1 Department of Cell and Molecular Physiology, 2 Department of Biology, 3 Center for Gastrointestinal Biology and Disease, and 4 Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Insulin-like growth factor I (IGF-I) may promote survival of putative stem cells in the small intestinal epithelium. Mitosis and apoptosis were quantified in crypts of nonirradiated and irradiated IGF-I transgenic (TG) and wild-type (WT) littermates. The mean apoptotic index was significantly greater in WT vs. TG littermates. After irradiation, apoptotic indexes increased, and WT mice showed a more dramatic increase in apoptosis than TG mice at the location of putative stem cells. After irradiation, no mitotic figures were observed in WT crypts, whereas mitosis was maintained within the jejunal epithelium of TG mice. The abundance and localization of Bax mRNA did not differ between nonirradiated littermates. However, there was more Bax mRNA in TG vs. WT mice after irradiation. Bax mRNA was located along the entire length of the irradiated crypt epithelium, but there was less Bax protein observed in the bottom third of TG mouse crypts compared with WT littermates. IGF-I regulates cell number by stimulating crypt cell proliferation and decreasing apoptosis preferentially within the stem cell compartment.
Bax; mitosis; stem cells; crypts
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INTRODUCTION |
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INSULIN-LIKE GROWTH FACTOR I (IGF-I) regulates somatic growth in pre- and postnatal life (1, 2). The small intestinal epithelium, one of the most rapidly proliferating systems in the body, is a target organ for IGF-I action (20, 31, 32). Systemically administered IGF-I acts in an endocrine manner to increase the mass of small bowel mucosa and crypt cell proliferation (11, 18, 31, 32, 37, 43, 48). In addition, IGF-I is locally expressed throughout the gastrointestinal tract (12, 21). Changes in local IGF-I expression correlate with changes in mucosal mass, suggesting that locally expressed IGF-I also exerts paracrine and/or autocrine actions on bowel growth and function (45, 48, 49).
Evidence is accumulating (28, 30, 41, 44) that IGF-I not only stimulates cell proliferation but can also protect against apoptosis in many cell types in vitro. In cultured cells, IGF-I protects against apoptosis due to cytotoxic agents (41) or serum deprivation (28, 30, 44). There are fewer reports that address the action of IGF-I on apoptosis in vivo. However, it is known that systemically administered IGF-I decreases apoptosis in rat liver after cutaneous thermal injury (13). In addition, transgenic (TG) mice that overexpress IGF-I in the brain exhibit decreases in apoptosis of cerebellar neurons (6), and IGF-I null mice have increased apoptosis of cochlear neurons (5).
The antiapoptotic actions of IGF-I have been shown to occur through several mechanisms. IGF-I is known to decrease expression of the mitochondrial-associated cell death protein Bax as well as to increase expression of the antiapoptotic proteins Bcl-xL and Bcl-2 (6, 26, 28, 40, 41, 44). Inhibition of apoptosis by IGF-I has also been shown to occur through suppression of caspases, a family of cell death-promoting enzymes (14), as well as IGF-I-induced phosphorylation and inactivation of the proapoptotic protein Bad (6, 9, 10, 30).
Irradiation and cytotoxic drugs dramatically increase the low level of spontaneous apoptosis in intestinal crypts (33-36). Spontaneous apoptosis of intestinal crypt cells is believed to protect against the survival and expansion of genetically damaged cells (33-36). Irradiation increases apoptosis and is also known to cause cell cycle arrest in intestinal crypts (7), an effect that limits proliferation of genetically damaged cells. Factors, such as IGF-I, that prevent apoptosis and induce intestinal growth could therefore, increase cancer risk (4). Indeed, epidemiological data indicate a positive correlation between circulating levels of IGF-I or the structurally homologous IGF-II and the risk of colorectal cancer (15, 38, 39). Although antiapoptotic actions of IGF-I have been noted (19, 29) in colon cancer cells in culture, to date the effect of IGF-I on spontaneous or irradiation-induced apoptosis of intestinal crypt cells in vivo has not been examined. Determining whether IGF-I alters crypt cell apoptosis in vivo is relevant to the potential risks of therapeutic strategies aimed at promoting increased intestinal mucosal growth and/or elevating levels of circulating or locally expressed IGF-I (18, 20, 22, 31, 32, 37, 48, 49).
