, 35, 36) reported no effect of I/R on VEGF mRNA levels in the kidney, although a recent report by Villanueva et al. did report an upregulation of VEGF protein by 24 h. While reasons for the discrepant results are unclear, there remains a need to evaluate VEGF expression over longer time courses with relevance to renal regeneration.

Our data suggest there is inhibition of VEGF mRNA within 24 h of I/R and that protein and mRNA levels are impaired for up to 3 or 7 days postischemia, respectively. In addition, a moderate reduction in VEGF expression following I/R was also observed in our PCR array formats. The loss of VEGF appears to be due to the reduction in the content in proximal tubules. These observations are also consistent with another report by Yuan et al. (40) in a model of acute nephrotoxic AKI.

The observation that VEGF is repressed at times when many growth factors are activated may relate to the lack of a vascular repair or stabilization following I/R. An interesting question to consider is whether the reduction in VEGF expression represents the important observation or rather is it that there is no evidence for a substantial and persistent increase in the expression of VEGF? The lack of increased VEGF expression in the setting of acute renal I/R is clearly at odds with canonical signaling pathway in which hypoxia stimulates VEGF transcription through activity of the HIF1{alpha} transcription factor (26). In the current study, HO-1, another HIF1{alpha}-responsive gene induced by renal I/R (28, 39), was dramatically increased postischemia in the same samples used to measure VEGF expression. In previous studies, we also demonstrated that kidneys of postischemic rats were persistently hypoxic even up to 5 wk following recovery from I/R injury (3). Therefore, even the reestablishment of VEGF to basal levels could be considered an impaired response. Although speculative, we suggest that the restitution of VEGF to basal levels is insufficient or too late to mediate any effective vascular repair process. Additional studies are in progress to determine whether VEGF treatment might impart vascular repair following renal I/R.

In renal cell culture systems, VEGF expression is increased in response to hypoxia (25). In contrast, Sandner et al. (30) demonstrated that when rats are subjected to whole body hypoxia, there was an increase in the renal expression erythropoietin mRNA, but there was no corresponding increase in VEGF mRNA. Therefore, it appears that the mechanisms controlling VEGF expression in response to hypoxia are regulated differently in kidney in vivo than in cultured renal cells. This unexpected paradox may underlie the VEGF repression that has been described in many renal disease states associated with hypoxia (13, 14, 23, 27, 33). The nature of the discrepancy between VEGF responsiveness in kidney cultures vs. in the in vivo state has not been substantially addressed.

While we clearly observed a modest transitory reduction in renal VEGF-R1 expression by Western blot, the significance of this observation, if any, is not yet clear. It is perhaps interesting that the soluble VEGF-R1 (s-Flt) was identified in the plasma of postischemic animals. The soluble form of the flt-1 receptor results from the differential splicing of mRNA and is thought to act as a competitive inhibitor of VEGF thereby reducing its bioavailability (10). Its elevation in plasma was reported in patients with preeclampsia and has been reported in models of chronic kidney disease and hypertension (16, 19). Its elevation in the setting of renal I/R injury of the rat may relate to a further inhibition of VEGF activity in kidney or in the periphery and compromise vascular repair and function. Interestingly, TGF-β, a factor that is highly expressed in this model, stimulates the production of both pro- and anti-angiogenic factors including s-flt (24). However, we did not detect differences in s-flt mRNA in the kidney following I/R (data not shown) and the source of s-flt in the circulation remains unresolved.

The second goal of this study was to expand our characterization of molecules with potential vasculotrophic activity by initially employing a rat-specific microarray spotted with 70-mer oligonucleotides corresponding angiogenesis-related genes. However, because a large number of genes were not detectable, we also employed commercial PCR-based plate arrays. In general, although the sensitivity of the PCR format was superior, there were similarities between the two formats. Control genes with relevance to renal I/R were altered in both formats and the single most repressed experimental gene (aminopeptidase N) was the same in both formats. In addition, of those genes which were detectable in both formats, surprisingly few were altered by I/R injury. Although the PCR format identified a greater number of differentially expressed genes, the only gene identified using commercial PCR arrays which was not previously identified using spotted arrays or in previous publications was the repression of fibroblast growth factor receptor-3 (FgfR3).

