Regulation of Renal Transporters by Pro-inflammatory Cytokines in Human Proximal Tubular Epithelial Cells: Identification of the Perpetrator and Mechanisms

Introduction

Infection and inflammation elevate circulating pro-inflammatory cytokines that can affect renal drug clearance. Accordingly, we sought to (i) quantify the extent of modulation of renal drug-metabolizing enzymes and transporters (DMETs) by cytokines and (ii) identify the mechanism(s) underlying these effects.

Methods

Fresh primary human proximal tubular epithelial cells (PTECs) were cultured on extracellular matrix-coated Transwells. PTECs were exposed every 24 h, for 48 h, to IL-6, IL-1β, TNF-α, IFN-γ, IL-4, or IL-10 (0.1 or 1 ng/mL), individually or as a cocktail. mRNA expression of 25 renal DMETs was quantified by RT-qPCR. Individual activity of OAT1–4, OCT2, and OCTN1 was measured. To determine mechanisms of these effects, selective MAPK/NF-κB inhibitors (ERK [PD98059], p38 ^MAPK [SB203580], JNK [SP600125], and NF-κB [PDTC]), individually or as a cocktail, were used. IL-6, soluble IL-6 receptor (sIL-6Rα), and IL-6 + sIL-6Rα were used to probe endogenous/exogenous IL-6 classic versus trans-signaling.

Results

IL-1β was the predominant modulator, downregulating mRNA expression of OAT1–3, OCT2, OAT4, MATE2-K, MRP2, and OATP4C1, and upregulating mRNA expression of OCTN1 and MRP3. TNF-α downregulated OAT1–3 mRNA expression to an extent similar to IL-1β, but did not affect other transporters. Activity changes for the major uptake transporters mirrored mRNA directionality. MAPK/NF-κB blockade by the inhibitor cocktail reduced IL-6 secretion while completely reversing the IL-1β-driven downregulation of OAT1–3 mRNA. JNK inhibition alone restored OAT1/3 mRNA. Inhibition of p38 ^MAPK blunted OAT2 mRNA downregulation. OCTN1 mRNA induction required NF-κB. Downregulation of OAT4/OCT2 mRNA was largely MAPK/NF-κB-independent. IL-6 alone, sIL-6Rα alone, or IL-6 + sIL-6Rα did not reproduce IL-1β-driven changes in transporter mRNA.

Conclusions

IL-1β is the principal driver of cytokine-mediated regulation of human renal transporters in PTECs via JNK/p38 ^MAPK /NF-κB nodes. These mechanistic, exposure-verified data provide inputs for physiologically based pharmacokinetic predictions of renal secretory clearance and pathway-mediated drug interactions during inflammation.

Visual Abstract

Translational Statement

Systemic inflammation increases cytokine concentrations and alters drug pharmacokinetics. Yet, cytokine regulation of renal drug transporters remains poorly defined, even though the kidney clears many anti-infective drugs via active secretion. Using an optimized primary human proximal tubular epithelial cell model that preserves expression and function of major renal transporters, we found that IL-1β is the predominant cytokine that downregulates the mRNA and activity of OAT1–3, OCT2, and OAT4, while upregulating the mRNA and activity of OCTN1. We further showed that IL-1β-driven downregulation of OAT1/3 occurs through JNK signaling, OAT2 through p38 ^MAPK , and OCTN1 through NF-κB. These data provide quantitative inputs for physiologically based pharmacokinetic models to predict how inflammation alters renal transporter-mediated drug clearance, informing dose adjustment and risk assessment for disease-drug and drug-drug interactions in patients with inflammatory kidney disease or systemic infections. They also highlight signaling nodes where anti-inflammatory therapies might inadvertently modify renal drug transport.