Although some of the downstream mediators of spontaneous and irradiation-induced apoptosis have been identified in the small intestine, the in vivo effects of IGF-I on these pathways have not been determined. Several studies (8, 25) have demonstrated that p53, a tumor-suppressor gene product, is required for irradiation-induced crypt cell apoptosis. Other research (16, 17) has indicated that the expression of Bax increases after irradiation, suggesting that Bax plays an important role in the regulation of spontaneous or irradiation-induced apoptosis in the small intestine.
The goals of our study were to assess whether IGF-I alters spontaneous or irradiation-induced apoptosis and cell cycle arrest in the small intestine in vivo and if the effects of IGF-I are associated with altered expression of Bax mRNA or protein. TG mice, which express a metallothionein promoter-driven human IGF-I transgene, and wild-type (WT) littermates have proven a useful model to study the effects of excess circulating and locally expressed IGF-I on increased mucosal mass and crypt cell proliferation (27). We examined the frequency and localization of mitosis and apoptosis in the small intestine of irradiated and nonirradiated IGF-I TG and WT littermates and the abundance and localization of Bax mRNA and protein.
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MATERIALS AND METHODS |
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Laboratory animals. Mice overexpressing the human IGF-I transgene were established and maintained at the University of North Carolina (Chapel Hill, NC) as described previously (23, 27). IGF-I TG mice overexpress a fusion gene that consists of the mouse metallothionein I promoter, a rat somatostatin signal sequence to promote secretion, and a human IGF-Ea cDNA (23). We (27) have previously established that IGF-I TG mice have elevated circulating IGF-I and express the IGF-I transgene in villus enterocytes. IGF-I TG mice and their sex-matched WT littermates were 50 to 70 days old when used for the study. Because TG mice eat ~30% more than WT mice when food is unrestricted (27), the daily food intake of IGF-I TG mice was adjusted to that of WT littermates 4 days before the study to avoid any impact of differences in luminal nutrients. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of North Carolina; study protocols were in compliance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205].
Irradiation and tissue preparation.
Mice received 5 Gy of whole body irradiation delivered at 1 Gy/min with
a 137Cs source. The mice were killed under anesthesia
4 h after irradiation, because maximum apoptosis and cell
cycle arrest have been observed (33) in mice after this
dose of irradiation. The proximal jejunum (defined as one-third of the
small bowel beginning at the ligament of Treitz) was collected for
extraction of RNA and rapidly frozen in liquid nitrogen and stored at
80°C until analysis. An adjacent 1-cm piece of jejunum was
processed for protein extraction; the mucosa was scraped and placed in
lysis buffer (50 mM HEPES, 150 mM NaCl, 20 mM sodium pyrophosphate, 100 mM NaF, 1.5% Triton X-100, and 10 mM EDTA). Three pieces of adjacent
jejunum (each 0.5-cm long) were collected for histological analyses.
Tissue for morphological analyses of apoptotic or mitotic cells was
fixed in Carnoy's fixative for 30 min followed by dehydration in 70%
ethanol and paraffin embedding. Tissue used for terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling
(TUNEL) or immunohistochemistry was fixed in 10% formalin for 4 h, dehydrated in 70% ethanol, and embedded in paraffin. The tissue for
in situ hybridization was embedded in OCT compound (Miles, Elkhart,
IN), frozen in isopentane at
40°C, and stored at
80°C until sectioning.
