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REGULAR ARTICLES |
*
Nephrourology Unit, Institute of Child Health, University College London
Medical School, London, United Kingdom
Regeneron Pharmaceuticals, Inc., Tarrytown, New York.
Correspondence to Dr. Hai Tao Yuan, Nephrourology Unit, Institute of Child Health, University College London Medical School, 30 Guilford Street, London WC1EN 1EH, United Kingdom. Phone: 0171 242 9789; Fax: 0171 916 0011; E-mail: h.yuan{at}ich.ucl.ac.uk
Abstract
Abstract. The Tie-2 receptor tyrosine kinase transduces embryonic onic endothelial differentiation, with Angiopoietin-1 (Ang-1) acting as a stimulatory ligand and Ang-2 postulated to be a naturally occurring inhibitor. Expression of these genes was sought during mouse kidney maturation from the onset of glomerulogenesis (embryonic day 14 [E14]) to the end of nephron formation (2 wk postnatal [P2]), and during medullary maturation into adulthood (P8). Using Northern and slot blotting of RNA extracted from whole organs, these three genes were expressed throughout the experimental period with peak levels at P2 to P3. By in situ hybridization analysis at E18, P1, and P3, Ang-1 mRNA was found to localize to condensing renal mesenchymal cells, proximal tubules, and glomeruli in addition to maturing tubules of the outer medulla. In contrast, Ang-2 transcripts were more spatially restricted, being detected only in differentiating outer medullary tubules and the vasa recta bundle area. Using in situ hybridization and immunohistochemistry, Tie-2 was detected in capillaries of the nephrogenic cortex, glomerular tufts, cortical interstitium, and medulla including vessels in the vasa recta. Using Western blotting of protein extracted from whole organs, Tie-2 protein was detected between E14 and P8 with tyrosine phosphorylated Tie-2 evident from E18. These data are consistent with the hypothesis that Tie-2 has roles in maturation of both glomeruli and vasa rectae.
Each adult kidney receives 10% of the cardiac output, a high blood flow supplying glomerular capillaries, which ultrafilter plasma, and also peritubular capillaries, including the vasa rectae, which contribute to the countercurrent system (1,2) The mouse metanephros forms on embryonic day 11 (E11) when the ureteric bud penetrates avascular renal mesenchyme (3,4). The bud branches into collecting ducts, and mesenchyme differentiates into nephrons (glomerular, proximal tubule, and loop of Henle epithelia). Capillaries are prominent in metanephric stroma at E12 and the first vascular glomeruli form at E14 (4). In mice, 80% of glomeruli form after birth, a process completed by 2 wk postnatal (5). Vasa rectae mature alongside loops of Henle, which grow into the medulla even after nephron generation has ended (6). The focus of study is currently on the molecules that direct differentiation of these microcirculations (4,7).
Various classes of molecule are implicated in embryonic endothelial development, including transcription factors (8) and cell adhesion molecules (9). Importantly, phenotypes of null mutant mice indicate that the sequential expression of specific growth factors and their receptor tyrosine kinases (RTK) determine survival, proliferation, differentiation, and morphogenesis of endothelia in vivo (10,11). One RTK group includes vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) and VEGFR-2 (Flk-1/KDR), which are, respectively, expressed from mouse E7.5 and E7.0 in yolk sac blood islands and endothelial precursors of the aorta and heart (12). VEGFR-2 is required for endothelial formation, whereas VEGFR-1 modulates vessel assembly (13,14). Vascular endothelial growth factor (VEGF), VEGFR-1, and VEGFR-2 are expressed by uninduced mouse metanephric mesenchyme (15) and later in murine (15,16,17) and human (18) kidney development. Functional experiments implicate VEGF in glomerulogenesis in vivo (19) and in hypoxia-driven metanephric endothelial proliferation in vitro (20).
Another class of endothelial RTK is called Tie (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor similar domains) (21,22). The onset of Tie-1 and Tie-2 (previously called Tek) expression postdate VEGFR-2 but precede von Willebrand factor (12,23,24). Tie-1 is an orphan RTK, and null mutant mice die in late gestation with impaired vessel integrity (25,26). The gene is expressed in mouse metanephric mesenchyme from the inception of nephrogenesis and in interstitial and glomerular capillaries at later stages of development (15).
