1,25(OH)2 vitamin D (1,25D) is tightly regulated in kidney proximal tubules (PT) by FGF23, PTH, and 1,25D itself, and is markedly disturbed in diseases involving these hormones. The 1,25D catabolic enzyme CYP24A1 is elevated in response to FGF23 and 1,25D, whereas PTH functions to suppress its expression, as well as  reciprocally control the anabolic enzyme CYP27B1, providing precise 1,25D regulation. FGF23’s actions require high affinity binding to its co-receptor Klotho, which is localized to both PT and distal tubule epithelial cells, thus posing a significant challenge in identifying the PT-specific mechanisms directing

Emmanuel Solis¹, Kayleigh N. Jennings¹, Yamil Marambio¹, Sheng Liu¹, Jun Wan¹, Mark B. Meyer², Kenneth E. White^1
1Indiana University School of Medicine, Indianapolis, IN, United States; and ²University of Wisconsin-Madison, Madison, WI, United States.

FGF23-mediated 1,25D regulation. To this end, 10X Multiome on kidneys from mouse models of gain/loss of FGF23 function was used to isolate novel and differentially expressed genes at the single single-cell level. WT mice injected with FGF23 had increased expression of several transcription factors (TF), with one TF specifically co-localizing exclusively in PTS1-2 cells with Cyp24a1 and Cyp27b1; whereas KL-KO mice highly suppressed its mRNA and promoter accessibility (>90%; p<0.05). In collaboration with Dr. Mark Meyer’s group at the University of Wisconsin-Madison we characterized this TF, and showed that FGF23 induced TF nuclear protein accumulation in megalin-positive PT cells at 1-4 h as determined by IF and nuclei isolates. Notably, expression of this TF was repressed in the presence of PTH, delineating a dynamic regulatory role between these hormones. Validation was determined in vitro, with TF regulon activity confirmed downstream of FGF23 in HEK-mKL cells. Further, native TF protein was dynamically induced by FGF23 in HEK-mKL cells, whereas 1,25D alone had no effect. In vitro, a human cDNA increased CYP24A1 (4-6 fold; p<0.01) in HEK-mKL and RPTEC cells, and shRNA blocked FGF23 increases in CYP24A1. In summary, we isolated a PT-specific TF activated by FGF23 but not 1,25D that increases Cyp24a1, and loss of function resulted in aberrant control of key 1,25D metabolizing enzymes. Thus, we identified a novel component directing balanced control of systemic 1,25D, opening new connections for strategies critically needed for vitamin D- and FGF23-related disorders.