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*
Division of Mammalian Development, National Institute for Medical
Research, London, United Kingdom
Division of Protein Structure, National Institute for Medical Research,
London, United Kingdom
Nephro-Urology Unit, Institute of Child Health, London, United
Kingdom
Correspondence to Professor Adrian S. Woolf, Nephro-Urology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Phone: 44-20-7242-9789 x0712; Fax: 44-20-7916-0011; E-mail: a.woolf{at}ich.ucl.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Galectin-3 is a two-domain molecule with an N-terminal proline-rich domain and a C-terminal carbohydrate recognition domain (1,2). Galectin-3 binds to simple ß-galactosides such as lactosamine, Galß1-4GlcNAc, and with higher affinity to polylactosamine chains (6,7,8). Addition of sialic acid, N-acetylgalactosamine or fucose substituents to the terminal galactose of a polylactosamine chain enhances galectin-3 binding still further (6,7,8). Amino acid residues Arg139 and Ser232 in the carbohydrate-binding pocket of galectin-3 are essential for binding to extended oligosaccharides (9).
Galectin-3 is expressed by hamster metanephroi (10) and during human nephrogenesis (11). In human ureteric bud branch tips, galectin-3 immunolocalizes to apical epithelial surfaces, but, as this lineage matures, it appears in a less-polarized distribution in cytoplasm and cell surface membranes, with high expression in fetal medullary collecting ducts. Postnatally, galectin-3 is downregulated but remains expressed by intercalated cells. The protein is expressed apically in ureteric bud—derived cysts in human dysplastic kidneys (11); the aberrant hyperproliferation of these epithelia correlates with expression of PAX2, a potentially oncogenic transcription factor, and BCL2, a survival molecule (12). Similar upregulation and location of galectin-3 occurs in collecting duct cysts in human autosomal recessive polycystic kidneys (11).
In culture, galectin-3 is synthesized and secreted by Madin Darby canine kidney (MDCK) cells (13). These form cysts in collagen gels, and galectin-3 co-localizes with laminin on the basal surface of these structures (14). Cyst growth is modulated by perturbing galectin-3 activity: blocking antibodies increase their expansion, whereas exogenously added galectin-3 slows their expansion. Ricin-resistant MDCK cells, which fail to transfer galactose residues during synthesis of glycoconjugates and lack extracellular galectin-3 receptors, undergo enhanced cystogenesis and abnormal morphogenesis (15). Therefore, in vitro, MDCK cell growth is regulated by galectin-3 interactions at basal membranes. In normal renal development, the changing distribution of the protein could regulate collecting duct growth while expression in cysts may reflect a failure of ureteric bud lineage maturation. We describe galectin-3 expression in mouse metanephroi and examine the bioactivity of this lectin in organ culture. Our evidence supports the hypothesis that galectin-3 modulates ureteric bud branching.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Reverse Transcription-PCR
RNA was extracted from organs, as described
(19,20).
Reverse transcription-PCR (RT-PCR) galectin-3 primers were as follows:
5'-AGC TTA TCC TGG CTC AAC TG-3' for sense primer, corresponding
to nucleotides 405 to 424 of mouse galectin-3 (GENEMBL X16074), and
5'-CGG AGG TTC TTC ATC CGA TG-3' for the antisense primer,
corresponding to nucleotides 853 to 872. ß-Actin primers were as
described (19). Thirty-five
cycles of amplification were performed: 94°C for 1 min, 60°C for 1
min, 72°C for 3 min, and finally 72°C for 7 min. Seven µ1 of each
reaction was electrophoresed through a 1% agarose gel and 467-bp galectin-3
and 540-bp ß-actin products sought after ethidium bromide staining.
Negative controls included reactions with water substituted for RNA or reverse
transcriptase.