Angiopoietin-1 (Ang-1) is a ligand for the RTK called Tie-2 (27). Although Ang-1 does not cause significant endothelial proliferation in culture (27), it does induce sprouting in vitro, an activity synergizing with VEGF (28). Ang-1 (29) and Tie-2 (25,30) null mutant mice die by E12 with a homogenization of vessel caliber and poor branching. In addition, endothelia were separated from sparse pericytes and growth-retarded myocardium. It was speculated that Tie-2 signaling caused endothelial stabilization with secondary maturational effects on differentiating pericytes and cardiomyocytes, effects elicited by endothelial-derived molecules including platelet-derived growth factor-BB, heparin-binding epidermal growth factor, and neuregulin (31). Ang-2, a homologue of Ang-1, binds Tie-2 but does not cause tyrosine phosphorylation in cultured endothelia (32). In endothelia, Ang-2 antagonizes Ang-1-induced Tie-2 phosphorylation, while Ang-2 overexpression in vivo disrupts embryonic vessel formation and causes cardiac defects resembling Tie-2 and Ang-1 null mutants (32). Furthermore, Witzenbichler et al. (33) found that Ang-1 was chemotactic for endothelial cells and that Ang-2 blocked this effect. Hence, Ang-2 may be a naturally occurring antagonist of Ang-1.
Evidence is emerging about the sites of angiopoietin expression. Davis et al. (27) localized Ang-1 mRNA in embryonic mice to cells near developing endothelia, which themselves expressed Tie-2, with a similar pattern in embryonic myocardium and endocardium. Maisonpierre et al. (32) reported that Ang-2 transcripts localized to areas in mouse embryos "likely to be endothelial cells or closely associated pericytes" with an expression pattern more spatially restricted than Ang-1. It has also been reported that subsets of endothelial cells express Ang-2 in vitro, whereas smooth muscle cells express Ang-1 and Ang-2 (33,34). These reports are consistent with the paracrine modulation of Tie-2 by both Ang-1 and Ang-2, with the additional possibility of an autocrine action of Ang-2 on endothelia.
To date, angiopoietin expression during renal ontogeny has not been investigated. The experiments in our current study provide novel information on Ang-1, Ang-2, and Tie-2 expression during mouse kidney maturation.
Materials and Methods
Reagents
Reagents were obtained from Sigma Chemical Co. unless otherwise
specified.
Tissues
We studied a normal mouse strain (CD1) with the day of the vaginal plug
designated E0. In these mice, the metanephros forms on E11 and the first
glomeruli with primitive capillary loops forms on E14
(4). New layers of nephrons
continue to be generated postnatally for 1 to 2 wk. The ages examined in the
study were E14, E15, E16, E17, E18 and postnatal weeks 1, 2, 3, 4, and 8 (P1,
P2, P3, P4, and P8).
Northern and Slot Blotting for Angiopoietin and Tie Genes
Total RNA was isolated with Tri-Reagent from kidneys at 10 time points
(E14, E15, E16, E17, E18, P1, P2, P3, P4, and P8). Separate pools of 10 to 20
embryonic kidneys were used for the prenatal stages, and a single organ was
used for each postnatal time point, with the whole experiment performed in
triplicate. For Northern blotting, 20 µg of total RNA was electrophoresed
in 1% formaldehyde-denatured agarose gel in 1 x
3-[N-morpholino]propanesulfonic acid buffer, transferred onto Hybond-N
membrane (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), and
fixed with UV-Stratalinker (Stratagene, La Jolla, CA). For slot blotting, 10
µg of denatured total RNA (in a total volume of 200 µl) was transferred
onto pre-wet Hybond-N membrane using Bio-Dot SF apparatus (Bio-Rad,
Hert-fordshire, United Kingdom) and fixed with UV-Stratalinker. Plasmids with
mouse cDNA inserts were: Tie-2 (1.2 kb; kindly provided by W. Risau,
Max-Planck-Institute, Bad Nauheim, Germany), Tie-1 (238 bp; isolated from
metanephric cDNA, with identity confirmed by sequencing;
(15)), as well as Ang-1 (560
bp) and Ang-2 (680 bp) (Regeneron Pharmaceuticals, Tarrytown, NY). Inserts
were isolated after digestion with appropriate restriction enzymes, and random
primer labeling was performed with Prime-a-Gene labeling system (Promega).
Unincorporated labeled-dCTP was removed by using a push-column (Stratagene).
