Increased Renal Angiopoietin-1 Expression in Folic Acid-Induced Nephrotoxicity in Mice
David A. Long*,
Adrian S. Woolf*,
Toshio Suda and
Hai T. Yuan*
*Nephro-Urology Unit, Institute of Child Health, University College London, London, United Kingdom; and Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan.
Correspondence to Dr. Hai T. Yuan, Nephro-Urology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK. Phone: 00-44-207-905-2196; Fax: 00-44-207-916-0011; E-mail: h.yuan{at}ich.ucl.ac.uk
ABSTRACT. Growth factors affect epithelial regeneration afteracute renal injury, but less is known regarding the expressionof vascular growth factors in this setting. A mouse model offolic acid (FA)-induced nephrotoxicity was used to study theexpression of angiopoietins (Ang), factors that bind the Tie-2receptor and modulate endothelial growth. Tubular damage wasdetected 1 d after FA administration; in the next 14 d, mosttubules regenerated but patchy atrophy, with interstitial fibrosis,was also observed. Ang-1 immunostaining was detected betweencortical tubules and in the vasa rectae of vehicle-treated animals.FA-induced nephropathy was associated with the acquisition ofAng-1 immunostaining in renal arterial walls and in a subsetof injured cortical tubules that failed to stain with periodicacid-Schiff stain, which indicated that they were distal tubules.Renal Ang-1 protein levels were significantly increased afterFA administration, compared with time-matched control values,as assessed by Western blotting. Capillaries between regeneratingtubules expressed both Tie-2 and platelet-endothelial cell adhesionmolecule. A subset of these endothelia expressed proliferatingcell nuclear antigen, whereas capillary proliferation was absentin control samples. Therefore, FA-induced nephropathy is associatedwith increased Ang-1 protein expression in renal epithelia andarteries. In addition, Tie-2-expressing capillaries near damagedcortical tubules undergo proliferation. Further experimentsare required to establish whether these events are functionallyrelated.
Acute renal failure among human patients is associated withhigh mortality rates of 30 to 50% (1). The pathogenesis is oftenmultifactorial, involving ischemia and nephrotoxins; morphologically,"acute tubular necrosis," with flattened epithelia and tubulardilation, is a common feature (1). Folic acid (FA) induces dose-dependentnephrotoxicity in mice and rats, with the rapid appearance ofFA crystals within renal tubules and subsequent acute tubularnecrosis, followed by epithelial regeneration (2–5). Onoccasion, these effects are accompanied by renal cortical scarring(6). Although the damaging effects of FA have been attributedto microscopic tubular obstruction, Fink et al. (7) observedthat alkalinization of urine, via coadministration of NaHCO3,decreased crystal deposition but proximal tubular lesions stilloccurred, consistent with a direct nephrotoxic effect of FA.FA-induced nephropathy involves upregulation of molecules characteristicof kidney development, including the Pax-2 transcription factor(8) and the hepatocyte growth factor receptor (9); an imbalanceof cell survival and death molecules, such as Bcl2 and Bax (10);induction of inflammatory cytokines, such as tumor necrosisfactor- (10); and invasion by monocytes/macrophages (3). Mostanimal studies of acute renal failure have focused on tubulardamage and regeneration, and this has led to the testing oftherapies that aim to enhance epithelial recovery via the administrationof growth factors (11).
Each adult mammalian kidney receives 10% of the cardiac output,a high blood flow supplying glomerular and cortical peritubularcapillaries as well as the vasa rectae. A sustained reductionin renal blood flow occurs in experimental models of acute renalfailure (1,12,13), but there is little information availableregarding structural changes in the renal microvasculature inthis setting. Specific molecules, including growth factors actingthrough receptor tyrosine kinases, direct embryonic vessel differentiation(14). This study focuses on one such signaling system mediatedby the angiopoietin (Ang) ligands, which act via Tie-2, a memberof the Tie (tyrosine kinase containing immunoglobulin-like loopsand epidermal growth factor-similar domains) receptor tyrosinekinase family.