Analysis of apoptosis and mitosis. Paraffin-embedded jejunal tissue was cut transversely at 4 µm and stained with Mayer's hematoxylin and eosin. Analyses of apoptosis were performed on hematoxylin and eosin-stained sections and restricted to well-oriented crypts that contained Paneth cells at the base, a lumen, and clearly defined cells aligned with the base of adjacent villi. The morphological identification and quantification of apoptotic crypt cells were based on distinctive morphological features described elsewhere (see Ref. 46 for review). Morphological features of apoptosis include nuclear margination, chromatin and cytoplasmic condensation, shrinkage from neighboring cells, and the formation of apoptotic bodies due to nuclear and cytoplasmic fragmentation. Small apoptotic bodies clustered at a single position were regarded as one apoptotic cell. The position of apoptotic cells along the depth of the intestinal crypt was recorded as described previously (34) with cells at the base designated as position 1. Mitotic figures were counted within the same crypt sections used for morphological scoring of apoptosis. All scoring was performed twice by a single investigator unaware of the mouse genotype or treatment. Scoring was performed on two sections from the same specimen that were more than 50 µm apart to avoid counting the same apoptotic or mitotic cell twice. Scoring was also verified by a second investigator.
TUNEL labeling of apoptotic cells. The TUNEL method was used on segments of irradiated jejunum to verify quantitation of apoptosis by morphological methods. Jejunal tissue specimens fixed in 10% formalin and embedded in paraffin were cut transversely at 8 µm. Detection of DNA fragmentation in apoptotic cells was determined by the TUNEL method as described in the handbook from Intergen (Purchase, NY). Briefly, slides were deparaffinized, treated with proteinase K and 3.0% hydrogen peroxide, and incubated with TdT and digoxigenin-labeled nucleotides for 1 h at room temperature. Slides were washed and then treated with a peroxidase-conjugated digoxigenin antibody (Intergen). Visualization was accomplished using diaminobenzidine (DAB). Slides were counterstained with hematoxylin, dehydrated in ethanol, and coverslipped. As a positive control, sections were incubated with DNase I before the TdT reaction. Negative controls were processed in the same manner as test slides except that TdT was omitted. Cells labeled by the TUNEL assay were quantified using the same method as described for morphological analysis of apoptotic and mitotic cells.
RNA isolation and Northern blot hybridization assay of Bax mRNA.
Total RNA was isolated from proximal jejunum by the guanidine
isothiocyanate-CsCl method. Aliquots of total RNA (15 µg) were denatured with glyoxal and dimethyl sulfoxide, size fractionated on 1%
agarose gels, and transferred to Gene Screen (New England Nuclear,
Boston, MA). Hybridization was performed using standard conditions and
an antisense Bax RNA probe labeled with [32P]UTP. The
antisense probe was synthesized by in vitro transcription from
linearized plasmid template comprising rat Bax cDNA subcloned into
pGEM4Z. The Bax plasmid was a generous gift from Dr. J. L. Tilly
(Johns Hopkins University, Baltimore, MD) (42). After washing and exposure to PhosphorImager screens, each blot was stripped
and rehybridized with a rat -actin complementary RNA probe (Ambion,
Austin, TX). Abundance of mRNA was calculated on a scanning
densitometer using NIH Image software (National Technical Information
Service, Springfield, VA). Abundance of Bax mRNA in each sample was
normalized to the abundance of invariant
-actin mRNA to control for
minor differences in RNA loading.
In situ hybridization histochemistry. In situ hybridization was performed using frozen sections (10 µm) as previously described (27). Briefly, sections were fixed with 4% paraformaldehyde, treated with proteinase K, and dehydrated before hybridization. Hybridization was performed at 55°C for 18 h in buffer containing [35S]UTP-labeled rat Bax antisense RNA probe (2 × 106 cpm/slide) prepared from the same template cDNA used for Northern blot. All sections were treated with RNase A (200 mg/ml) after hybridization and washed with 0.5× SSC (75 mM NaCl and 7.5 mM sodium citrate, pH 7.0) at 55°C. The sections were dehydrated, air dried, and exposed to Kodak NTB-2 emulsion at 4°C for 14 days. After developing, sections were counterstained with hematoxylin. Sections prepared as negative controls were treated with RNase A before hybridization or hybridized with a control Bax sense RNA probe.
