Gentamicin and vancomycin are nephrotoxic antibiotics that are a major cause of drug-induced acute kidney injury (AKI), an abrupt and potentially fatal loss of kidney function. AKI occurs in nearly 30% of exposed patients and increases healthcare costs by causing substantial morbidity and mortality. The lack of a unifying mechanism for proximal tubule cell injury, a major target for damage, complicates the discovery of effective therapeutic agents. The goal of this project is to discover the signals that mediate antibiotic-induced proximal tubule cell injury and to re-purpose existing therapeutic reagents. We hypothesize that computational analysis of an unbiased, broad-based shRNA screen will identify the ?best fit? mechanism(s) of antibiotic-induced AKI and that mechanistically-targeted drugs will ameliorate renal cell injury and AKI. Preliminary shRNA screening of gentamicin-exposed human proximal tubule cells detected 226 differentially expressed signal genes. Pathway analysis of these signal genes identified the Unfolded Protein Response (UPR) as the ?best fit? mechanism of gentamicin-induced cellular injury and guided the selection of several potentially therapeutic drug targets. The Unfolded Protein Response (UPR) is a mechanism that integrates stress-induced protein misfolding with protein degradation, mitochondrial injury and cell death. Preliminary data confirmed that gentamicin exposure caused enhanced protein ubiquitination (a marker of protein degradation), luciferase dysfunction, mitochondrial fragmentation, decreased ATP content and death of human proximal tubule epithelial cells. Chemical induction of Hsp70, a protective chaperone protein that regulates UPR at a proximate step, markedly reduced protein ubiquitination, prevented mitochondrial fragmentation, and dramatically improved human renal cell survival. In this proposal, we intend to use this methodology to describe the mechanism of vancomycin-induced AKI, to identify effective therapeutics that prevent vancomycin-induced injury and to determine the extent of protection in an in-vivo vancomycin model. We also intend to continue mechanistically-focused experiments to determine the extent of UPR pathway manipulations in our in vivo models of antibiotic-induced AKI. In-vivo measures of the UPR pathway activation will be correlated with urinary biomarkers, BUN and cystatin c, histologic injury score, inflammation and animal survival. This strategy provides a rational approach to identifying targeted therapeutics by combining unbiased genetic screening and computational analysis with in-vitro and in-vivo pharmacologic manipulations. Our strategy identifies UPR as a unifying mechanism of gentamicin-induced proximal tubule cell injury, identifies a UPR-targeted drug that improves cell survival after injury and has the potential to identify both mechanisms and therapeutics for vancomycin-induced injury.

Public Health Relevance

Acute kidney injury (AKI) is often caused by commonly prescribed antibiotics. AKI increases mortality and is a clinically relevant healthcare challenge that occurs in 2.5 million intensive care unit admissions within the United States each year. Our goal is to identify the signal pathways that mediate kidney damage in response to these common antibiotics and to use existing, pathway-targeted reagents to reduce antibiotic-associated AKI and mortality by identifying new treatments to prevent and treat it.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZDK1)
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Maric-Bilkan, Christine
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Boston University
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United States
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