The interpretation of the potential significance of these results is confounded by multiple factors. For example, the expressed proteins may have limited access to the vascular space and may participate in other activities of renal injury or repair independent of the vasculature. For example, CCl2, CxCl1, Mdk are soluble cytokines that have been characterized as proinflammatory and their inhibition in renal I/R ameliorates leukocyte infiltration and resultant kidney damage (21, 31). Nevertheless, they are also considered proangiogenic and thus these results are not consistent with our suggestion that angiogenic factors are repressed following I/R and must be considered in light of the overall reduction in blood vessel density in this setting. In contrast, the identification of MMP2 confirms our previous report on this molecule, in which we previously suggested that it may impart the activation of the VEGF inhibitor, angiostatin (6).

We also attempted to characterize the expression of other potential proangiogenic factors. Aminopeptidase N was downregulated and although it is a secreted molecule, its predominant localization to the renal brush border makes it difficult to suggest that it may influence vascular homeostasis in the setting of I/R. In addition, Smad-1 was localized in tubular epithelium of the postischemic kidney (not shown) consistent with the report of Villanueva et al. (37), suggesting that its activity likely post-I/R relates to tubular repair rather than influencing vascular stability.

We suggest that ADAMTS-1 warrants further consideration as potential mediator of renal vascular stability. ADAMTS-1 is a soluble matrix metalloprotease molecule that inhibits angiogenesis by mechanisms that may relate to direct sequestration of VEGF or by activating release of anti-angiogenic thromospondin-derived peptides (17, 18). Immunohistochemistry suggested that ADAMTS-1 is enhanced in rat proximal tubules after I/R and it was clearly expressed in HK-1 proximal tubule cells. However, we were unable to detect effects of simulated ischemic injury on the level of ADAMTS-1 in cultured HK-1 cells. This observation may speak to the fundamental differences between culture system and in vivo models. Regardless, as VEGF from the tubular system is considered to provide trophic support to adjacent vascular cells (15), one could envisage that ADAMTS-1 secretion from proximal tubules could have a destabilizing effect on adjacent vascular cells, either directly or indirectly by inhibiting VEGF. However, since ADAMTS-1 expression has not, to our knowledge, been described in renal diseases, the pathophysiological role of ADAMTS-1 remains to be determined.

Taken together, the current study supports the viewpoint that angiogenic factors are altered in the postischemic milieu of the kidney. Although no consistent pattern of pro- vs. anti-angiogenic factors emerges, it is primarily important that the anticipated increase in VEGF is compromised postischemia and there is evidence of potential inhibitors of VEGF activity such as circulating s-Flt and renal expression of ADAMTS-1. Thus the relative inhibition of the VEGF system, by VEGF reduction and increased in inhibitory factors, provides justification for the pursuit of VEGF-directed therapies in the setting of renal I/R as a possible means to affect vascular stability and influence both short-term and chronic renal function.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-63114 (to D. P. Basile) and AG-024179 (to A. R. Parrish).


   ACKNOWLEDGMENTS
 
The authors thank Dr. A. S. Greene for important discussions regarding the design of microarrays, P. Glocka (Medical College of Wisconsin) for assistance in printing slides, and D. McNew (Indiana University Microarray Core Facility) and A. Bowser for assistance in expression studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. P. Basile, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Drive MS 334, Indianapolis, IN 46202 (e-mail: dpbasile{at}iupui.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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E. C. Leonard, J. L. Friedrich, and D. P. Basile
VEGF-121 preserves renal microvessel structure and ameliorates secondary renal disease following acute kidney injury
Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1648 - F1657.
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