Oncogene (1998) 17, 661 ± 666
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
http://www.stockton-press.co.uk/onc
SHORT REPORT
Sex di€erence in immunostaining of RET in the adult mouse kidney
SY Chan and DCW Lee
Department of Paediatrics, the University of Hong Kong, Queen Mary Hospital, Hong Kong
The c-ret proto-oncogene encodes a receptor tyrosine
kinase which is important for the development of the
kidney and the enteric nervous system. During nephrogenesis, c-ret is expressed in the ureteric bud epithelium
and later in its derivative, the collecting duct. This takes
place during 11 ± 17.5 days post-coitum (d.p.c.) in the
mouse and our immunohistochemical study showed that
the RET protein co-localized with the transcript. At
18.5 d.p.c. the kidney is fully di€erentiated. At
18.5 d.p.c., 1 week and 10 weeks old, RET was found
in the proximal convoluted tubules, which is formed from
the condensed mesenchyme. This suggests that c-ret may
also play a role in kidney function. For the 10 weeks old
kidney, RET immunostaining in male was concentrated
on the basolateral side while female had a stronger
staining in the whole cell. Furthermore, cytoplasmic
staining was observed in male whereas both cytoplasmic
and nuclear staining was found in female. c-ret transcript
was detected by RT ± PCR, and in situ hybridization
showed its expression throughout the kidney. The reason
for the sex-speci®c staining and the role of RET in
kidney function remain to be determined.
Keywords: c-ret; RET; kidney
The c-ret proto-oncogene, a member of the receptor
tyrosine kinase (RTK) gene superfamily, was ®rst
identi®ed by transfection of human T-cell lymphoma
DNA into NIH3T3 ®broblast (Takahashi et al., 1985).
Similar to other RTK, the c-ret protein (RET) is
characterized by an extracellular ligand binding
domain, a transmembrane domain and a cytoplasmic
tyrosine kinase domain. In addition, the extracellular
domain of RET contains homology to the intermolecular binding region of the cadherin family, although
no adhesive properties was found (Iwamoto et al.,
1993).
Mutations in c-ret are implicated in a number of
human diseases. In papillary thyroid carcinomas,
somatic rearrangements result in constitutive activation of c-ret. Germ-line point mutations in c-ret are
associated with several dominantly inherited cancer
syndromes including multiple endocrine neoplasia types
2A and 2B (MEN2A, MEN2B) and familial medullary
thyroid carcinoma (FMTC). These cancer syndromes
seem to be caused by gain-of-function mutations in cret. On the other hand, loss-of-function mutations in cret are associated with Hirschsprung disease, which is
the most common among congenital defects a€ecting
Correspondence: SY Chan
Received 2 January 1998; revised 11 March 1998; accepted 11 March
1998
the development of the enteric nervous system
(reviewed by Pasini et al., 1996).
Besides playing a role in enteric neurogenesis, c-ret is
important for kidney development. In mice homozygous for a null mutation in c-ret, enteric neurons
were absent. Besides, renal agenesis or severe dysgenesis occurred which was characterized by reduced
numbers of glomeruli, proximal and distal tubules,
reduced branchings of the ureter, absence of mature
collecting ducts and large regions of undi€erentiated
mesenchyme (Schuchardt et al., 1994). The phenotype
is similar to that of mice homozygous for a null
mutation in glial-cell-line-derived neurotrophic factor,
GDNF (Moore et al., 1996; Pichel et al., 1996; SaÂnchez
et al., 1996). Concurrently, binding of GDNF through
GDNFR-a to RET was shown biochemically, which
resulted in autophosphorylation of RET (Jing et al.,
1996; Treanor et al., 1996).
During kidney development, the c-ret transcript was
localized to the nephric duct at 8.5 ± 10.5 days postcoitum (d.p.c.), the ureteric bud epithelium at 11 ±
11.5 d.p.c. and the growing tips of the renal collecting
ducts at 13.5 ± 17.5 d.p.c. in mice (Pachnis et al., 1993;
Avantaggiato et al., 1994). In contrast the GDNF
transcript was expressed in the metanephric mesenchyme (Hellmich et al., 1996). This suggests the
involvement of GDNF/RET in mesenchymal-epithelial inductive interactions. From immunohistochemical
studies in 11.5, 13 and 16.5 d.p.c. rat embryonic
kidneys, RET was localized to the same areas where
RNA expression was found in mice. However, RET is
no longer detected in the two week-old or adult rat
kidneys (Tsuzuki et al., 1995).
