Low renal mineralocorticoid receptor expression at birth contributes to partial aldosterone resistance in neonates

Abstract

The human neonatal period is characterized by renal immaturity with impaired capacity to regulate water and sodium homeostasis, resembling partial aldosterone resistance. Because aldosterone effects are mediated by the mineralocorticoid receptor (MR), we postulated that this hormonal unresponsiveness could be related to low MR expression in the distal nephron. We measured aldosterone and renin levels in umbilical cord blood of healthy newborns. We used quantitative real-time PCR and immunohistochemistry to analyze the expression of MR and key players of the mineralocorticoid signaling pathway during human and mouse renal development. High aldosterone and renin levels were found at birth. MR mRNA was detected in mouse kidney at d 16 postcoitum, peaking at d 18 postcoitum, but its expression was surprisingly very low at birth, rising progressively afterward. Similar biphasic temporal expression was observed during human renal embryogenesis, with a transient expression between 15 and 24 wk of gestation but an undetectable immunoreactive MR in late gestational and neonatal kidneys. This cyclic MR expression was tightly correlated with the evolution of the 11beta-hydroxysteroid dehydrogenase type 2 and the epithelial sodium channel alpha-subunit. In contrast, glucocorticoid and vasopressin receptors and aquaporin 2 followed a progressive and sustained evolution during renal maturation. Our study provides the first evidence for a low renal MR expression level at birth, despite high aldosterone levels, which could account for compromised postnatal sodium handling. Elucidation of regulatory mechanisms governing MR expression should lead to new strategies for the management of sodium waste in preterms and neonates.

Figures

Figure 1. Ontogenesis of mMR, mMRα, αENaC and 11βHSD2 mRNA expression during mouse kidney development

(A, B, D, E): Relative mRNA expression levels were determined using qPCR at various developmental stages as follow: E15–19: 15–19 days postcoitum. D0: day of birth. D2–15: second to fifteenth postnatal day. D0 was used as reference for statistical analysis: * P<0.05, ** P<0.01, *** P<0.001. Results, expressed as the ratio of attomoles of specific gene/femtomoles of 18S, are mean ± SEM (E15 n=6, E16, n= 8, E18 n=10, E19 n=12, D0 n=15, D2 n=6, D4 n=6, D8 n=6, D15 n=6). (C, F): Correlations between the relative expression of mMR versus mMRα (C) or 11βHSD2 (F) were obtained by Spearman regression analysis. r represents the correlation coefficient.

Figure 2. Immunodetection of MR, GR and AQP2 during renal development of murine fetus and neonates

(A–F): Immunoreactive MR protein is absent at E15 (A) but begins to be detected at E16, most notably in the nuclei of cortical collecting duct cells (B, see inset, arrow; see also Supplemetnal Fig 2). MR expression is rather weak at E18 and D0 but increases afterwards at D8 and in the adult kidney. (G–L): GR protein is already present at E15 (G), in the nuclei (see inset) of endothelial, interstitial and epithelial cells, in different segments of the nephron and in the glomeruli (H–L). (M–R): A strong AQP2 immunostaining is observed at the apical membrane of the cortical collecting duct cells from E16 to adulthood (N–R). E15, E16, E18: 15, 16 and 18 days postcoitum. D0: day of birth. D8: eighth post natal day. Original magnification: x20. Insets: x100. gl: glomerulus; dct: distal convoluted tubule; ccd: cortical collecting duct.

Figure 3. Western blot analysis of MR expressin during murine renal development

Twenty μg of protein from kidney homogenates at different developmental stages (E18, 18 days postcoitum, D0: day of birth. D8: eighth postnatal day, A: adult) were loaded on 7.5% SDS-PAGE followed by direct immunoblotting with anti-MR 39N antibody (1/2000). α-tubulin was used as loading control. MR was normalized to α-tubulin protein levels after digitalization on a gel scanner using QuantityOne software (Bio-Rad, Marnes-la-Coquette, France). Results are means ± SD of 4 independent determinations and are expressed relative to MR expression measured at day 0, arbitrarily set at 1.

Figure 4. Ontogenesis of hMR mRNA expression during human kidney development

(A): Relative hMR expression was determined using qPCR in fetal kidney samples at various gestational ages. Each point represents the mean of three independent determinations of hMR expression, performed in triplicate in a given sample (see materials and methods), and normalized by hMR expression in one adult kidney sample (0.04 ± 0.08 amol/pmol of 18S, mean ± sem, n=11). (B, C): Correlations between the expression of hMRα isoform versus αENaC (B) or 11βHSD2 (C) were obtained by Spearman regression analysis. r represents the correlation coefficient.

Figure 5. Histology and MR, GR and AQP2 immunoreactivity in human fetal kidneys

(A–D): H&E staining at various stages of fetal renal development. At 10 gestational weeks (GW), the human kidney is clearly immature (A). At 40 GW (D), glomeruli and nephron formation is achieved, but some podocytes conserve an immature cubic shape (inset, D). (E–H): MR immunodetection is observed at specific stages of renal development. Immunoreactive MR is transiently detected in distal collecting ducts at 19 GW, as illustrated by a positive staining, mostly in the nuclear compartiment (inset, F). In contrast, no renal MR expression is found at 10 GW (E) and between 30 to 40 GW (G–H). (I–L): The GR protein is immunodetected at all developmental stages, in the nuclei of endothelial, interstitial and epithelial cells, in different nephronic segments and the glomeruli. (M–P): AQP2 is not expressed at 10 GW (M) whereas a strong immunostaining is detected at the apical membrane of the cortical collecting duct cells from 15 to 40 GW (N,O and P). Original magnification: x20. Insets: x100. gl: glomerulus; dct: distal convoluted tubule; ccd: cortical collecting duct.

Figure 6. Histology and MR, GR and AQP2 immunoreactivity in human neonatal and adult kidneys

(A–C): H&E staining at various stages of postnatal renal development. Podocytes have reached a mature flattened shape at 11 months postnatal (inset, B). (D–F): MR immunodetection is observed at specific stages postnatal renal development. No MR protein is detected from birth until 11 postnatal months (D). Afterwards, a MR immunoreactivity is readily detected at 11 months after birth (E) as in the adult kidney (F). (G–I): GR is weakly expressed during the 2–10 months postnatal period (G). Afterwards, GR protein is strongly expressed in the nuclei of endothelial, interstitial and epithelial cells, in different parts of the nephron and the glomeruli (H and I). (J–L): A strong AQP2 immunostaining is detected at the apical membrane of the cortical collecting duct cells from birth to adulthood. Original magnification: x20. gl: glomerulus; dct: distal convoluted tubule; ccd: cortical collecting duct.