ARTICLE |
Correspondence to: Pangala V. Bhat, Lab. of Nutrition and Cancer, Centre de Recherche du CHUM, Pavillon Hôtel-Dieu, 3850 St-Urbain Street, Montréal, Québec, Canada H2W 1T8..
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Summary |
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We have recently characterized a cytosolic aldehyde dehydrogenase from rat kidney that functions as a retinal dehydrogenase (RALDH) and have cloned the corresponding gene. RALDH catalyzes the oxidation of retinal to retinoic acid, which regulates cell growth and differentiation by activating retinoic acid receptors. In situ hybridization demonstrates that RALDH mRNA expression is prominent in kidney in 2-day-old rats, is detected in lung and in epithelia of several tissues, but is not found in liver tissue. Retinal dehydrogenase activity peaks in kidney at Day 2 after birth and decreases gradually until adulthood, correlating well with RALDH expression. Weaker activity is also detectable in lungs but not in liver. Notably, distribution patterns of RALDH in kidney tissues are dramatically altered during postnatal development (P). From P0 to P6, hybridization is essentially concentrated within the marginal nephrogenic zone of the cortex. Expression progresses to deeper cortical layers from P12 to P16 and is intense in the medulla at P42, and focal expression is still detectable in the cortex. Immunocytochemical localization of RALDH in neonatal kidney shows staining mostly in cortical zone convoluted tubules and in adult rat shows staining in segments of distal and proximal tubules. These data suggest an important role for RALDH in modulating retinoic acid levels in different cell types during rat kidney development. The changing patterns of RALDH expression mirror stages of nephron formation in the developing rat kidney, strongly suggesting a central role for RALDH and thus for retinoids in controlling kidney development. (J Histochem Cytochem 46:10251032, 1998)
Key Words: kidney epithelium, retinoic acid, in situ hybridization, immunocytochemistry, anatomic distribution
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Introduction |
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Retinoic acid (RA) plays a fundamental role in vertebrate cell differentiation (
Effects of RA are mediated by nuclear receptors that are ligand-dependent transcriptional regulators (, ß, and
) and retinoid X receptors (RXR
, ß, and
). Hypoplasia or agenesis of kidneys of mice deficient for expression of combinations of several RAR isoforms was recently reported, supporting a role for RA in kidney development from early steps in metanephros formation (
RA is biosynthesized from retinol by two sequential reactions, with retinal as an intermediate (
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Materials and Methods |
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Animals
SpragueDawley rats were purchased from Canadian Breeding farm (Québec, Canada). Fetuses were delivered by caesarean section on Day 19 of pregnancy (-2 days). For Northern blot, Western blot, and enzyme assay analyses, the organs from fetuses (n = 40) or young animals (n = 80) were pooled (465 individual organs), snap-frozen in liquid nitrogen, and stored at -80C before analysis. Usually the tissues were from 19-day embryo (E19) and postnatal rats on Days 0, 2, 6, 12, 16, 22, 35, and 42 (P0P42). For in situ hybridization, 24 newborn rats were cooled on ice and decapitated. The kidneys and liver were dissected, frozen at -35C in isopentane, and cut into 14-µm sagittal sections. For anatomic studies, four newborn rats (P2) were cut across the whole body. For immunocytochemistry, the paraffin tissue sections were from collection of four rat kidneys fixed by heart perfusion with Bouin's fluid (
Preparation of Enzyme and Incubations
Enzyme activity was measured on the basis of in vitro formation of RA from retinal in the presence of cytosolic preparations. Supernatant fractions from tissue homogenates at 100,000 x g were prepared as described earlier (
Northern Blot Analysis
Northern blot analysis was carried out with [32P]-dCTP-labeled cDNA probes generated from whole RALDH cDNA as reported earlier (
Western Blot Analysis
Tissue samples were homogenized in 1.0 ml of 100 mM ice-cold Tris buffer (pH 8.0) containing 3 mM EDTA, 1 µg/ml leupeptin and pepstatin, 0.5 mM PMSF. The homogenates were centrifuged at 10,000 x g for 10 min and the supernatants were collected. Protein was quantified by the method of
cRNA Probes
The anti-sense and sense uridine 5'-[-[35S]-thio]triphosphate and cytosine 5'-[
-[35S]-thio]triphosphate-labeled complementary RNA probes were generated by in vitro transcription using T3 and T7 RNA polymerase, respectively, as described (
In Situ Hybridization
In situ hybridization was carried out with cryostat tissue cuts using a technique already described for other mRNAs (
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Immunocytochemistry
For immunocytochemistry, ABC staining was performed using the Vectastain ABC kit (Vector Laboratories; Burlingame, CA) according to the manufacturer's instructions. The antibody to RALDH was diluted 1:300 for overnight incubation at 4C. For control, the immunocytochemistry staining was completely blocked after the preadsorption of antibody to RALDH with an excess (10-6 M) of purified protein.