Histochemistry
Organs were fixed in 2% paraformaldehyde, paraffin embedded, and sectioned
at 4 µm. After quenching endogenous peroxidase with 3% hydrogen peroxide in
methanol for 30 min, sections were incubated at 37°C with
anti—galectin-3 antibodies followed by secondary antibody and a
streptavidin-biotin-peroxidase system
(11). Negative controls
comprised omission of primary antibody, substitution with preimmune serum, or
preabsorption of primary antibody with recombinant galectin-3. Nuclei were
stained with hematoxylin or methyl green, and some samples were stained with
periodic acid-Schiff (PAS) to define proximal tubule brush borders. Cultured
metanephroi were processed by three methods. Some were fixed in ice-cold
methanol for 10 min, washed in phosphate-buffered saline (PBS) containing 0.1%
Triton X-100, and reacted with calbindin-D28K (1:200) or laminin antibodies
(1:1000) at 4°C. They were incubated with secondary antibodies
(TR-conjugated for calbindin D28K and FITC-conjugated for laminin) for 4 h,
defining ureteric bud branches (calbindin-D28K positive/laminin
positive) and mesenchyme-derived nephrons (calbindin-D28K
negative/laminin positive). Others were exposed to FITC-DBA
(50 µg/ml in PBS) at 4°C overnight. These samples were mounted in
Vectashield (Vector Laboratories, Burlingame, CA) and viewed with a confocal
laser scanning microscope (Leica, Heidelberg, Germany) or a Zeiss Axiophot
epifluorescence microscope (Oberkochen, Germany). Other cultured metanephroi
were fixed in paraformaldehyde, paraffin embedded, and sectioned, as described
above.
Organ Culture
Embryonic day 11 (E11) and E12 metanephroi were explanted onto Millicell-CM
filters (Millipore Corporation, Bedford, MA) or 1 µm Nuclepore filters
(Corning Costar Corporation, Cambridge, MA) at the interface between air/5%
CO2 atmosphere and medium and cultured at 37°C in control
medium comprising Dulbecco's modified Eagle's medium (DMEM)-F12, 5% fetal calf
serum, 1 mM glutamine, and antibiotics, as described
(19,20).
In one set of experiments with E11 or E12 organs, medium was supplemented
with galectins (1 µM) as follows: (1) recombinant hamster
galectin-3 (21); (2)
a recombinant protein, 
1-93, containing the C-terminal domain but
lacking most of the N-terminal domain
(22): this fragment retains
carbohydrate-binding specificity comparable to full-length galectin-3;
(3) a mutant protein, SS, of full-length galectin-3 in which
mutagenesis of residue Arg139 to serine abolishes the preferred binding of
galectin-3 to extended oligosaccharides
(9); and (4)
galectin-1, a molecule that constitutively lacks the critical Arg139 residue
in its binding pocket and exhibits a similar binding specificity to the SS
mutant
(7,8).
A medium change, with or without additives, was performed every day during the
organ culture period. Metanephroi were examined daily under a dissecting
and/or phase-contrast microscope for up to 4 d. In some experiments, exogenous
galectin-3 was removed after 2 d by washing in PBS, and culture continued for
an additional 2 d in control medium. Branch tips and nephrons formed in
culture were examined by whole-mount immunostaining: the numbers of branch
tips in each group were expressed as a mean ± SD. To examine directly
the effects of galectins on mesenchymal differentiation, we placed isolated
mesenchymes from single E11 metanephroi adjacent to the dorsal edge of an E11
spinal cord segment (23) and
incubated them in the presence or absence of exogenous galectins (1 µM).
Isolated mesenchymes cultured without spinal cord failed to differentiate into
nephrons (not shown).
We also examined effects of galectin-3 blocking antibody on E12 organs cultured for 7 d. We used dilutions of antibody (1:20 to 1:50) known to be bioactive in MDCK cyst assays (14); preimmune rabbit antiserum, in the same dilutions, was used for controls. A medium change was performed every day during the organ culture period. Preliminary experiments demonstrated that culture of E16 to 17 organs, which already express abundant galectin-3 protein, resulted in necrosis within 24 h because of their large size; hence, they were not suitable for blocking experiments. Therefore, we elected to explant E12 organs and culture them for up to 1 wk. This longer-term culture strategy using earlier-stage organs permitted ex vivo differentiation of nephrons and collecting ducts and upregulation of galectin-3 expression by collecting ducts with good tissue viability, providing a "window of opportunity" in which galectin-3 antisera could be added and potential effects could be recorded. Explants were photographed at 3 and 7 d and examined in paraffin-embedded sections at 7 d. Ureteric bud branch tips and glomeruli were not quantified because these explants acquired considerable complexity over a week in culture.