Blots were prehybridized with Quick-Hybsolution (Stratagene) at 65°C for
30 min and hybridized with specific probes at 65°C for 2 h. After
hybridization, the filters were washed twice with 2 x SSC at room
temperature for 30 min and once with 0.1 x SSC/0.1% sodium dodecyl
sulfate (SDS) at 65°C for 30 min. X-films were exposed to filters for 12
to 48 h at -70°C. The equality of loading for Northern blotting was
confirmed by visualizing 28S and 18S rRNA. For slot blotting, 28S rRNA signals
were obtained by hybridizing the blots with 28S oligonucleotide (10 pmol/ml)
as described (35). The signal
intensity relative to 28S rRNA of each sample was measured with an image
analysis program (Phoretix 1D, Newcastle upon Tyne, United Kingdom), and the
ratio of target signal to 28S rRNA of E14 was used as arbitrary value 1. The
slot blotting results were expressed as the mean and SD of the three assays
performed at each time point. Because of the probable different affinities of
the angiopoietin and Tie probes to their target mRNA, as well as the variation
in labeling efficiency of different probes, it was not possible to compare the
absolute amounts of transcripts between genes.
Tie-2 Immunoprecipitation and Western Blotting for Tie-2 and
Phosphotyrosine
We currently have no antibodies available for Ang-1 and Ang-2 and therefore
these experiments were restricted to Tie-2 and the phosphorylated RTK.
Separate pools of 10 to 20 embryonic kidneys were used for the prenatal stages
(E14, E16, E17, and E18), and a single organ was used for each postnatal time
point (P1, P2, P3, P4, and P8), with the whole experiment performed in
triplicate. Kidneys were homogenized in radioimmunoprecipitation assay buffer
(30 µl/ml of 2.2 mg/ml aprotinin; 10 µl/ml of 10 µg/ml
phenylmethylsulfonyl fluoride; 10 µl/ml of 100 mM sodium orthovanadate) at
4°C, and the supernatants were collected by centrifugation at 13,000 rpm
for 30 min. Supernatants were used for protein determination (BCA protein
assay; Pierce, Rockford, IL). To 0.2-mg protein samples (in 0.5 ml), 0.2 µg
of rabbit antibody raised against amino acids 1103-1122 in the carboxy
terminus of human Tie-2 (Santa Cruz Biotechnology, Santa Cruz, CA) was added
and incubated at 4°C for 2 h. After this, 8 µl of Protein A-agarose
(Santa Cruz Biotechnology) was added and incubated at 4°C with agitation
for at least 4 h. Beads were washed with radioimmunoprecipitation assay buffer
four times and collected by centrifugation at 2500 rpm for 5 min at 4°C.
After the final wash, the supernatant was aspirated and discarded, and the
pellet was resuspended in 30 µl of 1 x electrophoresis buffer.
Samples were boiled for 3 min and separated on an SDS-8% polyacrylamide gel
electrophoresis gel. Proteins were transferred to nitrocellulose membranes
(Amersham Pharmacia Biotech) by electroblotting (Bio-Rad). Blots were blocked
overnight at 4°C with 5% (wt/vol) fat-free milk powder, 0.3% (vol/vol)
Tween 20 in phosphate-buffered saline, and then incubated with rabbit
anti-human Tie-2 antibody (1:1000 in blocking solution) for 2 h at 4°C.
After washing in blocking solution, blots were incubated for 30 min with
horseradish peroxidase-conjugated second antibodies diluted 1:1500 in blocking
solution. Blots were washed three times with blocking solution and once with
phosphate-buffered saline. Immunocomplexes were developed using an enhanced
horseradish peroxidase/luminol chemiluminescence reagent (Du Pont New England
Nuclear, Boston, MA). Proteins were sized with Rainbow markers (Amersham
Pharmacia Biotech). After visualizing the immobilized proteins, the antibody
complex was stripped by rocking the blot in 200 mM glycine, 200 mM NaCl, pH
2.3, for 2 h at room temperature. The blot was then reprobed with
antiphosphotyrosine antibody (PY-99, Santa Cruz) using the same procedure
described above.
