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Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for&#160;chronic kidney disease include ...

University of Washington

Small molecules targeting aberrant anaplastic lymphoma kinase (ALK) are active against ALK-positive non-small-cell lung cancers and neuroblastoma. Several targeted tyrosine kinase inhibitors (TKIs) have been shown to interact with polyspecific organic cation transporters (pOCTs), raising concerns about potential drug-drug interactions (DDIs). The purpose of this study was to assess the interaction of ALK inhibitors with pOCTs and the impact of substrate-dependent inhibition on the prediction of DDIs. Inhibition assays were conducted in transporter-overexpressing cells using meta-iodobenzylguanidine (mIBG), metformin, or 1-methyl-4-phenylpyridinium (MPP+) as the substrate. The half-maximal inhibitory concentrations (IC) of brigatinib and crizotinib for the substrates tested were used to predict their potential for in vivo transporter mediated DDIs. Here, we show that the inhibition potencies of brigatinib and crizotinib on pOCTs are isoform- and substrate-dependent. Human OCT3 (hOCT3) and multidrug and toxin extrusion protein 1 (hMATE1) were highly sensitive to inhibition by brigatinib and crizotinib for all three tested substrates. Apart from hMATE1, substrate-dependent inhibition was observed for all other transporters with varying degrees of dependency; hOCT1 inhibition showed the greatest substrate dependency, with differences in IC values of up to 22-fold across the tested substrates, followed by hOCT2 and hMATE2-K, with differences in IC values of up to 16- and 12-fold, respectively. Conversely, hOCT3 inhibition only showed a moderate substrate dependency (IC variance < 4.8). Among the substrates used, metformin was consistently shown to be the most sensitive substrate, followed by mIBG and MPP+. Pre-incubation of ALK inhibitors had little impact on their potencies toward hOCT2 and hMATE1. Our results underscore the complexity of the interactions between substrates and the inhibitors of pOCTs and have important implications for the clinical use of ALK inhibitors and their DDI predictions.

Interaction of ALK Inhibitors with Polyspecific Organic Cation Transporters and the Impact of Substrate-Dependent Inhibition on the Prediction of Drug-Drug Interactions.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs,  = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs;  = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1, tumor necrosis factor- (TNF-), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1, TNF-, and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation.

Dysregulation of Human Hepatic Drug Transporters by Proinflammatory Cytokines.

Pregnancy results in significant changes in drug pharmacokinetics (PK). While previous studies have elucidated the impact of pregnancy-related hormones (PRH) on mRNA or protein expression and activity of major hepatic metabolizing enzymes, their effect on hepatic drug transporters remains largely unexplored. Therefore, we investigated the effect of a cocktail of PRH on the mRNA expression and activity of hepatic transporters.

The effect of pregnancy-related hormones on hepatic transporters: studies with premenopausal human hepatocytes.

Cannabidiol (CBD) or delta-9-tetrahydrocannabinol (THC) can inhibit multiple CYPs and UGTs in vivo and/or in vitro. CBD, but not THC, is also a time-dependent inhibitor of CYP3A, CYP1A2, and CYP2C19. We showed that a single 640 mg oral dose of CBD inhibits oral midazolam plasma clearance by 56%, whereas others found no interaction of chronic CBD with midazolam. These data can be explained if chronic CBD induces CYP3A enzymes. To investigate if CBD or THC induces CYP enzymes or transporters, we treated 4 lots of human hepatocytes for 72 hours with in vivo relevant concentrations of CBD (42 nM, 420 nM) or THC (250 nM, 700 nM). Then, mRNA expression and CYP activity were measured using quantitative polymerase chain reaction and liquid chromatography-tandem mass spectrometry, respectively. CYP3A4 mRNA was significantly induced to 7.3-, 11.1-, and 3.3-fold by CBD (420 nM) and 14.8-, 5.9-, and 3.1-fold by THC (700 nM) in 3 of the 4 lots. CYP3A activity was significantly induced 3.39- and 3.28-fold by low (42 nM) and 2.4- and 2.3-fold by high (420 nM) CBD concentrations, respectively, in 2 lots, and 2.3-fold by THC (700 nM) in 1 lot. Rifampin (10 μM) significantly induced CYP3A mRNA and activity across all lots. CBD (420 nM) significantly induced CYP1A2 and CYP2B6 mRNA (but not activity) in 2 lots. No significant induction of other CYPs, UGTs, or transporters was observed. Incorporation of CBD E and EC of CYP3A4 mRNA induction (without scaling by rifampin mRNA induction) into a CBD physiologically-based pharmacokinetic model successfully captured the lack of the observed chronic CBD-midazolam drug interaction. SIGNIFICANCE STATEMENT: Time-dependent inhibition and induction of CYP3A enzymes by cannabidiol (CBD) is a plausible explanation for the significant CBD-midazolam pharmacokinetic interaction after single-dose CBD administration and the absence of such an interaction after multiple-dose CBD administration.

Cannabidiol and Δ9-tetrahydrocannabinol induce drug-metabolizing enzymes, but not transporters, in human hepatocytes: Implications for predicting complex cannabinoid-drug interactions.

Renal clearance of drugs mediated by transporters can be affected by diseases (eg, inflammation due to infections), physiological changes (eg, pregnancy), or drug-drug interactions. To elucidate the transporters involved, the magnitude of effect, and the underlying mechanisms, human proximal tubular epithelial cells could be exposed to potential perpetrators (eg, cytokines, pregnancy-related hormones or the interacting drug), and the activity of transporters quantified. A crucial prerequisite for such studies is the identification of selective substrates or substrate-inhibitor pairs for each renal transporter. Using transporter-transfected mammalian cells and membrane vesicles, we systematically evaluated the selectivity of 6 substrates (or substrate-inhibitor pairs) for the major uptake and efflux renal transporters. Cidofovir, levocetirizine, and ergothioneine were found to be selective substrates of the organic anion transporter (OAT) 1, 4, and organic cation/carnitine transporter 1, respectively. Nicotinic acid was transported by OAT2, but also by OAT1 and 3, though to a lesser extent. Probenecid did not selectively inhibit OAT1/3-mediated uptake of nicotinic acid, but quercetin did, allowing selective measurement of OAT2 activity. Interestingly, nicotinic acid was also transported by the endogenous monocarboxylate transporter 1 in HEK293 cells. Glycochenodeoxycholic acid sulfate was transported by OAT3 and multidrug resistance-associated protein 2 (MRP2), with MRP2 selectively inhibited by cyclosporine A, allowing selective measurement of OAT3 activity. Atenolol was transported by organic cation transporter 2 and multidrug and toxin extrusion proteins 1 and 2-K, with multidrug and toxin extrusion proteins activity selectively inhibited by mitoxantrone, allowing selective measurement of organic cation transporter 2 activity. SIGNIFICANCE STATEMENT: These findings provide a framework for measuring the in vitro activity of individual uptake transporters in primary human proximal tubular epithelial cells. By applying our proposed methodology, researchers can quantify how various factors (eg, cytokines, pregnancy-related hormone, drug interactions) modulate individual renal uptake transporter activity in proximal tubular epithelial cells.

Yik Pui Tsang

Department of Pharmaceutics,
School of Pharmacy,
Department of Pharmaceutics, School of Pharmacy

University of Washington

Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Yik Pui Tsang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmaceutics,School of Pharmacy,Department of Pharmaceutics, School of Pharmacy

University of Washington

Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Department of Pharmaceutics,
School of Pharmacy,
Department of Pharmaceutics, School of Pharmacy