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BACKGROUND Loss-of-function mutations of vitamin D-24 hydroxylase have recently been recognized as a cause of hypercalcaemia and nephrocalcinosis/nephrolithiasis in infants and adults. True prevalence and natural history of this condition are still to be defined. METHODS We describe two adult patients with homozygous mutations and six related heterozygous carriers. Mineral and hormonal data in these patients were compared with that in 27 patients with stage 2-3 chronic kidney disease and 39 healthy adult kidney donors. RESULTS Probands had recurrent nephrolithiasis, chronic hypercalcaemia with depressed parathyroid hormone (PTH) and increased 1,25(OH)(2)D levels; carriers had nephrolithiasis (two of six), hypercalciuria (two of six) and high or normal-high 1,25(OH)(2)D (four of four). Corticosteroids did not reduce plasma and urine calcium levels, but ketoconazole did, indicating that 1,25(OH)(2)D production is not maximally depressed despite coexisting hypercalcaemia, high 1,25(OH)(2)D and depressed PTH, and that 1,25(OH)(2)D degradation through vitamin D-24 hydroxylase is a regulator of plasma 1,25(OH)(2)D levels. Both probands had vascular calcifications and high bone mineral content. One developed stage 3b renal failure: in this patient 1,25(OH)(2)D decreased within normal limits as glomerular filtration rate (GFR) fell and PTH rose to high-normal values, yet hypercalcaemia persisted and the ratio of 1,25(OH)(2)D to GFR remained higher than normal for any degree of GFR. CONCLUSIONS This natural model indicates that vitamin D-24 hydroxylase is a key physiologic regulator of calcitriol and plasma calcium levels, and that balanced reduction of 1,25(OH)(2)D and GFR is instrumental for the maintenance of physiologic calcium levels and balance in chronic kidney diseases.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

Although the diagnosis of Gitelman syndrome (GS) and Bartter syndrome (BS) is now feasible by genetic analysis, implementation of genetic testing for these disorders is still hampered by several difficulties, including large gene dimensions, lack of hot-spot mutations, heavy workup time, and costs. This study evaluated in a cohort of patients with genetically proven GS or BS diagnostic sensibility and specificity of a diuretic test with oral hydrochlorothiazide (HCT test). Forty-one patients with GS (22 adults, aged 25 to 57; 19 children-adolescents, aged 7 to 17) and seven patients with BS (five type I, two type III) were studied; three patients with "pseudo-BS" from surreptitious diuretic intake (two patients) or vomiting (one patient) were also included. HCT test consisted of the administration of 50 mg of HCT orally (1 mg/kg in children-adolescents) and measurement of the maximal diuretic-induced increase over basal in the subsequent 3 h of chloride fractional clearance. All but three patients with GS but no patients with BS and pseudo-BS showed blunted (

We report a novel missense mutation N124K in the extracellular calcium receptor (CaR) identified in two related subjects with the phenotypic features of autosomal dominant hypocalcemia (ADH). Expression of the N124K mutant receptor created by site-directed mutagenesis and transfected into HEK-293 cells was comparable with that of the wild-type (WT) receptor and two other mutant receptors N118K and L125P identified in subjects with ADH. Functional characterization by the extracellular Ca2+ ion ([Ca2+]0)-stimulated phosphoinositide (PI) hydrolysis in transfected HEK-293 cells showed that the N124K mutant receptor was left-shifted in Ca2+ sensitivity. This biochemical gain-of-function is comparable with that seen in other missense mutations of the CaR identified in subjects with ADH. We tested a series of missense substitutions (R, Q, E, and G) in addition to K for N124 and found that only the N124K mutation and to a much lesser extent N124R caused a left shift in Ca2+ sensitivity. Thus, a specific substitution, not merely a mutation of the N124 residue, is required for receptor activation. The N124K mutation is one of eight naturally occurring mutations in subjects with ADH identified in a short segment A116-C129 of the CaR extracellular domain (ECD). We present a hypothesis to explain receptor activation by mutations in this region based on the recently described three-dimensional structure of the related metabotropic glutamate type 1 receptor (mGluR1).

A dopamine-inhibited adenylate cyclase has been demonstrated in anterior pituitary gland of adult female rats, lactating and not lactating. This inhibitory effect was completely GTP dependent. In contrast, in the adenohypophysis of male rats, dopamine had no detectable effect on adenylate cyclase activity. In female rats the inhibition of the enzyme appears mediated by specific dopaminergic receptors: the effect of dopamine was mimicked by the dopaminergic agonists apomorphine and the ergot derivative CH 29–717, while norepinephrine was much less potent. On the other hand, the dopaminergic antagonists trifluoperazine and sulpiride competitively antagonized the dopamine inhibition of the adenylate cyclase. The possibility that the dopamine-inhibited enzyme is located in mammotrophs appears supported 1) by its observation in the female rat pituitary, which contains this type of cells in much larger proportion than the male gland (33–38% vs.