Identification of putative gene-based markers of renal toxicity - Genomics and Risk Assessment Mini-Monograph

Environmental Health Perspectives, March 15, 2004 by Rupesh P. Amin, Alison E. Vickers, Frank Sistare, Karol L. Thompson, Richard J. Roman, Michael Lawton, Jeffrey Kramer, Hisham K. Hamadeh, Jennifer Collins, Sherry Grissom, Lee Bennett, C. Jeffrey Tucker, Stacie Wild, Clive Kind, Victor Oreffo, John W. Davis, II, Sandra Curtiss, Jorge M. Naciff, Michael Cunningham, Raymond Tennant, James Stevens, Bruce Car, Timothy A. Bertram, Cynthia A. Afshari

This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicanrs. For example, although puromycin is thought to specifically promote injury of the podocytes in the giomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologicaUy. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity.

Key words: biomarkers, cisplatin, gentamicin, microarrays, nephrotoxicity, proximal tubule, puromycin, toxicogenomics. Environ Health Perspect 112:465-479 (2004). doi:10.1289/txg.6683 available via http://dx.doi.org/ [Online 15 January 20041

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Renal toxicity commonly occurs after administration of xenobiotics and a variety of pharmaceutical agents. The process is typically initiated by a toxic injury to tubular epithelial cells in various nephron segments or by injury to specific cell types in the glomerulus. The initial injury is often followed by cellular proliferation and repair that attempts to restore normal renal function (Toback 1992). Changes in the expression of mRNA specifically expressed in the injured kidney cells are some of the earliest events that accompany renal injury. This is accompanied by changes in the expression of other genes that contribute either to cellular repair or recovery of renal function or in those that mediate fibrosis and further pathology of the kidney (Matejka 1998; Norman et al. 1988; Safirstein et al. 1990). For example, elevations in the expression of heme oxygenase I (HO-1), kidney injury molecule-1 (KIM-1), clusterin, thymosin beta 4, osteopontin, and several growth factors have been reported in various models of renal injury (Hammerman 1998a, 1998b; Huang et al. 2001; Ichimura et al. 1998; Yoshida et al. 2002).

Recent application of microarray-based gene profiling has provided some unique insights into compound-specific and toxicity-related gene expression changes in the liver (Hamadeh et al. 2002a, 2002b; Waring et al. 2001a, 2001b), and several laboratories have applied similar techniques to identify gene expression changes related to nephrotoxicity (Huang et al. 2001; Nagasawa et al. 2001; Yoshida et al. 2002).

This study, conducted as a consortium collaboration with the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) (Kramer et al. 2004; Pennie et al. 2004), compares kidney gene expression profiles induced by cisplatin and gentamicin, compounds that injure the proximal tubule, and puromycin, which produces proteinuria by selectively damaging podocytes, a key component of the glomerular filtration barrier to plasma proteins. Conventional clinical chemistry and histopathological or ultrastructural findings confirmed the induction of tubular or glomerular injury in these studies. Through comparison of the expression profiles derived from kidneys exposed to these three nephrotoxicants at multiple doses and time points (associated with varying types and severity of toxicity), we hypothesized that we could identify patterns of gene expression that reflect different types of nephrotoxicity. An important outcome is the identification of potential gene-based markers of nephrotoxicity and evidence that one can detect region-specific renal toxicity using microarray technology. Additional functional genomics studies may afford the opportunity to validate the proposed novel gene-based markers of nephrotoxicity, which in part may improve current sensitivity to assessing xenobiotic-induced nephrotoxicity.

Materials and Methods

General

This study was conducted in accordance with Good Laboratory Practices. All animals were handled and treated in accordance to the NIH Guide for the Care and Use of Laboratory Animals (NIH 1999). Experiments were performed on approximately 8- to 9-week-old male (approximately 250-300 g) Sprague-Dawley rats Crl:CD (SD)IGS BR VAF (abbreviated SD) purchased from Charles River Breeding Laboratories (Raleigh, NC). Food [Purina Rodent Chow no. 5002 (Purina Milling Inc., St. Louis, MO), or Certified Rodent Diet 18% no. 5LG3, (PMI Feeds, Richmond, IN)], except for study-defined fasting procedures, and water were available ad libirum.