Development and Application of Human Renal Proximal Tubule Epithelial Cells for Assessment of Compound Toxicity

Abstract

Kidney toxicity is a major problem both in drug development and clinical settings. It is difficult to predict nephrotoxicity in part because of the lack of appropriate in vitro cell models, limited endpoints, and the observation that the activity of membrane transporters which plays important roles in nephrotoxicity by affecting the pharmacokinetic profile of drugs is often not taken into account. We developed a new cell model using pseudo-immortalized human primary renal proximal tubule epithelial cells. This cell line (SA7K) was characterized by the presence of proximal tubule cell markers as well as several functional properties, including transporter activity and response to a few well-characterized nephrotoxicants. We subsequently evaluated a group of potential nephrotoxic compounds in SA7K cells and compared them to a commonly used human immortalized kidney cell line (HK-2). Cells were treated with test compounds and three endpoints were analyzed, including cell viability, apoptosis and mitochondrial membrane potential. The results showed that most of the known nephrotoxic compounds could be detected in one or more of these endpoints. There were sensitivity differences in response to several of the chemicals between HK-2 and SA7K cells, which may relate to differences in expressions of key transporters or other components of nephrotoxicity pathways. Our data suggest that SA7K cells appear as promising for the early detection of renal toxicants.

Keywords: Apoptosis; Mitochondrial membrane potential (MMP); Nephrotoxicity; SA7K cells; Transporters.

Fig. (1)

Growth, morphological and functional characterization of SA7K cells. (A) Normal epithelial morphology of SA7K cells, including dome formation; (B) Expression of the proximal tubule marker aminopeptidase N (CD13) in SA7K cells; (C) Extended population doublings in ZFN-modified SA7K versus HPTC cells; (D) Uptake of Albumin-FITC; (E) Response to Parathyroid Hormone (PTH); (F) GGT Activity in HPTC versus SA7K cells. Scale bar: 50 µm.

Fig. (2)

Expression profile of transporters. (A) HPTC versus SA7K cells; (B) SA7K versus HK-2 cells. The mRNA levels of nine known proximal tubule transporters were measured by RT-PCR in HPTC, SA7K and HK-2 cells.

Fig. (3)

Functional Characterization of Uptake and Efflux Transporter Activity in SA7K cells. (A) Uptake of PAH by OAT-1 and inhibition by probenecid; (B) Uptake of amantadine by OCT2 and inhibition by doxepin; (C) Inhibition of MDR1-mediated efflux of digoxin by verapamil; (D) Inhibition of MRP2-mediated efflux of SN-38 by MK571. Substrate concentrations used were 10 µM in each experiment.

Fig. (4)

Concentration response curves of selected compounds in HK-2 and SA7K cells. (A) Effect of ochratoxin A on cell viability; (B) Effect of campothecin on caspase 3/7; (C) Effect of doxorubicin on MMP.