1Harry B. and Aileen Gordon Diabetes Research Laboratory, Molecular Diabetes and Metabolism Section, Department of Pediatrics, 2Breast Center, and 3Department of Pathology, Baylor College of Medicine, Houston, Texas
Submitted 16 November 2004 ; accepted in final form 7 March 2005
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ABSTRACT |
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AKR1A; maturation; development; alternative transcripts
The aldehyde reductase promoter has been characterized. The combination of two transcription factors, STAF and Sp1 family factors, both of which are consistent with ubiquitous expression, drives basal expression of the aldehyde reductase gene (9). However, multitissue RNA analysis in mice and humans and analysis of enzyme activity in various rat and murine tissues show that the level of aldehyde reductase is significantly higher in the kidney than in other organs (3, 7, 20). This implies a special role for aldehyde reductase in the kidney in addition to the general metabolic role it plays in all organs and possibly during embryonic development (3).
In the present report, we establish that the expression of aldehyde reductase in several organs is driven by a distinct mechanism separate from that used ubiquitously. A unique transcription start site is utilized primarily in the kidney, resulting in the expression of a transcript with a short 5'-untranslated region (UTR). Expression of this short transcript in the murine kidney starts during the second postnatal week and grows in intensity up to the week 6, when it reaches the adult level. Appearance of this transcript is accompanied by corresponding increases in aldehyde reductase activity and protein, with the maximum increase occurring during week 3. In the kidney, aldehyde reductase expression is limited to the proximal convoluted tubules and the parietal epithelium of Bowmans capsule. We conclude that the high expression of aldehyde reductase via a specific transcription mechanism is a part of the process of tubular maturation. In the human kidney, the long and the short mRNA also mediate aldehyde reductase expression, but the induction of aldehyde reductase expression occurs prenatally. Administration of the aldehyde reductase inhibitor AL-1576 to adult mice causes an increase in urinary glucuronate level and the corresponding decrease in vitamin C output, thus substantiating the role of aldehyde reductase in the kidney cortex in the pathways of inositol and glucuronate metabolism and vitamin C synthesis.
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MATERIALS AND METHODS |
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Animals. C57BL/6 mice were purchased from Harlan and maintained in the Texas Childrens Hospital Feigin Center vivarium in accordance with an approved animal protocol. Aldehyde reductase inhibitor AL-1576 (Alcon Laboratories) was mixed with food, and the dosage was calculated based on average food consumption of 7 g/day. A cage of three male 4-mo-old mice received a dose of 20 mg·kg1·day1, and a similar cage of female mice received 10 mg·kg1·day1. Animals were eating similarly prepared food but without inhibitor for 1 wk before the beginning of the experiment, and their consumption of food and water was monitored. Control urine samples were collected on the last 2 days before the start of inhibitor administration. Mice had access only to food containing inhibitor for 4 days. Spot urine samples were collected every day of the experiment in the morning and in the evening. Consumption of food and water was measured every day using special tubes for collection of spilled water. Actual food consumption during the experiment varied between 5 and 7 g·mouse1·day1, giving an actual inhibitor dose received between 14.3 and 20 mg·kg1·day1 for a calculated dose of 20 mg·kg1·day1 and similar for a dose of 10 mg·kg1·day1. After 4 days, food was substituted again for regular food with water, and urine was collected on the days indicated. Urine samples were frozen at 20°C immediately after collection.
Measurements of metabolites in urine. The glucuronate concentration in urine was measured spectrophotometrically using a carbazole procedure as previously described (13). Ascorbic acid (vitamin C) was measured by the dipyridyl reaction (24). Creatinine was measured using an Infinity kit from Sigma according to the manufacturers instructions.