In Situ Hybridization for Ang-1, Ang-2, and Tie-2
Kidney sections were analyzed in longitudinal and cross-sectional planes in
organs harvested at E18, P1, and P3 from three animals at each stage. Mouse
Tie-2, Ang-1, and Ang-2 cDNA plasmids were linearized with restriction
enzymes, and sense and antisense uridine triphosphate-digoxigenin-labeled
riboprobes were prepared using linearized plasmid cDNA as template, the
appropriate RNA polymerase, and the conditions recommended in the Dig RNA
labeling kit (Boehringer Mannheim, Sussex, United Kingdom). Tie-2 transcript
was subjected to a limited alkaline hydrolysis to produce a 400-bp probe.
In situ hybridization was performed as described
(36) with minor modifications.
Paraffin-embedded tissue sections (7 µm) were dewaxed by treatment with
Histoclear (DiaMed, Windham, ME), treated with proteinase K (20 µg/ml) at
37°C for 10 min, and post-fixed in 4% paraformaldehyde. Sections were
covered with 50 µl of prehybridization mix (50% vol/vol formamide, 5x
SSC, 1x Denhardt's reagent, heat-denatured salmon sperm DNA 0.1 mg/ml,
and 10% wt/vol dextran sulfate) for 30 min at 65°C, followed by 50 µl
of the same mixture containing the digoxigenin-labeled riboprobe. A glass
cover-slip was applied and hybridization was allowed to occur at 65°C
overnight. Sections were washed at 65°C with 25% formamide in 2x SSC
for 1 h, 1x SSC and 0.1% SDS for 30 min, and 0.1x SSC and 0.1% SDS
for 30 min. Hybridized probe was detected by incubation with antidigoxigenin
antibody (1:1000) conjugated to alkaline phosphatase, followed by the
chromogen solution, nitroblue tetrazolium, and
5-bromo-4-chloro-3-indolylphosphate toluidine. This technique produced a
blue/purple in situ hybridization signal. Slides were washed and
mounted with Citifluor (Chemical Labs, London, United Kingdom). In some
samples, counterstaining with eosin was performed to facilitate cellular
localization of the in situ hybridization signal; however, this was
only feasible when the signal was intense because counterstaining was found to
obscure weaker in situ signals. Controls run in parallel with each
experiment included tissue sections that were incubated in hybridization mix
without riboprobe added or hybridized in an identical manner with
digoxigenin-labeled sense riboprobe. It should be noted that the preparation
of sections for the in situ technique resulted in tissue details that
were somewhat less distinct compared with the other histologic techniques
described in this article.
Immunohistochemistry for Tie-2, CD34, and Tamm-Horsfall
Glycoprotein
Organs were fixed in 4% paraformaldehyde, and 8-µm paraffin sections
were treated with trypsin (1 mg/ml) for 10 min at 37°C. Endogenous
peroxidase was quenched with 3% H2O2 in methanol for 30
min at room temperature, and sections were blocked in 10% goat serum with 0.1%
Tween 20. They were reacted with rabbit anti-human Tie-2 (1:2000; Santa Cruz),
a rat anti-mouse monoclonal antibody to the endothelial molecule CD34
(37) (1:50; Pharmingen, San
Diego, CA), and a sheep anti-human antibody to the loop of Henle molecule
Tamm-Horsfall glycoprotein
(38) (1:50; Europa
Bio-products, Ltd., Cambridge, United Kingdom). Bound antibodies were detected
with a streptavidin-biotin peroxidase system (ABC kit; DAKO, High Wycombe,
United Kingdom). This procedure produced a brown positive signal. Controls
included omission of the primary antibodies and preincubation of Tie-2
antibody with Tie-2 peptide (Santa Cruz). Some sections were counterstained
with hematoxylin, eosin, or periodic acid-Schiff to enhance, respectively,
cell nuclei, cytoplasm, or proximal tubule brush border.
Results
Northern and Slot Blotting
Figure 1A depicts a Northern
blot of gene expression at 10 stages from E14 to P8. As assessed by ethidium
bromide staining for 28S and 18S rRNA, the loading of all lanes was similar.
Major transcripts were observed for Ang-1 (4.1 kb), Ang-2 (2.8 kb), Tie-1 (4.2
kb), and Tie-2 (4.5 kb) at sizes similar to those reported in other studies
(32,39),
with the four genes expressed at all times points studied.
