Published online before print August 21, 2006, doi: 10.1161/01.HYP.0000237973.64711.e2
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(Hypertension. 2006;48:717.)
© 2006 American Heart Association, Inc.
Original Articles |
Adult Hypertension in Intrauterine Growth-Restricted Offspring of Hyperinsulinemic Rats
Evidence of Subtle Renal Damage
From the Departments of Medicine (M.B.), Pathology (T.B., I.A.), Psychiatry (T.G-D.), and Bioinformatics Center (Y.S.), Hadassah-Hebrew University Medical Center, Mount-Scopus, Jerusalem, Israel; Ein-Kerem (Y.S.), Jerusalem, Israel; and the Institute of Pathology (M-L.G., N.K.), Ruprecht-Karl University, Heidelberg, Germany.
Correspondence to Michael Bursztyn, Hypertension Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, Mount Scopus, PO Box 24035, Jerusalem 91240, Israel. E-mail bursz{at}cc.huji.ac.il
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Abstract |
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In humans, intrauterine growth-restricted newborns are prone to develop hypertension as adults. We studied a rat model of pregnancy-induced hypertension associated with intrauterine growth restriction (IUGR) produced by chronic administration of insulin. Fetuses of hyperinsulinemic dams (HDs) were smaller than those of normal dams (5.1±0.4 g versus 5.6±0.1 g, respectively; P<0.05). At 16 weeks of age, tail-cuff systolic blood pressure was measured, the rats were placed in metabolic cages and euthanized, and the kidneys were examined. Male but not female offspring of HDs (n=9) had higher blood pressure than normal-pregnancy offspring (n=12; 148±11 mm Hg versus 118±14 mm Hg; P<0.004). In contrast to other models, there was no difference in ours in the number and volume of glomeruli. However, there were significantly greater glomerular, tubulointerstitial, and vascular damage indices in the kidneys of male HD offspring versus controls (2.01±0.34 versus 1.08±0.16, 1.80±0.34 versus 0.76±0.12, and 2.13±0.81 versus 0.78±0.16, respectively; P<0.0001), with similar tubulointerstitial findings in females. Increased expression of collagen type IV, a kidney damage marker indicating fibrosis, was found in the tubulointerstitium. This may be associated with downregulation of bone morphogenetic protein 6, a presumptive antifibrogenic agent, at the end of gestation. In conclusion, male offspring of HDs displayed IUGR and adult hypertension accompanied by several indices of renal fibrosing damage, mainly in the renal tubulointerstitium. Our findings suggest that there is >1 pathway of fetal programming leading from IUGR to development of hypertension in later life.
Key Words: gene expression • hypertension, experimental • insulin • renal disease • pregnancy
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Introduction |
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Almost 2 decades ago, Barker and Osmond1 showed that low birth weight predicts higher adult life morbidity and mortality and suggested that adult cardiovascular diseases, including hypertension,2 may be of fetal origin. These observations have been confirmed worldwide, although not without reservations.3 Because hypertension is a trait of multifactorial nature with both genetic and environmental influences, uterine environment maybe easily confused with inheritance. Several animal studies confirmed the observation that unfavorable fetal environment causing fetal malnutrition, induced by restriction of food, protein, or uteroplacental blood flow, leads not only to intrauterine growth restriction (IUGR) but also to hypertension in adult offspring.3,4
Nevertheless, the relevance of undernutrition and low-protein diet to populations in industrial countries is limited. In such populations, one of the major causes of IUGR and reduction of placental blood flow is hypertension in pregnancy, which was reported in numerous epidemiologic and clinical studies to be related to maternal insulin resistance and hyperinsulinemia.5 Several years ago, we developed an animal model6 in which exogenous hyperinsulinemia in pregnant rats was consistently associated with IUGR.7–9
In animal models studied so far, hypertension of the adult offspring is associated with renal abnormalities, such as reduced glomerular number and increased apoptosis.3,4,10 In this study, we tested the hypothesis that IUGR resulting from maternal hyperinsulinemia will evolve into hypertension in the adult offspring, characterized by the renal abnormalities and investigated molecular events occurring in utero, which eventually lead to kidney damage.
