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DEVELOPMENT AND TISSUE PLASTICITY
David Geffen School of Medicine, Mattel Children's Hospital at University of California-Los Angeles, Los Angeles, California 90095-1752
Submitted 15 April 2003 ; accepted in final form 14 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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intrauterine growth retardation; Bcl-2; Bax; DNA methylation; epigenetics
Nephrogenesis involves the rapid remodeling of structures, which requires massive apoptosis (12, 34). Moreover, maternal protein restriction reduces glomeruli number in association with increased metonephric apoptosis on day 13 of gestation (73). The gene expression of apoptosis-related molecules Bcl-2 and Bax responds to the hypoxia, acidosis, and hypoglycemia that characterize the IUGR in utero environment, which is induced by uteroplacental insufficiency. Bcl-2 is an antiapoptosis protein that attenuates the effects of cytochrome c release from the mitochondria and counters the effects of the proapoptosis protein Bax (29, 33). Bcl-2 and Bax contribute to the signaling pathways that activate caspase-3 (casp-3), which is necessary for the chromatin condensation and DNA fragmentation that characterize apoptosis (28, 60). We hypothesized that induction of IUGR in the rat would reduce glomeruli number, increase renal apoptosis, and alter mRNA levels of apoptosis-related proteins.
To test this hypothesis, we induced IUGR using a rat model of uteroplacental insufficiency. Uteroplacental insufficiency causes asymmetric IUGR, in which brain growth is spared relative to body weight and is associated with the hypertensive disorders of pregnancy. These disorders are the most common medical complications of pregnancy and lead to serious perinatal mortality and morbidity (74). The IUGR human fetus endures in utero hypoxia, acidosis, hypoglycemia, altered levels of growth factors, and hypoinsulinemia (15-17). Similarly, when induced via bilateral uterine artery ligation of the pregnant dam, IUGR induces an identical response in the late gestation rat fetus (52, 54, 70). In this well-characterized and widely published rat model of asymmetrical growth retardation, IUGR pups are 20-25% lighter than the sham-operated control animals, and birth weights are normally distributed within and among litters (9, 36-41, 53, 54, 64). Litter size does not significantly differ between control and IUGR groups (36).
We quantified glomeruli number in sham-operated control (Con) and IUGR term rat pups (N0), as well as 21-day-old juvenile rat pups (J21). Apoptosis and the mRNA levels of the Bcl-2 and Bax were quantified in the N0 rat pups. Interestingly, Gobe et al. (21) found that renal Bcl-2 expression was associated with IGF-1 expression. The authors of this study speculated that this phenomenon was due to the promotion of distal tubule survival allowing the expression of growth factors. We therefore similarly followed up our findings of altered Bcl-2 expression by measuring renal IGF-1 mRNA levels.
p53 negatively regulates Bcl-2 expression and positively regulates Bax expression (63, 76). To investigate a mechanism through which the altered intrauterine milieu may affect Bcl-2 and Bax mRNA levels, we quantified mRNA levels of the transcription factor p53 and found significant differences between control and IUGR animals. Because promoter and exon 5-8 CpG methylation affects p53 mRNA levels, we determined CpG methylation of these regions using methylation-sensitive restriction enzymes and real-time PCR (57-59). Moreover, because p53-dependent apoptosis occurs in the absence of DNA methyltransferase 1 (DNMT1), we quantified mRNA levels of this enzyme (27).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Nephron number. The UCLA Brain Research Institute Microscopic Techniques Core Facility processed, sectioned, stained, and assisted in data acquisition for this study. Kidneys of sham-operated and control animals were fixed in 0.1 M sodium phosphate-buffered formaldehyde (ph 7.4, 4% formaldehyde) and embedded in Paraplast Plus (Fisher Scientific, Pittsburgh, PA). Sections (0.6-µm thick) were stained with Gills hematoxylin (Fisher Scientific, Pittsburgh, PA) and eosin y solution alcohol with phloxin (Sigma, St. Louis, MO) (n = 4 litters of control for both Con and IUGR).
Counting glomeruli in one kidney per animal was considered sufficient on the basis of the findings of Hinchliffe et al. (25, 26), which show similarities between the left and right kidneys. Three longitudinal sections that contained both poles and renal pelvis were used by an observer blinded to the study groups to count the total number of glomeruli per section using high-power magnification (x400) and the modified method of McVary and Maizels (3, 10, 48). Previous reports demonstrate that this technique is reliable and suggest that it accurately represents nephrogenesis (48).
