Chemical-induced nephrotoxicity is a major cause of acute kidney injury. My dissertation reveals that all-trans-retinoic acid (ATRA) affords cytoprotection against renal cell injury. Pretreatment with ATRA (25 μM, 24 hr) affords selective cytoprotection against p-aminophenol (PAP), iodoacetamide (IDAM), and 2-(glutathion-S-yl)-hydroquinone-induced necrosis. In contrast, pretreatment of cells with ATRA provides no protection against cisplatin-induced apoptosis. Inhibition of protein synthesis blunts ATRA-mediated cytoprotection, suggesting that critical cell survival signaling pathways are activated prior to toxicant exposure. Oxidative stress is a major contributor to cellular damage. To investigate the mechanism(s) by which ATRA affords cytoprotection, we determined its effects on ROS generation using a DCFDA assay. ATRA did not alter PAP or MGHQ-induced ROS levels. Moreover, ATRA had no effect on GSH levels nor Nrf2 expression, suggesting that other cytoprotective mechanisms are engaged by ATRA. Elevated ROS disrupt endoplasmic reticulum protein folding guided by the molecular chaperone Grp78. During ATRA-mediated pretreatment, the ER stress proteins Grp78 and p-eIF2α were induced (2-fold) in a time-dependent manner (24 and 4 hr respectively). In addition to influencing organelle stress proteins in the ER, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT with maximum levels achieved at 30 min. Moreover, induction of these stress kinases was observed at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Inhibition of p-ERK with PD98059 reduced the ability of ATRA to provide protection against PAP toxicity, implying a role for p-ERK and downstream target genes in the protective effects of ATRA. Gene ontology analysis of a microarray experiment of cells treated with ATRA revealed that ATRA rapidly (0.5, 1 hr) induced growth factors and genes involved in cell proliferation, with subsequent (4, 8, 12 hr) induction of genes involved in ribosome biogenesis, DNA replication and repair, and cell cycle regulation. Complementary data from a cell stress protein array and western blot analyses indicated that ATRA induced HIF1α 3-fold at 8 hr. Furthermore, the microarray data indicated the HIF1α target gene BHLHE40 (which encodes a basic-helix-loop-helix protein involved in cell differentiation) was increased 3-fold. As ATRA induced genes that were associated with cell proliferation, related assays were employed. ATRA had a small effect on cell cycle distribution demonstrated by an increase in the population of cells in the S and G2 phases between 8 and 24 hr. In addition, ATRA markedly increased total DNA content and cell number at 24 hr suggesting that mitogenic/proliferative effects contribute to ATRA cytoprotection. The present studies indicate that a signaling cascade of proteins downstream of p-ERK associated with mitogenesis work cooperatively to afford ATRA protection against renal cell injury. Understanding the mechanism of ATRA-mediated cytoprotection will provide insights into the development of novel therapeutic strategies for renal pathological conditions.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/625452 |
| Date | January 2017 |
| Creators | Sapiro, Jessica M., Sapiro, Jessica M. |
| Contributors | Klimecki, Walter T., Klimecki, Walter T., Briehl, Margaret M., Zhang, Donna D., Monks, Terrence J. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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