Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis

Abstract

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12‐month‐old progeny from a male mouse that had been treated with the chemical mutagen N‐ethyl‐N‐nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm‐Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome‐wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3‐B3 and whole‐exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain‐containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T) mice, when compared with wild‐type (Brd4+/+) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.

Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2

“…1G and H ). Bone metabolism was assessed by whole body dual-energy X-ray absorptiometry (DXA), and by measurement of the pro collagen type 1 N-terminal pro-peptide (P1NP) and C-terminal cross-linking telopeptide of type 1 collagen (CTX-1) bone turnover markers, as reported ( 18 , 19 ). Bone mineral content (BMC) corrected for body weight, BMD, and bone turnover in male and female Ap2s1 +/L15 mice were not significantly different to those observed in age- and sex-matched WT mice ( Table 2 and Supplementary Material, Table S1 ).…”

“…C-terminal cross-linking telopeptide of type 1 collagen (CTX-1) was measured using a mouse-specific ELISA (Biorbyt Ltd). Procollagen type 1 N-terminal propeptide (P1NP) was measured by an enzyme immunoassay (EIA) (Immunodiagnostic Systems) ( 18 ).…”

Ap2s1mutation in mice causes familial hypocalciuric hypercalcemia type 3

“…Phenotype of mice harboring the Ap2s1 mutation, p.Arg15Leu. Adult Ap2s1 +/L15 mice, aged [12][13][14][15][16][17][18][19][20][21][22] weeks, showed no gross morphological abnormalities, although male Ap2s1 +/L15 mice had a significantly reduced body weight when compared to age-matched WT male litter-mates, whereas female Ap2s1 +/L15 mice had a normal body weight (Table 2). Activities such as eating, drinking, grooming, moving and interacting with cage-mates were observed to be similar between Ap2s1 +/L15…”

“…Urine biochemical analysis showed that female Ap2s1 +/L15 mice had significantly reduced 24 hour urine calcium excretion, whereas male Ap2s1 +/L15 mice showed a significantly increased fractional excretion of phosphate, but no alterations of urine calcium excretion when compared to WT mice (Table 2, Figure 2G-H). Bone metabolism was assessed by whole body dual-energy X-ray absorptiometry (DXA), and by measurement of the pro-collagen type 1 N-terminal pro-peptide (P1NP) bone turnover marker, as reported (18,19). Bone mineral content (BMC) corrected for body weight, BMD, and bone turnover in male and female Ap2s1 +/L15 mice were not significantly different to those observed in sexmatched WT mice (Table 2 and Supplemental Table 1).…”

“…1,25-dihydroxyvitamin D by a two-step process involving purification by immunoextraction and quantification by enzyme immunoassay (Immunodiagnostic Systems); and FGF23 using a two-site ELISA kit (Kainos Laboratories), as described (19). Procollagen type 1 N-terminal propeptide (P1NP) was measured by an enzyme immunoassay (EIA) (Immunodiagnostic Systems) (18).…”