MDCK Assays
MDCK type II cells, trypsinized from monolayer cultures, were resuspended
in ice-cold 2.4 mg/ml collagen type I solution at 1 x 105
cells/ml and dispensed into multiwell plates (1 ml/well; Costar). After
gelation at 37°C for 45 min, cells were grown in DMEM with 10% fetal calf
serum for up to 2 mo (14).
Some cultures were grown throughout with galectins, as described in the
Results section. Cyst growth was assessed by measuring the maximum diameter of
randomly selected spherical cysts, as described
(14). Results for each
experimental group, comprising 10 fields in triplicate wells, were expressed
as mean ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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The E11 metanephros contained mesenchyme surrounding a just-branched ureteric bud, while at E12, mesenchymal condensates had formed near branch tips; galectin-3 immunostaining was absent in these stages (not shown). At E16, immunostaining was noted in tubules in the superficial cortex and in larger diameter tubules in the deep cortex (Figure 2, A and B). In the E17 nephrogenic zone (Figure 2C), stems of ureteric bud branches expressed galectin-3, whereas their tips had barely detectable staining. Deep in the E17 cortex, collecting ducts expressed galectin-3 (Figure 2D), some with cytoplasmic or weak basal staining, others with a subset of cells with intense cytoplasmic immunostaining. Large medullary collecting ducts displayed intense galectin-3 staining basally (Figure 2E). Epithelia lining the E19 papilla also expressed the lectin (Figure 2F). The impression that galectin-3 immunostaining was restricted to the ureteric bud/collecting duct lineage was supported by PAS counterstaining, which confirmed that proximal tubules were galectin-3 negative (not shown). No immunostaining was noted in glomeruli. Postnatally, galectin-3 protein was detected in a uniform apical distribution in cortical tubules that were PAS negative and that were narrow in diameter, i.e., they were not proximal tubules; these galectin-3—expressing cortical tubules were often located adjacent to glomeruli, suggesting that they were distal convoluted tubules. Galectin-3 was also immunolocalized to deep medullary collecting ducts and the urothelium of the renal papilla (not shown).
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Effects of Galectin-3 Added to Organ Culture
E11 metanephroi in control medium underwent extensive morphogenesis over 4
d: the ureteric bud, which had branched once in vivo, produced
approximately five further branching events over this period, and mesenchyme
formed numerous nephrons. In cultures that had been treated with 1 µM
galectin-3, a decrease in ureteric branch tips was visible after wholemount
immunostaining (Figure 3). In a
typical quantified experiment assessed at 3 d, there were 86 ± 13 tips
in control cultures versus 23 ± 5 (27% of controls) in
lectin-treated cultures (Figure
4A). With the use of daily inverted microscope observations of
individual organs, an inhibitory effect on branching of galectin-3 was visible
after 2 d (not shown). Moreover, the inhibition was largely reversible: after
2 d of galectin-3 treatment followed by 2 d without exogenous lectin, five of
five explants had recovered and the mean branching was 80% of the controls
(Figure 3; data not shown).
Although not formally quantified, qualitatively similar results were obtained
with E12 metanephric cultures after visualizing ureteric bud derivatives with
FITC-DBA (not shown). We examined the properties of galectin-3 required for
branching inhibition (Figure
4A). 1-93 protein (1 µM), containing the C-terminal
domain but lacking most of the N-terminal domain of galectin-3
(22), significantly inhibited
branching. In contrast, neither the galectin-3 SS mutant
(9) nor galectin-1 inhibited
branching at the same concentrations. We also tested these galectins, at the
same concentrations used in metanephric cultures, in the MDCK cyst assay
(Figure 4B). Galectin-3
effectively slowed cyst expansion. The carbohydrate recognition fragment
1-193 protein (22) was
also inhibitory, whereas, at equivalent concentrations, the galectin-3 SS
mutant or galectin-1 was not.