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Results |
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RALDH Is Expressed Predominantly in the Kidney of Newborn Rats
Whole body sections of 2-day-old rats were hybridized with cRNA probes corresponding to RALDH anti-sense or sense sequences (Figure 1AC). Control sense probes produced nonspecific background (Figure 1C). RALDH mRNAs are highly expressed in kidney and can be detected in several other tissues, such as retina, olfactory neuroepithelium in the olfactory turbinates (OT), midbrain (MidBr), the primordia of upper and lower molars (Mol), lungs (Lu), stomach (St), and intestine (Int), but neither in liver (Li) nor in adrenals (Adr) (Figure 1).
Levels of RALDH Activity, RALDH Expression in Rat Tissues During Development
Retinal dehydrogenase activity is already detectable in cytosolic extracts from rat kidney during late intrauterine development, but its levels of expression rise abruptly around birth (Figure 2), reaching the highest levels postnatally between Days 2 and 16 (P2P16). Subsequently, retinal dehydrogenase activity in kidney declines progressively, reaching levels comparable to those observed before birth at P42. In lung, retinal dehydrogenase activity is below the detection limit at birth but increases progressively from P10 (Figure 2). RALDH expression was monitored between P2 and P42 by Northern blot and Western blot analyses (Figure 3 and Figure 4). Sequencing of cDNA fragments obtained by RT-PCR confirmed that Northern blot signals correspond to RALDH mRNAs in these tissues and are not generated by cross-hybridization with other aldehyde dehydrogenases (
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In marked contrast with expression patterns of human or mouse ALDHs, which are highly expressed in liver (
Developmental Gene Expression Patterns of RALDH in Rat Kidney
Results of in situ hybridization, Northern and Western blot assays and enzymatic assays establish that kidney is a major site of RALDH expression. To investigate the cell specificity of this expression, kidney tissues at Stages P0, P2, P6, P12, P16, and P42 of postnatal development were hybridized with RALDH riboprobes (Figure 5). Overall, RALDH mRNA levels changed markedly with time in postnatal kidneys. Intensity of hybridization to the RALDH probe increased from P0 to P12, followed by a decline from P16 to adulthood levels of P42 (Figure 5AF), correlating to a large extent with results obtained by Northern blot assay (Figure 3).
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Marked changes in spatial distribution patterns of RALDH expression could be observed over the time period studied. On P0, the hybridization signal was essentially concentrated within the marginal zone of the cortex, with much lower intensity in the medulla (Figure 5A). This spatial distribution pattern remained essentially unchanged until P6 (Figure 5AC). On P12, increased RALDH mRNA levels appeared in the deeper cortical zone and in the medulla (Figure 5D). On P16 there was a clear rearrangement of the hybridization pattern, with stronger hybridization signal in the deep cortical layers than at the periphery (Figure 5E). Intensity of hybridization per cell in the medulla was strongest at P42, with persistence of focal expression in the cortex (Figure 5F). Particularly high levels of RALDH transcripts were present in the inner band of the medulla containing the thin segments of Henle's loops, whereas much less labeling was seen in the center medullary band containing essentially the descending and ascending limbs of Henle's loops. Control sense probes produced nonspecific background (data not shown).
