Expression patterns of the aquaporin gene family during renal development: influence of genetic variability

Abstract

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney.

Fig. 1

Influence of genetic background on the expression of AQP isoforms in kidneys of adult C57BL/6J and CD-1 mice. a Real-time RT-PCR quantification of AQP isoforms (AQP0 to AQP12) in C57 vs. CD-1 mouse adult kidneys (n = 5 individual kidneys for each genotype; *P < 0.05). The values are expressed in [target/reporter gene (Gapdh) ratio] × 10³. b Immunostaining for AQP2 (a–d), Ser256 pAQP2 (e–f), and AQP1 (g–h) in adult kidneys from CD-1 (left panels) and C57 (right panels) mice. The representative micrographs show a higher staining intensity for AQP2 and pAQP2 in the collecting ducts (outer medulla) of the CD-1 kidneys, whereas the staining intensity for AQP1 in the proximal tubules and medullary structures (including descending vasa recta and descending thin limbs of long loop nephrons) is similar (g–h). Bars 50 μm

Fig. 2

In situ hybridization for AQP isoforms in developing mouse kidneys. a Digoxigenin-labeled antisense probes detected AQP1, AQP7, and AQP11 mRNAs essentially in the cortex (proximal tubules) and faintly in the medulla, whereas AQP2, AQP3, and AQP4 mRNAs are detected in collecting ducts in developing (E17.5) and mature (AD) C57 mouse kidneys. b No signal was obtained with the antisense probes for AQP5, AQP9, AQP10, and AQP12

Fig. 3

mRNA expression pattern of AQP isoforms during mouse kidney development and maturation: influence of the strain. Messenger RNA quantification of proximal (a AQP1, AQP7, and AQP11) and distal (b AQP2, AQP3, AQP4) AQP isoforms in fetal (E13.5 to E18.5, blue bars), postnatal (P1 to P28, orange bars), and adult (Ad, black bar) kidneys of C57 (n = 4) and CD-1 (n = 5) mouse strains. The mRNA expression levels were first normalized by Gapdh at every developmental stage, then adjusted to the adult level set at 100%. Significant differences (P < 0.05) between the relative abundance of AQP genes in C57 and CD-1 kidneys are indicated for the appropriate time points

Fig. 4

mRNA expression pattern of tubular markers during mouse kidney development and maturation: influence of the strain. Messenger RNA quantification of proximal (a NaPiIIa, megalin, and OAT1) and distal (b AVPR2 and α-ENaC) genes in fetal (E13.5 to E18.5, blue bars), postnatal (P1 to P28, orange bars), and adult (Ad, black bar) kidneys of C57 (n = 4) and CD-1 (n = 5) mouse strains. The mRNA expression levels were first normalized by Gapdh at every developmental stage, then adjusted to the adult level set at 100%. Significant differences (P < 0.05) between the relative abundance of AQP genes in C57 and CD-1 kidneys are indicated for the appropriate time points

Fig. 5

Expression and maturation of AQP1, AQP2 and AQP3 in the developing mouse kidney: immunoblotting and glycosylation analyses. a Representative immunoblotting analyses of AQP expression in membranes fractions of fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult kidneys of C57 vs. CD-1 mouse strains. Equal amounts (20 µg) of protein were loaded in each line, and the blots were probed with anti-AQP1 (1:5,000), anti-AQP2 (1:1,000), anti-AQP3 (1:5,000), and anti-β-actin (1:10,000) antibodies. The AQP bands of about 24 to 28 kDa correspond to the non-glycosylated core, whereas bands of between 37 and 50 kDa correspond to the glyosylated isoforms. b Biochemical analysis of AQP1, AQP2, and AQP3 proteins during nephrogenesis. Immunoblot analysis of membrane fractions from CD-1 kidneys at E16.5, P7 and P28 stages. Samples were treated with endoglycosidase F (PNGase F) or endoglycosidase H (Endo H) and equal loads (15 µg) of deglycosylated and non-deglycosylated proteins were analyzed using the same antibodies than in a

Fig. 6

Segmental distribution of AQP1 during CD-1 mouse nephrogenesis. a–d Immunostaining for AQP1 (1:200) at E17.5. AQP1 immunoreactivity is detected in some medullary descending thin limb (dtl) and developing proximal tubules (dp) at E17.5. Glomeruli (g) and collecting duct (cd) are unstained. Luminal surface of blood vessels (v) are strongly stained. e–h. Postnatal increase in the density of AQP1-positive tubule profiles in the cortex and medulla at P7 (e), P21 (f), and in adult (g–h). Scale bars 200 µm

Fig. 7

Segmental distribution of AQP2 during CD-1 mouse nephrogenesis. a–c Immunostaining for AQP2 (1:200) at E17.5. AQP2 immunoreactivity is detected in collecting ducts (cd). Glomeruli (g) and developing proximal tubules (dp) are negative. d–f Postnatal increase in AQP2 staining in the medulla, located in collecting ducts profiles at P1 (d), P7 (e), and P21 (f). g–i Adult kidneys with AQP2 staining in the collecting ducts (cd) (g, h) with negative proximal tubules (pt). Note the negative intercalated cells (asterisk) in the collecting ducts of mature kidney (i). Scale bars 200 µm

Fig. 8

Response to water loading in C57 and CD-1 adult mice. Six pairs of mice were injected i.p. with 100 μl/g BW of sterile water. In comparison with C57 mice, the CD-1 mice showed a significantly delayed excretion of water up 2 h after the load, with a compensatory increase at later stages (*P < 0.05)