Renal Tubular Transport of Drugs in Healthy and Diseased Kidney

Abstract

In this thesis work, I applied traditional and innovative bioengineering approaches to develop an in vitro model of renal proximal tubule with human proximal tubule epithelial cells (PTECs) that is particularly suited for drug transport studies. The use of human PTECs has thus far been hindered by the lack of complete understanding of their cell morphology and functioning in vitro. At the initial stage of our development, freshly isolated human PTECs were successfully expanded through conventional culturing, which afforded an adequate cell source. When passaged PTECs were grown on permeable supports, they maintained their polarity, barrier integrity, transporter expression (mRNA and protein immunocytochemistry) and activity. We then developed a novel 3D culture of human PTECs in a Nortis microphysiological system (MPS). Under continuous flow, PTECs in the MPS sustained long term viability up to 28 days; they also maintained the expression of prototypical proximal tubule markers (e.g. E-Cadherin, aquaporin 1, SGLT2, γ-glutamyl transpeptidase), cellular structures (e.g. cilia), and critical synthetic and metabolic functions. The next key step of development involved co-culturing of human PTECs with human umbilical vein endothelial cells (HUVECs) in a dual-channel Nortis MPS to establish a vascularized proximal tubule MPS. This system is uniquely designed to investigate the multiple steps involved in the renal tubular secretion of compounds; it allows for independent vascular (basolateral) and tubular (apical) perfusion and provides multiple readouts, including fluorescence microscopy and effluent analysis. Regular fluorescent and 2-photon imaging technique were applied to track a fluorescently labelled dextran from its entry into and leakage from the vessel, diffusion through the interstitium, and ultimately its passage across the PTEC tubule; in fact, PTEC was shown to be the limiting barrier in dextran’s translocation across the tubulo-interstitium. Through a series of radiolabeled solute experiments, permeability across the PTEC tubule was demonstrated to be several-fold higher for p-aminohippuric acid (PAH) than mannitol and dextran, both of which are paracellular permeability markers. Importantly, these findings yielded a favorable prediction of PAH renal clearance upon in vitro−to−in vivo scaling. Thus, our vascularized proximal tubule MPS appears to be a promising in vitro model for investigations of renal tubular drug secretion. The last part of my thesis work focused on alterations in renal secretory clearance of drugs in renal impairment. Dosing adjustments in chronic kidney diseases (CKD) have traditionally relied on serum creatinine as a biomarker of glomerular filtration rate (GFR). However, a number of drugs associated with adverse drug events in CKD undergo renal secretion, a process localized to the proximal tubule. In an evaluation of the available literature to-date, I showed that the renal clearance for some drugs decline less rapidly than (Group A), more rapidly than (Group B), or proportionately to (Group C) creatinine clearance in advancing kidney disease. It is important to note that, in healthy subjects, all these drugs are extensively secreted via the proximal tubule and minimally reabsorbed along the entire tubule. These findings point to the complexity in the handling of drugs by the proximal tubule in CKD, and challenge the traditional assumption that secretory clearance decline in parallel with glomerular filtration rate, whereby creatinine clearance can serve as a guide for drug dosing adjustments.