Figure 1B shows a densitometric
analysis of gene expression measured by slot blotting after standardization to
28S rRNA. Based on the mean standardized levels of three separate experiments,
the transcript levels peaked at P2 to P3 for Ang-1, Ang-2, and Tie-2, and at
P2 for Tie-1. Thereafter, the levels of mRNA of all four genes decreased. The
relative rise of transcript levels for Ang-1, Tie-1, and Tie-2 between E14 and
P2 was approximately twofold but was 25-fold for Ang-2. No direct comparisons
could be made between mRNA levels of these genes because the signals generated
in slot blotting are determined not only by transcript levels, but also by the
individual hybridization efficiency and radioactive labeling of each
probe.
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Western Blotting for Tie-2 and Phosphotyrosine
Figure 2 depicts Western
blotting for Tie-2 and the tyrosine phosphorylated protein after
immunoprecipitation with an anti-Tie-2 antibody. Antibody to Tie-2 detected a
band at 140 kD, as described for other tissues
(24), at all stages examined
(E14, E16, E17, E18, P1, P2, P3, P4, and P8). To investigate the in
vivo activation of Tie-2, we stripped and reprobed the same blots with a
phosphotyrosine antibody, and a definite band for tyrosine phosphorylated
Tie-2 was detected from E18 onward. Although no attempt was made to formally
measure the intensity of bands, the patterns described above were the same in
three separate experiments using proteins extracted from pools of E14 to E18
metanephroi or single organs at postnatal stages.
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Overview of in Situ Hybridization
Analyses were performed to localize Ang-1, Ang-2, and Tie-2 mRNA at E18,
P1, and P3. Figures
3,4,5,6,7,8
depict results that are representative of three sets of experiments. Sense
ribroprobes for the genes generated low background signals, which were used to
assess the significance of signals on adjacent tissue sections that were
generated by the appropriate antisense probes. Because of the numerous ages
and tissue regions analyzed, we have shown select, but representative, data
for Ang-1, Ang-2, and Tie-2 sense probes.
Table 1 summarizes the main
sites of gene expression, which are described in detail below.
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In Situ Hybridization at E18
At E18, the outer cortex of the kidney contained condensing mesenchymal
cells adjacent to ureteric bud branch tips and nephron precursors including
vesicles and primitive glomeruli (Figure
3A). Ang-1 mRNA was expressed by condensing renal mesenchyme,
whereas ureteric bud branch tips did not express significant levels of this
transcript (Figure 3, B and C).
A faint signal for Ang-1 also localized over primitive glomeruli and proximal
tubules deeper in the cortex (not shown). No specific signal was observed in
the nephrogenic zone with the antisense Ang-2 probe
(Figure 3D) when compared with
the Ang-2 sense probe control (not shown). Tie-2 transcripts were identified
in cells located between primitive tubules, in configurations consistent with
differentiating capillaries (Figure
3E). Using the current methodology, Tie-2 mRNA could not be
detected over primitive glomeruli (Figure
3E). When sections of the nephrogenic zone were reacted with CD34
antibody, weak positive signals were detected between developing tubules
(Figure 3F). At E18, the outer
medulla contained loose associations of loops of Henle growing toward the
inner medulla. Thick ascending limbs immunostained for Tamm-Horsfall
glycoprotein, as did the terminal portion of descending limbs
(Figure 4, A and B). These
loops were flanked by collecting ducts and CD34-positive capillaries
(Figure 4, C and D). The
diameter of all of these tubules was relatively uniform compared with their
mature derivatives. Ang-1 mRNA was expressed by most tubules in this zone
(Figure 4E), whereas Ang-2
transcripts were restricted to a subset of structures
(Figure 4F). Tie-2 mRNA
localized to capillaries aligned in parallel to tubules
(Figure 4G). Sense control for
Tie-2 are shown in Figure 4H.
No significant signal was found in sense controls for Ang-1 and Ang-2 (not
shown).
In Situ Hybridization at P1
By P1, the nephrogenic zone was no longer prominent, and cortex and
medullary structures had become more distinct with aggregations of loops of
Henle noted in the outer medulla. In the deep cortex, Ang-1 transcripts were
prominent in proximal tubules (Figure 5, A
and B) with no significant signal generated by the Ang-1 sense
probe (data not shown). A weak signal for Ang-1 was also noted in cells in the
outermost cortex and glomeruli (not shown). Using in situ
hybridization, Ang-2 mRNA was not detected in the epithelia of the cortex
(Figure 5C), while signals for
Tie-2 were found in interstitial capillaries
(Figure 5D) and in the
endothelial lining of cortical arteries
(Figure 5D).
