Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets

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1 Clinical Science (1994) 86, (Printed in Great Britain) 217 Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets Simon C. LANGLEY and Alan A. JACKSON Department of Human Nutrition, University of Southampton. (Received 27 Mayla September 1993; accepted 14 September 1993) Southampton, U.K. 1. Possible associations between maternal nutrition in pregnancy and non-communicable diseases of adulthood were assessed using a rat model. Rats were habituated to diets containing a range of protein levels (18, 12, 9 and 6% by weight), over a 14 day period, before mating. The low protein diets were maintained throughout pregnancy. Lactating mothers and their offspring were transferred to a standard chow diet (20% protein). 2. Pregnant rats demonstrated a graded response to the diets, with those fed 9 and 6% protein tending to consume less energy and gain less weight than 18% protein fed controls. Litter size and newborn death rates were not significantly altered by the low protein diets. 3. Offspring of 12 and 9% protein fed dams were grossly normal, gaining weight at a similar rate to those born to 18% protein fed control rats. Offspring of the 6% protein fed dams were smaller than pups from all other groups, over a 21 week period. 4. At 9 weeks of age, systolic blood pressure was determined in the offspring. All offspring from the three low protein groups were found to have significantly elevated blood pressure (15-22 mmhg) relative to the control group. An inverse relationship between maternal protein intake and the systolic blood pressure of the offspring was observed. Blood pressure remained elevated in the offspring of the 9 and 6% protein fed dams until 21 weeks of age. The observed hypertension was associated with increased pulmonary angiotensin-converting enzyme activity in the low protein groups. 5. The data are consistent with the hypothesis that poor maternal nutrition in pregnancy may irreversibly impair aspects of physiological and biochemical function in the fetus. This has potential adverse consequences for the later health of the offspring. INTRODUCTION Exposure of the fetus to maternal malnutrition is a well-documented causal factor for intrauterine growth retardation, in both humans and animals C1-43. Low protein diets in particular have been associated with altered placental growth and composition [S, 61, and fetal growth and organ development [7-91. Epidemiological studies in humans have demonstrated an association of ischaemic heart disease, hypertension and impaired glucose tolerance in adults, with low birthweight and altered placental growth [lo-12]. These changes are observed in childhood [ 131, and are amplified with increasing age. The data from these studies suggest that poor maternal nutrition during pregnancy may lead to metabolic and morphological changes in the fetus, with lifelong consequences. Animal models of the interaction between maternal diet and fetal development are frequently based upon extremes of dietary variation, for example starvation [I41 or very low (&I%) [6, 151 or very high (24%-30%) [7-91 protein diets, and do not consider the effects of an inadequate diet to which the mother has become habituated. Our previous studies have considered the effects of maternal low (6%) and intermediate (12%, 9%) protein diets on rat fetus and placenta. A complex interaction of dietary protein and fetal and placental growth has been observed [16]. In the present paper we report the induction of hypertension in the rat through fetal exposure to maternal undernutrition. The possible role of alterations to the renin-angiotensin system, in this model, is also demonstrated. METHODS Twenty-four virgin female Wistar rats (initial weight 2W225 g) were housed individually in wiremesh cages, and were maintained at 24 C on a 12 h light cycle. The rats were given free access to synthetic diets containing 18, 12, 9 or 6% protein by weight (Table I). The protein source was casein and all diets contained 5g/kg methionine to avoid sulphur deficiency. All animals had free access to water. After 14 days of feeding the diets the animals were mated. Feeding of synthetic diets continued throughout the mating period, which varied from 1 to 7 days, and until the delivery of the pups. Within 12h of giving birth the mothers were placed on a standard laboratory chow diet (CRMX; 20% pro- Key words: fetus, hypertension, protein deficiency. Abbreviation: ACE, angiotensiruonvening enzyme. Correspondence: Dr S. C. Langley, Department of Nutrition, University of Southampton. Bmett Crescent East, Southampton SO9 3TU. U.K.

