Administration of retinoic acid to pregnant mice increases the number of fetal mouse glomeruli

Abstract

The prevalence of chronic kidney disease (CKD) is increasing worldwide, and CKD is a serious global health problem. Low glomerular number is one of the risk factors for CKD; therefore, the glomerular number is associated with the risk of CKD. Increasing the glomerular number above normal levels may reduce the risk of CKD. It has been reported that, in vitro, the addition of retinoic acid (RA) to the culture medium increases the glomerular number. However, there is no report of an increase in glomerular number above normal levels with the addition of RA in vivo. In this study, RA (20 mg/kg) was administered intraperitoneally to pregnant mice once at embryonic day (E) 10.5, E12.5, E14.5, or E16.5. The fetuses were harvested at E18.5 and fetal mouse kidneys were evaluated. Fetal kidney volume and weight were significantly increased in the E16.5 group compared to the control group. The total glomerular number in the E16.5 group was also approximately 1.46 times higher than that in the control group. In summary, we established a method to increase the glomerular number in the fetal kidney by administration of RA to pregnant mice at E16.5. These results will facilitate the investigation of whether CKD risk is reduced when the glomerular number increases above normal.

Keywords: CKD, chronic kidney disease; Chronic kidney disease; E, embryonic day; GA, glomerular area; GV, glomerular volume; Glomerular number; Mouse fetus; RA, retinoic acid; Retinoic acid.

Graphical abstract

Fig. 1

Body weight, kidney weight, and kidney volume in fetuses treated with retinoic acid The control group received corn oil intraperitoneally at E10.5. Experimental groups received retinoic acid (RA) intraperitonially once at E10.5, E12.5, E14.5, or E16.5. Fetuses were harvested at E18.5, and the fetal body weight, kidney volume, and kidney weight were compared. (A) Fetal weight, kidney volume, and kidney weight were measured in all the fetuses (n = 33 fetuses in the control group, 53 fetuses in the E10.5 group, 40 fetuses in E12.5 group, 44 fetuses in the E14.5 group, and 37 fetuses in the E16.5 group). There was no difference in fetal body weight between the control group and the RA groups. (B) Kidney volume was compared after normalizing it to fetal body weight. The E16.5 group had a larger kidney volume than the control group (6.82 ± 0.19 mL/g vs. 8.31 ± 0.18 mL/g, p < 0.0001). (C) Kidney weight was compared after normalizing it to fetal body weight. The E14.5 group (8.45 ± 0.12 mg/g vs. 8.91 ± 0.16 mg/g, p = 0.0364) and the E16.5 group (8.45 ± 0.12 mg/g vs. 9.895 ± 0.16 mg/g, p < 0.0001) had significantly higher kidney weight than the control group. ns: not significant, *P < 0.05, ****P < 0.0001. The error bars represent standard errors of the mean (SEMs). RA: Retinoic acid, E: Embryonic day.

Fig. 2

Fetal kidneys dissected at E18.5 (A–E) Images showing fetal kidneys dissected at E18.5 of the control group (A) and the E10.5 (B), E12.5 (C), E14.5 (D), and E16.5 (E) groups. All images are kidneys taken from fetuses closest to the average fetal body weight (scale bar, 2 mm). The kidneys in the E16.5 group were macroscopically larger than those in the control group. RA: Retinoic acid, E: Embryonic day.

Fig. 3

Histological analysis and glomerular number per unit area (A, B) Micrographs of fetal kidneys after hematoxylin-eosin staining. The largest cross-sectional slice of kidney in the E16.5 group (B) was larger than that of the control group (A) (scale bar, 0.5 mm). (C) There was no significant difference in the glomerular number per unit area between the control group and the E16.5 group (n = 3 kidneys in the control group and 3 kidneys in the E16.5 group, 25.98 ± 1.66 pieces/mm² vs. 29.23 ± 1.83 pieces/mm², p = 0.2083). (D) There was no significant difference in the glomerular volume between the control group and the E16.5 group (n = 15 glomeruli in the control group and 15 glomeruli in the E16.5 group, 16.16 ± 1.42 × 10⁴ μm³ vs. 16.25 ± 1.12 × 10⁴ μm³, p = 0.9606). ns: not significant. The error bars represent standard errors of the mean (SEMs). RA: Retinoic acid, E: Embryonic day.

Fig. 4

Glomerular number per kidney The total glomerular number per kidney in the E16.5 group was significantly higher than that in the control group (n = 3 kidneys in the control group and 3 kidneys in the E16.5 group, 1557 ± 175.2 vs. 2270 ± 126.6, p = 0.0300). *P < 0.05. The error bars represent standard errors of the mean (SEMs). RA, retinoic acid; E, embryonic day.