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Methods |
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Female Wistar rats (weight, 200 to 280 g) were housed in regular cages (4 rats to a cage) and maintained on standard rat chow (Kofflok) containing 56% grain-derived carbohydrate, 20% protein, 13% moisture, 5.5% cellulose, 3% fat, 0.8% calcium, 0.6% phosphorus, and 0.3% NaCl, with free access to tap water. They were maintained on a 12-hour light/dark cycle. All of the animals were handled and housed according to the guidelines and manual of the Committee for the Care of Laboratory Animals of the Hebrew University-Hadassah Medical School and with its written approval. The rats were divided in 2 groups: normal pregnant rats and pregnant rats treated chronically with insulin.
Insulin-treated rats received a 2-mm segment of a 7-mm sustained-release insulin implant (Linplant, Linshin Canada Inc) designed to deliver 
2 U/day for >40 days by means of a 12-gauge hypodermic needle under brief ether anesthesia. A pellet was implanted subcutaneously 1 week before mating and a second one on day 7 of pregnancy. The 2 pellets, with a moderate amount of insulin in each, allow the gradual development of hyperinsulinemia without severe hypoglycemia. Control rats underwent sham implantations under the same conditions.6–8
Day 1 of gestation was documented by the presence of spermatozoa in the vaginal smear. Plasma glucose, fructosamine, and insulin were measured at the end of pregnancy.
The dams were allowed to give birth, and the newborn rats were not handled in order not to affect maternal care. They were weaned at 3 weeks of age and put in cages of 4 rats on standard rat chow. After habituation to systolic blood pressure (SBP) measurements at 11 weeks of age, SBP was measured at 12 and 16 weeks.
Offspring of 12 hyperinsulinemic dams (HDs) and 9 controls were used for the study, which was carried out as follows: rats were weighed and SBP was measured (prewarmed) by the tail-cuff method (IITC, Life Sciences). All of the measurements (
10 per rat) were averaged as recommended.11 Another set of pregnant rats (6 control and 12 insulin-treated) had their SBP measured on day 21 of gestation.
Metabolic Cages Study
Rats were housed in individual Nalgene metabolic cages for 24 hours for habituation. During the following 24 hours, urine was collected to measure the excretion of Na+, K+, creatinine, and protein.
After the metabolic cage study, the rats were anesthetized with intraperitoneal pentobarbital. A laparotomy incision was made, the aorta was exposed, and blood was collected into prechilled lithium-heparin-containing test tubes. The kidneys were dissected, weighed, and bihalved along the coronal plane. The tissue was fixed in 4% buffered formalin, processed, and embedded in paraffin.
In another experiment, the pregnant rats were killed on day 22, the usual day of delivery, and the number and weight of the fetuses were determined. The fetuses were dissected: 1 kidney was fixed in 4% buffered formalin, and the other kidney of each fetus was snap-frozen in liquid nitrogen and stored at –80°C.
