Kathryn L Pellegrini*, Cory V Gerlach*, Florin L Craciun, Krithika Ramachandran, Vanesa Bijol, Haydn T Kissick, and Vishal S Vaidya. 2016. “Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis..” Toxicol Appl Pharmacol, 312: 42-52. Abstract

*Authors equally shared

Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250mg/kgi.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (>2-fold, p<0.05), we chose to further investigate miR-18a-5p, which is expressed during the acute stage of the injury; miR-132-3p, which is upregulated during transition between acute and fibrotic injury; and miR-146b-5p, which is highly expressed at the peak of fibrosis. Using qRT-PCR, we confirmed the increased expression of these candidate miRNAs in the folic acid model as well as in other established mouse models of acute injury (ischemia/reperfusion injury) and fibrosis (unilateral ureteral obstruction). In situ hybridization confirmed high expression of miR-18a-5p, miR-132-3p and miR-146b-5p throughout the kidney cortex in mice and humans with severe kidney injury or fibrosis. When primary human proximal tubular epithelial cells were treated with model nephrotoxicants such as cadmium chloride (CdCl2), arsenic trioxide, aristolochic acid (AA), potassium dichromate (K2Cr2O7) and cisplatin, miRNA-132-3p was upregulated 4.3-fold after AA treatment and 1.5-fold after K2Cr2O7 and CdCl2 treatment. These results demonstrate the application of temporal small RNA sequencing to identify miR-18a, miR-132 and miR-146b as differentially expressed miRNAs during distinct phases of kidney injury and fibrosis progression.

Cory V Gerlach, Siba R Das, David C Volz, William H Bisson, Siva K Kolluri, and Robert L Tanguay. 2014. “Mono-substituted isopropylated triaryl phosphate, a major component of Firemaster 550, is an AHR agonist that exhibits AHR-independent cardiotoxicity in zebrafish..” Aquat Toxicol, 154: 71-9. Abstract
Firemaster 550 (FM550) is an additive flame retardant mixture used within polyurethane foam and is increasingly found in house dust and the environment due to leaching. Despite the widespread use of FM550, very few studies have investigated the potential toxicity of its ingredients during early vertebrate development. In the current study, we sought to specifically investigate mono-substituted isopropylated triaryl phosphate (mITP), a component comprising approximately 32% of FM550, which has been shown to cause cardiotoxicity during zebrafish embryogenesis. Previous research showed that developmental defects are rescued using an aryl hydrocarbon receptor (AHR) antagonist (CH223191), suggesting that mITP-induced toxicity was AHR-dependent. As zebrafish have three known AHR isoforms, we used a functional AHR2 knockout line along with AHR1A- and AHR1B-specific morpholinos to determine which AHR isoform, if any, mediates mITP-induced cardiotoxicity. As in silico structural homology modeling predicted that mITP may bind favorably to both AHR2 and AHR1B isoforms, we evaluated AHR involvement in vivo by measuring CYP1A mRNA and protein expression following exposure to mITP in the presence or absence of CH223191 or AHR-specific morpholinos. Based on these studies, we found that mITP interacts with both AHR2 and AHR1B isoforms to induce CYP1A expression. However, while CH223191 blocked mITP-induced CYP1A induction and cardiotoxicity, knockdown of all three AHR isoforms failed to block mITP-induced cardiotoxicity in the absence of detectable CYP1A induction. Overall, these results suggest that, while mITP is an AHR agonist, mITP causes AHR-independent cardiotoxicity through a pathway that is also antagonized by CH223191.