As endothelia differentiate, the onset of Tie-2 expression postdatesvascular endothelial growth factor (VEGF) receptor 2 expressionbut precedes maturity (14). Platelet-endothelial cell adhesionmolecule (PECAM) is coexpressed with Tie-2 during this process(15). Accordingly, Tie receptors modulate the growth of precursorsthat have entered the endothelial lineage (16). Ang consistof an amino-terminal coiled-coil domain mediating the formationof dimers and multimers and carboxy-terminal fibrinogen-likedomains mediating differential effects on Tie-2 phosphorylation(17). Ang-1 binds Tie-2, causing endothelial cell survival andsprouting (18,19). Ang-1 overexpression in vivo produces large,numerous, highly branched vessels (20), and the factor preventscapillary leakage during inflammation by stabilizing PECAM aswell as inhibiting tumor necrosis factor--induced leukocytetransmigration out of capillaries (21,22). Both Ang-1 and Tie-2null-mutant mouse embryos exhibit abnormal vascular networks(16,23). Ang-2 antagonizes Ang-1-induced Tie-2 phosphorylation,and Ang-2 overexpression in vivo causes developmental defectsresembling those of Tie-2 and Ang-1 null mutants (24).
In this study, we determined the expression of Ang-1, Ang-2,and Tie-2 at sequential stages after the induction of FA-inducednephropathy in mice, using immunohistochemical and Western blottinganalyses. Our results demonstrate that Ang-1 is upregulatedin this setting.
Reagents
Reagents were obtained from Sigma Chemical Company (Poole, Dorset,UK) unless otherwise specified. We used a rabbit antibody toa 20-amino acid sequence in the amino-terminus of mouse Ang-1(Alpha Diagnostic International, San Antonio, TX) for immunohistochemicaland Western blotting analyses; a rabbit antibody to a 20-aminoacid sequence in the amino-terminus of mouse Ang-2 (Alpha DiagnosticInternational) for Western blotting (it should be noted thatthe Ang-1 and Ang-2 immunizing peptides have no significanthomology); rat anti-mouse F4/80 macrophage antigen (Serotec,Raleigh, NC) for immunohistochemical analyses; rat anti-mousePECAM (Pharmingen, San Diego, CA) for immunohistochemical analyses;biotin-conjugated mouse anti-human proliferating cell nuclearantigen (PCNA) (Pharmingen) for immunohistochemical analyses;rat anti-mouse Tie-2 (25) for immunohistochemical analyses;and rabbit anti-human Tie-2 (Santa Cruz Biochemicals, SantaCruz, CA) (26) for Western blotting.
Experimental Model
Eight-week-old, male, CD1 mice (Charles Rivers Mouse Farms,Margate, Kent, UK) were administered FA (240 mg/kg) in vehicle(0.2 ml of 0.3 M NaHCO3) or vehicle only by intraperitonealinjection. This FA dose reliably induced severe nephrotoxicity,as assessed by histologic examinations, but was associated witha morbidity rate of <5% for the experimental period (5).Six control kidneys were analyzed before FA or vehicle administration.Kidneys were collected at 1, 2, 3, 7, and 14 d, with six FA-treatedand three vehicle-treated animals at each time point. Left kidneyswere used for immunohistochemical analyses and right kidneysfor Western blotting.
Immunohistochemical Analyses
Organs were fixed in 4% paraformaldehyde. Five-micrometer paraffinsections were dewaxed and rehydrated; some were treated with20 µg/ml proteinase K or proteinase, depending on theantibody used. Endogenous peroxidase was quenched with 3% H2O2for 30 min, and sections were blocked with 10% fetal calf serum,0.2% bovine serum albumin, and 0.1% Tween-20 in phosphate-bufferedsaline (PBS) (pH 7.4). Sections were reacted overnight withanti-Ang-1 (1:1000), anti-F4/80 (1:4000), anti-PECAM (1:1000),or anti-Tie-2 (1:2000) antibodies. Preliminary experiments failedto generate convincing immunohistochemical signals with anti-Ang-2antibody; therefore, Ang-2 analysis was confined to Westernblotting (see below). Bound primary antibodies were detectedwith an EnVision kit (Dako, High Wycombe, UK) and appropriatesecondary antibodies (Vector Laboratories, Burlingame, CA).Brown color was generated by using a diaminobenzidine substrate.Negative controls comprised the omission of primary antibodyor, for Ang-1, prereaction of primary antibody with an excess(10:1, wt/wt) of the immunizing peptide. Nuclei were counterstainedwith hematoxylin. Some sections were additionally stained withperiodic acid-Schiff (PAS) stain, which colors proximal tubulebrush borders pink. As assessed by using [3H]thymidine incorporation,whole-kidney proliferation peaks 1 to 2 d after FA administration(5). For assessment of in situ endothelial cell proliferation,sections were probed with antibodies to both PECAM and PCNA;the latter is a DNA polymerase--associated protein expressedin S phase (27). PECAM immunostaining was performed first, asdescribed above. Sections were then microwaved in citric acid(1.9 g/L, pH 6.0) for 5 min, and endogenous biotin was maskedby using an avidin-biotin blocking kit (Vector Laboratories).Slides were incubated overnight at 4°C with biotin-conjugatedmouse anti-human PCNA (1:50) and were reacted with streptavidin-conjugatedhorseradish peroxidase followed by a diaminobenzidine metal-enhancedsolution, resulting in a blue signal for PCNA.
