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Departments of Pathology (G.H., H.S., T.S., J.T., Y.M., H.N.), Internal Medicine (G.H., N.H., T.T.), and Surgery (K.F.), Tohoku University School of Medicine, Sendai, Japan; and the Laboratory of Molecular Hypertension, Baker Medical Research Institute (Z.S.K.), Melbourne, Australia
Address all correspondence and requests for reprints to: Gen Hirasawa, M.D., Department of Pathology, Tohoku University School of Medicine, 2–1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: g-hirasawa{at}patholo2.med.tohoku.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mineralocorticoids are also thought to play some role during fetal development. 11ßHSD2 expression has been previously explored in various mouse fetal organs (9, 10), ovine fetal kidney and lung (11), and several human fetal tissues (12, 13, 14). Recently, a correlation has been reported between the expression of MR and electrolyte transport in rat fetal gastrointestinal epithelial cells (15, 16). However, relationships between 11ßHSD2 and MR expression throughout human fetal gestation have not been well characterized. Recently, Suzuki et al. reported the expression of 11ßHSD2, glucocorticoid receptor (GR), and MR in human lung at various stages of development (17). Therefore, in this study we examined the immunolocalization of MR and 11ßHSD2 in various human fetal organs to determine their possible biological significance in human fetal development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal tissues without significant histopathological abnormalities were retrieved from surgical pathology files of Tohoku University Hospital (Sendai, Japan). This study protocol was approved by the ethics committee of Tohoku University School of Medicine (Sendai, Japan). The gestational age of the tissues ranged from 4–40 weeks. Tissue samples had been routinely fixed in 10% neutral formalin for 24–48 h at room temperature and embedded in paraffin wax.
Primary antibody
The generation and characterization of the primary antibodies for 11ßHSD2 (HUH23) and MR (MINREC4) have been described previously (4, 18). Briefly, HUH23 is an immunopurified polyclonal antibody raised in rabbits against a synthetic peptide corresponding to the last 16 amino acid residues of human 11ßHSD2. The polyclonal antibody MINREC4 was raised in rabbits against a synthetic fusion protein corresponding to 167 amino acids of the N-terminal region of the human renal MR. Application of these antibodies to immunohistochemistry was reported previously (8, 19). MINREC4 did not cross-react with GR (20, 21). Van Steensel et al. reported that GR and MR were present in separate components in nuclei of rat hippocampal neurons using MINREC4 as the primary antibody in immunohistochemistry (20). In addition, immunohistochemical analysis of MR in human kidney using MINREC4 demonstrated immunoreactivity in distal tubules and collecting ducts, but not in proximal tubules where GR is located (19).
Immunostaining
Immunohistochemical analysis was performed employing the streptavidin-biotin amplification method using a Histofine Kit (Nichirei, Tokyo, Japan) and was described in detail previously (8, 19). The HUH23 antibody was used at a final concentration of 5 µg/mL, and MINREC4 was used at a dilution of 1:600. The antigen-antibody complex was visualized with 3,3'-diaminobenzidine solution [1 mmol/L DAB and 50 mmol/L Tris-HCl buffer (pH 7.6), and 0.006% H2O2] and counterstained with methyl green. Adult human kidney tissues were used as positive controls for 11ßHSD2 and MR. For negative controls, preimmune rabbit serum was used instead of primary antibodies, and no specific immunoreactivity was detected in these sections. Relative immunoreactivity was classified as follows: 0, none; 1, weak; 2, moderate; and 3, marked.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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None of the three components, including pronephros, mesonephros,
and metanephros, demonstrated immunoreactivity for 11ßHSD2 and MR at
gestational weeks 8 and 11. From 14 weeks to term (n = 9), both
11ßHSD2 and MR immunoreactivities were detected in the distal
tubules, collecting tubules, and thick ascending loop of Henle (Fig. 1, 1A-B).
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From 8–30 weeks gestation (n = 5), during the
pseudoglandular and canalicular phases, columnar epithelium in the
terminal bronchiolar budding component demonstrated moderate
immunoreactivity for both 11ßHSD2 and MR (Fig. 1
, 2A-B). From 11–39 weeks gestation
(n = 9), immunoreactivity of 11ßHSD2 and MR was weak to moderate
in the tracheal and bronchial ciliated epithelial cells.
Skin
From 16–21 weeks gestation (n = 3), peridermal cells and the
intermediate layer of the epidermis were strongly positive for both
11ßHSD2 and MR. However, a single basal layer of the epidermis did
not demonstrate any immunoreactivity (Fig. 1, 3A-B). Bulbous hair follicles were
moderately positive for both 11ßHSD2 and MR.
Gastrointestinal tract
The ciliated epithelium of the foregut (11–21 weeks gestation;
n = 7) was moderately positive for both 11ßHSD2 and MR. In
particular, the luminal surface of these epithelial cells demonstrated
both 11ßHSD2 and MR immunoreactivities. The gastric mucosa of the
fetus was developed into superficial and deep zones, with maturation of
the proximal midgut. More intensive immunoreactivity of both 11ßHSD2
and MR was detected in mucous cells of the deep zone than in those of
the superficial zone (14–36 weeks gestation; n = 10; Fig. 1, 4A-B). Columnar epithelial cells of the
superficial zone were weakly or moderately positive for both proteins
(14–36 weeks gestation; n = 15).