Histomorphometric Analysis of Indices of Renal Damage (Glomerulosclerosis and Tubulointerstitial and Vascular Damage)
This study was performed on all 9 male and 6 female offspring of HD and 12 male and 6 female offspring of control dams. Five-micron paraffin sections of kidneys from male offspring aged 16 weeks were placed on double-frosted slides. The degree of sclerosis within the glomerular tuft, which served as an index of progression, was determined in periodic acid-Schiff stain (PAS) stained paraffin sections, using the semiquantitative scoring system proposed by El Nahas et al.12 Briefly, using light microscopy at x400 magnification, the glomerular score of each animal was derived as the mean of 100 glomeruli, and the severity of glomerulosclerosis was expressed on an arbitrary scale from 0 to 4 as described.12,13 Tubulointerstitial and vascular damage was assessed in PAS-stained paraffin sections at x100 magnification using a similar scoring system.14 The tubulointerstitium was scored completely by 2 investigators blinded to the animal groups. Masson trichrome stain for collagen fibers was performed in selected cases to demonstrate fibrosis in areas with increased expression of collagen type IV (see below). The glomerular geometry, including area and volume density of the renal cortex and medulla, as well as the number of glomeruli per area, was measured using a Zeiss eyepiece (Integrationsplatte II, Zeiss Co) and the point counting method at a magnification of x400 as described in Weibel15 and Amann et al.16
Immunohistochemistry
As for damage indices, this study was performed on all 9 male and 6 female offspring of HD and 12 male and 6 female offspring of control dams. Immunohistochemical staining was performed, using antibodies against proteins investigated previously in association with renal damage. These included cytokines such as transforming growth factor (TGF) ß1 (anti-TGFß1 rabbit polyclonal antibody, sc 146, Santa Cruz Biotechnology, diluted 1:40), platelet-derived growth factor antibody (polyclonal goat, Upstate Biotechnology, diluted 1:50), and collagen type IV (rabbit polyclonal, 2150-1470, diluted 1:25, BioTrend, Cologne, Germany), which is indicative of renal sclerosis. To avoid nonspecific cross-reaction of tissue components with antibody, a biotin-streptavidin detection system conjugated with alkaline phosphatase was used (BioGenex). Fast Red was applied as chromogen (DAKO Cytomation). Negative controls were carried out by omitting the primary antibody. The sections were analyzed according to a semiquantitative scoring system in a blinded fashion (x200 magnification). The intensity of staining was ranked according to an arbitrary scale, grading from 0 to 4 according to the area of positive staining: 0: no expression, score 1: weak expression, score 2: moderate expression, score 3: strong expression, and score 4: extremely strong expression. At least 30 high-power fields that were chosen randomly were scored separately by 2 investigators blinded to the animal groups.
Biochemistry
Sodium, potassium, glucose, creatinine, and fructosamine serum levels were determined by standard laboratory techniques, and the insulin serum level was determined using an Insik-5 radioimmunoassay kit (Sorin Biomedica). Within- and between-assay coefficients of variation are <6.2% and <6.6%, respectively, and sensitivity was 12 pmol/L. As a preliminary study to point out molecular changes occurring in utero, which may lead to renal damage postnatally, we screened the gene expression profile of the fetal kidneys on day 22 of gestation.
Gene Expression Profile of Fetal Kidneys
Total RNA was isolated using an RNA STAT-60 kit (Tel-Test, Inc) according to the manufacturer’s instructions. The total RNA of 5 kidneys from each group of fetuses, HD and control, was hybridized to Affymetrix GeneChips (Rat Expression Set 230A) according to the manufacturer’s instructions at the Department of Biological Services, the Weizmann Institute of Science.
Quantitative Real-Time PCR
RNA Isolation
Frozen fetal kidneys of 5 fetuses of HDs and 5 controls, other than those used for the microarray experiment (biological replicates), were homogenized in 1 mL of TriPure reagent (Roche), using a Polytron homogenizer (Kinematika AG). Total RNA was extracted using TriPure isolation reagent (Roche), according to the manufacturer’s instructions, followed by digestion with RNase-free deoxyribonuclease I (Invitrogen) to remove potential trace amounts of residual genomic DNA. RNA concentration was measured in a UV1101 spectrophotometer (WPA) at 260 nm, and the quality of samples was determined by analysis on 1% agarose electrophoresis gels. Purified total RNA was stored at –80°C until further processing.