Terminal deoxynucleotidyl transferase uridine nick end-label assay. The UCLA Brain Research Institute Microscopic Techniques Core Facility processed, sectioned, stained, and assisted with data acquisition for this study. The facility dehydrated and dewaxed tissue samples. Apoptotic nuclei were labeled using the terminal deoxynucleotidyl transferase uridine nick end-label technique (TUNEL) via the In Situ Death Detection Kit, POD (Roche, Nutley, NJ). A light hematoxylin stain was used for tissue orientation after the DAB rinses.
A quantification of apoptosis using the TUNEL assay was performed using a modification of the technique described by Malik et al. (46). In brief, an observer blinded to the study group counted the total number of apoptotic nuclei per high-power field (HPF) in six nonoverlapping regions per section (x400). Previous reports demonstrate that the TUNEL technique correlates well with electron microscopy and accurately represents apoptosis in the developing rat kidney (30).
Casp-3 activity. We used the Apoalert CPP32/casp-3 colorimetric assay kit to quantitate N0 renal casp-3 activity as previously described (42). This kit measures the proteolytic cleavage of the chromophore 
-nitroanilide from a DEVD tetrapeptide sequence. In brief, kidneys were homogenized in chilled lysis buffer and subsequently centrifuged to precipitate cellular debris. The supernatant was then transferred to a new microcentrifuge tube. Fifty microliters of reaction buffer containing DTT was added to each reaction, and then 1 mM of conjugated substrate was added to each tube. The reaction was incubated at 37° for 1 h. Absorbencies were read at 405 nm, and a standard curve was generated using a positive control of CPP32 chromogenic substrate. Negative controls were generated by running a reaction without conjugated substrate and with the casp-3 inhibitor DEVD-fmk, respectively.
RNA isolation. Total RNA was extracted from N0 kidneys and quantified in triplicate using ultraviolet absorbance (11). Gel electrophoresis confirmed the integrity of the samples. RNA was treated to DNase (Ambion, Austin, TX).
Real-time PCR. Kidney mRNA levels of Bcl-2, IGF-1, Bax, and p53 were measured using real-time PCR as previously described (41). cDNA was synthesized from 0.5 µg of DNase-treated total RNA as described above. Target (Bcl-2, IGF-1, Bax, p53, DNMT1) primers and probes were designed using Primer Express Software (Applied Biosystems, Foster, CA) (Table 1); target probes were labeled with fluorescent reporter dye FAM. Before the performance of real-time PCR, all primer pairs were tested with serial Mg2+ and primer concentrations to determine the optimal reaction conditions and to demonstrate the specificity of each primer pair. Reporter dye emission is detected by an automated sequence detector combined with ABI Prism 7700 Sequence Detection System software (Applied Biosystem). An algorithm normalizes the reporter signal (Rn) to a passive reference and multiplies the standard deviation of the background Rn in the first cycles by a default factor of 10 to determine threshold CT. CT has a linear relation with the logarithm of the initial template copy number (24). Real-time PCR quantification is then performed using the Taqman glyceraldehyde-3-phosphate dehydrogenase controls. Before the use of GAPDH as a control, serial dilutions of cDNA are quantified to prove the validity of using GAPDH as an internal control. Relative quantification of PCR products are then based on value differences between the target and GAPDH control using the comparative CT method (49). Cycle parameters were 55°C x 5 min, 95°C x 10 min, and then 40 cycles of 95°C x 15 s 
58°C x 60 s. For every sample, each PCR reaction was performed on three separate occasions; in each set of reactions, every sample was present in triplicate.
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p53 promoter CpG methylation. The methylation status of the p53 promoter region was determined using the technique described by Pogribny and James (58). We modified the technique by using real-time PCR vs. conventional PCR and added an internal control. The internal control was a segment of DNA from the hexokinase I promoter that contains no CpGs. In brief, 10 µg of N0 control and IUGR DNA were treated with the methylation-sensitive restriction enzymes Hha I, Aci I, and BstU I as per the manufacturer's instructions (New England Biolabs, Beverly, MA). Real-time PCR was subsequently performed using a total volume of 20 µl and the Sybr Green PCR Master Mix per the manufacturer's instructions (Applied Biosystems). Table 1 contains the primer sequences. Cycle parameters were 55°C x 5 min, 95°C x 10 min, and then 40 cycles of 95°C x 30 s 55°C x 60 s 72°C x 60 s. Relative quantification of PCR products was then based on value differences between the target and non-CpG internal control using the comparative CT method (49). As described above, each sample was analyzed in triplicate and performed on three separate occasions. Single bands of the appropriate size were verified with 10x excess the optimal primer concentrations.
