Abstract
Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5 682P/+) and homozygous (Trpv5 682P/682P) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5 682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D3 concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5 682P/+ and Trpv5 682P/682P mice consistent with a trafficking defect. In addition, Trpv5682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.
Highlights
Kidney stone disease affects 12% of men and 5% of women by the seventh decade of life and has a recurrence rate of,10% per annum [1]
Genome-wide association studies, aiming to reveal gene variants contributing to polygenic traits,in Icelandic and Dutch populations identified susceptibility risk variants in the claudin 14 (CLDN14) gene for hypercalciuric nephrolithiasis, [10] and a study of Swiss renal calcium stone formers has reported an association between an ancestral haplotype defined by the nonsynonymous polymorphisms of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6), which resulted in a gain-of-function and absorptive hypercalciuria [11]
Our studies have identified a novel mouse model, HCALC1, for autosomal dominant hypercalciuria harbouring a S682P mutation in TRPV5
Summary
Kidney stone disease (nephrolithiasis) affects 12% of men and 5% of women by the seventh decade of life and has a recurrence rate of ,10% per annum [1]. Studies of monogenic (i.e. familial) forms of hypercalciuric nephrolithiasis have identified: an association between the human soluble adenylyl cyclase and an autosomal dominant form of absorptive hypercalciuria [12,13]; gain-of-function mutations of the calcium-sensing receptor in autosomal dominant hypocalcaemia with hypercalciuria [2,14]; mutations of the sodiumphosphate co-transporter solute family 34 member 3 (SLC34A3), in an autosomal recessive form of hypophosphataemic rickets with hypercalciuria [2,15]; mutations of the chloride/proton antiporter, CLC-5, in Dent’s disease, an X-linked recessive form of hypercalciuric nephrolithiasis [16]; and mutations of the bumetanide-sensitive sodium-potassium-chloride cotransporter (NKCC2), the renal outer-medullary potassium channel (ROMK), and the voltage-gated chloride channel, CLC-Kb in autosomal recessive forms of Bartter’s syndrome type I-III, respectively, which are associated with hypercalciuria [15] These latter studies have been successful as large families with the disorder were available. We report the identification of an ENU-induced mouse mutant model for autosomal dominant hypercalciuria, HCALC1, due to mutation of the transient receptor potential cation channel, subfamily V, member 5 (Trpv5) gene
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