cDNA Synthesis
One microgram of RNA was reverse transcribed using SuperScript II First-Strand Synthesis System for RT-PCR (Gibco BRL) in a total reaction volume of 20 µL. After being denatured together with 1 µL of hexanucleotide random primers at 65°C for 5 minutes, RNA samples were incubated at 42°C for 50 minutes with 1 µL of reverse transcriptase (RT), 1 µL of 10 mmol/L dNTPs, 4 µL of 10a RT buffer, 1 µL of RNase inhibitor (RNaseOUT), and 2 µL of 0.1 mol/L dithiothreitol in a thermal cycler. The reaction was stopped by heating to 70°C for 15 minutes. Samples in which RT was omitted were also subjected to PCR as negative control samples to confirm that no genomic DNA contamination occurred.
Real-Time PCR
The mRNA was quantified using real-time quantitative PCR performed in a GeneAmp 7500 Real Time PCR System, with Sequence Detection System software version 2.0 (Applied Biosystems). The RT reaction was performed using assay by demand TaqMan RT probes for bone morphogenetic protein 6 and GAPDH and reagents (Applied Biosystems) according to the manufacturer’s protocol. Briefly, a computer algorithm normalizes the signal to an internal reference dye and calculates the threshold cycle number. The amount of fluorescence was normalized by endogenous control (GAPDH) and calculated by the change in the threshold cycle method according to the manufacturer’s instructions (Applied Biosystems).
Statistical Analysis
Means±SD are presented. Student t test, ANOVA, and Mann-Whitney test were applied as necessary (Microsoft Excel 2002). Fetus data were averaged per dam, and then the group mean was calculated from the average per dam. The results were considered significant when 2-sided P
0.05.
Genes from the Affymetrix GeneChips were selected by the Significance Analysis of Microarray scoring method17 that uses multiple testing to guarantee a low false-discovery rate. Further analysis was carried out using the Spotfire Decisionsite 8.1.1 package. Genes with P<0.01 for the t test/ANOVA treatment comparison were considered to be differentially expressed. Fold changes in gene expression were calculated by dividing the mean intensity signals from fetal kidneys of fetuses born to HDs by the mean intensity signals from control fetuses.
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Results |
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Dams
HDs (n=19; weight: 380±18 g) had a significantly higher insulin level than normal pregnant controls (n=9; weight: 382±49 g; 792±252 pmol/L versus 168±71 pmol/L; P<0.0001), and a lower glucose (4.4±1.5 mmol/dL versus 6.7±1.7 mmol/L; P=0.0012) and fructosamine (112±46 µmol/L versus 156±61 µmol/L; P<0.05) level. SBP of HDs and control dams was 109±14 mm Hg and 112±14 mm Hg, respectively, not significantly different.
Fetuses
Fetuses of HDs were significantly smaller and had smaller placentas compared with fetuses of normal pregnant dams, and litter size did not differ (Table 1).
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Offspring
The offspring were not weighed before weaning, and by that time all of the "catch-up" growth had already occurred, and there was no difference in body weight between the 2 groups (Figure 1). Because no significant blood pressure effect was found in females, only results of males are presented in this figure. By week 16, offspring females of HD weighed 234±12 g, and the offspring of controls weighed 244±11g.
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SBP was significantly higher in the male offspring of HDs at both week 12 and 16 (Figure 2). Therefore, only males underwent the metabolic cages study. In the metabolic cages (at week 16), the male offspring of HDs drank less water and excreted less urine compared with controls but had similar 24-hour urinary excretion of electrolytes and protein (table 2), as well as similar electrolytes serum levels.
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Histomorphometric Analysis of Offspring Kidneys
There were significantly higher tubulointerstitial (Table 3 and Figure 3) and vascular damage indices in both male and female offspring of HDs and higher glomerular damage index in the males (Table 3 and Figure 3). Increased expression of tubulointerstitial collagen type IV was also demonstrated by immunohistochemical staining in both the male and female offspring and tubulointerstitial TGFß1 in the female offspring of HD (Table 3). Number and volume of glomeruli were similar in the 2 experimental groups in both males and females.