Western blot analysis. Protein was isolated from jejunal mucosa. Aliquots of total protein (30 µg) were separated on a 14% acrylamide gel and transferred to an Immobilon-P membrane (Millipore). Immunoblots were blocked in 4% milk in Tris-buffered saline (TBS) overnight at 4°C. After washing in TBS with 0.05% Tween, primary antibody (Bax, Santa Cruz Biotech; N-20 or actin, Jackson Immunoresearch) was added in TBS for 2 h at room temperature. Blots were then washed and treated with a horseradish peroxidase-conjugated donkey anti-rabbit antibody (Jackson Immunoresearch) for 30 min and placed in enhanced chemiluminescence mix.
Localization of protein using immunohistochemistry. Formalin-fixed paraffin sections of jejunum were cut at 8 µm for immunohistochemistry. Coded sections from TG and WT littermate pairs were mounted on the same slides and processed simultaneously to eliminate interslide variability. Slides were deparaffinized, blocked with 3% hydrogen peroxide followed by 10% normal goat serum, and washed in PBS. Primary antibody (Bax, Santa Cruz Biotech; N-20) was added to sections in PBS containing 1% normal goat serum and Triton X-100 overnight for 18 h. Slides were then washed in PBS with Triton X-100 and treated with a biotinylated goat anti-rabbit antibody (Vectastain) for 60 min. Slides were incubated with avidin-biotin complex (Vectastain) for 60 min, and labeling was visualized using DAB. Slides were counterstained with hematoxylin, dehydrated in ethanol, coverslipped, and analyzed using light microscopy. Positively stained cells within the crypt epithelia were quantified as described for apoptotic and mitotic cells in Analysis of apoptosis and mitosis.
Statistical analyses. Values are expressed as means ± SE. Statistical comparisons of means were performed using the Mann-Whitney U-test. P < 0.05 was considered to be statistically significant. A two-factor ANOVA was also used to determine whether there was significant interaction between irradiation and IGF-I overexpression on Bax mRNA and protein expression.
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RESULTS |
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Apoptotic indexes.
In both nonirradiated and irradiated WT mice, apoptosis
assessed by morphological criteria or TUNEL was confined to the crypts with no detectable apoptosis in villus enterocytes. The mean
apoptotic index (the sum of apoptotic cells in all crypts
counted per total number of cells in all small intestinal crypts
counted) was calculated from both hematoxylin-stained and TUNEL-labeled
tissue from all TG and WT animals (Table
1). The mean apoptotic index in
nonirradiated IGF-I TG mice was significantly lower than that of WT
littermates (P < 0.05), indicating that the frequency
of spontaneous apoptosis was decreased in IGF-I TG mice. At
4 h after exposure to 5 Gy, apoptotic indexes were markedly
increased in both WT and IGF-I TG mice; however, there were
significantly fewer apoptotic cells per total cells counted in
irradiated IGF-I TG mice than in WT littermates (Fig.
1 and Table 1). To determine whether the
decreased apoptotic index in TG mice was partially due to the fact
that the number of cells per crypt was greater in IGF-I TG mice, we also compared the absolute number of apoptotic cells per crypt. There were significantly fewer apoptotic cells per crypt in IGF-I TG mice than in WT littermates in both nonirradiated and irradiated groups (Table 1, P < 0.05), confirming that both
spontaneous and irradiation-induced apoptosis occurred less
frequently in IGF-I TG mice than in WT littermates.
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Mitotic indexes.
The mitotic index (the sum of mitotic cells counted in all crypts per
total number of cells in all crypts counted) was significantly greater
in nonirradiated IGF-I TG mice than in WT littermates (Table
2, P < 0.05). At 4 h after 5 Gy of irradiation, no mitotic figures were observed within
the crypts in any sections of jejunum from WT mice. In contrast,
irradiation did not completely eliminate mitotic figures in IGF-I TG
mice. Irradiated TG mice maintained a higher number of mitotic cells
per crypt than nonirradiated WT mice (Table 2). The greatest percentage
(63%) of mitotic cells was located in the bottom third
(positions 1-5) of the crypts in irradiated
TG mice with peaks at the third (19%) and fifth (19%) cell positions.