The PCR amplifies a 418-bp segment containing the 85-bp basal promoter region (Fig. 1) (6). The targeted segment of DNA contains four Aci I, one Hha I, and one BstUI methylation-sensitive restriction sites. CpG methylation at these sites prevents restriction enzyme cleavage and thus permits amplification of the PCR product; in contrast, if the site is unmethylated, cleavage prevents amplification. Previous studies demonstrate that the methylation status of these sites either affects transcription or becomes altered in preneoplastic states (58, 59, 75).
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Exon 5 to exon 8 CpG methylation. Because hypomethylation of exon 5-8 is associated with increased p53 expression, methylation status of this region was determined using the modified technique of Woodson et al. (57, 75). Previous studies previously validated this technique for examining methylation within specific regions of the p53 gene (65). We modified the technique by using real-time PCR, adding the same internal control as described above. In brief, we treated 10 µg of DNA with the methylation-sensitive restriction enzymes Hpa II, Msp I, Aci I, and Hha I. Msp I cleavage is not affected by CpG methylation and therefore acts as a control for the Hpa II. Real-time PCR was subsequently performed using a total volume of 20 µl and the Sybr Green PCR Master Mix per the manufacturer's instructions. Table 1 contains the primer sequences. Cycle parameters were 55°C x 5 min, 95°C x 10 min, and then 40 cycles of 95°C x 30 s 60°C x 30 s 72°C x 60 s. Each sample was analyzed as described for the p53 promoter. The targeted region of DNA for exon 5-6 contains two Hpa II sites and one Aci site. The targeted region of DNA for exon 7-8 contains one restriction site for each methylation-sensitive restriction enzyme.
Statistics. All data presented are expressed as mean percent of control ± SE. ANOVA (Fisher's protected least significant difference) and the Student's unpaired t-test determined statistical significance.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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TUNEL assay. Apoptotic nuclei in N0 kidneys were quantified as described by Malik et al. (47) (n = 6 litters). Wide-spread mesenchymal apoptosis, and to a lesser extent tubular apoptosis, characterized both control and IUGR animals. Uteroplacental insufficiency significantly increased the number of apoptotic nuclei in IUGR N0 kidneys to 
180% of control values [control 5.7 ± 0.5 TUNEL-positive cells/HPF; IUGR 10.5 ± 0.5 TUNEL-positive cells/HPF (P < 0.01); quantification, Fig. 2B; representative photograph, Fig. 3]. No difference in the relative histological distribution of the apoptotic nuclei was noted.
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Casp-3 activity. The nuclear morphological changes that occur with apoptosis, as well as DNA fragmentation, require casp-3 (28). Uteroplacental insufficiency significantly increased casp-3 activity in IUGR kidneys to 201 ± 28% (P < 0.01) of control values (Fig. 4A).
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mRNA levels of Bcl-2, IGF-1, Bax, and p53. Uteroplacental insufficiency significantly reduced N0 IUGR renal Bcl-2 and IGF-1 mRNA levels to 49 ± 11.5 (P < 0.01) and 38 ± 7.0 (P < 0.01) percent of control values, respectively (Fig. 4B). Control value SEs were 8.4 and 9.2% for Bcl-2 and IGF-1, respectively.
In contrast, real-time RT-PCR revealed that Bax and p53 mRNA levels were significantly increased to 122 ± 9.1 (P < 0.05) and 195 ± 23.6% (P < 0.01) of control values, respectively (Fig. 4B). For Bax and p53, control value SEs were 3.5 and 11.6%, respectively.
CpG methylation of the p53 promoter. Hha I, Aci I, and BstU I methylation-sensitive restriction enzyme sites characterize the p53 promoter (6, 58). Digestion with these enzymes followed by PCR determined the percent of methylation at these sites as described by James and colleagues (58). Uteroplacental insufficiency resulting in IUGR significantly reduced CpG methylation at the BstU I site (-179) to 43.7 ± 3% (P < 0.001) of control values (Fig. 5). This site is located in the rat p53 85-bp basal promoter region. No differences in DNA methylation were noted for either the Aci I sites (-410, -364, -304, -180) or the Hha I site (-450; Fig. 5; Ref. 6).