Western Blotting
Kidneys were homogenized in radioimmunoprecipitation assay buffer(30 µl of 2.2 mg/ml aprotinin, 10 µl of 10 µg/mlphenylmethylsulfonyl fluoride, and 10 µl of 100 mM sodiumorthovanadate per 1 ml of solution at 4°C. Supernatantswere collected after 30 min of centrifugation at 13,000 rpm,and protein concentrations were measured (BCA protein assay;Pierce, Rockford, IL). One hundred thirty micrograms of proteinwere denatured at 100°C for 5 min and separated on sodiumdodecyl sulfate-8% polyacrylamide gels. In parallel experiments,Coomassie blue staining was used to confirm that similar levelsof proteins had been loaded in each lane. Proteins were transferredto nitrocellulose membranes (Amersham Pharmacia Biotech, LittleChalfont, Buckinghamshire, UK) by electroblotting (Bio-Rad,Hemel Hempstead, Hertfordshire, UK). Blots were blocked for1 h with 5% (wt/vol) fat-free milk powder, 0.1% bovine serumalbumin, and 0.1% Tween-20 in PBS and were subsequently incubatedovernight at 4°C with anti-Ang-1 (1:1000), anti-Ang-2 (1:1000),or anti-Tie-2 (1:1000) antibodies. Blots were washed in PBSwith 0.2% Tween-20 and once in blocking solution. Blots wereincubated for 30 min with secondary antibodies, and bands weredetected by chemiluminescence analysis (Amersham Pharmacia Biotech).Negative controls comprised the omission of primary antibodiesor, for Ang-1 and Ang-2, prereaction of primary antibodies withthe respective immunizing peptides. Proteins were sized withRainbow markers (Amersham Pharmacia Biotech). Preliminary experimentsindicated that these protocols resulted in major bands of 100kD for Ang-1, 50 and 75 kD for Ang-2, and 160 kD for Tie-2;in three separate sets of experiments, the intensities of thesebands were densitometrically measured and standardized for proteinloading by normalization with respect to the major (approximately)50-kD band observed with Coomassie blue staining of replicatesamples.
Statistical Analyses
Levels of individual proteins were statistically compared betweenFA-treated and time-matched, vehicle-treated, control samples(n = 3 for each group) by using the Mann-Whitney U test, withdifferences being considered significant at P < 0.05.
In vehicle-treated animals, faint linear Ang-1 immunostainingwas detected between the cortical tubules, probably in capillaries(Figure 1A). One to 3 d after FA administration, flattened corticalepithelia and dilated tubule lumina were noted (Figure 1, Bto D). Glomerular tufts were spared, although Bowman’sspaces were prominent (Figure 1, C and D). FA-induced nephropathywas associated with the acquisition, 1 to 3 d after nephrotoxinadministration, of Ang-1 immunostaining in a subset of injuredcortical tubules (Figure 1, B and C); these tubules tended tobe of smaller caliber than Ang-1-negative tubules, suggestingthat they were distal segments. Ang-1 protein was also detectedbetween damaged cortical tubules (Figure 1C). No significantstaining was detected after prereaction of antibody with theimmunizing peptide (Figure 1D). By 14 d, most cortical areashad regenerated and Ang-1 immunostaining was downregulated (Figure 1E).Ang-1 immunoreactive protein became detectable in the wallsof small cortical arteries in the first 3 d after FA administration(Figure 1B), an appearance that was maintained in atrophic andfibrotic areas (Figure 1F). To better define the minor subsetof cortical tubules that contained Ang-1 immunoreactive proteinafter the induction of nephrotoxicity, we probed sections withanti-Ang-1 antibody and counterstained the sections with hematoxylinand PAS stain (Figure 2). Most damaged tubules could be observedto have a thin brush border and were therefore proximal tubules;they did not exhibit immunostaining for Ang-1. A minority oftubules did not have brush borders, as assessed by PAS staining,but did exhibit immunostaining for Ang-1 and were thus mostlikely to be distal tubules.
