The klotho gene encodes a novel type I membrane protein of β-glycosidase family and is expressed principally in distal tubule cells of the kidney and choroid plexus in the brain. These mutants displayed abnormal calcium and phosphorus homeostasis together with increased serum 1,25-(OH)₂D. In kl^−/− mice at the age of 3 wk, elevated levels of serum calcium (10.9 ± 0.31 mg/dl vs. 10.0 ± 0.048 mg/dl in wild-type mice), phosphorus (14.7 ± 1.1 mg/dl vs. 9.7 ± 1.5 mg/dl in wild type) and most notably, 1,25-(OH)₂D (403 ± 99.7 mg/dl vs. 88.0 ± 34.0 mg/dl in wild type) were observed.

Reduction of serum 1,25-(OH)₂D concentrations by dietary restriction resulted in alleviation of most of the phenotypes, suggesting that they are downstream events resulting from elevated 1,25-(OH)₂D. We searched for the signals that lead to up-regulation of vitamin D activating enzymes. We examined the response of 1α-hydroxylase gene expression to calcium regulating hormones, such as PTH, calcitonin, and 1,25-(OH)₂D₃. These pathways were intact in klotho null mutant mice, suggesting the existence of alternate regulatory circuits. We also found that the administration of 1,25-(OH)₂D₃ induced the expression of klotho in the kidney. These observations suggest that klotho may participate in a negative regulatory circuit of the vitamin D endocrine system, through the regulation of 1α-hydroxylase gene expression.