Figure 6 depicts the outer
medulla at P1. In Figure 6A,
loops of Henle were evident, often with their "U" turns in this
region. Ascending limbs stained for Tamm-Horsfall glycoprotein, as did the
terminal portion of the thinner, descending limbs. The region was rich in
CD34-positive capillaries aligned in parallel and at right angles to tubules
(Figure 6B). A weak signal for
Ang-1 mRNA was detected in diverse tubules in this region
(Figure 6, C and D), while an
intense signal for Ang-2 was restricted to the thinnest of these structures
(Figure 6, E and F), which were
therefore likely to represent thin limbs of loops of Henle derived from the
outermost nephrons or maturing vasa rectae. Tie-2 transcripts were expressed
in capillaries running parallel to the tubules
(Figure 6, G and H) and by
inner medullary capillaries (not shown).
In Situ Hybridization at P3
In the P3 cortex, Ang-1 was detected in glomerular tufts with weaker
signals in Bowman's capsules and proximal tubules
(Figure 7, A and B). Although
all cells of the glomerulus appeared positive versus the Ang-1 sense
probe (Figure 7A), the highest
signal was localized to cells at the periphery of the tuft, where podocytes
reside. As for E18 and P1, Ang-2 expression could not be detected in epithelia
of the P3 cortex (not shown). Tie-2 transcripts were detected in cells inside
glomerular tufts, where endothelia and mesangial cells reside
(Figure 7, C and D). For
comparison, CD34 immunostaining of endothelial cells within glomerular tufts
is depicted in Figure 7E. Tie-2
transcripts were also detected in arterial endothelia
(Figure 7F). In the outer
medulla, the mature arrangement of the vasa recta was evident at this stage
(Figure 8). Ascending limbs of
loops of Henle, collecting ducts, and interbundle capillaries surrounded vasa
recta bundles. In rodents, these bundles contain three structures
(1,2):
(1) descending vasa recta capillaries comprised of endothelium and
surrounding pericytes; (2) ascending vasa recta capillaries comprised
of highly fenestrated endothelia lacking pericytes; and (3)
descending thin limbs of loops of Henle derived from short-looped nephrons
that incorporate into the outer part of the vasa recta bundle.
Figure 8A shows Tamm-Horsfall
immunostained tubules surrounding the vasa recta bundle region, while
Figure 8B shows CD34
immunostained capillaries in the vasa recta and in an interbundle plexus. The
antisense probe for Ang-1 produced a barely significant signal within the
bundle in cells closely associated with capillaries
(Figure 8, C and D). In
situ hybridization for Ang-2 showed strong signals in the bundle region
in cells closely associated with vasa recta capillaries
(Figure 8E) and in thin tubules
on the periphery of the bundle (Figures 8,
E and F), which may represent descending thin loops of Henle.
Strong signals for Tie-2 transcripts were detected closely associated with
capillaries of the vasa recta, but Tie-2 transcripts were sparse or absent in
the interbundle region (Figure 8, G and
H).
Immunohistochemistry for Tie-2
At E18, linear Tie-2 immunostaining was noted in an interstitial
distribution in the nephrogenic cortex and in cores of immature glomeruli
(Figure 9A). It is probable
that Tie-2 immunohistochemistry was more sensitive than in situ
hybridization because the latter method did not detect Tie-2 in primitive
glomeruli (Figure 3E).
Tie-2-positive, spindle-shaped cells were detected in the E18 fetal medulla.
Some appeared in a loose network and may have represented developing capillary
plexi (Figure 9B). In the P1
outer medulla, Tie-2 immunostaining was detected in capillaries aligned
alongside tubules (data not shown). In the P3 cortex, Tie-2 protein was
detected in the cores of glomerular tufts, where capillaries and mesangial
cells are located, and in capillaries between tubules
(Figure 9C), with no
significant staining when the antibody was preabsorbed with immunising peptide
(Figure 9D). At P3, Tie-2
immunostaining was prominent in capillaries of the vasa rectae in the outer
medulla as shown in longitudinal section
(Figure 9E).