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Analysis of Affymetrix GeneChips using Significance Analysis of Microarray identified 99 of 15 923 probe pairs that were differentially expressed; of these, 12 known genes and 20 expressed sequence tags were upregulated, and 32 genes and 35 expressed sequence tags were downregulated. The results are accessible at the National Center for Biotechnology Information gene expression and hybridization array data repository (www.ncbi.nlm.nih.gov/geo/; GEO accession no. GSE3215).
Bone morphogenetic protein 6 (BMP6), from the TGFß superfamily, was one of the genes that was found downregulated in the microarray experiment. We chose to corroborate this finding because of the presumptive role of this gene product in renal fibrosis. RT-PCR validated the downregulation of BMP6x66%±22% (P<0.03) in the kidneys of HD fetuses.
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Discussion |
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We found that male rats with IUGR induced by maternal hyperinsulinemia develop significantly higher blood pressure by 23 to 30 mm Hg at 12 and 16 weeks of age, compared with offspring of control dams. Females did not have higher blood pressure. Similar findings were found in other models of IUGR.4,18–23 In offspring of rats with reduced uterine perfusion by clipping the aorta and uterine arteries, hypertension occurred at a young age followed invasively. The hypertensive (both male and female) offspring, however, maintained their lower fetal weight.18 In another study (adhering to a somewhat different protocol of uterine artery ligation), there was no difference in blood pressure. However, in adult offspring of uterine artery ligated dams, which have caught up in growth when studied, the blood pressure was salt sensitive on exposure to increased salt intake.19 Our rats were not weighed until they were weaned nor was their blood pressure measured, because we did not want to interfere (by handling) in mother-pup relations. During this period, however, catch-up growth had already occurred, and the weight difference between the groups, apparent in the fetuses, was no longer present.
A study of restricted diet during most of the pregnancy in rats caused IUGR and hypertension, despite an early catch-up growth.20 Restriction of maternal dietary protein according to a variety of protocols from before mating to late pregnancy was similarly associated with IUGR and hypertension mostly in male offspring.19–21 In some other studies of protein restriction, adult hypertension was found, but IUGR was not reported.3 Also, the findings in terms of IUGR or blood pressure differed by the degree of maternal dietary protein restriction, and the effect on IUGR and subsequent hypertension was greater with greater reduction in dietary protein, at 6% versus 9% when compared with a standard 18% protein diet.22 Other methodologic differences may account for some of the inconsistencies in the literature, for example, time of assessment, gender, and so forth.4 For example, in the study of Kwong et al,21 males developed hypertension at 4 weeks but females did not, and in the study by Ozaki et al,20 females developed hypertension by week 14 but not week 8, unlike the earlier blood pressure rise in the males.20 Thus, the lack of blood pressure elevation in our female offspring may be because of the fact that they were not studied at an appropriate age. Such a phenomenon could be explained, in part, by differences in sex-specific expression of the angiotensin II type 2 receptor.23 In the males, however, hypertension was already well established by 12 weeks of age and possibly earlier.
We investigated the kidneys of the offspring of HDs, searching for changes associated with the development of hypertension. We found morphological evidence of subtle renal damage in the tubulointerstitial compartments, similar to the renal changes accompanying hypertension in humans and many animal models of hypertension as reviewed recently by Johnson et al.24 Nevertheless, the morphological changes were generally mild and to a large extent tubulointerstitial, and this could be a reason for the absence of differences in protein excretion between the 2 groups of offspring. There were significantly higher tubulointerstitial and vascular damage indices in both males and females offspring of HDs, as well as increased expression of collagen type IV in the tubulointerstitium. However, the level of significance of tubulointerstitial damage was much higher in the males than in the females. Glomerular damage index, as well as TGFß1,was significantly higher in the males only, possibly secondary to hypertension, and TGFß1 was significantly expressed in the tubulointerstitium of the females.