Although RET was not detected in adult rat
kidneys, alternative spliced transcripts were detected
in the adult human kidney by using reverse transcription-polymerase chain reaction (RT ± PCR; Ivanchuk et
al., 1997). Here we report the study of c-ret expression
in embryonic, neonatal (day 7) and adult mouse
kidneys. Transcription was detected at all stages by
RT ± PCR. Surprisingly we found a sex-di€erence in
RET expression in the proximal convoluted tubules
(PCT) of adult kidneys.
RT ± PCR
Total RNA was extracted from kidneys of 14.5 d.p.c.
and adult ICR mice by the method of Chomczynski
and Sacchi (1987) and reverse transcribed by Superscript II reverse transcriptase (Gibco). c-ret cDNA
was ampli®ed by mh-ret 1 (5'-GAA AAC GCC TCC CAG AGT GAG; 2287 ± 2307) and mh-ret 2 (5'GGA GAT GAG GTC ACC CAT GGT; 2562 ± 2542).
The ampli®ed 276 bp fragment corresponds to the
kinase region of the mouse c-ret gene (Iwamoto et al.,
Immunostaining of RET in the mouse kidney
SY Chan and DCW Lee
662
1993). The 14.5 d.p.c. embryonic kidney served as a
positive control. The level of c-ret expression in the
adult kidney was found to be relatively lower than
that of the 14.5 d.p.c. kidney (Figure 1). The
speci®city of the PCR product was con®rmed by
PstI digestion to produce a 100 bp and a 176 bp
fragment (not shown).
In situ hybridization
ICR embryonic kidneys at 12.5, 14.5 and 18.5 d.p.c.,
neonatal (1 week old) and adult kidneys (10 weeks old)
of both sexes were employed. We also included kidneys
from one pregnant ICR, two of each sex of C57BL/6N
adult mice. In situ hybridization was performed
according to Wilkinson and Green (1990) using a cret riboprobe derived from pmcret7 (Pachnis et al.,
1993). Sections were examined after 7 days exposure.
In the positive controls, c-ret was detected in the
epithelial cells of the ureteric bud at 12.5 d.p.c. and the
growing tips of the collecting duct at 14.5 d.p.c.
(Figure 2a) and 18.5 d.p.c. (Figure 2b). The signal at
18.5 d.p.c. was relatively weaker than that of
14.5 d.p.c. In contrast, the c-ret transcript was found
ubiquitously in the neonatal (Figure 2c) and the adult
kidney.
Di€erent expression patterns of RET were found
between the developing and the adult kidneys. Staining
was completely abolished when the RET antibody was
preabsorbed with the RET peptide, indicating the
speci®city of staining. Cytoplasmic staining of RET
was found in the ureteric bud epithelium at 12.5 d.p.c.
and this is consistent with previous in situ hybridization
studies (Pachnis et al., 1993; Avantaggiato et al., 1994).
At 14.5 d.p.c., c-ret RNA was expressed in the growing
tips of the collecting duct at the periphery of the
kidney, but absent from the centrally located collecting
ducts which are more matured than those at the
periphery. In contrast, we found RET immunostaining
in some of the more centrally located collecting duct in
addition to the developing collecting duct. We also
Immunohistochemistry
The immunohistochemical staining was performed
with streptavidin-biotinylated peroxidase complex
(Dako), using one of the antibodies: (1) rabbit
polyclonal antibody against RET (Santa Cruz, Ret
C-19) 1 mg/ml, which recognizes the tyrosine kinase
domain at amino acids 784 ± 801. This antibody has
been shown to identify the two di€erent isoforms
(150 kDa and 170 kDa) of RET in Western analysis
of Xenopus embryo and mouse ®broblast extracts
(Durbec et al., 1996; Takahashi et al., 1993); (2) rabbit
polyclonal antibody against rat glycoprotein gp330
(kind gift from Dr RT McCluskey, Harvard Medical
School, Massachusetts General Hospital, Charlestown,
USA), which is found on the apical cell surface of
PCT (Bachinsky et al., 1993). The procedures for
immunohistochemical studies were described before
(Lee et al., 1998).