The remainder of mitotic cells within irradiated TG mouse crypts were
located in the middle (33%) and upper third (4%) of the crypts.
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Expression and localization of Bax mRNA.
To determine whether IGF-I overexpression had an effect on expression
of mitochondrial-associated apoptotic proteins, the amount of Bax
mRNA was quantified in jejunum from each group of mice (Fig.
3). The expression of Bax mRNA was
significantly increased after irradiation in both IGF-I TG and WT mice.
The abundance of jejunal Bax mRNA did not differ between IGF-I TG and
WT littermates in the nonirradiated groups and was significantly
greater in IGF-I TG mice than in WT mice after irradiation. A
two-factor ANOVA revealed that irradiation and genotype have a
significant effect (P < 0.05) on Bax mRNA expression.
However, there was no significant interaction between these two
variables.
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Expression and localization of Bax protein.
The abundance of Bax protein in the entire mucosa from IGF-I TG and WT
mice is shown in Fig. 5. Although there
was no significant difference in protein levels, Bax was found in TG
and WT mice both before and after irradiation.
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DISCUSSION |
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The results of this study demonstrate that chronic IGF-I excess results in lower levels of spontaneous and irradiation-induced apoptosis in small intestinal crypts in vivo. Although IGF-I is known to reduce apoptosis in a number of tissues in vivo (3, 6, 13), this has not previously been demonstrated in the bowel. While spontaneous levels of apoptosis in WT mice were low, as reported previously (33), they were even lower in TG mice, and apoptotic cells in TG mice were located primarily at the base of the crypts where putative stem cells are located.
Apoptosis induced by -irradiation in the small intestine has
been extensively studied (8, 16, 25, 33, 35).
Consistent with previous observations (33), irradiation in
WT mice resulted in a more than 10-fold increase in apoptosis
with peak apoptosis occurring at positions 3 and
4, the likely position of crypt stem cells. Irradiation also
induced a 10-fold increase in apoptotic cells over spontaneous
levels in IGF-I TG mice, demonstrating that there is a population of
cells not protected from apoptosis by IGF-I. However, the
overall number of apoptotic cells or apoptotic cells per crypt
in IGF-I TG mice was 30-40% of the number observed in WT mice, as
assessed by morphological criteria. Independent evaluation of
apoptosis by TUNEL validated the lower levels of irradiation-induced apoptosis in TG mice (50% of WT mice),
although the relative level of apoptotic cells was somewhat
different. Given that apoptotic cells exhibiting morphological
features of apoptosis may not contain sufficient DNA capable of
TUNEL labeling, it is not surprising that there is a difference in
apoptotic indexes derived from quantitation using these two
methods. The similarity of our results in WT mice by both TUNEL and
morphological criteria to previous results (33) validates
the scoring system used in this study to compare IGF-I TG and WT mice.
Intriguingly, the difference in the percentage of apoptotic cells
undergoing apoptosis between IGF-I TG and WT mice was much greater at positions 1 through 4 than at higher
positions within the crypt. This indicates that IGF-I preferentially
promotes survival of cells located at the base of the crypts. Because
the lower positions in the crypts correspond to the putative stem cell
population (33), IGF-I-mediated survival of stem cells
after -irradiation might facilitate crypt regeneration and at the
same time enhance clonal expansion of genetically damaged stem cells
and predispose to neoplasia. The mechanism whereby IGF-I may
preferentially affect stem cells is not defined, but it has been
reported that IGF-I receptor abundance may be higher in the crypts than
in the villi (see Ref. 22 for review).
