Background: Tubular atrophy and interstitial fibrosis are well-recognized sequelae of chronic proteinuria; however, little is known regarding the molecular pathways activated within tubulointerstitium in chronic proteinuric nephropathies. Methods: To investigate the molecular mechanisms of proteinuria-associated tubulointerstitial (TI) disease, doxorubicin nephropathy was induced in rats. Progression of disease was monitored with weekly urinary biomarker assays. Because histopathology revealed multifocal TI injury, immunodirected laser capture microdissection was used to identify and isolate injured proximal tubules, as indicated by kidney injury molecule-1 immunolabeling. Adjacent interstitial cells were harvested separately. Gene expression microarray, manual annotation of gene lists, and Gene Set Enrichment Analysis were performed. A subset of the regulated transcripts was validated by quantitative PCR and immunohistochemistry. Results: Severe proteinuria preceded tubular injury biomarkers by 1 week. Histology revealed multifocal, mild TI damage at 3 weeks, which progressed in severity at 5 weeks. Affymetrix microarray analysis revealed tissue-specific regulation of gene expression. Manual annotation of gene lists, gene set enrichment analysis, and urinary biomarker assays revealed similarities to pathways activated in direct TI injuries. This suggests commonalities amongst the molecular mechanisms of TI injury secondary to proteinuria, ischemia-reperfusion, and nephrotoxicity.
As the genomes of mammalian species become sequenced and gene functions are ascribed, the use of differential gene expression (DGE) to evaluate organ function will become common in the experimental evaluation of new drug therapies. The ability to translate this technology into useful information for human exposures depends on tissue sampling that is impractical or generally not possible in man. The possibility that the DGE of nucleated cells, reticulocytes, or platelets in blood may present the necessary link with target organ toxicity provides an opportunity to correlate preclinical with clinical outcomes. Adriamycin is highly effective alone and more frequently in combination with other chemotherapeutic agents in the treatment of a variety of susceptible malignancies. Adriamycin-induced cardiomyopathy was examined as an endpoint to measure the utility of DGE on whole blood as a predictor of cardiac toxicity. Statistically significant gene changes were observed between relevant blood and cardiac gene profiles that corroborated the accepted mechanisms of toxicity (oxidative stress, effects on carnitine transport, DNA intercalation). There were, however, clear indications that other target organs (bone marrow and intestinal tract) were affected. The divergent expression of some genes between the blood and the heart on day 7 may also indicate the timing and mechanism of development of the cardiomyopathy and confirm current therapeutic approaches for its prevention. The data demonstrate that whole blood gene expression particularly in relation to oxidative stress, in conjunction with standard hematology and clinical chemistry, may be useful in monitoring and predicting cardiac damage secondary to adriamycin administration. Appendices A & B, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre = journal&issn = 0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.
As the genomes of mammalian species become sequenced and gene functions are ascribed, the use of differential gene expression (DGE) to evaluate organ function will become common in the experimental evaluation of new drug therapies. The ability to translate this technology into useful information for human exposures depends on tissue sampling that is impractical or generally not possible in man. The possibility that the DGE of nucleated cells, reticulocytes, or platelets in blood may present the necessary link with target organ toxicity provides an opportunity to correlate preclinical with clinical outcomes. Adriamycin is highly effective alone and more frequently in combination with other chemotherapeutic agents in the treatment of a variety of susceptible malignancies. Adriamycin-induced cardiomyopathy was examined as an endpoint to measure the utility of DOE on whole blood as a predictor of cardiac toxicity. Statistically significant gene changes were observed between relevant blood and cardiac gene profiles that corroborated the accepted mechanisms of toxicity (oxidative stress, effects on carnitine transport, DNA intercalation). There were, however, clear indications that other target organs (bone marrow and intestinal tract) were affected. The divergent expression of some genes between the blood and the heart on day 7 may also indicate the timing and mechanism of development of the cardiomyopathy and confirm current therapeutic approaches for its prevention. The data demonstrate that whole blood gene expression particularly in relation to oxidative stress, in conjunction with standard hematology and clinical chemistry, may be useful in monitoring and predicting cardiac damage secondary to adriamycin administration. Appendices A & B, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.
Peroxisome proliferator chemicals, acting via the peroxisome proliferator-activated receptor-α ( Pparα ), are potent hepatic mitogens and carcinogens in mice and rats. To test whether Pparα is required for hepatic growth in response to other stimuli, we studied liver regeneration and hepatic gene expression following partial hepatectomy (PH) of wild-type and Pparα -null mice. Pparα -null mice had a 12- to 24-hour delay in liver regeneration associated with a delayed onset and lower peak magnitude of hepatocellular DNA synthesis. Furthermore, these mice had a 24-hour lag in the hepatic expression of the G 1 /S checkpoint regulator genes Ccnd1 and cMyc and increased expression of the IL-1β cytokine gene. Hepatic expression of Ccnd1 , cMyc , IL-1r1 , and IL-6r was induced in wild-type mice, but not Pparα -null mice, after acute exposure to the potent Pparα agonist Wy-14,643, indicating a role for Pparα in regulating the expression of these genes. Expression of the fatty acid ω-hydroxylase gene Cyp4a14 , a commonly used indicator gene for Pparα activation, was strongly induced in wild-type mice after hepatectomy, suggesting that altered hepatocyte lipid processing may also contribute to the impaired regeneration in mice lacking the Pparα gene. In conclusion, liver regeneration in Pparα -null mice is transiently impaired and is associated with altered expression of genes involved in cell cycle control, cytokine signaling, and fat metabolism. (Hepatology 2002;36:544-554.)
Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 microM), lauryl sulfate (260 microM), dimethylsulfoxide (1.28 M), cycloheximide (62.5 microM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 microM), alloxan (130 mM), adriamycin (40 microM), hydrogen peroxide (4 mM), and heat stress (45 degrees C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide a useful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21(waf1/cip1), GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0-1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation between the biochemical and transcriptional measures of oxidative stress. We conclude that it is essential to measure the mechanism of interest directly in the test system being used when assessing gene expression as a tool for toxicology. Tables 1-15, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.
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