Journal of Histochemistry and Cytochemistry, Vol. 46, 1025-1032, September 1998, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

Changing Patterns of Renal Retinal Dehydrogenase Expression Parallel Nephron Development in the Rat

Pangala V. Bhata, Mieczyslaw Marcinkiewiczb, Yuan Lia, and Sylvie Mader1,c
a Laboratory of Nutrition and Cancer, Centre de Recherche du CHUM
b Laboratory of Molecular Neuroendocrinology, Institut de Recherches Cliniques de Montréal and Department of Medicine, Université de Montréal
c Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada

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..


  Summary
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Materials and Methods
Results
Discussion
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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 46:1025–1032, 1998)

Key Words: kidney epithelium, retinoic acid, in situ hybridization, immunocytochemistry, anatomic distribution


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Retinoic acid (RA) plays a fundamental role in vertebrate cell differentiation (Roberts and Sporn 1984 ) and in the embryonic development of the vertebrate limb and central nervous system (Maden et al. 1989 ; Smith et al. 1989 ). In addition, anomalies of the genitourinary tract are often observed in the offspring of vitamin A-deficient dams, and these defects can be prevented by vitamin A administered to the mother at times coinciding with metanephros formation (Wilson and Warkany 1948 ; Wilson et al. 1953 ). Vitamin A derivatives can stimulate metanephros organogenesis in organ culture in controlling nephrogenic induction processes and ureteric bud patterning (Vilar et al. 1996 ). RA is also a potent stimulator of tubulogenesis when added to renal cell cultures (Humes and Cieslinski 1992 ).

Effects of RA are mediated by nuclear receptors that are ligand-dependent transcriptional regulators (Kastner et al. 1994 ). Two families of retinoid receptors have been identified, the RA receptors (RAR{alpha}, ß, and {gamma}) and retinoid X receptors (RXR{alpha}, ß, and {gamma}). 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 (Lohnes et al. 1994 ; Mendelsohn et al. 1994 ).

RA is biosynthesized from retinol by two sequential reactions, with retinal as an intermediate (Bhat et al. 1988a ; Lee et al. 1991 ). We previously reported high levels of RA and RA-synthesizing NAD-dependent dehydrogenase (RALDH) activity in the rat kidney (Bhat and Lacroix 1991 ; Labrecque et al. 1995 ). The purpose of this study was to investigate the tissue specificity of RALDH expression patterns and the cell specificity of its expression in kidney. We show that RALDH is highly expressed in kidney after birth and that its expression progresses from peripheral to deeper cortical layers and to the medulla during post-natal kidney development. Nephron formation is an ongoing process in rat after birth, and is initiated from the metanephric blastema in the peripheral cortical layers. Therefore, RALDH patterns of expression mirror the different stages of nephron formation, suggesting a specific role for this enzyme in rat kidney development.


  Materials and Methods
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Introduction
Materials and Methods
Results
Discussion
Literature Cited

Animals
Sprague–Dawley 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 (4–65 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 (P0–P42). 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 (Meyer et al. 1996 ).

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 (Bhat et al. 1988a ). In a standard assay, 1.2 mg of supernatant protein was incubated at pH 7.5 with 141 µM retinal in the presence of NAD (564 µM) and dithiothreitol (DTT, 1.61 mM) at 37C for 1 h in a final volume of 258 µl. The details of the control incubations, the extraction of the reaction products, and the expression of the enzyme activity have been described earlier (Bhat et al. 1988a ). The reaction products were separated and quantitated by HPLC as described earlier (Bhat et al. 1988b ).

Northern Blot Analysis
Northern blot analysis was carried out with [{alpha}32P]-dCTP-labeled cDNA probes generated from whole RALDH cDNA as reported earlier (Bhat et al. 1995 ), using a random priming kit (Prime-it; Stratagene, La Jolla, CA). Total tissue RNAs were isolated with the TRI pure isolation reagent (Boehringer Mannheim; Indianapolis, IN) using the manufacturer's protocols. Total RNA 15 µg was separated by electrophoresis in a 1.2% formaldehyde–agarose gel and blotted into nylon membranes. The blots were prehybridized and hybridized at 68C using Quickhyb reagent (Stratagene). After hybridization, blots were washed twice with 2 x SSC, 0.1% SDS at room temperature for 15 min. The final wash was with 0.1 x SSC, 0.1% SDS at 65C for 30 min. Actin (2.0 KB; Oncor, Gaithersburg, MD) was used as a control probe.