Induction of the proapoptotic protein Bax appears to be a key mediator of irradiation-induced crypt cell apoptosis (16, 17). As expected, irradiation induced Bax mRNA expression in both WT and IGF-I TG mice. Despite the fact that there were significantly fewer apoptotic cells in crypts from IGF-I TG mice compared with their WT littermates, the abundance of Bax mRNA was greater in IGF-I TG mice than WT mice as indicated by Northern blot analysis. Semiquantitative in situ hybridization histochemistry also indicates that Bax mRNA was induced at higher levels in IGF-I TG mice in all crypt compartments, including cells at the base of the crypts. Thus irradiation induces Bax mRNA in basal crypt cells despite the presence of elevated IGF-I. IGF-I therefore, appears to exert antiapoptotic actions by mechanisms downstream of transcription of the Bax gene or independent of Bax expression.
IGF-I is known (6, 41, 44) to exert its protective action on several cell types in culture by repressing the expression of Bax protein. Semiquantitative analysis of Bax protein using immunocytochemistry indicated that irradiated IGF-I TG mice had significantly less Bax protein in cells at the base of jejunal crypts compared with WT mice. The basal area of jejunal crypts contains stem cells and is also where the most potent protective effects of IGF-I on apoptosis were observed. Therefore, our findings raise the intriguing possibility that IGF-I acts to protect these stem cells in part by decreasing the accumulation of Bax protein expression in this selected cell population. Together, our results indicate that IGF-I may decrease apoptosis of crypt stem cells through posttranscriptional effects on Bax expression such as decreased translation or increased degradation of protein. Other studies (24, 47) have also indicated that protection from apoptosis is sometimes associated with a decrease in Bax protein expression despite an increase in expression of Bax mRNA. The results of our study are strikingly similar to results from Chrysis et al. (6) who showed that chronic overexpression of IGF-I in the cerebellum results in protection from apoptosis, associated with decreased Bax protein abundance despite elevated Bax mRNA.
Exposure to irradiation or DNA-damaging agents causes mammalian cells to arrest at the G1/S boundary, that is the G2 phase of the cell cycle (7). G2 arrest results in a decrease in the mitotic index. In the small intestine, temporal G2 arrest, also referred to as mitotic delay, occurs at 1 h/Gy in the midcrypt and 2.5 h/Gy in the crypt base (7). Our observation that no mitotic figures were present in crypts from WT mice 4 h after 5 Gy of irradiation are consistent with complete G2 phase arrest. In contrast, IGF-I TG mice exhibited incomplete arrest at the G2 stage of the cell cycle because their crypts exhibited significant numbers of mitotic cells. Interestingly, most of the mitotic cells observed in IGF-I TG mice were found in the lower third of the crypt compartment in the stem cell region. This supports the hypothesis that IGF-I not only preferentially promotes survival of this cell population after exposure to irradiation but also permits continued proliferation.
Our current findings do not exclude other proapoptotic or antiapoptotic proteins as mediators of IGF-I protection against crypt cell apoptosis. In fact, it seems likely that all the mediators of IGF-I antiapoptotic action have not yet been defined. Evaluation of other potential mediators is worthy of future investigation.
In summary, chronic overexpression of IGF-I resulted in 1) decreased spontaneous apoptosis, 2) decreased irradiation-induced apoptosis, 3) decreased expression of Bax protein within the stem cell region, 4) reduced irradiation-induced mitotic delay in small intestinal crypts, and 5) preferential protection of the putative stem cell population.
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ACKNOWLEDGEMENTS |
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We thank K. McNaughton, K. Poore, and J. Simmons for contributing to the completion of this work. We also acknowledge the use of facilities in the Microscopy Services Laboratory in the Department of Pathology and the DNA synthesis core of the Lineberger Cancer Center. H. R. Wilkins is a fellow in the Seeding Postdoctoral Innovators in Research and Education program.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-40247 (P. K. Lund) and Minority Opportunities in Research Division of the National Institute of General Medical Sciences Grant GM-000678.
Address for reprint requests and other correspondence: H. R. Wilkins, Dept. of Cell and Molecular Physiology, CB 7545, Univ. of North Carolina, Chapel Hill, NC 27599-7545.
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.
10.1152/ajpgi.00019.2002
Received 16 January 2002; accepted in final form 14 March 2002.
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