2 218 S. C. Langley and A. A. Jackson Table I. Composition of synthetic diets. Diet was provided to the animals as balls (6&1OOg dry weight), dried for 24h at 60 C. Snowflake is starch. Solkafloc is fibre (SDS Ltd). Composition (% by wt.) Dietary protein content... 18% I 2% 9% 6% Casein Snowflake Solkaflcc Sucrose Choline Mineral mix (AIN-76) Vitamin mix (AIN-76) Methionine Corn coil tein). The animals remained on this diet throughout the suckling period. Two rats, one on the 6% protein diet and one on the 12% protein diet, failed to conceive. The pups were weaned at 4 weeks of age on to chow and were housed in pairs until 9 weeks of age. Systolic blood pressure was measured in a total of 52 conscious female offspring, by recording tail vein pulses (Blood Pressure Monitor; Linton Instrumentation, Diss, Norfolk, U.K.) Rats were placed in a darkened perspex restraint tube, maintained at 27 C. A 15mm cuff was placed over the tail and inflated to 300mmHg, and pulses were recorded during deflation at a rate of 3mmHg/s. Blood pressure was determined in triplicate for each animal, with the average systolic pressure being recorded. Preliminary studies using a mixed group of control and low-protein-exposed animals indicated that there was no requirement for training the rats, a coefficient of variation of 3% (range 1-9%) being noted for six animals measured on six separate occasions. Determinations of blood pressure were repeated at 12, 15 and 21 weeks of age, in the same hands. Pulmonary angiotensin-converting enzyme activity (ACE) was determined in a group of the animals when aged 9 weeks. The rats were killed using carbon dioxide and the lungs were rapidly excised and frozen in liquid nitrogen. Activity was determined by the method of Hayakiri et al. [17], based upon the hydrolysis of hippuryl-l-histidyl-l-leucine to hippuric acid. Activities were expressed as units/ mg of protein, with lung protein being determined by the method of Smith et al. [18]. One unit corresponds to the production of 1 pmol of hippuric acid/min at 37 C. Statistical analysis All analyses were carried out using the SPSS/PC statistics package (4.01). At the first level analysis of differences between groups were sought using simple one-way analysis of variance. To explore in greater detail the interaction among diet, blood pressure Table 2. Effects of low and intermediate protein diets during pregnancy on maternal weight gain and energy intake. Virgin female rats were fed synthetic diets for 14 days before mating and during pregnancy. Body weight and food intake were monitored daily over a 40 day period. Data are means + SEM for n observations. Statistical significance: *P <0.05. **P<O.Ol compared with all other groups. Analysis of variance for pregnant rats: body weight, P<O.OOOI. F= (3.21); energy intake, P<O.OOOI, F=22.53 (3.21). Wf. (8) Energy intake (kj/day) Dietarygroup Initial Mating Final Prepregnancy Pregnant 18%Casein 228k4 256k w+22 (n = 6) IZ%Casein 231 +_3 159k k I1 573k I l8 (n = 5) %Casein I7 345k 17' 481 f9' * (n = 6) 6%Casein 232k6 245k IS " 379k9" (I** (n = 5) and age, a further analysis of variance was conducted adjusting for the effects of these co-variates. RESULTS Effects of low protein diets on maternal growth and energy intake Body weights of the rats were not significantly altered by feeding low protein diets during the prepregnancy period of 14 days (Table 2), although the weight gain in this period did tend to be lower in rats fed 9, or 6% casein. During pregnancy, the weight gain of the 18 and 12% casein fed rats was significantly greater than that seen in the 9 and 6% casein fed groups. Energy intakes of pregnant rats fed each of the diets did not alter significantly during pregnancy, relative to the pre-pregnancy period (Table 2). Energy intakes of the 18 and 12% casein fed rats were not significantly different. The 6% casein fed group consumed approximately 50% less energy than the 18 and 12% casein fed groups. The 9% casein fed group consumed approximately 30% more energy than the 6% casein fed group, but 15-20% less than the 18 and 12% casein fed groups (P <0.05). The food intakes on a g of feed/day basis were significantly reduced in the 6% casein fed group (22f3g/day), and tended to be lower in the 1TX (25+6g/day) and 9% (25*4g/day) casein fed groups, relative to 18% casein fed controls (28.5 f 3.5 g/day). As a consequence the reductions in protein intake relative to the 18% casein fed group were 42%, 56% and 75% for the 12, 9 and 6% casein fed groups, respectively. Litter sizes were not significantly altered by the dietary manipulations ( 18% casein, 10.3 f 1.5 pups/ litter; 12% casein, 1 I.6 k0.8 pups/litter; 9% casein 10.0 k 0.7 pups/litter; 6% casein, pups/ litter). Mortality rates among the pups did not change significantly in response to dietary changes,

