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Development of a Microphysiological System to Assess Vitamin D Metabolite Disposition in the Renal Proximal Tubule
Author(s) -
Chapron Brian Dennis,
Jaklic Alenka,
Phillips Brian,
Kelly Edward,
Himmelfarb Jonathan,
Thummel Kenneth
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.719.4
Subject(s) - renal cortex , reabsorption , microbiology and biotechnology , chemistry , epithelial polarity , kidney , homeostasis , epithelium , metabolite , medicine , endocrinology , biology , biochemistry , cell , pathology
The development of a 3‐dimensional microphysiological system (3D MPS), incorporating human proximal tubule epithelial cells (PTECs), represents a major step towards a more faithful in vitro recapitulation of the in vivo renal tubular environment. Such a model would enable investigators to explore the complex mechanisms underpinning drug‐precipitated alterations in renal function. With this goal in mind, PTECs were isolated from human renal cortex and cultured in the Nortis MPS platform under conditions of fluid sheer stress. Polarization of PTECs in the 3D MPS was demonstrated by the enrichment of apical proteins along the media‐perfused luminal interface and localization of Na/K ATPase along the basolateral cell surfaces abutting the extracellular matrix. Electron micrographs further revealed the presence of microvilli and basolateral interdigitations, hallmark structural features of PTECs in vivo . PTECs cultured in the MPS system exhibited glucose reabsorption, glutathione reclamation, ammoniagenesis and organic anion secretion, key physiologic functions of the renal tubular epithelium. The renal proximal tubular epithelium is also the major site for conversion of 25OHD 3 to bioactive 1α,25(OH) 2 D 3 and inactive 24,25(OH) 2 D 3 . The 1α,25(OH) 2 D 3 produced in PTECs exerts VDR‐dependent endocrine effects that are important in regulating systemic mineral homeostasis. To demonstrate vitamin D functionality, the cells were cultured with 25OHD 3 , which resulted in the formation of quantifiable levels of 1α,25(OH) 2 D 3 and 24,25(OH) 2 D 3 . When challenged with exogenous 1α,25(OH) 2 D 3 , there was a linear dose‐dependent accumulation of CYP24A1 mRNA. This was accompanied by dose‐dependent induction of 24‐hydroxylase activity (EC50 ~ 56 nM). In vivo , systemically available 25OHD 3 is tightly bound to vitamin D binding protein (DBP), which is filtered through the glomerulus and reabsorbed by PTECs via megalin‐mediated endocytosis. Thus, a successful model for drug‐mediated perturbations of renal vitamin D homeostasis would require the demonstration of megalin‐mediated uptake of DBP. PTECs cultured in the 3D MPS displayed positive ICC staining for megalin. Cells cultured in DBP‐supplemented media or in fetal bovine serum (FBS)‐supplemented media both exhibited comparable 24‐hydroxylation activity. However, there was a substantial time‐delay (24–36 hrs) of 24‐hydroxylation clearance in cells incubated with DBP‐supplemented media, as compared to cells incubated with FBS‐containing media. This suggests rapid DBP‐dependent sequestration of 25OHD 3 as a result of high affinity ligand binding, followed by megalin‐dependent uptake of 25OHD 3 ‐DBP into PTECs and subsequent hydroxylation. Future studies will evaluate the effects of drugs and uremic toxins on these critical renal cellular functions in order to better understand the disruption of mineral homeostasis by drugs and renal disease. Support or Funding Information This work was supported in part by grants from NIH: R01 GM63666, UH2 TR000504 and TL1 TR000422.