Superficial epithelial cells of the small intestine, including the distal midgut, were weakly positive for 11ßHSD2 and MR (11–40 weeks gestation; n = 5). On the other hand, epithelial cells of the hindgut during the postnatal period demonstrated no immunoreactivity for either 11ßHSD2 or MR before 39 weeks gestation (15–36 weeks gestation; n = 8). Immunoreactivity of both 11ßHSD2 and MR in fetal colonic epithelium was clearly detected at term (38–40 weeks gestation; n = 2).
Pancreas
There was no immunoreactivity of 11ßHSD2 or MR in fetal pancreatic tissues throughout gestation (14–39 weeks gestation; n = 9).
Liver
From 8–27 weeks gestation, very weak immunoreactivity of 11ßHSD2, but not MR, was detected in hepatocytes at 8 and 20 weeks gestation among seven liver specimens available for examination. Bile ducts were immunohistochemically negative for both 11ßHSD2 and MR in these specimens.
Adrenal gland
11ßHSD2 was weakly detectable in the four fetal adrenal glands (8–21 weeks gestation) available for examination.
Other tissues
Umbilical cord, brainstem, cerebellum, cerebrum, spinal cord, spleen, heart (pericardium and myocardium), and thymus were negative for both 11ßHSD2 and MR throughout gestation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tubules of the fetal or metanephric kidney are thought to be functional at 9–12 weeks gestation, and reabsorptive functions involving the loop of Henle are generally considered to occur by 14 weeks gestation, although new nephrons continue to form until birth (25). In our study, both 11ßHSD2 and MR are detectable from approximately 14 weeks gestation in the human fetal kidney. These findings indicate that mineralocorticoids are also involved in fetal renal function. In the fetal respiratory tract, 11ßHSD2 and MR are both considered to play important roles in water and electrolyte absorption at ciliated epithelia and the budding component, and may also contribute to the local regulation of lung liquid involved in surfactant synthesis, as previously suggested by Suzuki et al. (17). Suzuki et al. also recently reported the presence of GR in these cells of human fetal lung (17). Glucocorticoids have been shown to modulate the amiloride-sensitive sodium channel via the GR in rat lung cells in primary culture (26), suggesting that multiple mechanisms contribute to fluid flux in the lung. Recently, gene deletion studies have shown no gross morphological changes in the lungs of MR-deficient mice, consistent with the existence of multiple, redundant systems for modulating lung fluid (27).
Close to term the human fetus swallows 200–450 mL/day amniotic fluid (28), a volume that is almost completely resorbed from the surface of the fetal gastrointestinal tract, resulting in the formation of meconium within the distal fetal colon (24, 29). The reabsorption of amniotic fluid in human fetal gastrointestinal tract has not been well characterized. The results of our present study indicate that the upper gastrointestinal tract, i.e. the foregut of the esophagus and the midgut of the stomach and small intestine, may regulate absorption of ingested amniotic fluid via mineralocorticoid action. Consistent with this proposal is our observation that both 11ßHSD2 and MR immunoreactivities decreased in the esophagus as the epithelium transformed from ciliated, in early gestation, to squamous in mid- to late gestation. On the other hand, 11ßHSD2 and MR immunoreactivities were not detected in the colon or hindgut until late gestation, whereas in the adult colon there are high levels of both proteins (4, 5, 8).
These results indicate that reabsorption of ingested amniotic fluid occurs predominantly in the foregut and a part of midgut in early to midgestation. The pivotal role of MR in controlling sodium homeostasis is underlined by the marked decreases in sodium channel function in mice lacking a functional MR gene (27).
Pancreatic exocrine ductal cells are also considered mineralocorticoid targets (19). However, neither 11ßHSD2 nor MR immunoreactivity was detected in these cells throughout gestation, consistent with the inactivity of these glands at this time. Induction of 11ßHSD2 and MR may be initiated by food intake and digestion in the neonate. There is evidence of high pancreatic 11ßHSD2 gene expression in the adult and in the rat by Western blot analysis (30, 31).
Cells of the periderm, which is present in the epidermis of all amniote embryos, have been postulated to be involved in the exchange of water, sodium, and possibly glucose between the amniotic fluid and the epidermis (24, 32). This study is the first to demonstrate the presence of both 11ßHSD2 and MR in human fetal skin and suggests that fetal skin exposed to amniotic fluid is also a mineralocorticoid target tissue, as is adult skin (33, 34). Finally, we observed relatively weak 11ßHSD2 immunoreactivity in fetal hepatocytes and adrenocortical cells. Neither of these tissues is known to express MR, but given the widespread distribution of 11ßHSD2 early in mouse development, it would suggest that glucocorticoid inactivation is also important during human development (9).
Received September 15, 1998.
Revised November 24, 1998.
Accepted November 25, 1998.
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