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Discussion
Before this report, only fragmentary information was available regarding Tie-2 expression in renal ontogeny, and no information was available for the angiopoietins. In 1994, Landels et al. (40) presented preliminary data reporting the detection of Tie-2 transcripts after amplification and sequencing of mouse E12 metanephric cDNA using degenerate primers to tyrosine kinase domains. Using Western blotting, Kee et al. (41) detected Tie-2 protein in perinatal rat kidney with levels variably downregulated in the adults, while Wong et al. (42) immunolocalized Tie-2 to postnatal rat glomeruli. Our current data provide a more comprehensive picture of Tie-2 expression in murine renal maturation and constitute the first report of angiopoietins in renal ontogeny. On the basis of these results, we hypothesize that the angiopoietins and Tie-2 may have roles in differentiation of glomeruli and vasa rectae.
By examining 10 time points from E14 to P8 using Northern blotting, we found that Ang-1, Ang-2, and Tie-2 were expressed during mouse kidney maturation and, as assessed by quantitative slot blotting, maximal levels of these mRNA occurred at P2 to P3. We noted that the relative increase in Ang-1 and Tie-2 levels between E14 and P2/P3 was approximately twofold, whereas Ang-2 increased approximately 25-fold. This temporal pattern is consistent with the concept that Ang-2 acts as a "brake" to fine-tune Tie-2 signaling, as outlined in the introductory remarks. Using Western blotting, we demonstrated that Tie-2 protein was expressed through the study period, and when we studied renal Tie-2 phosphorylation we found that a definite signal could be detected from E18 onward.
However, speculation about angiopoietin/Tie-2 signaling based on analyses of RNA and protein derived from whole organs may not be extrapolated to specific patterns of gene expression or receptor tyrosine kinase phosphorylation, which might occur in different regions within the organ. Therefore, we investigated the in situ expression of Ang-1, Ang-2, and Tie-2 during renal maturation by seeking gene transcripts and, additionally, Tie-2 protein at E18, P1, and P3. We found different tissue expression patterns for Ang-1, Ang-2, and Tie-2. Here, we summarize and discuss gene expression in the cortex and medulla separately.
During cortical development, Ang-1 mRNA first localized to condensing renal mesenchyme and thereafter, at P1-3, to maturing proximal tubules and glomeruli where expression was prominent in the outer cells of the tuft, i.e., presumptive podocytes. Of note, VEGF is also expressed by podocytes (43), where it may maintain fenestrations in adjacent capillaries. An in situ hybridization signal for Ang-2 could not be detected in epithelia of the renal cortex at any stage examined. Transcripts for Tie-2 were initially detected in interstitial areas between nascent epithelia of the nephrogenic cortex in locations where CD34 immunostaining was faint or absent. These observations are consistent with a hypothesis that Tie-2 is expressed earlier in the endothelial lineage than CD34. As the cortex matured, Tie-2 mRNA was detected inside glomerular tufts and in locations expected of interstitial capillaries. The patterns of Tie-2 transcripts as assessed by in situ hybridization were similar to those of Tie-2 protein, as assessed by immunohisto-chemistry. However, the latter method may be more sensitive because it detected Tie-2 in the cores of primitive glomeruli at E18, where in situ hybridization was negative. The exact glomerular cells that express Tie-2 will require clarification by immunoelectron microscopy, but we suggest that they may include both endothelia and mesangial cells. In this respect, it is notable that mesangial cells in vitro have been found to express certain RTK in common with endothelia including VEGFR-1 (44) and the hepatocyte growth factor receptor Met (45).
All tubules that traversed the E18 outer medulla expressed Ang-1 transcripts, and a less intense signal was detected in tubules in this region at P1. By P3, only a very faint signal for Ang-1 could be detected in the vasa recta bundle. At E18, Ang-2 mRNA was expressed in a subset of outer medullary structures of uncertain designation. However, by P1, Ang-2 expression was clearly localized to the thinnest of structures that traversed this region, strongly suggesting that they were descending loops of Henle or maturing vasa rectae. The impression was strengthened by the pattern at P3 when Ang-2 was expressed by thin tubules at the periphery of vasa recta bundles, where descending limbs reside in rodents (1,2). At this stage, Ang-2 was also expressed by cells within the bundle, which were closely associated with the vasa recta capillaries. We consider that these cells were most likely endothelia or supporting pericytes, although the resolution of the current technique precludes definitive cellular assignment. As assessed by in situ hybridization and immunohistochemistry, Tie-2-expressing cells were noted in the outer medulla. At E18 and P1, these cells were located in capillaries aligned parallel to the tubules. By P3, Tie-2 transcripts had become localized to the vasa recta capillaries. Of note, Tie-2 expression appeared absent from the interbundle region despite the presence of a CD34-expressing capillary plexus.
