Renal and Nonrenal 25-Hydroxyvitamin D-1α-Hydroxylases and Their Clinical Significance

Under normal circumstances, 1,25-dihydroxyvitamin D (1,25(OH)2D), the most biologically active metabolite of vitamin D, is synthesized in the proximal renal tubule from 25-hydroxyvitamin D (25(OH)D) by the enzyme 25(OH)D-1α-hydroxylase. Synthesis is enhanced by parathyroid hormone (PTH)1-3 and insulin-like growth factor-I4, 5 and is inhibited by calcium2, 6-8 and phosphorus.2, 9, 10 25(OH)D-1α-hydroxylase, a member of the cytochrome P450 enzyme family, was recently cloned from kidneys of mice lacking the vitamin D receptor (VDR) and kidneys of rats with vitamin D deficiency and found to contain heme- and sterol-binding domains and to have some homology with rat vitamin D-25-hydroxylase and rat 25(OH)D-24-hydroxylase.11, 12 25(OH)D and 1,25(OH)D are converted to 24,25(OH)2D and 1,24,25(OH)3D, respectively, by the enzyme 25(OH)D-24-hydroxylase, and 1,25(OH)2D is ultimately converted by a degradative pathway to more polar metabolites including calcitroic acid.13, 14 1,25(OH)2D3 induces 25(OH)D-24-hydroxylase,15 and studies in 25(OH)D-24-hydroxylase knockout mice indicate that 25(OH)D-24-hydroxylase is the major degrading enzyme for vitamin D metabolites.16 Animals lacking 25(OH)D-24-hydroxylase develop hypercalcemia, hypercalciuria, and nephrocalcinosis and die perinatally as a consequence of increased serum 1,25(OH)2D.16 Expression of 25(OH)D-1α-hydroxylase is suppressed by 1,25(OH)2D3 in kidneys from intact mice but not in kidneys from mice lacking the VDR.11 1,25(OH)2D therefore prevents hypercalcemia and its consequences in the kidney by inhibiting its own synthesis and by stimulating its own degradation as well as that of its precursor 25(OH)D, effects that are mediated by the VDR (Table 1). This is evident by the demonstration that 1,25(OH)2D3 both increases the rate of metabolic clearance of 25(OH)D3 in rats17 and completely prevents increases in serum 25(OH)D produced by vitamin D in human subjects.18 Mutations of 25(OH)D-1α-hydroxylase evidently cause vitamin D–dependent rickets type I, a disease characterized by impaired renal production of 1,25(OH)2D,19 since both the 25(OH)D-1α-hydroxylase gene and disease map to 12q13.1-p13.3 of chromosome 12 of the human genome.12 The disease is readily treated with physiologic doses of 1,25(OH)2D3 and requires pharmacologic doses of vitamin D or 25(OH)D3. 1,25(OH)2D modulates the intestinal absorption of calcium, and circulating 1,25(OH)2D varies inversely with calcium intake; values are increased in response to a low calcium intake and are reduced in response to a high calcium intake.20, 21 Serum Ca2+ itself modulates renal production of 1,25(OH)2D,7, 8 and it is likely that the effect of increased extracellular calcium to inhibit renal synthesis of 1,25(OH)2D is modulated not only by PTH but by the newly described calcium receptor that is present in kidney. The human receptor was cloned, sequenced, and found to consist of a large amino-terminal extracellular domain that binds calcium, a seven membrane–spanning domain and a cytoplasmic carboxy-terminal intracellar domain.22, 23 The calcium receptor is a member of a superfamily of G protein–coupled receptors and stimulates phospholipase C. It also is present in chief cells of the parathyroid glands and modulates secretion of PTH in response to changes in serum Ca2+.23 In the kidney, the calcium receptor also modulates the tubular reabsorption of calcium. Mutations of the gene for the calcium receptor are associated with three human diseases. Two are characterized by increased receptor activity, familial benign hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, and the third by diminished receptor activity, autosomal dominant hypocalcemia.23 In normal subjects, renal production of 1,25(OH)2D is tightly regulated in response to acute changes in vitamin D metabolism. In normal Caucasian and African-American adults and African-American adolescents, for example, increases in serum 25(OH)D produced by administration of vitamin D do not alter serum 1,25(OH)2D or serum calcium.24-26 A curious exception is the fact that administration of vitamin D is associated with significant increases in serum 25(OH)D, serum 1,25(OH)2D, serum calcium, and serum phosphorus (all of which remain within the normal range) in normal Caucasian adolescents.24 The reason for this exception is not known. However, serum 1,25(OH)2D is not tightly regulated in response to chronic changes in vitamin D metabolism. In both Caucasian and African-American men and women in the northern hemisphere, serum 25(OH)D and 1,25(OH)2D values are higher in the summer than in winter.27, 28 In contrast to renal synthesis, extrarenal synthesis of 1,25(OH)2D is not tightly regulated. In anephric patients, for example, serum 1,25(OH)2D varies with serum 25(OH)D,29 and serum 1,25(OH)2D increases in response to 25(OH)D3 to values that are within the low normal range.30 Peripheral macrophages cultured in serum-free medium from normal subjects and uremic patients who are on chronic dialysis synthesize 1,25(OH)2D.31 Production of the metabolite is significantly higher and degradation of the metabolite is significantly lower in peripheral blood macrophages from patients than in those from controls.31 In these studies, production in vitro of 1,25(OH)2D3 was not altered by PTH even at high concentrations of the hormone, and 