Figure 1. Angiopoietin-1 (Ang-1) immunohistochemical staining in renal cortex. Sections were counterstained with hematoxylin and reacted with anti-Ang-1 antibody (in D, the antibody was prereacted with the immunizing peptide). (A) A kidney from a vehicle-treated mouse demonstrated faint linear (arrow) Ang-1 immunostaining (brown) between tubules. (B) One day after folic acid (FA) administration, most epithelia were flattened. Small arteries (v) and a minor subset of tubules (*) exhibited immunostaining for Ang-1. (C) Three days after FA administration, Ang-1 immunostaining was observed in subsets of tubules (*) and undesignated cells (arrows) between tubules. The dilated Bowman’s space should be noted. (D) An area similar to that in C demonstrated no significant staining after prereaction of the antibody with the immunizing peptide. (E) By 14 d after FA administration, most areas regenerated and downregulated Ang-1. (F) Ang-1 was detected in vessels (v) in atrophic and fibrotic areas. Bars, 40 µm.
Figure 2. Ang-1 and periodic acid-Schiff (PAS) staining. A section through the outer cortex, 1 d after FA administration, was probed with anti-Ang-1 antibody and counterstained with hematoxylin and PAS stain. The flattened tubular epithelia should be noted. Most tubules have a thin pink brush border (arrows) and are therefore proximal tubules (pt); these do not exhibit immunostaining for Ang-1. A minority of tubules (*) do not have a brush border but do exhibit immunostaining for Ang-1 (brown); these are most likely distal tubules. Bar, 40 µm.
No gross structural arterial changes were noted after FA administration(Figure 3, A and B). Large cortical arteries in control andvehicle-treated mice demonstrated no Ang-1 immunostaining (Figure 3A),whereas Ang-1 was detected in arterial muscle layers inFA-treated organs (Figure 3B). The normal delicate pattern ofthe vasa rectae was disrupted after FA administration, and thesewere surrounded by tubules containing flattened epithelia (Figure 3, C and D).The morphologic features of this region returnedto normal by 7 d after FA administration (data not shown). Controlsamples exhibited Ang-1 immunostaining in vasa rectae (Figure 3C),but Ang-1 immunostaining became diffuse in this location1 to 3 d after FA administration (Figure 3D).
Figure 3. Ang-1 immunohistochemical staining in arteries and medulla. Sections were reacted with anti-Ang-1 antibody and hematoxylin. (A) No significant immunostaining was detected in a large cortical artery (a) from a vehicle-treated mouse. (B) Ang-1 immunostaining was noted in the muscular layer of a cortical artery (a) 7 d after FA administration. (C) A kidney from a vehicle-treated mouse demonstrated Ang-1 immunostaining in the vasa rectae (arrows). (D) Three days after FA administration, the vasa rectae were disorganized (arrows) and surrounded by tubules with flattened epithelia (*); diffuse Ang-1 immunoreactivity was apparent in this area. Bars, 40 µm.
Tie-2 immunostaining was detected in glomerular and corticalperitubular capillaries in control and vehicle-treated kidneys(Figure 4A). At all stages after FA administration, Tie-2 immunostainingwas detected in capillaries located between acutely damaged(Figure 4B) and regenerated (Figure 4C) cortical tubules. Tie-2capillary immunostaining was also detected in cortical regionsaffected by tubular atrophy and interstitial fibrosis (Figure 4D).Faint Tie-2 immunostaining was detected in the vasa rectaeof vehicle-treated animals (Figure 4E) and during the firstfew days after FA administration (Figure 4F); the signal appearedto be more prominent during recovery (Figure 4G).
Figure 4. Tie-2 immunohistochemical staining. Sections were reacted with anti-Tie-2 antibody and hematoxylin. (A to D) Cortex. (E to G) Outer medulla. (A) A low level of Tie-2 immunostaining (brown) was noted in capillaries (arrows) between tubules in a vehicle-treated animal. (B) Two days after FA administration, immunostaining was noted in capillaries (arrows) between damaged tubules (*). (C) Tie-2 immunostaining was detected in dilated capillaries between regenerated tubules 7 d after FA administration. (D) In atrophic and fibrotic areas, Tie-2 immunostaining was apparent in interstitial capillaries. (E) Tie-2 immunostaining was noted in the vasa rectae of a vehicle-treated mouse. (F) One day after FA administration, the outer medulla was disrupted, with flattened tubule epithelia (*) around vasa rectae that expressed Tie-2. (G) During regeneration, Tie-2 immunostaining was detected in vasa rectae. Bars, 40 µm.
