Fig 2: Quantification of DAB+ cells along the ES reveals staining gradients along the ES epithelium increasing from the iES to the eES. The y-axis shows the percentage of DAB+ cells relative to all ES epithelial cells. For cell counting, both the iES and eES were each divided into ten equal segments. The ticks on the x-axis correspond to each of the ten consecutive segments of the iES (left x-axis) and eES (right x-axis). The first two segments represent the endolymphatic duct epithelium. The values are the percentages of the total number of cells in the respective ES segment and are given as mean values ± SD from six animals. a Quantification of DAB+ cells for CaSR and the apical Ca2+ channels TRPV5 and TRPV6. b Quantification of DAB+ cells for the cytoplasmatic proteins SERCA1 and SERCA2. c Quantification of DAB+ cells for proteins located in the basolateral membrane

Fig 3: Hypercalciuria in HCALC1 ENU mutant mice and identification of a Trpv5 mutation.(A) Urine calcium/creatinine ratios in 23 G2 offspring of the HCALC1 founder male revealed that 10 of the 23 mice were hypercalciuric, consistent with an autosomal dominant inheritance. Bar, mean calcium/creatinine values. (B) Haplotype analysis of 89 G2 mice (39 hypercalciuric and 50 normocalciuric) was initially undertaken separately in the hypercalciuric and normocalciuric mice, as the penetrance of HCALC1 was unknown. Haplotype analysis of the hypercalciuric mice localised Hcalc1 to a 17.38 Mb interval on chromosome 6, flanked by rs13478688 and rs30110406 (broken double-headed arrow). Haplotype analysis using combined data for the hypercalciuric and normocalciuric mice identified the smaller interval, 11.94-Mb, flanked by rs13478709 and rs30110406 (solid double-headed arrow). The Hcalc1 locus is inherited with the C57BL/6J haplotype from the F1 founder male. Filled box, C57BL/6J allele; and open box, C3H/HeH allele. Number of mice observed for each haplotype is shown beneath each column. (C) DNA sequence analysis of Trpv5 identified a heterozygous T to C transition in codon 682 in hypercalciuric mice predicted to alter a wild-type serine (Ser) to a mutant proline (Pro). This mutation resulted in gain of a BsaJI restriction enzyme site that was used to confirm the presence of the mutation in the 39 hypercalciuric mice (n = 3 shown) and its absence in the 50 normocalciuric mice (n = 3 shown). wt, wild-type; m, mutant. (D) Amino acid sequence alignment revealed evolutionary conservation of the wild-type mouse TRPV5 serine (S) residue at codon 682 (arrowed) in 5 species, as well as in mouse TRPV6 (mTrpv6). Identical residues are shaded black and conservative changes are shaded grey.

Fig 4: Channel characteristics of wild-type and mutant TRPV5.(A) Whole-cell currents in TRPV5-WT (V5-WT) and TRPV5-682P (V5-S682P) injected Xenopus oocytes recorded in response to 300 ms test pulses to various potentials (from -100 to +60 mV in 10 mV increments). Holding potential, 0 mV (N = 5). (B) Mean current-voltage relationships for TRPV5-WT and TRPV5-682P channels (N = 5). These current-voltage relationships are similar to those reported for TRPV5 channels [62]. (C) Mean whole-cell tail currents measured in TRPV5-WT and TRPV5-682P injected Xenopus oocytes during test potentials applied in 10 mV increments from -70 to +40 mV after a pre-pulse to -100 mV in TRPV5-WT and TRPV5-682P channels (N = 5). (D) Time-dependent inhibition of TRPV-WT and TRPV5-682P whole-cell currents. Oocytes were stimulated every 1 s. The peak current amplitude was normalised to that recorded during the first pulse (N = 4). (E) Representative trace of Fura-2 ratio in HEK293 cells transiently transfected with an empty EGFP vector (mock), or EGFP-tagged TRPV5-WT or TRPV5-S682P. Cells expressing EGFP were selected and monitored for changes in intracellular Ca2+ levels when extracellular Ca2+ concentrations were varied from 1.4 mM Ca2+ to 0 mM Ca2+ (2 mM EDTA) and 1.4 mM Ca2+ which was facilitated by superfusion. (F) Fura-2 levels under resting conditions (t0), minimal Fura-2 ratio after EDTA treatment (tmin) and peak level (tmax) upon administration of 1.4 mM Ca2+ after EDTA treatment. Average data of cells transfected with the empty vector (n = 7), TRPV5-wt (n = 24) and TRPV5-S682P (N = 24) from at least three independent experiments. * p<0.05 for TRPV5-WT or TRPV5-S682P versus cells transfected with empty vector. # p<0.05 for TRPV5-S682P versus TRPV5-wt transfected cells.

Fig 5: Assessment of Renal Expression of Calcium Regulatory Genes and Proteins.Renal expression of (A) Trpv5, (B) Trpv6 and (C) Cyp24a1 in wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice (n = 6/group) were assessed by quantitative real-time PCR. All data were normalised to levels of the housekeeping gene Gapdh and wild-type values are expressed as 1. Histogram data are presented as mean ± SEM. #p<0.05, $p<0.01, compared to wild-type mice. (D) Western blot analysis of TRPV5, TRPV6, CYP24a1 and calbindin-D28K (CaBP28K) in kidneys of wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice. The α-1 subunit of the Na/K-ATPase and α-tubulin were used as housekeeping genes to normalize for equal loading. (E) Semi-quantitative densitometry analysis of CYP24a1. (F) Semi-quantitative analysis of CaBP28K. All histogram data are presented as means ± SEM. *p<0.05 and **p<0.02 compared to wild-type mice. (G) Immunohistochemical images of calbindin-D28K (CaBP28K) stained kidney sections from wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice. Scale bar = 200 µm.