Taken together, our descriptive data are consistent with the hypothesis that Ang-1 may have multiple roles in the maturation of the renal microcirculations. We speculate that renal mesenchymal cell-derived growth factor may be chemotactic for sprouting endothelia in the nephrogenic zone. Second, the expression of Ang-1 by proximal tubules may by important for the maturation of cortical interstitial capillaries that express Tie-2. Furthermore, since Ang-1 and Tie-2 are expressed in glomeruli, the factor may be important in the formation and/or maintenance of glomerular capillaries. Since in situ hybridization did not detect Ang-2 in forming glomeruli at any stage examined, a role cannot be ascribed to this factor in glomerulogenesis. It is possible, however, that more sensitive techniques might be required to detect Ang-2 transcripts in glomeruli or other locations in the cortex. Our results are also consistent with a theory that Tie-2 signaling is important for the development of the vasa recta capillaries. In particular, it was striking that the intensity of outer medulla in situ hybridization signal for Ang-1 fell between E18 to P3, whereas the signal for Ang-2 was strong at P3. Therefore, if Ang-2 acts as an antagonist of Ang-1 as outlined in the introductory remarks, we speculate that a changing balance of Ang-1 and Ang-2 may initially stimulate, and then terminate, Tie-2 signaling to modulate construction of the vasa recta microcirculation.
Although our results lead to speculation regarding the roles of the angiopoietins and Tie-2 in differentiation of the renal vasculature, definitive proof of their roles awaits functional data. Unfortunately, the relatively early embryonic death of Ang-1 and Tie-2 null mutant mice precludes analyses of the potential roles of these genes during metanephrogenesis. In the future, an analysis of the kidneys of chimeric mice constituted from null and wild-type cells, as has been described for Tie-1 (46), may be useful. In addition, further work is clearly needed to fully document the potential expression of the angiopoietins and Tie-2 in the early nephrogenic period, between E11 and E14.
Our current study further indicates that Tie-1 mRNA is expressed from E14, with levels peaking at P2 and expression downregulated thereafter. This is a pattern similar to Tie-2. Examination of Tie-1lcz/Tie-1lczn- chimeras have suggested a role for Tie-1 in metanephric vessel formation since mutant cells did not significantly contribute to renal vasculature (46). Loughna et al. (15) detected Tie-1 mRNA in renal mesenchyme from E10.5 to E15, and the fate of Tie-1-expressing cells was studied by transplantation of E11 Tie-1/LacZ organs into the neonatal nephrogenic cortex of wild-type mice (15), a site that facilitates growth of precursors into filtering glomeruli (47). Transgene-expressing glomerular and interstitial capillaries developed in these transplants, demonstrating that at least part of the renal vasculature can form in situ (15). Furthermore, in organ culture of E13 Tie-1/LacZ mouse metanephroi, Loughna et al. found that reporter gene expression was upregulated in hypoxia (48). Hypoxia has also been reported to upregulate VEGFR-1 and VEGFR-2 in murine metanephric organ culture (20,48), and it would be interesting to study the regulation of Tie-2 expression in this milieu. In this respect, it is known that endothelial Per-AHR-ARNT-Sim domain protein 1, an endothelial-specific transcription factor activated by hypoxia, can upregulate Tie-2 (49).
Of note, a human genetic disease has illuminated the function of Tie-2. Vikkula et al. (50) investigated two families with dominant inheritance of vascular malformations composed of a relative excess of endothelial cells versus pericytes. They identified a missense mutation of Tie-2 on chromosome 9p causing an arginine to tryptophan substitution in the kinase domain. This increased Tie-2 tyrosine phosphorylation in vitro (50). Human Ang-1 and Ang-2 genes respectively localize to 8q22 and 8p23, but mutations have yet to be implicated in disease. Other clues regarding pathogenetic roles of Tie-2 derive from studies of healing skin wounds (42), where Tie-2 expression was upregulated in neovessels. Capillary microcirculations are also implicated in kidney disease pathogenesis (7) and, in the future, it will be interesting to study the expression patterns of the angiopoietin and the Tie genes in these disorders.
Acknowledgments
Acknowledgments
This work was supported by British Heart Foundation Project Grant 96120 and the Kidney Research Aid Fund.
Footnotes
American Society of Nephrology
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