As assessed by Western blotting for Ang-1, relatively weak bandsat 50 and 70 kD (probably representing the monomer and a glycosylatedform, respectively) and a stronger band at 100 kD (most likelyan Ang-1 dimer) were detected in kidney samples from control,vehicle-treated, and FA-treated mice (Figure 5). These signalswere abolished after prereaction of the primary antibody withAng-1 immunizing peptide (data not shown). As assessed by densitometricanalysis of the 100-kD band, statistically significant upregulationof Ang-1 protein levels, compared with time-matched, vehicle-treated,control kidneys, was observed at 1, 3, 7, and 14 d after FAadministration, which was consistent with the increased Ang-1immunohistochemical signal observed for this nephropathy. Ang-2Western blotting demonstrated two major bands, at 50 and 70kD (Figure 5), which probably represent the monomer and a glycosylatedform, respectively (Alpha Diagnostic International data sheet).Ang-2 signals were of similar intensities in organs from vehicle-and FA-treated animals (Figure 5) and were abolished after prereactionof the primary antibody with Ang-2 peptide (data not shown).Tie-2 protein was detected at all times in both the FA- andvehicle-treated groups, with statistically significant upregulationat 14 d after induction of nephrotoxicity.
Figure 5. Western blotting. C0, untreated controls; C1, C3, C7, and C14, kidneys 1, 3, 7, and 14 d, respectively, after vehicle administration; F1, F3, F7, and F14, kidneys after FA administration. On the left, representative Western blots from three data sets are shown. On the right, densitometric analysis results are presented as means ± SEM (n = 3 for each group, at each time point) (), control; , FA). *P < 0.05 for FA-treated group versus time-matched, vehicle-treated group. (A) An Ang-1 blot shows minor bands at 50 and 70 kD (most likely representing the monomer and glycosylated monomer, respectively) and a major band at approximately 100 kD (most likely representing the dimer). Statistically significant upregulation of signal, with respect to the 100-kD band, after FA administration should be noted. (B) An Ang-2 blot shows major bands at 50 and 70 kD (probably representing the monomer and a glycosylated form, respectively). Signals were of similar intensities for the vehicle- and FA-treated groups. (C) A Tie-2 blot shows a band at 160 kD. Tie-2 was significantly upregulated at day 14 in the FA-treated group.
In all groups, PECAM protein was detected in glomerular andperitubular capillaries (Figure 6, A to D), in locations similarto those for Tie-2, as described above. In areas of corticalregeneration, peritubular capillaries immunostained for eitherTie-2 or PECAM appeared to have larger lumina than did capillariesfrom similar areas in vehicle-treated organs (e.g., compareFigure 4C with Figure 4A for Tie-2 and Figure 6, C and F, withFigure 6, A and E, for PECAM). Examination of 20 high-powerfields for each kidney from vehicle-treated mice revealed nocells expressing both PECAM and PCNA (Figure 6E). In contrast,all high-power fields for kidney cortex 2 or 3 d after FA administrationcontained one to 10 peritubular cells that coexpressed PCNAand PECAM (Figure 6F), consistent with capillary proliferation.Endothelial proliferation was not detected at 7 and 14 d (datanot shown).
Figure 6. Platelet-endothelial cell adhesion molecule (PECAM) immunostaining. Cortical sections were reacted with an anti-PECAM antibody (brown positive reaction). Sections in E and F were also immunostained with a proliferating cell nuclear antigen (PCNA)-specific antibody (blue positive reaction). Sections in A to D were counterstained with hematoxylin. (A) In a control kidney, PECAM immunostaining was observed in capillaries (arrows) between tubules and in glomeruli. (B) One day after FA administration, PECAM immunostaining was noted in distended capillaries (arrows) between damaged tubules (*). (C) Fourteen days after FA administration, PECAM was detected in capillaries (arrows) between regenerated tubules. (D) PECAM immunostaining was noted in fibrotic interstitium between atrophic tubules. (E) In control samples, PCNA-positive nuclei were absent from cortical capillaries immunostained for PECAM. (F) High-power fields for kidneys 2 to 3 d after FA administration showed proliferating nuclei (five are indicated by arrows) in PECAM-expressing capillaries. Other, PECAM-negative, proliferating cells were noted in tubules (arrowheads) and interstitium (unmarked). Bars: 40 µm in A to D; 20 µm in E and F.
