Maternal calcium intake during pregnancy and its impact on offspring blood pressure identifying the optimal rat strain for mechanistic studies

Abstract

Arterial hypertension is a major risk for disability and death globally. Hypertension is influenced by genetics, epigenetics and environmental factors. An inverse association between calcium intake and high blood pressure (BP) has been widely described. Several works have demonstrated that maternal calcium deficiency during pregnancy can lead to increased BP in offspring, highlighting the importance of maternal nutrition in foetal programming. Therefore, the aim of this review is to identify the most suitable rat strain for studying the effects of maternal calcium intake during pregnancy on offspring BP in adulthood. The literature search found articles involving Wistar, Sprague Dawley, and Wistar Kyoto rat strains. These strains are appropriate models for investigating the relationship between calcium intake and BP in normotensive rats. However, there is little evidence on the effects of calcium intake during pregnancy on offspring BP, suggesting the need of further research to elucidate the underlying mechanisms..

CONCEPTOS CLAVE

Qué se sabe sobre el tema.

Se ha reportado que la ingesta diaria de calcio esta inversamente relacionada con la presión arterial. También hay trabajos que hallaron que una baja ingesta de calcio durante el embarazo influye en la presión arterial de la descendencia.

Qué aporta este trabajo

Recopila las líneas de ratas empleadas para estudiar el mecanismo por el cual la ingesta de calcio materna influye en la presión arterial de la descendencia.

Divulgación

La hipertensión tiene una alta prevalencia a nivel mundial. Una adecuada ingesta de calcio se encuentra asociada a menores valores de presión arterial. La OMS recomienda la suplementación con calcio durante el embarazo para reducir los trastornos hipertensivos del embarazo. Se ha observado que la descendencia de madres suplementadas con calcio durante el embarazo presenta menores valores de presión arterial. Aún permanece desconocido el mecanismo por el cual se produce este efecto. El trabajo presenta los modelos en los cuales se ha estudiado el efecto del calcio dietario en la presión arterial, buscando cuál es el modelo más adecuado para estudiar estos mecanismos subyacentes a la hipertensión.

Introduction

Globally, arterial hypertension represents the greatest risk factor in terms of years of life lost due to disability and death, leading to significant costs in medical care. Preventing arterial hypertension is one of the most important strategies to reduce cardiovascular diseases incidence and burden, the leading global cause of death. ⁽¹⁾ However, the precise mechanisms underlying the development of hypertension have yet to be fully elucidated. Therefore, clarifying these underlying mechanisms is clinically essential to the development of new therapeutic strategies. (2, 3) It is known that the different aetiologies of hypertension are the product of dynamic interactions between genetics and environmental elements, leading to the alteration of biological pathways and ultimately to hypertension. (4, 5) A large genetic association study involving one million patients identified 535 new genetic loci related to blood pressure, demonstrating that hypertension has a multigenic nature. ⁽⁶⁾ Currently, there is substantial epidemiological evidence that intrauterine stress can "program" the later development of the disease. Several animal studies have linked maternal nutritional status from conception to weaning with persistent changes in cardiovascular and offspring metabolic function. (7, 8)

Several studies have found an inverse association between calcium (Ca) intake and blood pressure in rats, young individuals ⁽⁹⁾ and pregnant women. ⁽¹⁰⁾ Moreover, Ca supplements administration during pregnancy has been shown to be associated with a significant reduction in the incidence of preeclampsia, especially in communities with a calcium deficient diet. (10-12)

The mechanism by which low Ca intake produces an increase in blood pressure appears to be mediated by the interaction between calciotropic hormones and blood pressure regulators. Low Ca intake stimulates parathormone (PTH) secretion and increases the conversion of vitamin D into its active form (calcitriol). Both PTH and calcitriol raise intracellular calcium concentration, leading to the contraction of vascular smooth muscle cells and resulting in vasoconstriction. On the other hand, renin release is stimulated by both low Ca intake and PTH. Additionally, PTH increases the synthesis of angiotensin II and aldosterone, which also leads to vasoconstriction and increases renal water reabsorption, thereby elevating blood pressure. ⁽¹³⁾ However, the mechanism by Ca intake reduces the preeclampsia development is unknown.