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Most (17 of 23) E11 metanephroi that were treated with galectin-3 failed to
form nephrons, and in the minority of cases in which nephrons did form, their
numbers were reduced. Although nephrons were not formally quantified, this
effect is depicted in Figure 3. The 1-193 protein behaved similarly to intact galectin-3, but neither
the SS mutant nor galectin-1 had a discernible effect on nephron formation
(not shown). We also examined E12 explants cultured for 4 d: unlike small E11
explants, these organs were large enough to process reproducibly by paraffin
embedding and histology. In controls, ureteric bud branches were flanked by
nephron condensates and vesicles (Figure
5A), whereas glomeruli formed deeper in the explants
(Figure 5C). However, in organs
that were exposed to 1 µM galectin-3, ureteric branches appeared distorted,
and although condensed mesenchyme was observed, these organs lacked mature
nephrons (Figure 5, B and D).
To distinguish whether galectin-3 inhibited mesenchymal to epithelial
transformation directly or indirectly, we cultured isolated mesenchymes with a
potent inducer, the spinal cord. Cultures differentiated similarly in the
presence or absence of 1 µM galectin-3: after 3 d, all mesenchymes formed
laminin-positive epithelial tubules (Figure
6).
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Effects of Galectin-3—Blocking Antisera in Organ Culture
Control E12 explants in 1:20 preimmune rabbit antiserum (n = 25,
two experiments) grew over 7 d into increasingly complex structures that
contained numerous glomeruli with normal tubule morphogenesis
(Figure 7, A through C). By the
end of the experiment, galectin-3 immunohistochemistry revealed a similar
pattern to E16 to 17 organs in vivo (not shown). When organs were
cultured in 1:20 galectin-3 antiserum (n = 20, two experiments),
organs appeared less compact and the formation of structures that resembled
dilated tubules was noted at the periphery of the organ at 3 d
(Figure 7D); this malformation
was maintained until day 7 (Figure
7E). Figure 7F
depicts a tissue section on the last day of blockade, showing a dilated
epithelial tubule on the edge of the organ, resembling a small cyst,
surrounded by undifferentiated tissue lacking glomeruli. Most tubules in these
organs stained with DBA agglutinin (data not shown), suggesting that they were
ureteric bud-derived structures. When control medium was substituted for
blocking antisera on day 3 (n = 25, two experiments), relatively
normal growth resumed in the last 4 d of culture, with organs becoming more
compact with formation of glomeruli and normal caliber tubules
(Figure 7, G through I).
Qualitatively similar but lesser tubule malformations were generated with
1:100 blocking antisera (n = 10; not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Exogenous galectin-3 perturbed branching in E11 and E12 explants. This inhibition occurred at relatively low concentrations and was largely reversible, hence unlikely to be due to unspecific cytotoxicity. Perhaps addition of exogenous galectin-3 to basal surfaces of ureteric bud epithelium, analogous to the pattern of endogenous protein in maturing medullary collecting ducts, converts ureteric tips to a more trunklike fate, unable to produce or receive the inducing signals. This action might be mediated in several ways.
One possibility is that galectin-3 affects growth signaling pathways.
Candidate molecules that promote branching and that originate from ureteric
bud lineage epithelia include Wnt 11
(23) and bone morphogenetic
protein 7 (24), whereas
mesenchymal-derived signals include glial cell line—derived neurotrophic
factor (GDNF) (19), fibroblast
growth factor 7 (25), and
integrin 
8ß1 (26).
Such branch-inducing pathways might be inhibited by galectin-3, perhaps by
modulation of ureteric bud cell-surface receptors that bind these signaling
molecules. Another explanation for the effects of galectin-3 comes from the
observation that the lectin slows MDCK cyst expansion. Indeed, the inhibitory
actions on metanephric branching and MDCK cyst expansion are mediated by a
galectin-3 domain that permits high-affinity binding to glycoproteins that
carry extended glycan chains. Expansion and banching of the ureteric bud
epithelium and enlargement of MDCK cysts in gels probably require a delicate
balance between the relative strength of cell—cell and cell—matrix
adhesions. Of note, metanephric growth is tunicamycin sensitive
(27), implicating
glycosylation of adhesive proteins, whereas other experiments show that
galectin-3 binds matrix components, such as laminins
(22,28)
and cell-surface integrins including ß1 integrins
(29,30),
and these same classes of molecules are implicated in branching in
vivo and MDCK tubulogenesis in vitro
(16,31,32,33,34,35).
Therefore, extracellular galectin-3 might modulate metanephric growth by cross
linking laminins to membrane-bound receptors.