Because macrophages have been implicated in Ang-1 expression(28), sections were probed with anti-F4/80 antibody. In kidneysfrom vehicle-treated mice, positive cells were rarely detectedin the cortex (Figure 7A), whereas peritubular staining appearedto be moderately increased in the first few days after nephrotoxicity(Figure 7B) and during regeneration (Figure 7C) and was markedlyincreased in fibrotic and atrophic areas (Figure 7D).
Figure 7. Macrophage immunostaining. Cortical sections were probed with anti-F4/80 antibody and counterstained with hematoxylin. (A) A kidney from a vehicle-treated mouse contained sparse macrophages (brown). (B) Three days after FA administration, F4/80 immunostaining was prominent between damaged tubules. (C) In regenerated areas on day 14, only sparse macrophages remained. (D) In contrast, F4/80 immunostaining was prominent in fibrotic and atrophic locations. Bars, 40 µm.
This study demonstrates that FA-induced nephrotoxicity is associatedwith increased renal Ang-1 protein levels. Additionally, capillariesadjacent to damaged epithelia express Tie-2 and proliferate.To put these results in perspective, it is useful to brieflyreview what else is known regarding Ang and Tie genes in thekidney.
Tie-1, an "orphan receptor" homologue of Tie-2, is expressedby renal mesenchymal cells at the start of metanephrogenesis;these precursors differentiate into glomerular and interstitialcapillaries after transplantation of metanephroi into the renalcortex of neonatal mice (29,30). Metanephroi also express Ang-1,Ang-2, and Tie-2, with mRNA levels being downregulated in adultorgans (26). Ang-1 transcripts are localized to mesenchyme,maturing glomeruli, and cortical and medullary tubules, whereasarterial endothelia and capillaries express Tie-2 (26). Ang-2is expressed by walls of differentiating renal arteries, mesangialcells, and thin descending limbs of loops of Henle (26,31,32).These expression patterns for Ang and Tie genes suggest involvementin renal vascular maturation. Intriguingly, the observationthat tyrosine-phosphorylated Tie-2 is detectable in adult kidneys(26) is consistent with a yet-to-be defined role for this signalingsystem in mature organs. Indeed, in this study, Ang-1, Ang-2,and Tie-2 immunoreactive proteins were detected by Western blottingin control kidneys, with Ang-1 and Tie-2 immunoreactivity beingnoted between cortical tubules and in vasa rectae.
In the first few days after FA-induced nephropathy, the mostprominent sites of Ang-1 expression were a subset of damagedcortical tubules (which were judged to be distal tubules onthe basis of their size, distribution, and lack of PAS-stainedbrush borders) and renal arterial walls. The suggestion, basedon immunohistochemical findings, that Ang-1 protein was moreprominent in FA-induced nephrotoxicity was supported by ourdensitometric Western blot analyses, which demonstrated statisticallysignificantly increased Ang-1 levels from 1 to 14 d after FAadministration, compared with time-matched controls. If we acceptthe premise that Ang-1 protein is synthesized by epithelialand vascular smooth muscle cells, then it is possible to envisageparacrine signaling to nearby Tie-2-expressing endothelia. Itis notable that Zimmermann et al. (4) reported that a higherdose of FA (500 mg/kg) than used in these experiments inducedrenal vascular changes in rats, including fibrinoid medial lesionsof the arcuate and interlobular arteries; it is possible thatmore detailed examination of renal arteries in our study mighthave revealed subtle changes that were not evident in routinehistologic examinations.
Further experiments are needed to define stimuli that upregulateAng-1 in FA-induced nephropathy, using, for example, isolatedcells exposed to pathologic stimuli, including hypoxia/ischemia,stretch, and cytokines. Such stimuli have been reported to alterAng-2 and/or Tie-2 expression in vitro (32–34), althoughtheir effects on Ang-1 are presently unclear. It has been suggestedthat monocytes/macrophages express Ang at sites of inflammation(28). We immunolocalized renal macrophages and observed a modestincrease in the first few days after FA administration, withmore marked infiltration in atrophic and fibrotic areas laterin the course of the disease. Therefore, these cells representanother potential source of Ang-1 after nephrotoxicity.