In contrast to some other models of hypertension after IUGR, in which a reduced number of glomeruli associated with increased apoptosis was documented,3,4,25–30 there was no difference in the number of glomeruli at 16 weeks between offspring of HDs and controls in our model; neither was a significant difference in glomerular volume and density or in other histomorphometric measurements found.
Reduction in the number of glomeruli, long suspected as a cause of hypertension,31 confirmed recently by human autopsy studies,31–33 was not found in our IUGR rats. However, irrespective of the reduced number of nephrons, the kidneys have long been known to be an important, if not the primary, regulator of blood pressure,34 and IUGR could have additional effect aside from that on the glomeruli.3,4,35
Our findings suggest that in rats with IUGR because of maternal hyperinsulinemia, which are hypertensive at 16 weeks, kidneys have apparent signs of renal injury. It is difficult to assess to what extent this is a primary or secondary phenomenon. The glomerular and vascular changes that we observed suggest changes secondary to higher blood pressure in the areas directly exposed to it, that is, glomeruli and vasculature. However, in the females, although having somewhat similar histological changes mostly in the tubulointerstitium, there was no increase in SBP. This finding suggests that at least this part of the renal damage was the result of IUGR rather than having been induced by the hypertension. Nevertheless, females are known to be more resistant to a variety of renal injuries.36
Our preliminary results of the differential gene expression in the fetal kidneys yielded interesting findings, even if only partially analyzed. We found a reduction of BMP6 in the fetuses of HDs. BMP6 is closely related to BMP7, both being members of the TGFß superfamily and having similar biological functions by activating the same downstream pathways.37 Unlike BMP7, which is essential for early glomerular formation and, therefore, has been extensively studied, BMP6 is expressed in the kidney only toward late gestation.38 BMP6 has antiproliferative activity,39 and assuming a similar function to BMP7 as an antifibrogenic agent,40–42 its downregulation late in gestation may predispose to tubulointerstitial fibrosis in postnatal life. This hypothesis needs further investigation.
Tubulointerstitial fibrosis has been proposed as an antecedent of hypertension.24,43 In the reduction of perfusion pressure model of IUGR with adult hypertension, renal sympathetic denervation abolished the hypertension,4 also pointing to the relevance of the kidneys to the adult hypertension of rats with IUGR. The presence of tubulointerstitial fibrosis is compatible with the concept of intrauterine damage leading to the second stage of hypertension susceptibility of the unifying hypothesis of Johnson et al.23
Our model of hyperinsulinemia in pregnancy may differ from hyperinsulinemia of human pregnancy in the obese, because in human pregnancy it is associated with normal or elevated glucose levels,5 whereas in our hyperinsulinemic pregnant rats, it was associated with lower glucose levels.6–8 On the other hand, the changes in the NO system documented previously in our model are similar to processes occurring in hypertension associated with pregnancy in humans.5–8
Perspectives
We present a unique model of hypertension developing in rats born growth restricted to HDs. In this model, adult hypertension in the males is accompanied by renal changes similar to those in human hypertensive patients, as well as other animal models of hypertension. Unlike different models of IUGR leading to adult hypertension, in our model, nephron number was not reduced and glomerular volume not increased in the offspring; however, other kidney changes prevail, particularly tubulointerstitial fibrosing damage. Our findings suggest that there is >1 pathway of fetal programming leading from IUGR to development of hypertension in later life, conceivably depending on the type of insult causing the IUGR.
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Acknowledgments |
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The authors are indebted to Judith Mekler, whose dedicated technical assistance made this study possible.
Sources of Funding
This study was supported in part by an institutional compensatory grant of the Hadassah Medical Organization.
Disclosures
None.
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Footnotes |
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The first 2 authors contributed equally to this study.
Received April 26, 2006; first decision May 12, 2006; accepted July 14, 2006.
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