Diversity Outbred mice are a model for mechanistic understanding of susceptibility to kidney injury induced by valproic acid Haixia Lin, Shaoke Luo, Julia Tobacyk, Lascelles Lyn-Cook Jr., and Alison Harrill University of Arkansas for Medical Sciences, Little Rock, AR 72205 background VPA-induced idiosyncratic kidney injury Valproic acid (VPA) is widely used to treat epilepsy, migraines and bipolar disorders. VPA-induced idiosyncratic responses include liver injury and Fanconi syndrome caused by proximal renal tubular acidosis. The mechanistic link between individual susceptibility to toxicity response remains unknown. Genetic variation within the human population is one of potential factors contributing to VPA-induced idiosyncratic adverse effects. Average human renal cell lines lack genetic diversity; thus, utilization of a genetically diverse model may inform the mode of action of VPA-induced renal injury in susceptible patients. A B Representative images- VPA-induced kidney gross pathology C D Animal ID : 2358 BUN 46 mg/dL ALT 91 U/L AST 156 U/L Animal ID : 2058 BUN 35 mg/dL ALT 26 U/L AST 65 U/L Figure 3. VPA caused kidney injury by gross pathological examination. Severe hydronephrosis was observed in several animals by gross pathological examination in VPA treated group. Figure 1. Clinical chemistry biomarkers. Serum levels of ALT, AST, BUN, and TBIL were measured within the VPA-treated group and the vehicle group. Serum BUN in VPA treatment group was significantly increased compared to the vehicle group (p<0.0001) (Fig. 1C) which may indicate renal injury. There was no significant difference in liver specific biomarkers such as AST, ALT and TBIL between treatment groups (Fig. 1A-B; D). Diversity Outbred Animal ID : 2057 BUN 72 mg/dL ALT 27 U/L AST 65 U/L Collaborative Cross Diversity Outbred DO Initiation 144 CC lines between G2:F4 & G2:F12 Diversity Outbred (DO) mice are a population of outbred mice derived from eight genetic founder strains. DO mice have a high degree of genetic diversity exceeding the diversity of common SNPs in the human population. DO mice have been successfully utilized to model idiosyncratic drug-induced toxicities that are observed in clinical practice and to determine pharmacogenetic tests. Use of genetically diverse mouse populations in nonclinical safety screening may offer advantages to detecting sensitive individuals. Table 1. Gross pathology summary Pathol Changes Pale color Hydronephrosis # Affected Animals in VPA 12/49 (24%) 3/49 (6%) Clinical relevance: VPA is known to be associated with Fanconi syndrome (impairment of proximal tubule mediated reabsorption of sodium, bicarbonate, K, phosphate, glucose, amino acids, uric acid, low MW proteins and peptides). Figure 2. Plasma miRNA-122 abundance. The concentration of miR-122 in plasma was not significantly elevated in VPA-treated group compared to the vehicle group. Conclusions The DO mice provided a clinically relevant model for population differences in VPA-induced renal injury. VPA significantly increased serum BUN levels compared to the vehicle. There were no significant changes in AST, ALT, and TBIL. Several DO mice had pale kidneys and hydronephrosis which were not predicted well by classical inbred mouse strains (data not shown). Both sensitive and resistant DO mice were identified, providing a potential model for understanding the mechanisms behind idiosyncratic renal injury induced by VPA. Materials and Methods Drug regimen and necropsy. Female DO mice were orally administered VPA (500 mg/kg; N=49) or vehicle (water; N=50) for 7 days and sacrificed 24 hours following the final dose. During the necropsy, blood (serum and plasma) and tissues (liver and kidney) were collected. Clinical chemistry analysis. Serum blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine transaminase (ALT), and total bilirubin (TBIL) were analyzed. MicroRNA-122 measurement. The concentration of miRNA-122 was measured in the plasma using qRT- PCR using standard curve quantification. Histopathology. Left liver section was isolated from each animal, fixed in formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E), following standard protocols in the Experimental Pathology Laboratories (RTP, NC). Future Directions STEP 1: Drug-induced kidney injury is observed in clinical trials (pre-market) or in the clinic (post-market) Translation of Mouse Results to Human Population Clinical Observation of Toxicity Experimental Workflow Time (days) 1 2 3 4 5 6 7 8 Necropsy Endpoints of interest: Liver (histopathology) Kidney (RNA-seq) Serum (clinical chemistry) Plasma (miR-122 expression) Valproic Acid Dosing Regimen (500 mg/kg/day) STEP 2: Determine whether Diversity Outbred mice experience the clinically relevant adverse drug reaction Whole-Transcriptome Shotgun Sequencing (RNA-Seq) Gene Pathway Analysis STEP 3: Determine transcript expression differences that distinguish responder from nonresponder mice. STEP 4: Identify molecular pathways that directly affect susceptibility to VPA-induced kidney injury to inform the mode of action Genetically Diverse Mouse Population Acknowledgements: The authors wish to thank Gary Churchill and Dan Gatti (The Jackson Laboratory). Funding was provided by the US Food and Drug Administration (FDA BAA-12-00118) and the Burroughs Wellcome Fund Innovation in Regulatory Science Award. No official support or endorsement of the content of this poster by the FDA is intended or should be inferred.