Folic acid supplementation alleviates reduced ureteric branching, nephrogenesis, and global DNA methylation induced by maternal nutrient restriction in rat embryonic kidney

Abstract

We previously reported that maternal nutrient restriction (NR) inhibited ureteric branching, metanephric growth, and nephrogenesis in the rat. Here we examined whether folic acid, a methyl-group donor, rescues the inhibition of kidney development induced by NR and whether DNA methylation is involved in it. The offspring of dams given food ad libitum (CON) and those subjected to 50% food restriction (NR) were examined. NR significantly reduced ureteric tip number at embryonic day 14, which was attenuated by folic acid supplementation to nutrient restricted dams. At embryonic day 18, glomerular number, kidney weight, and global DNA methylation were reduced by NR, and maternal folic acid supplementation again alleviated them. Among DNA methyltransferases (DNMTs), DNMT1 was strongly expressed at embryonic day 15 in CON but was reduced in NR. In organ culture, an inhibitor of DNA methylation 5-aza-2 '-deoxycytidine as well as medium lacking methyl donors folic acid, choline, and methionine, significantly decreased ureteric tip number and kidney size mimicking the effect of NR. In conclusion, global DNA methylation is necessary for normal kidney development. Folic acid supplementation to nutrient restricted dams alleviated the impaired kidney development and DNA methylation in the offspring.

Fig 1. Effect of folic acid supplementation on the reduction in body weight and ureteric tip number induced by maternal nutrient restriction at embryonic day 14.

(A) After fetal weight was recorded, kidneys were isolated, fixed with methanol, and stained with anti-pancytokeratin antibody and anti-Six2 antibody. The number of ureteric bud tips was counted observing z-stack images by confocal microscopy. Kidney surface area was measured by ImageJ software and expressed in arbitrary units. The mean value of each litter was used for statistical analysis (ANOVA followed by Tukey’s test). CON, controls; NR, offspring of nutrient-restricted mothers; NRFA, offspring of nutrient-restricted mothers supplemented with folic acid. n = 3 litters, n = 4–6 animals per litter. Dots indicate the mean value of each litter. Bars represent ±SE. *, P<0.05 vs CON. (B) Representative images. Scale bar, 250 μm.

Fig 2. Effect of folic acid supplementation on the reduction in body weight, kidney weight, and glomerular number induced by maternal nutrient restriction at embryonic day 18.

(A) After fetal weight was recorded, kidneys were isolated and weighed. They were then fixed, sectioned, and stained with hematoxylin and eosin (representative figure) or PAS (glomerular counting). Glomerular number was counted as described in Materials and Methods. The mean value (body weight and kidney weight) or one value (glomerular count) of each litter was used for statistical analysis (ANOVA followed by Tukey’s test). CON, controls; NR, offspring of nutrient-restricted mothers; NRFA, offspring of nutrient-restricted mothers supplemented with folic acid. n = 4 litters, n = 9–15 animals per litter. Dots indicate the mean value (body weight and kidney weight) or one value (glomerular number) of each litter. Bars represent ±SE. *, P<0.05 vs CON; **, P<0.05 vs NRFA. (B) Representative images. Scale bar, 1 mm.

Fig 3. Effect of maternal nutrient restriction with or without folic acid supplementation on global DNA methylation of rat E18 embryonic kidney.

CON, controls (n = 4 litters); NR, offspring of nutrient-restricted mothers (n = 4 litters); NRFA, offspring of nutrient-restricted mothers. supplemented with folic acid (n = 3 litters). n = 9–15 animals per litter. The mean value of each litter was used for statistical analysis (ANOVA followed by Tukey’s test). Dots indicate the mean value of each litter. Bars represent ±SE. *, P<0.05 vs CON.

Fig 4. Expression of DNA methyltransferases in the kidney from embryonic day 15.

(A) Representative immunoblot. (B) Quantitative analysis. n = 3 litters. n = 10–15 animals per litter. Statistical analysis was performed with unpaired t test. DNMT, DNA methyltransferase; CON, controls; NR, offspring of nutrient-restricted mothers. Bars represent ±SE. *, P<0.05 vs CON.

Fig 5. Effect of 5-aza-2’-deoxycytidine in metanephric organ culture.

Embryonic day 13 metanephroi from normal dams were subjected to organ culture in the absence (CON) or presence of 5-aza-2’-deoxycytidine (Aza) for 3 days. Ureteric buds were visualized by whole mount staining with anti-pancytokeratin antibody. (A) Quantitative analysis. n = 1 litter, n = 6 kidneys. Statistical analysis was performed with ANOVA followed by Tukey’s test. Bars represent ±SE. *, P<0.05 vs CON. (B) Representative images. Scale bar, 250 μm.

Fig 6. Effect of folic acid deficient- and methyl donor-deficient medium in organ culture.

Embryonic day 13 metanephroi from normal rats were subjected to organ culture in DMEM (CON), DMEM deficient in FA (FAD), or DMEM deficient in FA, choline, and methionine (MDD) for 3 days. Ureteric buds were visualized by whole mount staining with anti-pancytokeratin antibody. (A) Quantitative analysis. n = 3 litters, n = 10–11 kidneys. Statistical analysis was performed with ANOVA followed by Tukey’s test. Bars represent ±SE. *, P<0.05 vs CON; **, P<0.05 vs FAD. (B) Representative images. Scale bar, 250 μm.