3 ~~~ Hypertension induced by fetal expure to low protein diet 219 Table 3. Effects of fetal exposure to maternal low protein diets on subsequent growth of offspring. Body weights of pups born to dams fed diets of differing protein content were determined on a weekly basis for 6 weeks. with a final measurement at 21 weeks. Data are meansf SEM for n = observations (male and female rats). From 9 weeks all rats were female (n = 3-10), Statistical significance: *P<O.OI compared with other groups; fpco.05 compared with the 12% protein group; $P<O.O5 corn pared with the 9X protein group. Age (weeks) Body Wt. k) Maternal dietary protein intake... l8x I2X 9% 6% I IS f f f O.5t 25.0 f I.O 35.0 f I.Ot 38.5 f I.O f 69.0 f I f f f f 2.5 2M.Of f8.O f f f f f f f0.3t 24.0 f f I.O 64.0 f I f f f O It f f f 0.3' 18.0 f 0.5* 28.0 f I f I.5' 79.0 f 2.5' i 12.0 f f 4.0* f 3.0' f 5.01 U8.0 f 3.0' Table 4. Effects of fetal exposure to maternal low protein diets on systolic blood pressure in female adult rats. The female offspring of pregnant dams fed varying levels of protein throughout gestation were allowed to grow up for 9 weeks, at which point blood pressure was determined in the conscious animals, as described in the text. Data are meansf SEM for n observations. At 9 weeks diet significantly influenced blood pressure: F=6.84 (3,48). PtO.001. For all ages: f= (3,ll5), P < Abbreviation: CI, confidence intervals. Maternal dietary protein intake Systolic blood pressure (mmtlg) 9 weeks of age All ages n Mean 95X CI n Man 95X CI 18% IS , I33 128,IUI I 2% , ,154 9% , X II I59 150, I "1 but tended to be higher in those born to 6% casein fed dams (1 8% casein, 10.7 f 8.2% mortality; 12% casein, 18.9f 14.6% mortality; 9% casein, 18.5 f 16.4% mortality; 6% casein, 40.0 f 24.5% mortality). Effects of maternal low protein diets on the growth of offspring The body weights of the offspring born to dams fed the various levels of protein are shown in Table 3. At all ages studied the pups born to the 6% casein fed dams weighed significantly less than pups in all other dietary groups. Pups born to the 12% casein-fed dams were significantly heavier than the offspring of 18% casein fed dams until the age of weaning (4 weeks). Although smaller throughout the period of monitoring, the pups of 6% casein fed dams displayed a similar rate of growth to that seen for the pups of the 18, 12 and 9% casein fed dams, on a gain per unit body weight per day basis. Effects of maternal low protein diets on systolic blood pressure in adult offspring The effects of fetal exposure to maternal low protein diets on systolic blood pressure were determined in the female offspring at 9 weeks old (Table 4). Offspring of all three groups of low protein fed dams were found to have significantly higher systolic blood pressure than the offspring of the 18% casein group. The increase in the offspring of the 6% casein group was of greatest magnitude (22 mmhg higher than the offspring of the 18% casein group). Mean systolic blood pressure for the dietary groups was observed to be inversely related to average maternal protein intake (r= -0.52, P<0.005) as shown in Fig. 1, and a lesser relationship between maternal energy intake and systolic blood pressure was also noted (r= -0.33, P<O.O5). The interaction between maternal dietary protein intake and systolic blood pressure in the offspring at 9 weeks of age was statistically highly significant. This interaction was seen to persist at all further time points, with the offspring of the 18% protein fed group having a blood pressure which was highly significantly lower than that of all other groups (least squares procedure, multiple range test). For the offspring of all dietary groups there was a decrease in blood pressure with age, which was significant (Fig. 2). There was a highly significant interaction among diet, blood pressure and age (F=3.12, P<O.001). When the effect of age was adjusted for, there was a statistically significant, but numerically small, effect upon the interaction of diet and blood pressure. After adjusting for the effect of age, the interaction between diet and blood pressure m

4 220 S. C. Langley and A. A. Jackson I00 I J Age of offspring (weeks) Fig. 2. Systolic blood pressure of female adult offspring over the age range 9-21 weeks. The female offspring of pregnant dams fed varying levels of protein throughout gestation were allowed to grow up for 9 weeks, at which point blood pressure was first determined in the conscious animals, as described in the text Further measurements were made at 12. I5 and 21 weeks of age a, 18% protein group, A, 12% protein group. v, 9% protein group. +, 6% protein group Table 5. Effects of fetal exposure to maternal low protein diets on pulmonary ACE activity in female adult rats. The female offspring of pregnant dams fed varying levels of protein throughout gestation were allowed to grow up for 9 weeks, at which point pulmonary ACE activity was determined as described in the text Data are means f SEM for n observations Statistical significance *P< 0 01 compared with the 18% protein group Maternal dietary protein intake 18% (n = 7) 12% (n=7) 9% (n=7) 6% (n = 