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 Bradford 1976 using bovine serum albumin (BSA) as a standard. Fifteen and 20 µg of total protein from kidney and lung, respectively, were boiled in sample buffer, separated by 8% SDS-PAGE, and transferred to Hybond nitrocellulose (Amersham; Poole, UK). Blots were blocked with 0.05% Tween-20 in 5% BSA before incubation with antibodies. Immunoreactive protein was detected with an ECL detection kit. The antibody (in the form of serum) used for Western blot analysis was raised against phenobarbital-induced aldehyde dehydrogenase (kindly supplied by Dr. Ronald Lindahl). This antibody recognizes RALDH but no other cytosolic ALDHs, such as mouse and human ALDH.

cRNA Probes
The anti-sense and sense uridine 5'-[{lambda}-[35S]-thio]triphosphate and cytosine 5'-[{lambda}-[35S]-thio]triphosphate-labeled complementary RNA probes were generated by in vitro transcription using T3 and T7 RNA polymerase, respectively, as described (Marcinkiewicz et al. 1994 ). The cDNA RALDH sequence (nucleotides 250–1827) was inserted into the Bluescript PB SK+ plasmid with EcoR1 and HindIII sites (Bhat et al. 1995 ). The cDNA was linearized with AvaII to yield an RALDH anti-sense riboprobe of 597 BP (specific activity 269 Ci/mmol: 1 Ci = 37 GBq) or RALDH sense (control) riboprobe of 980 BP (specific activity 442 Ci/mmol).

In Situ Hybridization
In situ hybridization was carried out with cryostat tissue cuts using a technique already described for other mRNAs (Marcinkiewicz et al. 1994 ), except that the hybridization solution contained 200 mM DTT. All tissue slides from different ages were hybridized in the same experiments and exposed 4 days for X-ray film autoradiography or 30 days for emulsion autoradiography. After in situ hybridization, the tissue sections were stained with hematoxylin and eosin and viewed under dark- and lightfield illumination. Some nonspecific impressions were found in the emplacement of bones and skin, mostly due to mechanical stress produced during X-ray film exposure (Figure 1C). Autoradiographs produced with photographic NTB-2 emulsion were free of such nonspecific artifacts.



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Figure 1. Distribution of RALDH mRNA on P2 in representative section through whole body. In situ hybridization signals obtained with anti-sense (as) riboprobes detected on X-ray film are shown as white spots on dark field (A,B). Control hybridization with sense (ss) riboprobes showing nonspecific background and impressions at bones and skin sites (C). Bone impressions are indicated with dots (A–C). Adr, adrenals; Int, intestine; Ir, iris; Ki, kidney; Li, liver; Lu, lungs; MidBr, midbrain group of cells; Mol, molars; OT, olfactory turbinates; Ret, retina; St, stomach. Bar = 1 cm.

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.


  Results
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Materials and Methods
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 1A–C). 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 (P2–P16). 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 (Bhat et al. 1995 ). Changes in RNA levels were followed by corresponding changes in protein concentrations. Variations in the levels of RALDH expression (Figure 3 and Figure 4) between P2 and P42 mainly parallel evolution of aldehyde dehydrogenase activity both in kidney and lung, although slight discrepancies between enzyme activity, mRNA expression, and protein expression can be observed (Figure 2 and Figure 4). This could be due either to the relative stability of RALDH mRNA and protein or to the presence of other retinal-oxidizing enzyme activity or inhibitors in the crude cytosolic preparations.



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Figure 2. Retinal dehydrogenase activities in tissues of rats during development. {circ}{circ}, kidney; {bigtriangleup}{bigtriangleup}, lung; {bullet}{bullet}, liver.



