ARTICLE |
Correspondence to: Amrik Sahota, Dept. of Genetics, Nelson Laboratories, Rutgers University, 604 Allison Road, Piscataway, NJ 08854-8082. E-mail: sahota@nel-exchange.rutgers.edu
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Summary |
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Kidney androgen-regulated protein (Kap) is the most abundant protein in the mouse kidney, but its function is unknown. We previously observed a significant decrease in Kap mRNA expression in whole kidney tissue from male mice with adenine phosphoribosyltransferase (APRT) deficiency and 2,8-dihydroxyadenine (DHA) nephrolithiasis. The disease phenotype is more severe in male mice and is age-dependent. To identify the cellular basis for differential Kap expression, we used in situ hybridization (ISH) and reverse transcription-polymerase chain reaction ISH (RT-PCR ISH) to identify the cell types expressing this mRNA in paraffin-embedded kidney sections. In 1-month-old wild-type male mice, Kap was detected primarily in S3 proximal tubule segments, but expression was very low in female mice. In 1-month-old APRT-deficient male mice, Kap expression was decreased significantly and was undetectable in female mice. Kap mRNA was not detected in 3- or 6-month-old mice using our standard ISH protocol, but we observed intense cytoplasmic staining in S3 proximal tubules in wild-type male mice of these age groups using an improved RT-PCR ISH procedure. Our studies demonstrate age-, gender-, and APRT genotype-dependent changes in Kap mRNA expression in mouse kidney. Kap expression is under multihormonal control, and hormonal changes in DHA-induced nephrolithiasis may account for the decreased Kap expression in APRT-deficient mice.
(J Histochem Cytochem 50:16631669, 2002)
Key Words: adenine phosphoribosyltrans- ferase deficiency, 2,8-dihydroxadenine neph- rolithiasis, kidney androgen-regulated, protein, in situ hybridization, reverse transcription-poly- merase chain reaction in situ, hybridization
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Introduction |
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The major phenotypic features of human adenine phosphoribosyltransferase (APRT) deficiency are 2,8-dihydroxyadenine (DHA) nephrolithiasis and, in some cases, acute or chronic renal failure (
As a first step in understanding the role of Kap in DHA-induced nephrolithiasis, we examined, by in situ hybridization (ISH) and RT-PCR ISH, the expression of Kap mRNA in mouse kidney sections. These findings were then correlated with the renal histological changes in these mice. Here we show that there are age-, gender-, and APRT genotype-related differences in Kap mRNA expression. Decreased Kap expression in APRT-deficient mice may be due, at least in part, to DHA-induced hormonal changes.
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Materials and Methods |
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Preparation of Kidney Sections
All animal studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Strain 129 mice were used in these studies and 68-µm thick kidney sections were prepared as described previously (
In Situ Hybridization
Tissue sections were deparaffinized, rehydrated, equilibrated in 50 mM Tris-HCl buffer (pH 7.5), and then incubated with proteinase K (4 µg/ml) for 510 min at 37C in a humid chamber. The digestion conditions were optimized to allow probe access to intracellular mRNA but still maintain tissue integrity (
Tissue sections were pre-hybridized at 42C for 1 hr using ISH buffer (Ambion; Austin, TX), and then hybridized with digoxigenin-labeled probes for 1618 hr at the same temperature. The samples were sealed with Hybaid EasiSeals to prevent evaporation (Hybaid; Franklin, MA). The slides were washed (37C, 5 min) once in 2 x in situ wash solution (Ambion) and then twice in 1 x concentration of the same solution. For each tissue, adjacent sections were hybridized with the antisense and sense Kap probes. The probe concentration was 545 ng/µl (in a total volume of 60 µl) depending on message abundance, but we routinely used a probe concentration of 15 ng/µl. In addition to the sense probe, controls included omission of probe and omission of anti-digoxigenin antibody from the reaction mixture.