7) ACE activity (unitsimg of protein) 6.60 f f f I.45 I I I3* was highly statistically significant (F = 15.02, P<O.OOl), being 134, 147, 149 and 154 for the offspring of the l8')<, 12%, 9: and 67,, casein fed groups, respectively (multiple r2 412,). Pulmonary ACE activity (Table 5) was significantly higher in the offspring of the 6% casein fed group than in offspring of the 18'%, casein fed controls (81'%; increase), and tended to be elevated in the offspring of the 12 and 9%) casein fed groups (40 and 49';:) increases, respectively), although these changes failed to achieve statistical significance. DISCUSS10 N The present study has highlighted a striking physiological difference between animals fed identical diets from birth to adult, but exposed during fetal development to a range of maternal protein intakes. These findings are consistent with the concepts of metabolic programming [19], which propose that exposure to adverse influences during critical periods of growth may lead to irreversible changes in physiology and metabolic function. Human stillbirths associated with intrauterine growth retardation have been shown to have impaired renal development [20]. In the case of the offspring of the 6% casein fed group, it can be assumed that a pathological insult was sustained, as evidenced by their altered early growth pattern. The offspring of the 9 and l2x) casein fed dams, however, were grossly normal and appeared to have tolerated the low protein diets, as if in the normal physiological range. Recent observations in the human population suggest that inadequate nutrition during pregnancy may predispose the offspring to non-communicable disease in adulthood, including diabetes, cardiovascular disease and respiratory problems [1&-13]. As with the present study, the poor plane of nutrition suggested to underly these physiological changes does not constitute a pathological stress, but instead represents a sub-optimal state. The feeding of a diet very low in protein (6% casein) was found to significantly reduce the energy intake and weight gain of female rats both before and during pregnancy. Rats fed the 9% casein diet also exhibited reduced weight gain and tended to consume less energy. Diets deficient in one or more specific nutrients typically exert this effect on appetite and growth [21]. Although rats on the 9 and 6",, casein diets were, possibly, both protein- and energy-deficient, the principal dietary inadequacy was attributable to protein, intake of which was reduced by 752, in the 6':; casein fed group, as opposed to a 30% reduction in energy intake. Deficits of protein and/or energy did not have any significant effect on litter sizes and the ability of the rats to reproduce, as previously demonstrated [ 161. The early growth of the pups born to the rats on the various diets was also altered. Pups delivered to mothers fed the 6% casein diet were markedly smaller than all the other litters from the first week, and were then unable to catch up over a 21-week period. Pups in the litters of the 67" protein fed dams were noticeably less vigorous than normal in the first few days, and appeared more vulnerable to unexplained mortality. This mortality did not, however, appear to occur among weaker members of litters, the loss of two complete litters accounting for all the deaths of offspring of the 6",, protein fed group. The more rapid early growth of the pups of the 12:~;) casein fed dams may indicate alterations of the energy balance or appetite of these animals. At 9 weeks of age there was no evidence of changes in food intake (data not shown), although changes in the earlier phase of life cannot be excluded. Alterations to processes such as protein turnover, which are known to be influenced by dietary factors in adult and fetal animals [22], may significantly alter the magnitude of resting energy expenditure. Many animal models of experimentally induced hypertension require severe surgical intervention to achieve moderate alterations in blood pressure. Low

5 Hypertension induced by fetal exposure to low protein diet 221 birth weight induced in guinea pigs by ligation of uterine blood vessels [23] increased mean blood pressure in the offspring by no more than 10mmHg. Nephrectomy of the rat typically increases systolic blood pressure by 30 mmhg [24]. The important finding of the present study was that systolic blood pressure could be elevated by 15-16mmHg by relatively mild protein restriction during gestation. Pathological intervention with a 6% casein diet induced a comparable increase in blood pressure to that seen after nephrectomy of the rat [24], and treatment with noradrenaline or angiotensin I1 in the ovine fetus [25]. Fetal exposure to maternal low protein diets elicited highly significant increases in systolic blood pressure when measured in early adulthood. This elevation of systolic blood pressure persisted as the animals matured further. Smaller group sizes in the latter studies may explain the lack of any significant