Distribution of in situ hybridization for RALDH in the cortical region is presented for P2, P12, and P42 at higher magnification (Figure 5GI). In postnatal kidneys, strong in situ hybridization signal was detected in cells of convoluted tubules (large arrows in Figure 5GI). RALDH mRNAs are less abundant in mesenchyme cells (arrowheads in Figure 5G and Figure 5H) and are undetectable in the glomeruli or capillaries. However, some hybridization could be observed around glomeruli of newborn rats (small arrows in Figure 5G).
Immunocytochemical Localization of RALDH in Kidney of Developing and Adult Rat
Because most cytological detail was obscured by silver labeling, it was not possible to define clearly the types of cells expressing RALDH. We therefore sought to detect RALDH expression by immunocytochemistry. Immunocytochemical labeling was observed in the cortex and medulla of neonatal and adult rats (Figure 6). RALDH immunostaining was detected in newborn rats (P1P16) mostly in the cortical zone convoluted tubules (Figure 6AD), and the mesenchymal cells were negative. The pattern of RALDH immunoreactivity followed that of tubules extending into the deep renal structure. In the kidneys of newborn rats (P1P16), most if not all tubule structures were stained. RALDH immunostaining was also present both around and within glomeruli. Thus, cells of the parietal layer of Bowman's capsule are stained in P1 to P6 rats (Figure 6G and Figure H). By topography, immunostaining around glomeruli much resembled the in situ hybridization pattern observed in P2 (Figure 5H). Furthermore, podocytes within glomeruli were also immunostained (Figure 6G and Figure 6H). In contrast to neonatal kidney, in adult kidney only a fraction of tubules were stained (Figure 6E, Figure 6J, and Figure 6K), and no staining around or within glomeruli was seen (Figure 6E and Figure 6J). In the adult kidney, brown ABC staining could be observed mostly in the distal tubules showing strongly positive cells intercalated with clear negative cells (Figure 6J). RALDH expression was detected in the macula densa and in some proximal tubules. However, a proportion of convoluted tubules, as well as the glomeruli and capillaries, were not stained (Figure 6J). In the medulla, some collecting tubules displayed ABC staining (Figure 6K). Single ABC-stained cells, probable resident macrophages, were also seen in the medulla (Figure 6L). Controls with the antiserum blocked by preadsorption with the antigen were negative (Figure 6F), emphasizing the specificity of immunostaining.
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Discussion |
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RA, which is synthesized from retinol by a two-step oxidation process (
A number of studies have shown that Class I aldehyde dehydrogenases (ALDHs) have high activity for retinal oxidation (
RALDH expression patterns overlap with those described for the Class I alcohol dehydrogenase gene ADH-I (
Interestingly, RALDH expression patterns in mesenchymal and epithelial cells of the kidney marginal zone, with much less labeling in stromal tissue compartments of P0 rats (Figure 5A), are similar to those of the laminin A-chain in the 18-day-old mouse embryo (
In conclusion, the expression of RALDH in the epithelia of various tissues correlates with the well-known role of RA in induction and maintenance of epithelial cell differentiation (
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Footnotes |
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1 PVB and MM contributed equally to this work.
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Acknowledgments |
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Supported by grants from the Medical Research Council of Canada MT-13450 (PVB) and MT-12686 (MM). SM is a chercheur-boursière Junior I of the Fonds de la Recherche en Santé du Québec.
We wish to thank Ms J. Marcinkiewicz and Mr C. Charbonneau for their excellent work.
Received for publication July 24, 1997; accepted May 21, 1998.
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