The signal was detected as described in the manufacturer's instructions (Roche Molecular Biochemicals). Briefly, after post-hybridization washes, the slides were washed in washing buffer for 15 min and then immersed in blocking buffer for 30 min. The sections were incubated in a 1:300 dilution (in blocking buffer) of alkaline phosphatase-conjugated anti-digoxigenin antibody (sheep Fab fragments) for 2 hr at ambient temperature. The slides were then incubated in nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) at 37C for 1530 min, and the signal visualized as a purple precipitate. The slides were not routinely counterstained. To verify the identity of positively staining structures, selected sections were counterstained with methyl green and adjacent sections were routinely stained with hematoxylin and eosin.
The slides were examined using a Nikon Eclipse E800 microscope (Nikon; Melville, NY). For each section, five frames in a cross-configuration were captured using a x20 objective lens and a MagnaFire camera and software (Optronics; Goleta, CA). Images were processed using Scion Image for Windows (Scion; Fredrick, MD), a freely available version of NIH Image software (http://www.scioncorp.com and http://rsb.info.nih.gov/nih-image/). For each frame, 10 measurements of signal intensity were captured, again in a cross-configuration. Kidney sections from age- and sex-matched wild-type mice were used as controls. The results presented here are based on expression analyses in at least two sections from each of three to five animals of a given APRT genotype and gender. Morphometric measurements were also carried out using Scion Image software, and the data for wild-type and mutant mice were compared using the unpaired t-test.
RT-PCR ISH
We used indirect in situ RT-PCR (here referred to as RT-PCR ISH) for the cellular localization of Kap mRNA when this message could not be detected using our standard ISH protocol. RT-PCR ISH involves the incorporation of unlabeled nucleotides during PCR, followed by ISH using digoxigenin-labeled internal oligonucleotide probes (
The reaction mixture (50 µl per section) contained 5 µl of 10 x ExpeRT-PCR buffer (with MgCl2), 1 µl DMSO, 1 µl dNTP mix (final concentration 200 µM each), 300 nM forward and reverse Kap primers, 20 U RNase inhibitor, 15 U AMV reverse transcriptase, and 5 U Taq/Pwo mix. The sequences of the forward and reverse PCR primers were 5'-CTCAGTCTTTGACCAACTGGG-3' and 5'-CTGTGATGTCTGTGTTCTCAG-3', respectively. cDNA synthesis was carried out at 42C for 1 hr. The sections were then denatured at 90C for 1 min, followed by PCR amplification at 90C for 30 sec, 48C for 30 sec, and 70C for 1 min for six to ten cycles (
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Results |
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In Situ Hybridization
We detected Kap mRNA expression primarily in the S3 segments of proximal tubules and, to a lesser extent in the S1/S2 segments, in 1-month-old wild-type male mice (Fig 1A). There was a significant reduction in expression in 1-month-old APRT-deficient male mice (Fig 1B). Kap mRNA was detected mainly in S3 segments in 1-month-old wild-type female mice but expression levels were very low compared with male mice, and were undetectable in APRT-deficient female mice. We did not detect Kap mRNA in kidney sections from 3- or 6-month-old male or female mice of any APRT genotype using our standard ISH protocol (probe concentration 15 ng/µl), or even when the probe concentration was increased to 45 ng/µl.
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To determine whether the decrease in Kap expression in proximal tubules was the result of renal histological changes, we measured the number of proximal tubule sections per field, the number of nuclei, the number of glomeruli, and wall thickness of proximal tubules in kidney sections from 1-month-old wild-type and APRT-deficient male mice. Five x20 non-overlapping fields were counted in sections from each of three animals per group. There was no significant difference in the mean number of tubule sections or the mean number of nuclei between wild-type and APRT knockout mice. In APRT knockout mice, the number of glomeruli per field was approximately one half and the wall thickness of proximal tubules was approximately twofold compared with wild-type mice, and both these differences were highly significant (Table 1).
