The effect of maternal protein and folic acid intake on the developmental programming of adulthood diseases
thesisposted on 28.02.2017, 03:24 by Wood-Bradley, Ryan James
The leading causes of mortality and morbidity worldwide are cardiovascular disease (high blood pressure, high cholesterol and renal disease), cancer and diabetes. The development of these diseases is related to a complex interaction between adult lifestyle and genetic predisposition. Maternal nutrition can influence the fetal and early life environment and is known to be a risk factor for the future development of adult diseases. A growing body of large-scale human studies suggest that maternal malnutrition may impair organogenesis in the offspring, which can predispose these offspring to high blood pressure and renal dysfunction in adulthood. Studies in experimental animals have further illustrated the significant impact maternal diet has on offspring health. Many studies report changes in kidney structure (namely a reduction in the number of nephrons) in offspring of protein deprived dams. Although the early studies suggested that increased blood pressure was also present in offspring of protein restricted dams, this is not a universal finding and requires clarification. Importantly, to date, the literature offers little to no understanding of when in development these changes in kidney development occur, nor are the cellular and molecular mechanisms that drive these changes well characterised. Moreover, the mechanisms linking maternal nutrition and a suboptimal renal phenotype in offspring are yet to be discerned – one potential mechanism involves epigenetics. Epigenetics can be influenced by maternal diet, particularly the supply of micronutrients involved in methylation – specifically folate or folic acid. When folic acid has been added to protein deficient maternal diets in animal studies it has been shown to reverse the hypertension which may be programmed in offspring of rodents fed a sub-optimal maternal diet. This thesis explores the relationship between maternal protein deprivation and folic acid intake on rat kidney development, and the development of high blood pressure and renal dysfunction in the offspring. 1.1. MATERNAL PROTEIN RESTRICTION – IMPACT ON FETAL AND POSTNATAL GROWTH Fetuses developing in the face of maternal protein restriction (9% protein versus 20% protein in the control normal diet) showed minimal disruption in their growth trajectory except for an increased placenta:fetal ratio at embryonic day 20 (E20). Analysis of ureteric branching morphogenesis (an important driver of nephrogenesis) at E14.25 using whole metanephric organ culture found no effect of the low protein diet on branching, however a 14% reduction in nephron number was observed ex vivo from as early as E17.25 in low protein offspring. Postnatally, offspring of maternal protein deprivation demonstrated slower growth than controls, a 26% (female) and 17% (male) reduction in nephron endowment, but did not show any signs of cardiovascular (blood pressure, heart rate) or renal dysfunction (glomerular filtration rate, effective renal blood flow) in adulthood. 1.2. MATERNAL FOLIC ACID RESTRICTION OR SUPPLEMENTATION – IMPACT ON FETAL AND POSTNATAL GROWTH When maternal protein restriction was combined with folic acid restriction (<0.05mg/kg folic acid versus 5mg/kg folic acid in controls) the renal phenotype was very similar to that observed following maternal protein restriction alone. Offspring were of a similar weight to that of offspring exposed to maternal protein restriction, and likewise they had a similar nephron endowment, blood pressure and kidney function. Paradoxically, maternal folic acid supplementation (200mg/kg) combined with protein restriction in pregnancy lead to reduced kidney branching morphogenesis in the offspring (13% reduction in branch points and 12% reduction in ureteric tips). This finding was also observed in the metanephric organ culture system when exogenous folic acid was added to the media. 1.3. KIDNEY GENE EXPRESSION AT E14.25 Real-time polymerase chain reaction (RT-PCR) was used to assess expression of key kidney development genes following exposure to a maternal low protein diet with folic acid restriction or supplementation. Genes involved in the regulation of branching morphogenesis were analysed (Gdnf, Bmp4, Gfrα1) as were genes involved in mesenchyme to epithelial transition (Pax2 and Hnf4α). Female offspring exposed to maternal protein and folic acid restriction reduced expression at E14.25 of Pax2, Gdnf, Bmp4 compared with maternal protein restricted controls (no change in males), while male offspring had greater expression of Gfrα1. Female and male offspring exposed to maternal low protein and folic acid supplementation had reduced expression of Pax2, and females also had reduced expression of Gdnf and Bmp4 compared to controls. Analysis of genes involved in kidney development for epigenetic changes (altered methylation status) revealed no change in gene methylation despite the changes in levels of expression described above. Although Gfrα1 expression was negatively correlated with methylation status gene expression and methylation status of Gfrα1 was most closely correlated in offspring exposed to maternal low protein diet and was not different with the exposure of maternal folic acid intake (either supplementation or restriction). 1.4. OFFSPRING CARDIOVASCULAR FUNCTION – IMPACT OF MATERNAL FOLIC ACID SUPPLEMENTATION OR RESTRICTION Postnatal bodyweight of offspring exposed to maternal protein restriction with supplementation or restriction of folic acid did not differ compared with low protein controls, however bone mineral content was reduced at postnatal day 21 (PN21), PN180 and PN360 in offspring exposed to maternal low protein and folic acid restriction (LP-FA). Postnatal nephron endowment was significantly greater in LP-FA offspring at PN21. Compared with controls, neither cardiovascular nor renal function were altered in offspring exposed to maternal folic acid supplementation or restriction. 1.5. CONCLUSION The findings from this thesis suggest that maternal diet can have a significant impact on fetal development where a nephron endowment is programmed early, but this does not necessarily lead to adult disease despite the continued presence of a nephron deficit. It is likely that this scenario occurs because offspring did not exhibit catch-up growth, which prevented the development of high blood pressure and renal dysfunction. Understanding the impact of maternal diet on fetal development and offspring health is an effort targeted at reducing the global burden of non-communicable disease. The particular constituents of the maternal diet that have the greatest impact on offspring health are going to assist in developing effective and health-promoting diets. Folic acid has been shown to have beneficial effects on birth outcomes, but it is not able to compensate for macronutrient deficiencies in the maternal diet. While it has been suggested that folic acid supplementation may lead to an increased risk of developing asthma in childhood, the findings from the current study indicate reduced growth when folic acid supplementation is combined with low protein. There was no indication of disease onset in those offspring. Maternal diet is critical for offspring development, but poor maternal diet may not trigger disease onset without secondary insults.