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Abstract
The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysiological systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.
Highlights
In humans, conversion of 25-hydroxyvitamin D3 (25OHD3) to its bioactive form, 1α,25(OH)2D3, occurs primarily in the renal proximal tubule
3.2 D binding protein (DBP) and fetal bovine serum (FBS) differentially affect the regulation of cytochrome P450 24A1 (CYP24A1) activity by 1α,25(OH)2D3 DBP is considered essential for megalin-mediated uptake of 25OHD3 (Nykjaer et al, 1999)
We observed several key findings: (1) megalin plays a critical role in the delivery of DBP-bound 25OHD3 to the human proximal tubule, (2) megalin-mediated endocytosis of DBP-bound 1α,25(OH)2D3 is essential to achieve the maximal physiological response, and (3) elevated 1α,25(OH)2D3 levels do not induce, and may even suppress, megalin gene expression in human proximal tubule epithelial cells (PTECs), contrasting with previously published observations of induction in immortalized rat PTECs (Liu et al, 1998)
Summary
Conversion of 25OHD3 to its bioactive form, 1α,25(OH)2D3, occurs primarily in the renal proximal tubule It is a tightly regulated process, controlled by a number of intracrine and endocrine feedback loops (Wang et al, 2015; Dusso et al, 2005; Maiti and Beckman, 2007; Perwad et al, 2007). In order for the proximal tubule epithelial cells (PTECs) to sense and respond to systemic demands for more or less 1α,25(OH)2D3, both 1α,25(OH)2D3 and its metabolic precursor, 25OHD3, must gain intracellular access. Both 1α,25(OH)2D3 and 25OHD3 circulate tightly bound to vitamin D binding protein (DBP) (Bikle et al, 1985, 1986). Passive permeability of the unbound hormone and prohormone alone would yield low unbound intracellular concentrations (Dusso et al, 2005). Nykjaer et al (1999) conducted a series of experiments in megalin-knockout mice and reported that the major route by which 25OHD3 accesses murine PTECs is via megalin-mediated endocytosis of the DBP-bound
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