RNA isolation and Northern blots. Organs were collected immediately after euthanasia and frozen at 80°C for future RNA or protein isolation. Total RNA was isolated from the frozen tissues using either RNeasy minicolumns from Qiagen (Valencia, CA) or RNAzol from Tel-Test (Friendswood, TX). Both methods yielded RNA of identical quality. Northern blotting was performed by standard techniques. RNA samples were prepared with RNA-loading mix from Genhunter and separated on a denaturing agarose gel with 1% formaldehyde. RNA was transferred onto a positively charged Hybond-XL membrane (Amersham, Piscataway, NJ) and hybridized with corresponding probes in Expresshyb (Clontech, Palo Alto, CA) hybridization buffer according to the manufacturers protocol. The final washing step was performed at 55°C in 0.1x SSC. Bands were detected by autoradiography.
Ribonuclease protection assay. To generate the probe for mouse aldehyde reductase, a 266-bp template corresponding to the 3'-end of exon 1 and including the sequence from the intron downstream was generated by PCR on the subcloned genomic DNA. The T7 promoter sequence was attached to the antisense primer (5'-taatacgactcactatagggagaaggaaaccgaggtccagaaaca-3'), and the sense primer (rpa2) was 5'-gccctggatcctcagtactggagt-3'. The antisense RNA probe was transcribed from this template using a MAXIscript kit (Ambion, Austin, TX). The probe contained 70 bp of an exon 1 sequence and protected a 70-bp fragment from the long isoform and a 55-bp fragment from the short isoform of aldehyde reductase mRNA. The close size of the two fragments allowed for quantitative comparison of the abundance of the two RNA isoforms. The control template included in the ribonuclease protection assay (RPA) III kit (Ambion) was used to generate a mouse actin probe. The template for a human aldehyde reductase probe was generated by PCR on a human genomic DNA fragment using primers hrpa1a (5'-taatacgactcactatagggagaagtaaagaatcgaggccatct-3'; antisense) and hrpa2 (5'-ctcaccgctagacttaagctga-3'; sense). The human probe was also located at the 3'-end of exon 1 at a position corresponding to the mouse probe. These probes were hybridized with 3 µg of total RNA from organs or cell lines overnight at 42°C and further digested with RNAse A/T1 using the RPA III kit (Ambion). The protected fragments were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. Total RNA from human organs was purchased from Ambion.
Rapid amplification of cDNA ends. 5'-Rapid amplification of cDNA ends (RACE)-ready cDNA from the mouse kidney was purchased from Ambion. Two nested primers from the exon 23 area of the aldehyde reductase cDNA were used for PCR and identified the transcription start site for the short isoform. Their sequences are 5'-CAATGTACCGGTAGCCTGCGGTA-3' (inner primer) and 5'-cagaggcatcttctgtccagtgt-3' (outer primer). To identify the transcription start for the long isoform, an inner primer from exon 1 with a sequence 5'-tactgaggcaacagggcccgact-3' was used.
Activity measurements. Cytosolic protein was extracted from kidneys by disruption of tissue with polytron in a 5 mM phosphate buffer, pH 7.5, with 0.1 mM EDTA. Extracts were centrifuged at 10,000 g, and the supernatant was retained. Protein concentration was measured by the Bradford method using a protein assay reagent from Bio-Rad (Hercules, CA). Aldehyde reductase activity was measured by following NADPH oxidation at 340 nm in the presence of 45 mM D-glucuronate in 0.1 M phosphate buffer (pH 7.0). Control reactions without substrate (glucuronate) or enzyme (cell extract) were performed, and initial velocities were subtracted from the samples (if any). To confirm specificity of our measurement for aldehyde reductase, a specific aldehyde reductase inhibitor, AL-1576 (0.5 µM), was added in the course of the reaction.
Immunohistochemistry. Kidneys were cut in half and fixed in buffered formaldehyde (Fisher, Pittsburgh, PA) overnight and transferred to 70% ethanol the next day. Immunohistochemistry was performed on paraffin sections using a purified IgG fraction of aldehyde reductase antibodies at a concentration of 1.2 µg/ml. Antibodies against human recombinant aldehyde reductase were raised in rabbits as described previously (8). The IgG fraction was purified using a ImmunoPure Plus(A) IgG Purification Kit from Pierce (Rockford, IL). Goat anti-rabbit secondary antibody was from Vector (Burlingame, CA). Detection was performed using the ABC system (Vector) with horseradish peroxidase and 3-amino-9-ethylcarbazole (BioGenex, San Ramon, CA) as a chromogen. Slides were counterstained with hematoxylin.
