The Bioanalytical Core (Core B) will provide high quality, reproducible, low cost, automated high-throughput robotic analytical services of urine and kidney epithelial cells to the three Projects of the proposed Center. The systemic administration of antisense oligonucleotides (AOs) is currently being tested to alter gene expression in the treatment of several diseases (e.g. muscular dystrophy). GLP toxicology studies done by Center investigators have shown that AOs accumulate in renal proximal tubules, which become vacuolated and contain basophilic granules, and that this resolves with cessation of treatment (see Project 1). This is reminiscent of the minimal renal morphological alterations in acute kidney injury. Tests that predict the beneficial and toxic effects of AO drugs on renal tissues in a particular individual are currently limited. As a novel approach to renal biomarkers, we show data to suggest that renal proximal tubule cells (RPTCs) recovered from the urine can be used to predict the phenotype of individuals from whom the urine cells were collected. Services provided by Core B are divided into three specific aims.
In Specific Aim 1, RPTCs from urine from DMD patients from Project 1 will be banked, and tested after the investigator meeting in Year 3.
Aim 1 will also isolate RPTCs from rats and mice from Project 3 for development of testing methods. RPTCs will be grown in the polarized state using a patented 3-D cell culture method.
In Specific Aim 2, we will carry out biochemical, immunological, and histological marker studies of urine and RPTCs from preclinical models (Project 3;Years 1-5), and DMD patient urines (Project 1;Years 3-5). Assays will include kidney function, acute kidney injury (AKI) enzyme markers, renal proximal tubule markers, inflammatory markers, reactive oxygen species, and lipid peroxidation.
In Specific Aim 3, we will transfer robust validated novel biomarkers from Project 2 into Core B, and implement assays on pre-clinical and clinical samples. Pre-clinical biomarker discovery (animals from Project 3, assays from Project 2) will be critically evaluated in the investigator meeting in Year 3. Selected assays will be transitioned into Core B, or retained in Project 2 (if requiring MS/MS), and all pre-clinical samples from Project 3 analyzed. Novel biomarker assays may be implemented in the DMD drug and placebo samples from Project 1, as determined in the Year 3 investigator meeting. If successful, such studies could also be used to test the renal effects of any drug to determine the dose of a

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Specialized Center--Cooperative Agreements (U54)
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Special Emphasis Panel (ZHD1-DSR-A)
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Children's Research Institute
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Allegaert, Karel; Fanos, Vassilios; van den Anker, Johannes N et al. (2014) Perinatal pharmacology. Biomed Res Int 2014:101620
Tatem, Kathleen S; Quinn, James L; Phadke, Aditi et al. (2014) Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases. J Vis Exp :51785
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Allegaert, Karel; van de Velde, Marc; van den Anker, John (2014) Neonatal clinical pharmacology. Paediatr Anaesth 24:30-8
Allegaert, Karel; van den Anker, John N (2014) The addition of tramadol to a standard i.v. acetaminophen/morphine analgesia protocol in neonates: purposeful or just polypharmacy? Paediatr Anaesth 24:1189-90
Wu, B; Cloer, C; Lu, P et al. (2014) Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice. Gene Ther 21:785-93

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