Various roles can be postulated for Ang-1 after acute nephrotoxicity.Peters et al. (35) demonstrated that Tie-2 expression in breasttumors was correlated with microvessel growth, and Shyu et al.(36) reported that intramuscular expression of Ang-1 enhancedrevascularization in rabbit ischemic hindlimbs. Contemporaneouswith increased Ang-1 expression after FA administration, weobserved that renal cortical peritubular capillaries appearedto undergo remodeling. Although the dilated lumina of thesecapillaries could have been attributable to vascular stasis(e.g., caused by inflammatory cells), we also documented coexpressionof PCNA and PECAM, which represents evidence of endothelialproliferation. In control animals, peritubular endothelia didnot proliferate. Although glomerular endothelial cell turnoveris very low in normal adult kidneys, proliferation and apoptosisare upregulated in experimental glomerulopathies (37,38). Furthermore,there is preliminary evidence that peritubular capillary proliferationand remodeling associated with upregulated Tie-2 expressionoccur in mice after subtotal nephrectomy (39). Finally, we speculatethat Ang-1 might also limit capillary leakiness and inflammatorycell transmigration in nephrotoxicity (21,22).
The biologic effects of the Ang depend on an interplay betweenthe Ang themselves and VEGF signaling. In vitro, Ang-1 and VEGFsynergize to induce capillary sprouting (19). Furthermore, withabundant VEGF, Ang-2 disrupts vessels, facilitating sprouting;conversely, when ambient VEGF levels are low, Ang-2 causes vesselregression (40). Although Ang-2 was detected by Western blottingin this study, levels were unchanged during the course of nephropathy.Our unpublished observations demonstrated that VEGF immunostainingis not upregulated in the first few days of FA-induced nephropathybut becomes prominent at later stages in areas affected by atrophyand fibrosis (Long DA, Woolf AS, Yuan HT, unpublished observations).Recently, Kim et al. (41) reported that VEGF administrationaccelerated renal recovery in experimental thrombotic microangiopathy.In the future, it would be intriguing to explore the effectsof experimental up- or downregulation of Ang and VEGF on recoveryfrom FA-induced nephropathy.
In conclusion, our results provide preliminary evidence thatthe Ang/Tie-2 signaling system, which is normally prominentlyexpressed during development, may play a role during recoveryfrom renal injury. FA-induced nephropathy in mice is associatedwith upregulation of Ang-1 protein expression in epithelia andvessels of the renal cortex. At the same time, PECAM- and Tie-2-expressingcapillaries adjacent to damaged cortical tubules undergo proliferation.Further experiments are required to establish whether theseevents are functionally related.
Acknowledgments
This work was supported by Wellcome Trust Project Grant 058005,a Medical Research Council studentship, and the Kidney ResearchAid Fund.
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Received for publication January 12, 2001.
Accepted for publication May 25, 2001.
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Top
Abstract
Introduction
(10); and invasion by monocytes/macrophages (3). Most animal studies of acute renal failure have focused on tubular damage and regeneration, and this has led to the testing of therapies that aim to enhance epithelial recovery via the administration of growth factors (11). 
-associated protein expressed in S phase (27). PECAM immunostaining was performed first, as described above. Sections were then microwaved in citric acid (1.9 g/L, pH 6.0) for 5 min, and endogenous biotin was masked by using an avidin-biotin blocking kit (Vector Laboratories). Slides were incubated overnight at 4°C with biotin-conjugated mouse anti-human PCNA (1:50) and were reacted with streptavidin-conjugated horseradish peroxidase followed by a diaminobenzidine metal-enhanced solution, resulting in a blue signal for PCNA. 
), control; 
, FA). *P < 0.05 for FA-treated group versus time-matched, vehicle-treated group. (A) An Ang-1 blot shows minor bands at 50 and 70 kD (most likely representing the monomer and glycosylated monomer, respectively) and a major band at approximately 100 kD (most likely representing the dimer). Statistically significant upregulation of signal, with respect to the 100-kD band, after FA administration should be noted. (B) An Ang-2 blot shows major bands at 50 and 70 kD (probably representing the monomer and a glycosylated form, respectively). Signals were of similar intensities for the vehicle- and FA-treated groups. (C) A Tie-2 blot shows a band at 160 kD. Tie-2 was significantly upregulated at day 14 in the FA-treated group.