1,25(OH)2D3 inhibited its own synthesis and stimulated degradation of 1,25(OH)2D3 to more polar metabolites. Thus, activated macrophages may contribute to or be responsible for extrarenal production of 1,25(OH)2D in anephric patients or patients with renal failure who are undergoing chronic dialysis. Hypercalcemia and hypercalciuria occur in sarcoidosis and other granulomatous diseases and result from increased circulating 1,25(OH)2D.32-37 Characteristically, serum PTH is suppressed. Other diseases in which these abnormalities are found include tuberculosis, AIDS with Pneumocystis carinii pneumonia, AIDS with cytomegalovirus infection, disseminated candidiasis, leprosy, rheumatoid arthritis, silicone-induced granulomas, Wegerner's granulomatosis, Hodgkin's disease, lymphoma, histocytic lymphoma, T-cell leukemia, plasma cell granuloma, leiomyoblastoma, seminoma, and subcutaneous fat necrosis.34, 35 The combination of abnormal vitamin D and calcium metabolism also was described in patients with no apparent systemic illness.34 The finding of hypercalcemia caused by increased circulating 1,25(OH)2D in two anephric patients, one with sarcoidosis and the other with tuberculosis, provided the first evidence that the metabolite is produced at nonrenal sites in these diseases.36, 37 Later studies in patients with sarcoidosis indicated that γ-interferon is produced by alveolar macrophages and pulmonary T cells,38 1,25(OH)2D is produced by alveolar macrophages, and synthesis of 1,25(OH)2D is stimulated by γ-interferon.39-41 In sarcoidosis, glucocorticoids lower γ-interferon release by pulmonary mononuclear cells,38 and in sarcoidosis and related diseases, glucocorticoids lower serum 1,25(OH)2D and correct hypercalcemia.32-37 Production of 1,25(OH)2D by alveolar macrophages from patients with sarcoidosis is not regulated by calcium. This is evident clinically by the findings that serum 1,25(OH)2D is abnormally or inappropriately increased relative to the serum calcium in patients with hypercalcemia32-37 and that, in contrast to the response of normal subjects, serum 1,25(OH)2D is not diminished by increases in calcium intake in normocalcemic patients.26 Indeed, the lack of response of serum 1,25(OH)2D to changes in calcium intake is the most sensitive test yet developed for assessment of disease activity in patients with sarcoidosis and is evident even when the disease appears otherwise to be quiescent. In sarcoidosis and related diseases, synthesis of 1,25(OH)2D is not regulated, and serum 1,25(OH)2D varies with concentration of the substrate 25(OH)D. In patients with sarcoidosis, increases in serum 1,25(OH)2D and abnormal calcium metabolism often occur toward the end of summer after increased exposure to sunlight because of increases in serum 25(OH)D,32, 33 an exaggeration of the aforementioned seasonal changes in vitamin D metabolism that occur in normal subjects.27, 28 Further evidence of lack of regulation is the demonstration in normocalcemic patients with sarcoidosis that vitamin D administration increases serum 25(OH)D, serum 1,25(OH)2D, and serum calcium.38 No such changes occur in normal subjects when they are given vitamin D. 1,25(OH)2D3 stimulates 25(OH)D-24-hydroxylase activity, inhibits synthesis of 1,25(OH)2D3, and stimulates conversion of 1,25(OH)2D3 to more polar metabolites by cultured normal human peripheral blood monocytes.42 Higher doses of 1,25(OH)2D3 are required to stimulate 25(OH)D-24-hydroxylase activity and inhibit synthesis of 1,25(OH)2D by cultured normal pulmonary alveolar macrophages than by peripheral monocytes, and very modest and nonsignificant increases in polar metabolites are produced.42 Thus, pulmonary alveolar macrophages are less sensitive than peripheral blood monocytes to the effects of 1,25(OH)2D3 to modulate its own production and degradation. Further, 1,25(OH)2D3-induced increases in 25(OH)D-24-hydroxylase mRNA in normal human peripheral blood monocytes are inhibited by γ-interferon.34 Thus, γ-interferon acts not only to stimulate production of 1,25(OH)2D3 by macrophages but produces resistance to 1,25(OH)2D3 to stimulate its own degradation. In summary, regulation of synthesis and degradation of 1,25(OH)2D are different at renal and nonrenal sites. In the kidney, synthesis of the metabolite by 25(OH)D-1α-hydroxylase is tightly regulated by PTH and calcium, whereas in macrophages, synthesis is regulated by γ-interferon. Inhibition by 1,25(OH)2D of its own synthesis is more pronounced in kidney than in macrophages. Induction of 25(OH)D-24-hydroxylase and the degradative pathway by 1,25(OH)2D is more pronounced in kidney than in macrophages. Finally, lack of regulation by extracellular calcium of extrarenal synthesis of 1,25(OH)2D is the proximate cause of hypercalcemia and abnormal vitamin D metabolism in sarcoidosis and related diseases and can lead to nephrocalcinosis, nephrolithiasis, renal failure, and even death.34, 36, 37 In the northern hemisphere, patients are at greatest risk for hypercalcemia during the summer months after increased exposure to sunshine. Therefore, it is imperative that patients be carefully followed since hypercalcemia and its consequences can be successfully treated and prevented. This is accomplished by forcing fluids, limiting exposure to sunlight, reducing intake of vitamin D and calcium, and, if necessary, by administration of drugs such as prednisone or hydroxychloroquine.32-37