Furthermore, it has been demonstrated that low Ca intake during pregnancy results in offspring’s blood pressure increase in both animal models and humans. (14-16) A randomized clinical trial evaluated the effects of Ca supplements during pregnancy ⁽¹⁰⁾ on blood pressure of children of 7-year-old. This study showed that offspring of mothers who received Ca supplementation had a lower incidence of high blood pressure than the offspring of mothers who received placebo. ⁽¹⁷⁾ This same effect was observed in other clinical trials and in laboratory animals. ^(16,18–21) Rats whose mothers had Ca deficiency during pregnancy developed higher blood pressure during adulthood. ⁽¹⁶⁾ This observation is supported by epidemiological studies linking adverse foetal environment to an increased risk of hypertension in adults (Barker Hypothesis), (22, 23) and recent animal studies showing that hypertension can also be determined during foetal life by maternal protein restriction. ⁽²⁴⁾ Animal models fed with a protein-deficient diet during pregnancy have been widely used to study the mechanisms involved in this association, finding changes in gene expression. ⁽²⁵⁾ However, the effect of maternal Ca intake on the development of high blood pressure in the progeny has been poorly studied.

Therefore, the aim of this review is to elucidate the most suitable normotensive rat strain to carry out studies to perform research on the mechanisms involved in the effect of low maternal Ca intake during pregnancy on the offspring blood pressure in adulthood.

Methodology

The literature was searched on March and repeated on December 2023 and September 2024, using the databases of MEDLINE (National Library of Medicine) and Scopus. Blood pressure, calcium intake, calcium diet, rats, and offspring were used as keys terms.

The first search was done for “blood pressure” AND “calcium intake” OR “calcium diet” AND rats found 396 articles. A second search was performed in MEDLINE and Scopus with the following criteria: "blood pressure" AND ("Ca intake" OR “calcium diet”) AND rats AND offspring, eight studies were found.

A total of 404 papers were found and screened. After removing duplicated ones, 338 articles were selected from which 166 did not evaluate calcium effects on blood pressure, 21 were reviews, 11 were carried out in humans and 82 in hypertensive rats. The studies carried out with strains of normotensive rats were selected for full text screening (n=57). 6 studies were discarded because details of the experimental design were not available (three are written in Japanese, one is written in Chinese and two did not report the strain of rat used). 13 studies were discarded because blood pressure was not reported (n=10) or calcium was not administered in diet (n=3). Finally, Thirty Eight studies were selected (see Figura 1) .

Figure 1. Flowchart studies.

Results

38 studies evaluating the effect of Ca on blood pressure of normotensive rats were selected: 13 of them were carried out in Wistar rats, 14 in Sprague Dawley rats and 10 in Wistar Kyoto rats. All of them are models of healthy rats. Only one study uses other strains (Fischer 344, Wistar Furth or ACI rats) all of them are strains for same specific pathologies; they are not healthy rats.

The inverse association of Calcium intake and blood pressure was widely reported using different calcium content (from 0 to 4% of Ca) and time of administration (13±10, from 4 to 42 weeks). In Wistar rats, a significant reduction of blood pressure in the groups with higher calcium intake was shown in 9 out of the 13 studies, 10 out of the 14 in Sprague Dawley and 4 out of the 5 in Wistar Kyoto. The study done by Huie et al ⁽²⁶⁾ showed that a calcium restricted diet increases blood pressure in Fischer 344 rats but not in Wistar Furth or ACI rats. However, there are some studies that show a contradictory calcium effect on blood pressure. ^(27–29) Bukoski et al did not find an inverse relationship between calcium intake and blood pressure in the Wistar Kyoto rats. ⁽²⁷⁾ The other studies by Takaya et al. reported different effects of calcium on blood pressure in the offspring of mothers who received diets with varying calcium content during pregnancy and lactation, in a sex-dependent manner.