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CpG methylation of exon 5 through exon 8 and DNMT1 mRNA levels. Digestion with the methylation-sensitive restriction enzymes Hha I, Aci I, and Hpa II and real-time PCR determined relative CpG methylation within exons 5-6 and exons 7-8 (Fig. 6A). For exons 5-6, uteroplacental insufficiency significantly reduced methylation at the Aci I site without affecting the Hpa II sites. No product was amplified from the Msp I digest and no difference between control and IUGR was noted when exon 5-6 primers amplified an Hha I digest. The latter is expected because this region of DNA does not contain any Hha I sites. For exons 7-8, uteroplacental insufficiency significantly reduced methylation by 30-40% for all three restriction sites. In association with this relative hypomethylation, DNMT1 mRNA levels were also significantly decreased to 70 ± 5.2% (P < 0.05) of control values (Fig. 6B). The SE for DNMT1 control mRNA levels was 7.0%.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In this study, uteroplacental insufficiency reduced glomeruli numbers by 25% in the asymmetrically growth-retarded juvenile rat. Similarly, uteroplacental insufficiency and asymmetrical IUGR in rabbits induced a 25-30% decrease in nephron number, and asymmetrical IUGR in piglets reduced the nephron number index (total nephron number/body weight) by 20-30%; moreover, the change in nephron number caused a parallel drop in glomerular filtration rate (3, 4). Merlet-Benichou et al. (50) used partial unilateral artery ligation of the pregnant rat to induce IUGR, which reduced nephron number by 37% and glomerular filtration rate by 50%, respectively. Although the above studies clearly describe a clear relationship between IUGR and reduced nephron number, they do not demonstrate by what mechanism it occurs.
In contrast, both Welham et al. (73) and Vehaskari et al. (72) used low-protein diets to induce IUGR and subsequently demonstrated increased renal apoptosis. The former study found that increased metanephric apoptosis and decreased glomeruli characterized the IUGR rat. The latter Vehaskari study found a 28-29% reduction in glomeruli number in association with increased TUNEL staining in IUGR kidneys. Both of these studies are important because they established the initial link between renal apoptosis and IUGR; however, neither study determined whether protein deprivation affects expression of Bcl-2 and Bax.
The Welham and Vehaskari studies are also important because severe malnutrition during pregnancy is a concern in developing countries (5). In Western societies, however, uteroplacental insufficiency causes a significant majority of IUGR infants (5). Nephrogenesis depends heavily on apoptosis (12, 34). In the rat, renal apoptosis peaks late in gestation at approximately the same time that the present study induces IUGR. (46).
Bcl-2 and Bax are important because the molecular ratio of Bax/Bcl-2 acts as a cellular "rheostat" determining cellular flux toward or away from apoptosis (33, 55). The combination of decreased Bcl-2 mRNA, increased Bax mRNA, and increased casp-3 activity characterize other experimental models of increased renal apoptosis such as urinary outflow obstruction and experimental glomerulonephritis (63, 67, 77). Bcl-2 renal expression may be particularly important because it plays a role in mediating the formation of condensations of cells that are "committed" to differentiated structures (43). Moreover, a decrease in renal Bcl-2 mRNA promotes tubular apoptosis in hypokalemic nephropathy in rats and appears to threaten tubular production of key growth factors such as IGF-1, which is consistent with our findings of decreased renal IGF-1 mRNA levels (20, 31). Uteroplacental insufficiency and IUGR in the rat also cause a significant decrease in cerebral Bcl-2 mRNA levels (42). Bcl-2 may be a pivotal gene in the systemic IUGR response to uteroplacental insufficiency and may contribute to the close link between cerebral and renal injury in infants suffering asphyxia.
The reciprocal molecular relationship between Bcl-2 and p53 further highlights the significance of decreased renal IUGR Bcl-2 mRNA levels. Bcl-2 inhibits p53 transcriptional activity, and the status of Bcl-2 determines apoptotic homeostasis in renal cell carcinoma (18, 68). In turn, p53 downregulates Bcl-2 expression (8, 23, 76).
Multiple factors affect p53 gene expression, and acute insults stimulate posttranslational changes in accordance with the need to respond rapidly; however, p53 mRNA levels appear to be meaningful under the following three conditions (35). First, p53 mRNA levels regulate its protein expression during development (19). Second, chronic or moderate insults upregulate renal p53 mRNA and induce apoptosis (13, 51, 63). Third, altered p53 mRNA levels are linked to tumor progression and are associated with altered CpG methylation of the p53 gene (56, 57, 75).