The above experiments added galectin-3 during the earliest days of E11/12 metanephric growth in culture when there may be little, if any, endogenous galectin-3 protein. These studies represent a relatively pure, albeit artificial, situation of galectin-3 excess. Therefore, we attempted to ascertain whether endogenous galectin-3 had a role in metanephrogenesis by blocking its activity with antiserum when the protein was upregulated ex vivo during extended culture of E12 explants. We cultured E12 metanephroi for 7 d in dilutions of antiserum that enhance MDCK cyst expansion (14). Explant growth was altered in a reproducible and characteristic manner. Controls, exposed to preimmune sera, grew as compact structures with generation of normal caliber, slender tubules as well as glomeruli. In contrast, organs with galectin-3 blockade increased in area but became less compact and were composed of distorted tubules, some of which were abnormally large in cross section, superficially resembling small cysts. We believe that these are distorted ureteric bud branches, based on their peripheral distribution and that they bound DBA lectin, which are overgrowing, since our preliminary results demonstrated that >90% of nuclei in the walls of these structures express proliferating cell nuclear antigen (12). Because galectin-3 blockade was partially reversible, it was unlikely to be an unspecific toxic effect. We speculate that when the activity of endogenous metanephric galectin-3 is blocked, collecting duct epithelial growth is uninhibited and the normal pattern of branching morphogenesis is also lost.
Hence, an excess and also a deficiency of galectin-3 each leads to abnormal growth; other examples illustrate that not only "too little" but also "too much" of a molecule can perturb renal development. One example is the PAX2 transcription factor, a molecule expressed in the ureteric bud/collecting duct and early nephron lineages and downregulated as maturation proceeds. PAX2 deficiency leads to urinary tract growth failure. whereas overexpression is associated with another type of malformation, cyst formation (12,36,37). Another example is provided by RET, the GDNF receptor expressed by ureteric bud branch tips. When RET signaling is downregulated, e.g., by antibody blockade in organ culture (19), branching is deficient and nephron generation is also inhibited as a secondary effect. Conversely, when a constitutively active RET protein is expressed by the whole ureteric tree, another malformation is generated: nodules caused by deregulated branching (38).
The current study also demonstrates that exogenous galectin-3 downregulated mesenchymal to epithelial transformation in whole cultured metanephroi. However, it had no effect on tubules induced by spinal cord in isolated E11 renal mesenchymes. Thus, exogenous galectin-3 is likely to perturb mesenchyme induction in whole-organ culture indirectly, perhaps by impairing bud-derived mesenchyme growth factors (39). We also observed that antisera to galectin-3 prevented formation of glomeruli after 7 d of organ culture. We postulate that this specific effect is also likely to be secondary to perturbed bud development, especially because we failed to immunolocalize galectin-3 protein to the mesenchymal/nephron lineage.
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Conclusions |
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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8ß1 is critically important for epithelialmesenchymal
interactions during kidney morphogenesis. Cell88
: 603-613,1997[Medline]
3ß1 integrin has a crucial role in kidney and lung
organogenesis. Development 122:3537
-3547, 1996[Abstract]
2ß1 integrin expression results in reduced cyst formation, failure
of hepatocyte growth factor/scatter factor-induced branching morphogenesis and
apoptosis. J Cell Sci 108:3531
-3540, 1995[Abstract]
6ß1 is involved in kidney tubulogenesis in
vitro. J Cell Sci 109:2801
-2810, 1996[Abstract]
6 subunit is essential for epithelial polarization in developing kidney
tubules. J Cell Biol 111:1265
-1273, 1990This article has been cited by other articles:
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HOME
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EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
1-93
protein carbohydrate recognition domain (CRD), a mutant SS galectin-3 (Gal3
mutant) and galectin-1 (Gall). Means ± SD of ureteric bud branch tips
were counted in cultured explants (n = 11 for each set). (B) MDCK
cells were cultured as cysts in collagen type I gels for 1, 5, 10, 15, 20, and
25 d. Media contained additives as in A. The maximal diameter (mean ±
SD) of at least 10 separate cysts in triplicate cultures were measured.
Comparisons between controls and treated cultures at similar time points are
designated * P < 0.05 and ** P <
0.01 (t test) with unmarked bars representing no significant
difference.