Of the 38 selected studies, only 4 were done on pregnant rats and 5 on their offspring. The studies done in pregnant rats are descripted in Tabla 1 .

Table 1. Effect of Ca intake on blood pressure in pregnant rats. Description of studies where effect of maternal calcium intake during pregnancy on blood pressure were reported.

First author and year of publication	Strain	Ca content (g Ca/100g of aliment)	Treatment duration	Method of measure	Effect of Ca on blood pressure
Belizan 1981 [47]	Wistar	0,0.6%	12 weeks (9 weeks before pregnancy until 20 days of pregnancy)	Tail cuff method	Inverse association in systolic blood pressure
Aiko 1992 [48]	Wistar	0.02,1.2%	3 weeks	Tail cuff method	Inverse association in systolic blood pressure
Bogden 1995 [49]	Sprague Dawley	0.1, 0.5, 2.5%	Pregnancy and 1 week of lactation	Tail cuff method	Inverse association in systolic blood pressure
					Not dose dependent effect
Ezimokhai 1998 [50]	Wistar	0.9, 1.7 and 2.1%	12 weeks (8 weeks before pregnancy until delivery)	Tail cuff method	Inverse association in systolic blood pressure
					Not dose dependent effect

On the other hand, when the effect of Ca intake during pregnancy on blood pressure of offspring was studied, 5 articles were found. Four of them, from Takaya et al, ( 28-31) were carried out on Wistar rats and another on Wistar Kyoto rats. ⁽¹⁶⁾ Tabla 2 briefly describes studies.

Table 2. Effect of Ca intake during pregnancy on blood pressure of the offspring. Description of studies where effect of maternal calcium intake during pregnancy on blood pressure of offspring were reported.

Variable/Line	Wistar (Takaya et al) [28–31]			Wistar Kyoto (Bergel & Belizan) [16]
Maternal age	12 weeks			20 weeks
%Ca in maternal diet	0,0.9%			0, 0.5,0.8%
Maternal treatment duration	3 weeks of adaptation, mating, and pregnancy until weaning (21 days after birth)			4 weeks of adaptation, mating, and pregnancy until weaning 4 weeks after birth.
Pup treatment beginning	Birth			Birth
%Ca pup diet	0,90%			0,50%
Age of offspring blood pressure measure	28 weeks			52 weeks
Method of measure	Tail cuff method			Tail cuff method
Differences in offspring’s Blood pressure	Offspring [30,31]	Female offspring [29]	2°generation of offspring [28]	Offspring
					Without statistically significant differences in systolic blood pressure	Lower systolic blood pressure in female offspring from Ca deficient diet dams	Lower blood pressure in second generation of offspring from Ca deficient diet dams	Higher blood pressure in offspring of mother with low Ca diet than normal Ca diet.

Discussion

The effect of a low calcium intake during pregnancy on blood pressure of the offspring has been reported several times. In humans, observational studies (19-21) and clinical trials (17, 18, 32) have been reported. Two meta-analyses conclude that a low calcium intake during pregnancy predisposes offspring to higher blood pressure values. (33, 34) A recent study showed that calcium supplementation during pregnancy may lower systolic blood pressure in childhood and adolescence. ⁽³²⁾ However, no studies have elucidated the underlying mechanism.

Recent evidence suggests that exposure to certain environmental conditions during pregnancy may have significant effects on the development of susceptibility to chronic diseases in adulthood. ⁽⁸⁾ Epigenetic changes triggered in the foetal genome during pregnancy development appear to be one of the possible mechanisms for such effects beyond genomic changes. Among environmental factors, it is highlighted that maternal nutrition can induce persistent changes in the genome/epigenome of the offspring that may predispose them to potential diseases and risks in adulthood. ( 7, 35)

Maternal Ca deficiency during pregnancy can cause epigenetic changes that affect the regulation of gene expression and lead to different metabolic phenotypes in the offspring, such as insulin resistance and changes in lipid metabolism. (30, 36) In addition, Ca intake in pregnant women may be inversely related to high BP in their progeny. ⁽³⁷⁾ A low Ca prenatal diet is associated with higher BP in offspring. It is possible that the action of calcium-regulating hormones exerts a foetal programming of blood pressure. ⁽¹⁵⁾

