Importance of Extra-Renal CYP24A1 Expression for Maintaining Mineral Homeostasis

Calcium homeostasis involves a complex interplay between kidneys, parathyroid glands, intestine and bone. Specifically, 1,25(OH)(2)D(3) is a key calciotropic hormone which stimulates intestinal calcium absorption. A growing body of evidence suggests that circulating levels of 1,25(OH)(2)D(3) depend not only on its synthesis under the action of PTH in the kidneys, but also its catabolism by 24-hydroxylase, herein referred to as CYP24A1. The clinical importance of CYP24A1 has been demonstrated by human loss-of-function mutations, which lead to severe hypercalcemia due to exaggerated levels of 1,25(OH)(2)D(3). Despite its growing importance, little is known about its tissue-specific contributions to normal vitamin D metabolism. To explore the physiology of CYP24A1 and delineate renal-specific effects of CYP24A1 in calcium metabolism, we generated a mouse with constitutive kidney-specific deletion of Cyp24a1 (Six2(Cre)-Cyp24(flox)). Six2 marks the nephron progenitor population throughout nephrogenesis. We hypothesized that hypercalcemia as seen in CYP24A1 inactivating mutations is related to lack of both renal and extrarenal expression, and that renal deletion does not lead to severe hypercalcemia. To confirm Cyp24a1 deletion, we measured mRNA expression in the kidney using qPCR and RNA in situ hybridization. All mice were fed a standard commercial rodent diet and followed longitudinally for five months with interval calcium measurements. At time of termination, serum PTH levels were measured along with vitamin D-dependent calcium transporters as a functional measure of 1,25(OH)(2)D(3) action. Cyp24a1 expression was significantly knocked down in total kidneys from Six2(Cre)-Cyp24(flox) mice as compared to intestinal expression suggesting successful gene deletion. Compared to age-matched wildtype controls, Six2(Cre)-Cyp24(flox) mice were mildly but persistently hypercalcemic (diff between means= 0.46 mg/dL, p-value: 0.03, n=8 per group). As expected, 1,25D-dependent calcium transporters in the kidney (Calb1, Trpv5, Slc8a1, Atp2b1) and intestine (Trpv6, s100g) were all increased, consistent with increased systemic 1,25(OH)(2)D(3) activity. PTH levels were appropriately suppressed in the Six2(Cre)-Cyp24(flox) mice (diff between means=83 pg/mL, p-value 0.2, n=9 control, n=3 exp) as were renal cyp27b1 mRNA expression. These data suggest that renal CYP24A1 is important for systemic 1,25(OH)(2)D(3) regulation, but the lack of severe hypercalcemia supports critical contributions of extra-renal CYP24A1.