Figure 1 RT ± PCR analysis with c-ret (lanes 1 ± 3) and b actin
primers (lanes 4 ± 6). Templates used were: (2, 4) 14.5 d.p.c.
kidney cDNA; (3, 5) adult kidney cDNA; (1, 6) adult kidney
RNA. M: 100 bp ladders (Gibco)
Figure 2 In situ hybridization with c-ret anti-sense riboprobe.
Strong expression (arrows) was detected in the growing tips of the
collecting duct at 14.5 d.p.c. (a) and 18.5 d.p.c. (b), but localized
expression was lost in the neonatal kidney (c). Scale bar=0.05mm
Immunostaining of RET in the mouse kidney
SY Chan and DCW Lee
found RET in the ureter (Figure 3a and b). The
condensing renal vesicle and the metanephric mesenchyme were negative for staining.
In 18.5 d.p.c. kidneys, the expression site of RET
was di€erent from that of RNA. RET immunostaining
was observed in some segment of the PCT in the
subcortical region of the kidney but not in the growing
tips of the collecting duct at the periphery of the
kidney. Furthermore, there was cytoplasmic plus
nuclear staining (Figure 3c and d). In 7 days old
Figure 3 RET immunostaining in developing kidneys. Positive staining was found in: (a,b) 14.5 d.p.c. collecting ducts and ureter;
(c,d) 18.5 d.p.c. proximal convoluted tubules, PCT in the subcortical region, with strong cytoplasmic and nuclear staining as shown;
(e,f) 1 week old male with cytoplasmic staining in PCT; (g,h) 1 week old female with cytoplasmic and nuclear staining in PCT. Scale
bar in left panel=0.05 mm; in right panel=0.01 mm
663
Immunostaining of RET in the mouse kidney
SY Chan and DCW Lee
664
kidneys, RET staining was observed in some cells of
the PCT, not in the collecting duct. Nuclear and
cytoplasmic RET staining appeared in some cells of the
PCT in the female kidney (Figure 3g and h) whereas
mostly cytoplasmic staining was observed in the male
kidney (Figure 3e and f). This sex-speci®c pattern of
expression was obvious in adult kidneys, where no
nuclear staining was found in male.
To con®rm the sex-speci®c expression pattern, two
strains of mice, ICR and C57BL/6N, were studied.
Identical RET expression pattern was found in the
same sex in either strain. Staining was concentrated on
the basal side of the proximal tubule in male kidneys
(Figure 4a and c). However, cytoplasmic and nuclear
staining was observed in the PCT of female kidneys
(Figure 4b and d) including kidneys from a pregnant
mouse. In addition, the intensity of staining is
stronger in adult female than in adult male kidneys
(compare Figure 4a and b). It is interesting to note
that the early segment of the proximal tubule, as
identi®ed by its connection to the Bowman's capsule
and the tubular marker staining in the consecutive
section, was negative for RET staining (Figure 4a and
b arrowheads). This phenomenon was observed in
both sexes.
Discussion
Various studies have indicated that c-ret plays an
essential role in the development of the renal and the
nervous systems. Recently, c-ret transcripts were
detected in the adult human kidney (Ivanchuk et al.,
1997) and we also found expression in adult mouse
kidneys by RT ± PCR. Using in situ hybridization, we
found ubiquitous localization of the transcript in the
adult kidney. In our immunohistochemical studies,
RET was localized to the PCT at 18.5 d.p.c. and
Figure 4 Immunostaining in adult male (left panel) and female kidneys (right). Positive RET staining (a ± d) was found in PCT.
However, early segment of PCT, as identi®ed by its connection to the Bowman's capsule and positive gp330 staining (e,f
arrowheads), was negative for RET staining. Note that RET immunostaining was stronger in female and nuclear staining was
found. Scale bar in a, b, e, f=0.05 mm; in c, d=0.01 mm
Immunostaining of RET in the mouse kidney
SY Chan and DCW Lee
afterwards. This suggests that c-ret may also play a
role in the di€erentiated kidney.
