Biochemical and Functional Characteristics of Cultured Renal Epithelial Cells from Uninephrectomized Rats: Factors Influencing Nephrotoxicity LAWRENCE H. LASH, DAVID A. PUTT, and RUDOLFS K. ZALUPS Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan (L.H.L., D.A.P.); and Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia (R.K.Z.) Received August 14, 2000; accepted October 4, 2000 This paper is available online at http://jpet.aspetjournals.org ABSTRACT Primary cultures of renal proximal (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Daw- ley rats were established to characterize factors that are re- sponsible for the altered susceptibility to nephrotoxicants that occurs after compensatory renal cellular hypertrophy. Cells were grown in serum-free, hormonally defined medium and parameters were measured on days 1, 3, and 5 of primary culture. PT and DT cells from control and NPX rats appeared to maintain epithelial characteristics in culture, as shown by cyto- keratin staining, morphology, protein and DNA content, and enzyme activities. Activities of several glutathione-dependent enzymes, including -glutamyltransferase, glutathione S-trans- ferase, glutathione peroxidase, and -glutamylcysteine syn- thetase, were significantly greater in PT cells from NPX rats than in PT cells from control rats when factored by protein content. Rates of -methylglucose uptake across the basolat- eral and brush-border membranes and sodium-dependent up- take of glutathione across the basolateral membrane were 2- to 3-fold higher in PT cells from NPX rats than in PT cells from control rats. These results are consistent with the hypertro- phied phenotype being maintained in primary cultures of PT cells from NPX rats. The marked alterations in transport may play central roles in the delivery of nephrotoxicants to the target cell, and thus, increases the probability of chemically induced injury or death. These findings also suggest that these cell cultures may be useful for the study of biochemical processes associated with compensatory renal cellular hypertrophy. Reduced functional renal mass is a relatively common con- dition in humans that results from a host of factors, such as renal disease, surgery, or aging. Within a short period of time after a significant number of functioning nephrons has been reduced, the remnant renal tissue undergoes profound mor- phological and functional changes by mechanisms that still remain unclear (Meyer et al., 1996). In rodents, the acute hemodynamic, functional, and biochemical effects of compen- satory renal growth are nearly complete within 7 to 10 days after surgery (Zalups et al., 1987; Meyer et al., 1996). The cellular changes associated with uninephrectomy are most prominent in the proximal tubular (PT) region of the nephron (Meyer et al., 1996), and include cellular hypertrophy and increased cellular content of protein (Meyer et al., 1996), increased transport of sodium ions (Meyer et al., 1996), in- creased rates of mitochondrial electron transport (Harris et al., 1988), increased cellular synthesis and content of gluta- thione (GSH) and metallothionein (Zalups and Veltman, 1988; Zalups and Lash, 1990, 1994), and increased activities of several GSH-dependent enzymes (Lash and Zalups, 1994). There are toxicological implications of this hypertrophied state because rats that have undergone uninephrectomy and compensatory renal growth (NPX rats) exhibit altered sus- ceptibility to various nephrotoxicants, including inorganic mercury (Zalups and Diamond, 1987; Zalups and Lash, 1994; Zalups, 2000), analgesics (Mollard, 1976; Henry et al., 1983), and cadmium-metallothionein (Zalups et al., 1992). To study some of the biochemical properties of renal epi- thelial cells from NPX rats, we previously prepared suspen- sions of freshly isolated renal PT cells from NPX rats by collagenase perfusion and density-gradient centrifugation in Percoll, and showed that these cells retained their increased cell size, increased protein content, and increased activities of several enzymes (Lash and Zalups, 1992, 1994). We also prepared cells from the distal tubular (DT) region of the nephron to examine a nephron segment that is not the pri- This research was supported by National Institute on Environmental Health Sciences Grant R01-ES05157 (to L.H.L. and R.K.Z.) and R01-ES05980 (to R.K.Z.) and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK40725 (to L.H.L.). ABBREVIATIONS: PT, proximal tubular; GSH, glutathione; NPX, uninephrectomized; DT, distal tubular; AMG, -methylglucose; GDH, glutamate dehydrogenase; LDH, lactate dehydrogenase; GGT, -glutamyltransferase; GCS, -glutamylcysteine synthetase; GPX, glutathione peroxidase; GST, glutathione S-transferase; BBM, brush-border membrane; BLM, basolateral membrane. 0022-3565/01/2962-243–251$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 296, No. 2 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3220/877572 JPET 296:243–251, 2001 Printed in U.S.A. 243 at ASPET Journals on February 3, 2016 jpet.aspetjournals.org Downloaded from