Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality

Abstract

Absorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na⁺/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.

Figure 1

(A) Genotype and gender from pups from 9 litters born from two independent heterozygous Slc34a2 breedings. (B) body weight, (C) food intake, (D) water intake, (E) fecal output and (F) urinary output of wild type (n = 4) and heterozygous (n = 5) Slc34a2 rats fed standard chow (NPD) or challenged for 3 days with low Pi diet (LPD). Data are presented as means ± SEM.

Figure 2

(A) Fecal Pi, (B) fecal Ca²⁺, (C) urinary Pi, (D) urinary Ca²⁺ and (E) urinary creatinine of wild type (n = 4) and heterozygous (n = 5) Slc34a2 rats fed standard chow (NPD) or challenged for 3 days with low Pi diet (LPD). Data are presented as means ± SEM. Statistical significance was calculated by ANOVA-Bonferroni. *P ≤ 0.05 and ****P ≤ 0.0001.

Figure 3

(A) Plasma Pi, (B) plasma Ca²⁺, (C) plasma intact FGF-23 and (D) plasma 1,25(OH)₂ vitamin D₃ of wild type (n = 4) and heterozygous (n = 5) Slc34a2 rats fed standard chow (NPD) or challenged for 3 days with low Pi diet (LPD). Data are presented as means ± SEM. Statistical significance was calculated by ANOVA-Bonferroni. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Figure 4

Expression of NaPi-IIb in total membranes isolated from mucosa of (A) duodenum and (B) jejunum from wild type (n = 4) and heterozygous (n = 5) Slc34a2 rats challenged for 3 days with low Pi diet. The abundance of the cotransporter was normalize to the expression of actin. The ratio in WT rats was considered as 100%.

Figure 5

(A) Genotype of E18 embryos born from 3 heterozygous breedings. Embryonic (B) body weight, (C) representative images, (D) placental body weight and (E) amniotic fluid Pi concentration. Data are presented as means ± SEM. Scale bars in (C): 5 mm. Statistical significance was calculated by ANOVA-Bonferroni. ****P ≤ 0.0001.

Figure 6

Intestines from (A,C) wild type and (B,D) Slc34a2 homozygous E18 embryos stained with (A,B) H&E or with (C,D) a NaPi-IIb antibody. Scale bars: 100 µm in (A,B); 50 µm in (C,D).

Figure 7

Lungs from (A,C) wild type and (B,D) Slc34a2 homozygous E18 embryos stained with (A,B) H&E or with (C,D) a NaPi-IIb antibody. Scale bars: 100 µm in (A,B); 50 µm in (C,D).

Figure 8

Liver (A,B) and pancreas (C,D) from wild type (A,C) and Slc34a2 homozygous E18 embryos (B,D) stained with a NaPi-IIb antibody. Scale bars: 50 µm.

Figure 9

Kidneys from (A) wild type and (B) Slc34a2 homozygous E18 embryos stained with H&E. Scale bars: 250 µm.