The kidney is formed by reciprocal inductive
interactions between the ureteric bud and the
metanephric mesenchyme. The mesenchymal cells
induce the branching of the ureteric bud, which forms
the renal collecting system. The ureteric bud in turn
induces the mesenchyme to condense and form the
glomeruli and proximal and distal tubules (Saxen,
1987). The c-ret transcript was detected in the ureteric
bud epithelium at 11.5 d.p.c. and the growing tips of
the collecting duct at 13.5 ± 17.5 d.p.c. (Pachnis et al.,
1993) Consistently, we found RET in the cytoplasm of
the epithelial cells of the branching ureteric bud at
12.5 d.p.c. and the growing collecting ducts at
14.5 d.p.c. By 17.5 d.p.c., the mouse kidney is fully
di€erentiated and is able to function (Rugh, 1994). At
18.5 d.p.c., we found that c-ret expression in the
growing tips of the collecting duct has diminished.
RET immunostaining was found in the PCT in the
subcortical region of the kidney but not in the growing
tips of the collecting duct. This suggests that the
control of c-ret expression in the di€erentiated kidney
occurs at the level of translation. It will be interesting
to know which isoform of RET is expressed at di€erent
stages. In 1-week-old mouse kidneys, RET expression
was found in PCT. PCT was identi®ed by the apical
brush border in the tubular epithelial cells and positive
staining of gp330. Similarly, RET expression was
found in PCT in adult mice. However, it is also noted
that the early segment of PCT was negative for RET
staining. It is likely that RET plays a di€erent role
during development and in the functioning of the
kidney.
Although RET was found in the same segment of
the di€erentiated nephron in both sexes at 18.5 d.p.c.,
1-week postpartum and adulthood, we found a
di€erence in the staining pattern. In male, staining
was found in the cytoplasm, and is more concentrated
on the basal side in adult kidneys. The majority of
hormone and growth factor receptors are restricted to
the basolateral membranes of the renal tubule epithelia
to receive endocrine/paracrine signals from the
capillaries and the surrounding cells. For example,
epidermal growth factor receptor, a receptor tyrosine
kinase, is located exclusively to the basolateral
membranes of normal tubule epithelia (Harris, 1991).
Interestingly, we observed a stronger cytoplasmic
staining in the female than in the male adult kidney
in parallel experiments. Furthermore, in female
kidneys, nuclear and cytoplasmic staining was found
and the concentration of staining on the basal side was
less obvious. This phenomenon was con®rmed in two
di€erent strains of mouse and in pregnant female. The
reason for these di€erences in immunostaining is
unknown. Possibly this is caused by the binding of
ligand or sex-speci®c cytoplasmic factor to RET, and/
or the di€erent behaviour of di€erent isoforms of RET.
Related studies on other growth factors are discussed
below.
Nuclear translocation of ®broblast growth-factor
(FGF)-1 and -2, and heregulin has been reported
(reviewed by Prochiantz and Theodore, 1995). These
growth factors bind to a receptor tyrosine kinase. The
nuclear tracking of extracellular FGF-1 correlated
with the perinuclear association of the FGF receptor1a isoforms but not the FGF receptor-1b isoforms
(Prudovsky et al., 1996). In a di€erent study, an
intracellular binding protein which underwent a juxtanuclear localization coincident with the nuclear
translocation of FGF-2 was found (Kilkenny and
Hill, 1996). Furthermore, FGF receptor-1 was
detected in nuclei preparation upon FGF-2 treatment
of mouse ®broblasts (Maher, 1996). Using a variety of
techniques, other transmembrane receptors have been
shown to be translocated to the nucleus upon
stimulation by the ligands. These include insulin
receptors (Podlecki et al., 1987), epidermal growth
factor receptors (Rakowicz-Szulczynska et al., 1989;
Jiang and Schindler, 1990; Marti et al.,1991; Holt et
al., 1994), HER2 (Xie and Hung, 1994) and IL-1
receptors (Curtis et al., 1990). Similar experiments can
be done on nuclei preparation from female mouse
kidneys to con®rm the nuclear localization of RET and
to identify other protein(s) which may bind to RET
and directs its intracellular localization. Immunohistochemical localization of GDNF and neurturin, a
recently identi®ed ligand for RET (Buj-Bello et al.,
1997), may also shed light on the questions.
Acknowledgements
We thank Dr V Pachnis, NIMR, Mill Hill, London for the
gift of pmcret7; Dr RT McCluskey, Harvard Medical
School, USA for providing the gp330 antiserum; Prof L
Low, Department of Paediatrics, the University of Hong
Kong for reading the manuscript. This study was
supported by Committee on Research and Conference
Grants, the University of Hong Kong.
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