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Figure 3. Expression of RALDH mRNA in kidney (A) and lung (B) of rats at various stages of development. Northern blot of total RNA was carried out as described in Materials and Methods. Actin mRNA levels (2.0 KB) were used as internal standards.



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Figure 4. Expression of RALDH protein in kidney (A) and lung (B) of rats during development. RALDH protein was detected by Western blot, as described in Materials and Methods.

In marked contrast with expression patterns of human or mouse ALDHs, which are highly expressed in liver (Stewart et al. 1996 ), neither RALDH mRNA nor protein could be detected in liver (data not shown), correlating with the very low levels of retinal dehydrogenase activity in rat liver (Figure 2).

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 5A–F), correlating to a large extent with results obtained by Northern blot assay (Figure 3).



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Figure 5. In situ hybridization of RALDH in the kidney during postnatal development. In situ data from X-ray film are shown for P0 (A), P2 (B), P6 (C), P12 (D), P16 (E), and P42 (F). In situ hybridization data revealed by emulsion autoradiography in renal cortex are shown for P2 (G), P12 (H), and P42 (I). Small arrow in G indicates ALDH mRNA localized in proximal convoluted tubule near a glomerulus (Gl), unlabeled. Large arrows (G–H) within parenchyme containing proximal and distal tubules and collecting tubules. Cx, cortex; Gl, glomerulus; Med, medulla. Bars: E = 3 mm; I = 100 µm.

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 5A–C). 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 5G–I). In postnatal kidneys, strong in situ hybridization signal was detected in cells of convoluted tubules (large arrows in Figure 5G–I). 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 (P1–P16) mostly in the cortical zone convoluted tubules (Figure 6A–D), 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 (P1–P16), 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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Figure 6. Immunocytochemical localization of RALDH in kidney of newborn (P1, P2, P6, P12, and P16: A–D, G–I) and adult (Ad: E,F,J–L) rat under Nomarski optic (A–I) or after tissue staining with hematoxylin and eosin (J–L). ABC labeling detected in convoluted tubules in the cortex (large arrows: A–E) and some immunostaining in parietal cells of Bowman's capsule (medium arrows: G,H) and podocytes (thin arrows: G,H) in glomeruli (G1). (F) Control performed in adult kidney tissue after preadsorption of the antibody with an excess of purified RALDH showing lack of staining. (J) Higher magnification through the adult renal cortex. Brown ABC labeling is seen intracellularly within some tubules, composed mostly of cuboidal cells with round nuclei, and in macula densa (MD), whereas some other tubules (arrowheads) and the glomerulus (G1) are unstained. (K) ABC staining in collecting tubules (large arrows) in the medulla. (L) Single ABC-stained cells in the medulla (large arrows). Bars: AF = 100 µm; GL = 100 µm.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

RA, which is synthesized from retinol by a two-step oxidation process (Means and Gudas 1995 ) has a well-characterized role in the regulation of growth and differentiation of various cell types throughout life (Roberts and Sporn 1984 ) and of crucial embryonic developmental processes, including morphogenesis of the limb, skeletal patterning, and central nervous system organization (Niswander et al. 1994 ; Tickle and Eichele 1994 ). Studies performed on vertebrate embryos have demonstrated that endogenous RA is detectable in a distinct spatiotemporal pattern, suggesting similar regulation of the expression of enzymes involved in RA synthesis. In mouse embryos, RA and alcohol dehydrogenase (which catalyzes the oxidation of retinol to retinal) are first detectable in embryonic tissues at E7.5 during the primitive streak stage and are preferentially localized in posterior tissues, as well as in craniofacial tissues and retina, of E8.5 and E9.5 mouse embryos (Ang et al. 1996a ). RA is also detectable in mouse retina from E8.5 onwards (McCaffery et al. 1993 ). At later stages of mouse embryonic development, high levels of RA are found in the spinal cord (McCaffery and Drager 1994 ) and, by E16.5, in the kidney and intestine. Much lower levels of RA are found in the liver, heart, and brain (Ang et al. 1996b ). Therefore, endogenous RA levels in embryonic tissues are controlled by developmentally regulated mechanisms that are likely to include modulation of expression and/or activity of enzymes catalyzing RA synthesis.

