Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Abstract

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-l-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including γ-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of γ-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [${}^{14}\text{C}$]α-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na⁺+K⁺)-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker^® Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.

Introduction

Renal PT cells have large requirements for metabolic energy and for the maintenance of redox status because of the high activities of active transport processes and other energy-requiring, biosynthetic reactions. These cells also possess a unique combination of mechanisms for the handling of GSH, a principal cellular antioxidant and cosubstrate in numerous drug metabolism reactions. These mechanisms include carrier-mediated transport across the brush-border and basolateral plasma membranes and mitochondrial inner membrane, extracellular degradation by the BBM enzyme GGT, intracellular synthesis of GSH from precursor amino acids, and a full complement of GSH-dependent enzymes involved in drug metabolism and detoxification of reactive electrophiles and oxidants [1], [2].

To study the function and molecular biology of proteins of renal PT origin from the rat, a cell line derived from the rat proximal tubule is needed. NRK-52E cells are the natural choice for these studies because they are derived from normal rat kidney proximal tubules. Unfortunately, the biochemical function of these cells has not been characterized extensively, particularly with respect to mitochondrial function or GSH status. Woods and colleagues [3] used NRK-52E cells to study the up-regulation of GSH biosynthesis as an adaptation to chronic oxidative stress. These studies demonstrated the capability of the cell line to respond to conditioning with chronic exposure to an oxidant (by treatment with diethylmaleate) by increasing the production of GCS mRNA and protein. Miller et al. [4] used NRK-52E cells to study oxalate-induced apoptosis and necrosis. Their studies showed that oxalate causes both forms of cell death, thus demonstrating the utility of this cell culture model for studying such processes.

The goals of the present study were to determine activities of several key enzymes involved in cellular energetics and GSH metabolism, to compare these with those in rat renal proximal tubules, and to characterize the transport, metabolism, and subcellular distribution of transported GSH. Finally, the cytotoxicity of a renal PT toxicant that is known to target mitochondria was characterized to test the hypothesis that these cells respond similarly as the in vivo proximal tubule and that they are a suitable model for future studies on GSH transport and mitochondrial function.