Western blot analysis. Tissue protein extracts were separated on a 10% precast Novex gel with MOPS running buffer (Invitrogen, Carlsbad, CA), transferred electrophoretically on a nitrocellulose membrane, and hybridized with antibodies. Aldehyde reductase antibodies are described in the previous paragraph. Actin antibodies were purchased from Santa Cruz Biotechnology. Chemiluminescent detection was performed using the ECL Plus system from Amersham.
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RESULTS |
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Tissue distribution of aldehyde reductase activity and mRNA. Aldehyde reductase differs from other aldo-keto reductases in its preference for the negatively charged substrates. Of those, glucuronate is the specific substrate for aldehyde reductase that is not reduced in an NADPH-specific fashion by other enzymes (10, 23). In addition, it may be the physiological substrate for this enzyme, especially in the kidney. Therefore, we used glucuronate to assess aldehyde reductase activity in the cytosolic extracts from various mouse tissues. The specificity of the assay was confirmed by applying 0.5 µM (10x Ki) of the specific aldehyde reductase inhibitor AL-1576. We have previously shown that this inhibitor has a 13-fold higher affinity for aldehyde reductase than for aldose reductase, the most closely related member of the superfamily (8). Results presented in Table 1 indicate that kidney has 10-fold higher activity compared with the liver and intestine. Activity per milligram protein in the organs was compared with activity measured simultaneously with a known amount of purified aldehyde reductase. Based on this comparison, we estimated that aldehyde reductase represents as much as 1% of total cytosolic protein in the kidney. The same estimate is confirmed by Western blotting (Fig. 2A).
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The human kidney and liver were also tested for the expression of the short transcript (Fig. 2C). Similar to the mouse, the human kidney expressed a significant amount of the short transcript, about fourfold higher than that of the long one. The intensity of the small band in the liver is about fourfold lower than in the kidney, although the relative difference between the human organs is not as large as in the mouse.
Aldehyde reductase expression during kidney development. Temporal expression of aldehyde reductase mRNA, protein, and activity during prenatal and postnatal development was measured by RPA, Western blotting, and enzyme kinetics methods. Only the long ubiquitous isoform of the aldehyde reductase mRNA is present in ES cells and through the embryonic and newborn development until day 6 after birth. Starting from day 6, the short transcript appears and increases gradually in intensity, reaching its highest level in adulthood (Fig. 3A). This profile suggests that a separate mechanism for aldehyde reductase expression is turned on in the kidney during the second or third week of postnatal development.
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Aldehyde reductase localization in the kidney. As revealed by immunohistochemistry, aldehyde reductase is localized in the proximal tubules including the S1 segment, which is continued from the epithelial cells of Bowmans capsule (Figs. 4 and 5). Staining is distributed throughout the cytosol, but increased intensity is observed on the apical membrane (Fig. 4B). Significantly less staining is observed in the descending limb of the loop of Henle, and no staining is visible in the distal segments of the nephron, collecting ducts, or the glomeruli. The intensity of staining in murine kidney increases with age (Fig. 4A), suggesting that induced aldehyde reductase expression is a part of the process of tubular maturation.
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Role of aldehyde reductase in glucuronate metabolism. Aldehyde reductase participation in the pathways of inositol catabolism and vitamin C synthesis was suggested based on its substrate specificity for glucuronate (20, 26). To check whether aldehyde reductase participates in these pathways in vivo, we administered the aldehyde reductase inhibitor AL-1576 at 20 mg·kg1·day1 orally to mice for 4 days and measured urinary concentrations of glucuronate and vitamin C. Measurements were normalized by creatinine for correction of urinary dilution. Measurements did not differ between morning and evening collections; therefore, data obtained on a single day were combined. Urinary concentration of glucuronate increased almost sixfold after overnight administration of the inhibitor and continued to increase reaching maximum on day 4 (8.5-fold, P = 0.0039; Fig. 6). Similarly, vitamin C concentration started decreasing on day 1 of inhibitor administration and was minimal on day 4 (3.2-fold decrease, P = 0.0026; Fig. 6). After inhibitor withdrawal, the levels of metabolites returned to normal, suggesting that changes were reversible. A 10-mg/kg dose caused a similar effect but of a lesser magnitude [3.7-fold increase in glucuronate/creatinine ratio (P = 0.0015); 2.1-fold decrease in vitamin C/creatinine (P = 0.030)].