James and colleagues (57) used bisulfite mapping to demonstrate that hypomethylation of exons 6-7 increases p53 mRNA levels. With the use of a technique that has been previously validated for studying p53 methylation, the current study found a relative hypomethylation at the exon 6 Aci I site on the sense strand with the first set of p53 downstream primers, without detecting any difference in the Hpa II site (65). Similarly, with the use of a second set of downstream primers, the current study also found that uteroplacental insufficiency decreased methylation in the Hpa II sites (intron 6 and exon 7), the Aci I site (exon 7), and the Hpa I site (intron 7). Both this study and the James study note that loss of the cytosine methyl group was never 100% (59).
The James group is also responsible for the seminal papers that characterize the affects of CpG methylation on the rat p53 promoter (56, 58). In general, hypermethylation of CpG p53 promoter sites between -100 and -442 decreases p53 mRNA levels (56). In vitro methylation and transient transfection CAT assays demonstrate that CpG methylation affects p53 transcription, particularly at the -450 Hha I restriction sites (58). Isolated methylation of the BstU I site decreased p53 transcription by 20% (58). This latter site is significant because it is within the p53 85-bp basal promoter region, and we speculate that hypomethylation of this site and of exons 6-7 contributes significantly to the increased levels of p53 mRNA and increased apoptosis observed in this study (6). Our findings of decreased DNMT1 mRNA support this speculation, because Cre-mediated deletion of DNMT1 induces p53-dependent apoptosis (27). Moreover, because the BstU I site is methylated in normal tissue and becomes demethylated in preneoplastic tissue, it is intriguing that IUGR significantly increases the risk for nephroblastoma (58, 66).
Recent investigations demonstrate that promoter demethylation contributes to transcriptional regulation of specific genes (7, 22). It is now also clear that an active DNA demethylase exists (7, 14). The factors that regulate the expression of this demethylase are unknown; however, glutathione depletion causes genomic-wide DNA hypomethylation (44). The intrauterine environment associated with IUGR and uteroplacental insufficiency leads to oxidative stress, which is known to reduce renal glutathione levels (54, 62).
Caution is necessary when attempting to apply data from a rat model to human pathophysiology. The timing and impact of uteroplacental insufficiency experienced by humans range across a continuum, and the human life experience is confounded by both genetic and environmental variables. In contrast, the laboratory rat in this study is inbred and experiences a homogeneous diet and environment. The insult imposed on the fetal rat in this model of uteroplacental insufficiency is severe and occurs relatively late in gestation. However, when Hinchliffe et al. (25) assessed kidneys from IUGR and appropriately sized infants, they found that IUGR reduced the mean glomerular count by 35%. IUGR also increases the risk of the arterial hypertension and glomerulosclerosis in children with IgA glomerulonephritis (79).
In summary, uteroplacental insufficiency reduces nephron number and increases renal apoptosis in the IUGR newborn rat. The molecular mechanisms behind these findings include increased Bax and decreased Bcl-2 expression, which leads to increased casp-3 activity. Uteroplacental insufficiency also increases p53 expression in association with hypomethylation of its promoter and exon 6-7. Changes in CpG methylation may represent a mechanism through which an altered intrauterine environment predisposes the affected individual to adult onset diseases.
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DISCLOSURES |
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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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X. Ke, R. A. McKnight, Z.-m. Wang, X. Yu, L. Wang, C. W. Callaway, K. H. Albertine, and R. H. Lane Nonresponsiveness of cerebral p53-MDM2 functional circuit in newborn rat pups rendered IUGR via uteroplacental insufficiency Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2005; 288(4): R1038 - R1045. [Abstract] [Full Text] [PDF]
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Q. Fu, R. A. McKnight, X. Yu, L. Wang, C. W. Callaway, and R. H. Lane Uteroplacental insufficiency induces site-specific changes in histone H3 covalent modifications and affects DNA-histone H3 positioning in day 0 IUGR rat liver Physiol Genomics, December 15, 2004; 20(1): 108 - 116. [Abstract] [Full Text] [PDF]
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J. A Armitage, I. Y Khan, P. D Taylor, P. W Nathanielsz, and L. Poston Developmental programming of the metabolic syndrome by maternal nutritional imbalance: how strong is the evidence from experimental models in mammals? J. Physiol., December 1, 2004; 561(2): 355 - 377. [Abstract] [Full Text] [PDF]
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B. T. Alexander Intrauterine growth restriction and reduced glomerular number: role of apoptosis Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2003; 285(5): R933 - R934. [Full Text] [PDF]
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P < 0.001).