A number of studies have shown that Class I aldehyde dehydrogenases (ALDHs) have high activity for retinal oxidation (Lee et al. 1991 ; Lindahl 1992 ; Yoshida et al. 1992 ; Labrecque et al. 1995 ). RALDH, which was isolated from rat kidney, has 87% and 96% sequence identity with human and mouse Class I ALDHs, respectively (Bhat et al. 1995 ). The present study was aimed at characterizing RALDH expression patterns in developing and adult kidney. Three major tissue compartments are found in the developing kidney, i.e., mesenchyme, endothelium, and epithelium. Our histochemical results provide evidence for RALDH expression in neonatal and adult epithelial cells. Thus, by immunocytochemistry most epithelial cells forming convoluted tubules were positive in neonatal kidney, whereas a fraction were positive in adult kidney. Interestingly, other epithelial origin cells, such as cells of the parietal layer of Bowman's capsule and podocytes, were positive in neonatal kidney. RALDH immunoreactivity was found in distal convoluted tubules together with its integral component macula densa. Epithelia of some proximal tubules expressed RALDH at lower levels. Mesenchymal cells in postnatal kidney displayed less hybridization signal, and endothelial cells were negative. RALDH concentration in epithelia of distal and proximal tubules varies from one region to another, suggesting differences in local RA synthesis. During postnatal development, the marginal cortical zone displays more hybridization than the medulla until P6. This proportion is gradually reversed during later stages, from P12 until P42, leading to high levels of expression in the medulla. Spatial and temporal differences in RALDH expression may reflect variations in local needs of RA for cell replenishment and differentiation into polarized epithelial cells during development. Furthermore, RA may contribute to the maintenance of the epithelial character of cells in adult kidney.

RALDH expression patterns overlap with those described for the Class I alcohol dehydrogenase gene ADH-I (Vonesch et al. 1994 ; Ang et al. 1996b ), which can oxidize retinol into retinal. Strong and uneven in situ labeling of ADH-I RNAs was observed in renal cortex and the outer medullary band of adult mice. Labeling was absent in renal glomeruli and Bowman's capsule, and was distributed over the convoluted tubules and medullary rays. Therefore, in the renal cortex a good correlation exists in the distribution of transcripts between ADH-I and RALDH. However, a difference in the distribution of ADH-I and RALDH transcripts was noted in the medulla. ADH-I transcripts were predominant in the outer layer of the medulla, whereas RALDH transcripts were localized in the inner medullary band (Vonesch et al. 1994 ; and Figure 5F). These results suggest that renal tubules can synthesize retinoic acid from retinol in two steps, although further studies will be necessary to confirm that RALDH and ADH-I are co-expressed by the same cell types.

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 (Sorokin et al. 1990 ). The laminin B1 promoter contains a retinoic acid response element (Vasios et al. 1991 ). Retinoids can promote deposition of the A and B1 chains of laminin in vitro (Humes and Cieslinski 1992 ), which is essential for the conversion of embryonic mesenchymal cells into polarized, differentiated kidney epithelium (Sorokin et al. 1990 ; Klein et al. 1988 ). This suggests a direct mechanism for RALDH in promoting differentiation of tubule cells, through induction of laminin expression by locally produced RA.

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 (Wolbach and Howe 1925 ). Gradients of RA concentration play important roles in pattern formation (Tickle and Eichele 1994 ). Recently, it has been demonstrated that RA is a key regulator of renal organogenesis and controls nephrogenic induction processes and ureteric bud patterning (Vilar et al. 1996 ), which supports a role for RALDH as a patterning component in the developing kidney. High levels of RALDH expression in the postnatal kidney also suggest that this enzyme may play a key role in kidney maturation and function.


  Footnotes

1 PVB and MM contributed equally to this work.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
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