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DISCUSSION |
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Our tissue activity measurements of three organs reported here, namely, kidney, liver, and intestine, indicate that the level of activity differs significantly although aldehyde reductase is indeed present in all these organs. The level of aldehyde reductase in mouse kidney is 10 times higher than in liver and intestine. Based on the specific activity comparisons, aldehyde reductase represents 1% of total soluble protein in kidney, indicating that it has a major function in this organ. Immunohistochemical analysis reveals that aldehyde reductase level is very high in the proximal convoluted tubules and epithelial cells lining Bowmans capsule, much less in the descending limb of the loop of Henle, and completely absent from the distal parts of the nephron and collecting ducts. No aldehyde reductase is present in the glomerular tuft epithelium. Subcellular localization is cytosolic, with concentration on the apical membrane.
Table 1 shows that the murine kidney has very high ability to reduce glucuronate. Glucuronate is a specific substrate of aldehyde reductase that is not converted by other aldo-keto reductases. In the proximal tubules, glucuronate arises from inositol. It is remarkable that inositol catabolism occurs almost exclusively in the kidney cortex (26). The first enzyme of this pathway, inositol oxygenase (EC 1.13.99.1 [EC] ), converts inositol into glucuronate (26). This enzyme was cloned recently (4) and appeared identical in sequence with "renal-specific oxidoreductase" (36) (GenBank entry NM 017584). Yang et al. (36) showed by in situ hybridization that the enzyme localizes to the proximal tubular epithelium, the same cells where aldehyde reductase is found. Abundance of this enzyme in the kidney cortex increases dramatically during the first 3 wk of mouse life (18). Thus it appears that these two enzymes form a pathway active in the proximal tubules and their expression is coordinately regulated during development.
The study by Hoyle et al. (17) in 1992 showed that administration of compound AL1576 (which was then tested as an aldose reductase inhibitor) to rats resulted in an 11-fold increase in the urinary output of glucuronate. Later, in 1995 we showed that AL-1576 is a 10-fold better inhibitor of aldehyde than aldose reductase (8). Here, we report that AL-1576 administration to mice led to a ninefold increase in glucuronate and threefold decrease in vitamin C concentration in urine. Thus aldehyde reductase indeed participates in glucuronate metabolism and vitamin C production in vivo. Combined with the findings that aldehyde reductase is localized together with inositol oxygenase in the proximal tubular epithelium, we conclude that aldehyde reductase functions in the kidney as a participant in the inositol catabolism pathway. Inhibition of aldehyde reductase thus leads to increased wasting of glucuronate.
D-Glucuronate is converted by aldehyde reductase to L-gulonate, which further enters the pentose interconversions through the action of L-gulonate dehydrogenase, or is converted into vitamin C in animals by the consecutive action of gulono-3 lactonase and L-gulonolactone oxidase. The liver is considered a major site of vitamin C production due to hepatic localization of L-gulonolactone oxidase (humans cannot produce vitamin C due to the lack of this enzyme). Whereas the kidney cortex is the major source of upstream components of the pathway, aldehyde reductase and glucuronate, additional experiments are necessary to elucidate the tissue origin of glucuronate and vitamin C found in urine in our experiments.
A specific mechanism is employed to ensure high expression of aldehyde reductase in the kidney tubules. The kidney has two transcripts of aldehyde reductase that differ in their 5'-UTRs. The long transcript is found ubiquitously in mouse organs and in cell lines and contains a 319-bp-long 5'-UTR. We previously reported that the combination of factors STAF and SP drive the expression of a ubiquitous transcript (9). The tissue distribution of the ubiquitous transcript is consistent with these findings. It is present in every tissue examined, but the liver has lower level of this transcript consistent with the low level of STAF observed in this organ (1).