difference at 12 weeks old, and the apparent normalization of blood pressure in the offspring of the 12% casein fed group. The potential magnitude of this response in older animals and the underlying mechanism remain to be fully resolved. As the development of the fetal kidney is known to be impaired in human intrauterine growth retardation [23], and protein deficiency in rats [26] leads to altered renal function [27], a possible mechanism underlying the observed hypertension may be a dysfunction or dysregulation of the reninangiotensin system. The elevated ACE activities observed in the animals exposed to 6% casein in utero, and the tendency for a higher activity in 12 and 9% casein exposed rats, may suggest this mechanism may operate. However, as a wide range of metabolic alterations were noted in the littermates of these animals. (S. C. Langley et al., unpublished work), further study of the reninangiotensin system is required to establish the contribution of the elevated ACE activities to the hypertensive state. Alternatively, changes to the central nervous system, known to be induced in rat fetuses exposed to low protein diets [28], or the vascular system could mediate the observed hypertension. Linear regression analysis indicated a relationship between the reduced protein intakes of the mothers, and the systolic blood pressure of the offspring. Although there was evidence that the 6% casein diet had an anorectic effect, it would appear that this had little influence on the ensuing hypertension. As increased blood pressure was observed in the offspring of the 12% casein fed group, which had unchanged energy intakes, the primary factor underlying the observed hypertension must be assumed to be the maternal low dietary protein intake. Deficit of specific amino acids may, therefore, play a role in the observed alteration of physiological function. The fetus has a high demand for an unusual pattern of amino acids [29]. Protein deficiency has also been associated with disturbances of iron transport and anaemia, factors linked to altered development of the human feto-placental unit [3]. The relative effects of protein deficiency in the preconceptual period and during pregnancy on the later blood pressure of the offspring could not be determined in the present study. Recent observations by our group in collaboration with that of Hales would suggest that diet during the pregnancy period alone may be critical for the development of hypertension (S. C. Langley et al., unpublished work). In conclusion, the present study in which young female rats entered pregnancy on a poor plane of nutrition has illustrated that an impaired nutrient supply during fetal development may have significant physiological consequences in later life. These observations provide an important parallel to recent human physiological studies. REFERENCES I. Ariyuki F. Growth retardation induced in rat fetuses by maternal fasting and massive doses of ergocalciferol. J Nutr 1987; 117: Mello MAR, Cury L, Valle LBS, OliveirtFilho RM. Protein-calorie malnutrition in the young pregnant rat: factors involved in fetal growth impairment. Bra J Med Biol Res 1987; 20: 575-7, 3. Godfrey KM, Redman CWG, Barker DJP. Osmond C. The effect of maternal anaemia and iron deficiency on the ratio of fetal weight to placental weight. Br J Obstet Gynaecol 1991; I: Mellor DJ. Nutritional and placental determinants of foetal growth rate in sheep and consequences for the newborn lamb. Br Vet J 1983; 139: Wunderlich SM. 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6 222 S. C. Langley and A. A. Jackson 20. Hinchcliffe SA. Lynch MRJ. Sargent PH, Howard CV, van Zelzen D. The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obstet Gynaecol : Rose WC. The nutritive significance of the amino acids. Physiol Rev 1938; 18: Mayel-Afshar S, Grimble RF. Changes in protein turnover during gestation in the foetuses, placentas, liver, muscle and whole body of rats given a low protein diet. Bioc.him Biophys Acta 1983: 756: 182-W. 23. Persron E, Jansron T. Low birth weight is associated with elevated adult blood pressure in the chronically catheterized guinea-pig. Acta Physiol Scand 1992; 145: Coffman TM. Himmelstein S. Best C, Klotman PE. Post-transplant hypertension in the rat: effects of captopril and native nephrectomy. Kidney Int 1989; 36: 35-40, 25. Tangalakir K, Lumbers ER. Moritr KM. Towstoless MK. Wintour EM. Eflect of cortisol on blood pressure and vascular reactivity in the ovine fetus. Exp Phyriol 1992; n: Zeman FJ. Effects of maternal protein restriction on the kidney of the newborn young of rats. J Nutr 1968; 94: I 11-17, 27. Hall SM, Zeman FJ. Kidney function of the progeny of rats fed a low protein diet. J Nutr 1968: 95: Resnick 0, Morgan PJ. Hasson R, Miller M. Overt and hidden forms of chronic malnutrition in the rat and their relevance to man. Neurosci Biobehav Rev 1982: Jackson AA. Optimizing amino acid and protein supply and utilization in the newborn. Proc. Nutr Soc 1989; 4&