Interaction between hormones regulating phosphocalcic metabolism and controlling blood pressure are proposed as the mechanism by which Ca intake regulates blood pressure in an adult individual. Such changes involve an increase in intracellular calcium in arteriolar muscles resulting in vasoconstriction and increased blood pressure. There is also evidence of a bidirectional relationship between calcium regulatory hormones and renin angiotensin aldosterone system ⁽³⁸⁾ and epigenetic changes associated with hypertension have been shown too. ⁽³⁹⁾ Therefore, there is sufficient information to suggest that a low calcium intake during pregnancy could produce epigenetic alterations that modify the expression of genes linked to phosphocalcic metabolism and/or blood pressure control leading to higher blood pressure in adult life of the offsprings. In this regard, as shown in this review, there are only five studies carried out with experimental animals that research the effects of maternal calcium intake during pregnancy on the blood pressure of offspring. However, none of them explore the mechanisms involved. ^(16,28–31) Consequently, similar studies on the effect of maternal calcium intake and offspring blood pressure are needed to validate previous findings ⁽¹⁶⁾ and to explore the mechanisms involved.

In this review, we explore the rat strains useful for studying the mechanisms involved in the prenatal programming of offspring blood pressure induced by maternal calcium intake. We found that only Wistar strain or its variant, Wistar Kyoto, have been used to study the effects of maternal Ca intake during pregnancy; and no study of this type using Sprague Dawley rats was found. The Wistar or Sprague Dawley strains are widely used in basic research. Both strains are considered multipurpose models of healthy rats. (40, 41) The Wistar Kyoto rat is a strain of Wistar rats from Kyoto School of Medicine, belonging to the same group in which the spontaneously hypertensive rat (SHR) strain was developed. ⁽⁴²⁾ Only one study uses other strains: Wistar Furth (a rat strain used to study macrothrombocytopaenia, oncology, carcinogenesis, leukaemia and grand mal seizure), ⁽⁴³⁾ Fischer 344 (widely used in gerontology and in carcinogenicity testing programs), ⁽⁴⁴⁾ and the ACI (an animal model for congenital genitourinary anomalies). ⁽²⁶⁾

The results of the analysed studies indicate that Wistar, Sprague Dawley and Wistar Kyoto strains are suitable models for studying the effects of Ca intake on blood pressure in normotensive rats. Most studies show an inverse relationship between Ca intake and blood pressure. However, some studies report contradictory effects of Ca intake on blood pressure. (27-29)

For instance, Bukoski et al. did not find an inverse relationship between Ca intake and blood pressure in Wistar Kyoto rats. ⁽²⁷⁾ The authors suggest that this discrepancy compared to other reports could be due to basic nutrient composition of the diet used in their study. Specifically, the diet did not adhere to AIN guidelines for sodium and potassium content, both of which affect blood pressure. ( 45, 46) Other studies by Takaya et al reported the effect of maternal Ca intake on offspring blood pressure, similar to the findings of Bergel and Belizan, but with different results in similar models. Bergel and Belizan ⁽¹⁶⁾ used Wistar Kyoto rats and found higher blood pressure values in offspring of mothers with lower Ca intake (0%) compared to those with normal Ca intake (0.5%). Conversely, the studies of Takaya et al (28-31) using Wistar rats did not find an effect of maternal Ca intake on blood pressure in the first generation. However, in the second generation, offspring of mothers with low Ca intake showed a reduction in blood pressure values. Although the results of Bergel and Takaya seem contradictory, it is important to note that several differences in the experimental designs between the studies. These include the Ca concentration and the treatment duration, as well as the age at which blood pressure was measured in the offspring. Therefore, we could conclude that the Wistar Kyoto strain seems to be more suitable for studying the mechanism involved in the relationship between maternal Ca intake and offspring blood pressure.