Regulation of Human Renal Drug Transporters by Inflammatory Cytokines and Pregnancy-related Hormones in Primary Proximal Tubular Epithelial Cells

Abstract

Systemic inflammation and pregnancy both alter drug pharmacokinetics by changing physiology and modulating drug-metabolizing enzymes and transporters in eliminating organs. Regulation of hepatic enzymes in these states is relatively well described. However, the magnitude, direction, and mechanisms of renal transporter regulation remain poorly defined. This is despite clinical data suggesting that tubular secretory clearance can change independently of glomerular filtration rate and protein binding. Inflammation is characterized by elevated circulating pro-inflammatory cytokines, whereas pregnancy is accompanied by a distinct endocrine milieu with increased concentrations of multiple pregnancy-related hormones (PRHs). These soluble mediators are well known to regulate hepatic drug metabolizing enzymes and transporters (DMETs) and therefore represent plausible mechanistic drivers of renal transporter regulation. But mechanistic human data are lacking on whether and how inflammatory cytokines and pregnancy-related hormones (PRHs) regulate the major renal uptake and efflux transporters expressed in proximal tubular epithelial cells (PTECs). Published in vitro studies are further limited by non-selective transporter probes and by PTEC models that rapidly lose the expression and activity of key transporters. The overarching goal of this dissertation was to define how inflammatory cytokines and PRHs regulate human renal drug transporters in vitro, and to provide experimentally anchored parameters that can be incorporated into physiologically based pharmacokinetic (PBPK) models of inflammation, pregnancy, and their co-occurrence. The central hypothesis was that clinically relevant cytokine and hormone exposures cause coordinated, transporter-specific changes in mRNA expression and functional activity in human PTECs. To test this hypothesis, I pursued three specific aims: (1) establish an in vitro human PTEC system to quantify the mRNA expression and activity of individual renal transporters, including identification of selective substrate or substrate-inhibitor pairs for major uptake transporters; (2) quantify the effects of pro- and anti-inflammatory cytokines on renal transporter expression and activity; and (3) quantify the effects of trimester-matched PRH cocktails on renal transporter expression and activity. In Aim 1a, I systematically evaluated the selectivity of six candidate substrates (cidofovir, nicotinic acid, glycochenodeoxycholic acid sulfate [GCDCA-S], levocetirizine, ergothioneine, and atenolol) for the major renal uptake transporters (OAT1–4, OCT2, OCTN1/2) and for renal efflux transporters (P-gp, BCRP, MRPs, MATE1/2-K) using transporter-overexpressing HEK293 and MDCK-II cells and Sf9 membrane vesicles. Cidofovir, levocetirizine, and ergothioneine were confirmed as highly selective substrates of OAT1, OAT4, and OCTN1, respectively. Nicotinic acid was transported primarily by OAT2 but also by OAT1 and OAT3. GCDCA-S was transported by OAT3 and MRP2, and atenolol was transported by OCT2 and MATE1/2-K. Selective inhibitor combinations (e.g., nicotinic acid + quercetin for OAT2, GCDCA-S + cyclosporine A for OAT3, atenolol + mitoxantrone for OCT2) were identified to disentangle overlapping transport and establish selectivity towards OAT2, OAT3, and OCT2. These data provided a practical panel of substrates and substrate-inhibitor pairs that enables uptake transporter-specific activity measurements in primary human PTECs. In Aim 1b, I optimized an isolation and culture workflow for freshly isolated human PTECs seeded on Matrigel-coated Transwell inserts. This system preserved proximal tubule phenotype and maintained measurable OAT1–4, OCT2, and OCTN1 mRNA expression and activity over a defined 5–7-day assay window, overcoming the rapid dedifferentiation and loss of OAT activity observed previously in conventional flat-plate cultures. The combination of the selective substrates from Aim 1a and the optimized Transwell system provided a human-relevant platform for studying cytokine- and hormone-mediated regulation of renal transporters. In Aim 2, I first used plated PTECs to quantify how pathophysiologic concentrations of IL-6, IL-1β, TNF-α, and IFN-γ, tested individually and as a cocktail, altered renal transporter mRNA expression. Exposure to the cytokine cocktail for 48 h significantly downregulated mRNA expression of OCT2, OATP4C1, OAT4, MATE2-K, P-gp, and MRP2 and upregulated OCTN1 and MRP3, with IL-1β emerging as the main perpetrator. Building on these findings, I then used the Transwell human PTEC system to quantify both the mRNA expression of renal DMETs and the activity of uptake transporters (OAT1–4, OCT2, and OCTN1). IL-1β at 0.1 to 1 ng/mL robustly downregulated OAT1–3, OATP4C1, OCT2, OAT4, MATE2-K, MRP2, PEPT2, and endocytic receptors (cubilin, megalin), while inducing OCTN1 and MRP1/3. TNF-α reproduced the effects of IL-1β but only on OAT1–3. Functional assays showed concordant IL-1β-driven decreases in OAT1–3, OAT4, and OCT2 activity and increased OCTN1 activity. Mechanistic experiments with ERK, p38MAPK, JNK, and NF-κB inhibitors demonstrated that IL-1β suppresses OAT1/3 via JNK, OAT2 via p38MAPK, and induces OCTN1 via NF-κB. IL-6 classic and trans-signaling did not reproduce IL-1β-driven changes in transporter mRNA, despite IL-6 being a potent regulator for many hepatic DMET genes. These mechanistic, exposure-verified data can be used to inform PBPK models to predict renal secretory clearance and pathway-mediated drug interactions during inflammation. In Aim 3, I investigated whether PRHs drive the increased renal secretory clearance observed in pregnancy. Primary PTECs from three premenopausal female donors were exposed for 72 h to trimester-matched PRH cocktails (estrogens, progesterone, cortisol, testosterone, oxytocin, GH, and PGH) at physiologic (1×) or supraphysiologic (10×) concentrations, with medium refreshed every 24 h. At 1×, PRHs produced no significant changes in renal transporter, DME, or endocytic receptor mRNA expression or in uptake transporter activity, except for consistent downregulation of PEPT2. At 10×, selective mRNA changes emerged (e.g., induction of OAT1, OAT4, and MRP3; suppression of OCT2, OATP4C1, MATE2-K, MRP2, MRP4, and PEPT2), but these did not translate into measurable changes in OAT1–3, OAT4, OCT2, or OCTN1 activity. These findings argue against physiologic PRH concentrations as the primary driver of increased OAT-mediated secretion during pregnancy and point instead toward alternative mechanisms such as flow-dependent mechanotransduction and regulation by other hormones (e.g., prolactin, hCG). Together, this dissertation research delivers a validated workflow for quantifying individual renal transporter activities in human PTECs, a robust Transwell system that preserves proximal tubule transporter phenotype, and mechanistic, exposure-verified data of how inflammatory cytokines, but not physiologic PRHs, regulate renal drug transporters. These data can be integrated into PBPK models to predict inflammation-mediated changes in renal secretory clearance, and to refine dose selection for vulnerable populations experiencing acute or chronic inflammation, with or without pregnancy.