The short transcript has only a 64-bp 5'-UTR and is localized almost exclusively in the kidney. The level of the short transcript is at least 10-fold higher than that of the long one in the adult mouse kidney. Readily detectable amounts of the short transcript are also observed in the murine liver and intestine, but in these organs its level is several-fold lower than that of the long transcript.
The aldehyde reductase gene has a separate exon coding for the 5'-UTR (7). The short transcript results from the transcription initiation at the start site near the 3'-end of exon 1. This start site is 255 nt downstream from the previously characterized ubiquitous start site. The distance between the two start sites and the different patterns of expression of the short and long transcripts make us postulate that these transcripts are expressed by separate mechanisms and that different sets of transcription factors are involved. A similar system was reported by Lieberman and colleagues (19) for mouse -glutamyl transpeptidase. This gene appears to have seven different promoters and alternative start exons active in different tissues and at different stages of development (19). One of the promoters is also active exclusively in the kidney proximal tubules, where
-glutamyl transpeptidase plays important role in cysteine conservation (27).
Expression of the kidney-specific transcript of aldehyde reductase is regulated developmentally. In the mouse, it is absent from ES cells, embryonic and the newborn kidney, and appears for the first time on postnatal day 6, increasing in intensity afterward. Enzymatic activity of the whole kidney parallels the appearance of the short transcript, exhibiting a sigmoidal curve with a growth phase falling on the third week after birth. The increase in aldehyde reductase activity coincides in time to the process of tubular maturation, during which the gene expression pattern in tubules significantly changes (2, 14, 28). In accordance with this process, the intensity of immunostaining in the newborn proximal tubules is relatively low (Fig. 4). With age, both the total mass of the tubules and the staining intensity (reflecting aldehyde reductase amount per cell) increase, reflecting the maturation process in the kidney, changes in the transcription factor content, and a 10-fold increase in total aldehyde reductase activity from newborn to adult kidney. We conclude that synthesis of aldehyde reductase via a specific mechanism is a part of kidney tubular maturation.
The sharp rise in aldehyde reductase activity corresponds to the time of weaning, when the diet of mice changes from mothers milk to solid food. This shift is accompanied by the appearance of new metabolites and changes in the organism to adapt to new conditions. In the kidney tubules, the composition of phosphate, sodium, and potassium transporters changes during the process of maturation (14, 28, 31). Thus attainment of aldehyde reductase activity by the developing kidney may reflect the necessity and ability to process new metabolites arising from the consumption of new kinds of food and new metabolic requirements.
Histological localization of aldehyde reductase in the human and mouse kidney is very similar. The human kidney also contains short and long mRNA transcripts, with the short transcript prevailing. Mouse and human sequences contain 68% identity in the region surrounding the second transcription start site, with several putative transcription factor binding sites conserved. Thus the mechanism for aldehyde reductase expression in the human and in the mouse is similar, albeit expression in the human is not as kidney specific as in the mouse and the time frame for the induction of the short transcript might be different, because aldehyde reductase immunoreactivity was observed in the tubular epithelial cells of human kidney as early as in the 9-wk-old fetus.
The recent study by Stuart et al. (31) characterized changes in global gene expression during development and maturation of the rat kidney using high-density DNA array technology. Authors found a group of genes with marked expression during adulthood but not in the neonatal state. This group was enriched in transporters, detoxification enzymes, and antioxidative stress genes. Members of this group were strikingly more common in libraries from tissues characterized by the presence of branching ductal epithelial structures such as the kidney, lung, liver, and pancreas. Our findings place aldehyde reductase in this group. The multiplicity of genes with similar functions following the same developmental pattern suggests the possibility of a common mechanism used to regulate their expression. Detailed studies of the transcription mechanism of regulation of the aldehyde reductase kidney-specific transcript may thus reveal general features involved in the developmental program of a whole group of genes associated with tubular maturation and other processes related to epithelial differentiation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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