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We did not observe any nonspecific hybridization as demonstrated by the use of the corresponding sense probe, and this was confirmed using two additional indicators of hybridization specificity. First, no hybridization signal was observed using a mixture of unlabeled and labeled Kap antisense probes (in the ratio 100:1), indicating that the signal from the labeled probe was competed out by the unlabeled probe. Second, an antisense probe designed to hybridize to a different region of Kap mRNA was used and the hybridization pattern was identical to that obtained with the standard probe (data not shown).
RT-PCR ISH
The lack of Kap mRNA signal in mice older than 1 month may simply be due to the low sensitivity of standard ISH procedures. However, the application of several RT-PCR ISH protocols to kidney sections resulted in extensive tissue damage and loss of amplified material. This may possibly be due to extreme temperature fluctuations during the large number of PCR cycles that are typically used in these procedures and/or to uneven heat distribution over the slides (
We observed intense cytoplasmic staining primarily in S3 proximal tubule segments in 3-month and older wild-type male mice. Furthermore, a probe concentration of 12 ng/µl was sufficient for signal detection, and the signal intensity was comparable to that observed with 15 ng/µl probe concentration in the standard ISH assay, confirming specific amplification of Kap mRNA (Fig 2A). The signal was detectable with both the sense and antisense Kap probes, again demonstrating that cDNA synthesis had occurred. RT-PCR ISH controls were negative, demonstrating the specificity of the hybridization reaction (Fig 2B), and no signal was detected in the absence of RT-PCR, even at a probe concentration of 45 ng/µl (Fig 2C). Because of poor renal histology in APRT-deficient mice, there was variable nonspecific staining in kidney sections from these mice.
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Discussion |
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Kap represents the most abundant mRNA species in the mouse kidney, but its expression pattern is more complex than that of a number of other genes (e.g., ß-glucuronidase and ornithine decarboxylase) whose expression in the kidney is also induced by androgens (
Kap expression in the S3 segments is under the control of the pituitary hormone thyrotropin and of estrogens in females (
In our differential display and RT-PCR studies in APRT-deficient mice, we showed that the renal deposition of DHA leads to changes in the expression of several genes involved in tissue injury and that these changes were more pronounced in male mice (
Our ISH studies showed that Kap mRNA was expressed primarily in the S3 segments of proximal tubules in 1-month-old wild-type male mice and that expression levels were extremely low in wild-type female mice of this age group, confirming previous studies of Kap expression in the kidney (
There are differences in the progression of non-diabetic chronic renal disease among males and females (
Recent studies suggest that Kap interacts with cyclophilin B (CyPB) (
We did not detect Kap mRNA using our standard ISH protocol in kidney sections from wild-type or APRT-deficient mice older than 1 month. We therefore optimized an indirect in situ RT-PCR procedure (RT-PCR ISH) for detection of this mRNA. This procedure is inherently more specific than direct in situ RT-PCR, in which labeled nucleotides are included in the PCR step. RT-PCR ISH has been widely used to demonstrate the localization of low copy-number nucleic acid sequences within cells and to investigate the role of viruses in a variety of clinical conditions (
Previous studies have emphasized that a key parameter in RT-PCR ISH is the protease digestion step (
A comparison of Fig 1 and Fig 2 shows that tissue morphology after RT-PCR ISH is not of the same quality as in the standard ISH procedure. Therefore, one major limitation of RT-PCR ISH is that it is unlikely to be useful when the tissue to be examined is already extensively damaged, as in the case of, for example, 6-month-old APRT-deficient male mice. In these situations, reliable comparisons of gene expression between normal and diseased tissue are best made using solution RT-PCR or ISH/ immunohistochemical procedures that amplify the detection signal rather than the target mRNA (
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Acknowledgments |
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Supported by NIH grants DK38185, ES05652, and ES06096.
Received for publication June 3, 2001; accepted June 26, 2002.
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