FAM111A is dispensable for electrolyte homeostasis in mice

Figures

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  Figure 1. Mouse breeding strategy and genotyping. (A) The ZEN-UB1 cassette was inserted to replace the protein coding region of Fam111a in ES cells. Forward (Fw) and reverse (Rv) primers used for genotyping the mice are represented with arrows. Primer pair 1 recognises wildtype Fam111a and primer pair 2 recognises the ZEN-UB1 cassette. (B) PCR products showing the genotype of Fam111a+/+, Fam111a+/− and Fam111a−/− mice at the expected sizes. The full-length gel is presented in Supplementary Figure S1. (C) mRNA expression of Fam111a (i) in the kidney, (ii) proximal duodenum, (iii) distal colon and (iv) liver tissues confirming the genotype of Fam111a+/+, Fam111a+/− and Fam111a−/− mice 8–10 weeks old. n = 8 (4 males, 4 females) for Fam111a+/+ and Fam111a+/−, n = 7 (3 males, 4 females) for Fam111a−/− mice. For liver, n = 7 (3 males, 4 females) for Fam111a+/+ and n = 7 (4 males, 3 females) for Fam111a+/− and n = 7 (3 males, 4 females) for Fam111a−/− mice. Results were normalised to Gapdh expression (reference gene). Data are presented as mean ± standard deviation (SD). Significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05 compared to Fam111a+/+ group.

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  Figure 2. Kidney histology and gene expression of renal electrolyte transporters in Fam111a−/− mice. (A) Representative H&E staining of kidney tissues from Fam111a+/+ and (B) Fam111a−/− mice showing cortex (left) and medulla (right). Glomerular (white arrows) and tubular structures (black arrows) are indicated. (C) mRNA expression levels of (i) Trpm6, (ii) Trpm7, (iii) Cnnm2, (iv) Slc41a1, (v) Pthr, (vi) Cabp28k, (vii) Slc34a1, (viii) Slc34a3, (ix) Klotho, (x) Cyp24a1, (xi) Cyp27b1, (xii) Fgfr1, (xiii), Trpv5, (xiv) Prl1 and (xv) Prl2 in the kidneys of Fam111a+/+, Fam111a+/− and Fam111a−/− mice are similar. n = 8 (4 males, 4 females) for Fam111a+/+ and Fam111a+/−, n = 7 (3 males, 4 females) for Fam111a−/− mice. n = 7 (3 males, 4 females) for Fam111a+/+ mice for the Trpv5 and Slc34a1 genes. Results were normalised to Gapdh expression (reference gene). Data are presented as mean ± SD. Significance was determined using one-way ANOVA.

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  Figure 3. DCT area is unaltered in Fam111a−/− mice compared to Fam111a+/+ mice. Representative images of whole kidney slices (left, scale bars = 0.5 mm) and regions of interest in the cortex (overlay, scale bars = 50 μm) stained for NCC (magenta) and Parvalbumin (Parv, green) in (A) Fam111a+/+ and (B) Fam111a−/− mice. (C) (i) Percentage of NCC-positive area and (ii) Parv-positive area of total kidney slice area and (iii) the ratio of the NCC-Parv positive area in Fam111a+/+ and Fam111a−/− mice (n = 3 per group, all male mice).

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  Figure 4. Expression of intestinal Mg2+ and PO43− transporter genes of Fam111a−/− mice. (A) mRNA expression levels of (i) Trpm6, (ii) Trpm7, (iii) Cnnm4, (iv) Slc41a1 and v) Slc34a2 in the colon of Fam111a+/+, Fam111a+/− and Fam111a−/− mice are unaltered. (B) Duodenal mRNA expression of the phosphate transporter (i) Slc34a2 is significantly upregulated in Fam111a−/− mice, whereas expression of (ii) Slc20a1 and (iii) Slc20a2 is unchanged. n = 8 (4 males, 4 females) for Fam111a+/+ and n = 7 (3 males, 4 females) for Fam111a−/− mice. For colon Trpm6, Slc41a1 and Slc34a2 n = 8 for Fam111a+/− (4 males, 4 females). For colon Trpm7 and Cnnm4 n = 7 (3 males, 4 females) for Fam111a+/− mice, and for duodenal Slc34a2, Slc20a1 and Slc20a2 n = 7 (4 males, 3 females) for Fam111a+/− mice. Results were normalised to Gapdh expression (reference gene). Data are presented as mean ± SD. Significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05 compared to Fam111a+/+ mice.

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  Figure 5. Microstructural properties of the femur from Fam111a−/− mice are unaffected. (A) Trabecular and (B) cortical microarchitectural measurements in Fam111a+/+, Fam111a+/− and Fam111a−/− mice 8–10 weeks old. n = 7 (3 males, 4 females) for Fam111a+/+ and Fam111a+/− and n = 8 (4 males, 4 females) for Fam111a−/− mice. Data are presented as mean ± SD. Significance was determined using one-way ANOVA.

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  Figure 6. Parathyroid hormone levels and parathyroid histology in Fam111a−/− mice. (A) Parathyroid hormone levels in serum of Fam111a+/+ and Fam111a−/− mice. n = 4 for Fam111a+/+ and n = 3 for Fam111a−/− mice (all male mice). Data are presented as mean ± SD. Significance was determined using an unpaired t-test. (B) H&E staining of thyroid tissue showing normal parathyroid tissue in Fam111a+/+ and (C) Fam111a−/− mice. Parathyroid tissue is encircled by a dotted line.