Deanna L. Kroetz - US grants

DESCRIPTION: (Adapted from the Applicant's Abstract) Renal cytochrome P450-mediated eicosanoid production is an important determinant of integrated kidney function and renal vascular tone. In the spontaneously hypertensive rat (SHR), alterations in the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) by enzymes of the cytochrome P450 4A (CYP4A) subfamily have been associated with increases in blood pressure and changes in renal function. In the proposed studies the spontaneously hypertensive rat will be used to test the hypothesis that CYP4A-mediated hydroxylation of arachidonic acid is involved in the development of hypertension. Based on differences in renal arachidonic acid and omega-1 hydroxylase activity, Dr. Kroetz and her colleagues hypothesize that one or more of the CYP4A genes are differentially expressed in the hypertensive rat kidney as compared to its normotensive control. Gene-specific ribonuclease protection assays will be used to distinguish between the mRNA levels of CYP4A1, CYP4A2, CYP4A3 and CYP4A8 in the renal cortex and medulla, and to localize their expression to specific segments of the nephron. Expression patterns will be correlated with 20-HETE formation rates to assess the relative contribution of each CYP4A isoform in the generation of this prohypertensive eicosanoid. The specific aims of this proposal are: 1) to identify differences between hypertensive and normotensive rats in the developmental pattern of expression and the intrarenal distribution of mRNAs encoding cytochrome P450 4A enzymes in the renal cortex and medulla; 2) to establish the relationship between blood pressure and CYP4A expression by manipulating arachidonic acid omega-hydroxylase activity through induction of the CYP4A genes or mechanism-based inactivation of the CYP4A proteins; and 3) to characterize the metabolic profile for arachidonic acid oxidation by each of the renally expressed CYP4A proteins using an in vitro protein expression system. Knowledge of the renal CYP4A mRNA expression levels and distribution patterns in hypertensive and normotensive rats, the effect of modulation of CYP4A expression on blood pressure, and information about whether a given CYP4A gene product metabolizes physiological concentrations of arachidonic acid to prohypertensive metabolites will provide evidence for the involvement of the CYP4A family in the regulation of blood pressure. These data will provide a basis for investigating a similar mechanism for the pathophysiological changes associated with essential hypertension in humans. The long term goal of this program is to use this knowledge to develop therapies for targeted modification of blood pressure.

DESCRIPTION (Verbatim from Applicant's Abstract): The two major pathways of cytochrome P450 (CYP)-catalyzed arachidonic acid (AA) metabolism are omega-hydroxylation to form 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxidation which produces regio- and stereoisomeric epoxyeicosatrienoic acids (EETs). The EETs are further metabolized by epoxide hydrolases to the corresponding dihydroxyeicosatrienoic acids (DHETs). These CYP-derived eicosanoids are of interest since they are endogenous constituents of numerous tissues and posses a wide array of biological effects. The cell specific pattern of expression, relative abundance, and activity of the enzymes catalyzing these reactions will be a major determinant of the intracellular effect of these eicosanoids. In the kidney, CYP-derived eicosanoids have potent effects on renal vascular tone and tubular ion transport and have been implicated in the regulation of blood pressure. The overall goal of the proposed studies is to understand the molecular mechanisms controlling CYP-catalyzed AA metabolism in the rat kidney and the importance of renal CYP eicosanoid levels in blood pressure regulation. The specific aims of the proposed studies are (1) to examine the mechanistic basis of the antihypertensive effect of inhibition of AA omega-hydroxylase activity. Specifically, we will determine the isoform-specificity and potency of mechanism-based inhibitors of CYP AA omega-hydroxylases and evaluate their effect on blood pressure and vascular tone; (2) to determine the contribution of CYP4E isoforms to renal AA metabolism. CYP4F expression will be localized in the rat kidney and AA metabolism by the CYP4F isoforms will be characterized; (3) to isolate the major CYP2J epoxygenase expressed in the rat kidney. The cDNA encoding the CYP2J2 immunoreactive protein overexpressed in the spontaneously hypertensive rat kidney will be identified by expression cloning and functionally characterized; and (4) to examine the regulation of EET hydrolysis in the rat kidney. Specifically, we will determine the biochemical basis of EET hydrolysis in rat renal microsomes and the effect of soluble epoxide hydrolase inhibition on blood pressure and renal eicosanoid formation. The findings from these studies will lead to a comprehensive understanding of the expression and regulation of the major CYP and epoxide hydrolase enzymes involved in AA metabolism. A long term goal of these studies is to develop novel targeted therapeutics for the regulation of renal CYP eicosanoid production and blood pressure. Application of these principles for regulating CYP eicosanoid formation in other tissues is anticipated.

pharmacogenetics; pharmacokinetics; digoxin; genotype; twin /multiplet; gene expression; clinical research; questionnaires; human subject;

DESCRIPTION (provided by applicant): Soluble epoxide hydrolase (sEH) is a dual function Phase II metabolic enzyme that catalyzes the hydrolysis of both xenobiotic and endobiotic epoxides. sEH metabolism of xenobiotic epoxides often results in their detoxification and accelerated elimination, whereas that of endobiotic epoxides is generally associated with attenuation of epoxide biological properties. Endogenous substrates of sEH are unsaturated fatty acid epoxides, including epoxyeicosatrienoic acids (EETs), which are major products of cytochrome P450 (CYP)-catalyzed metabolism of arachidonic acid, an essential fatty acid nutrient. The hydrolysis of EETs to their corresponding dihydroxyeicosatrienoic acids by sEH has recently emerged as a key factor controlling the biological effects of EETs, including vasoactive, anti- inflammatory and anti-apoptotic effects. Recent preliminary data from our laboratory shows that chemical or genetic disruption of sEH activity protects against acute kidney injury induced by cisplatin treatment. Specifically, the protective effects of sEH inhibition are associated with decreased inflammation and a dramatic attenuation of apoptosis. The focus of this proposal is to understand the mechanistic basis for the renoprotection afforded by disruption of sEH activity. Three specific aims are proposed to test the overall hypothesis that inhibition of sEH protects against acute kidney injury. The first aim will identify the signaling pathways involved in the renoprotective effect of sEH inhibition in a cisplatin model of acute kidney injury. The role of NF-?B and PPAR? signaling will be examined, particularly with respect to the anti-inflammatory effects of sEH inhibition in acute kidney injury. The effect of sEH inhibition on the intrinsic mitochondrial apoptotic pathway will also be investigated. Studies proposed for the second aim will extend our findings in a cisplatin model of acute kidney injury to additional models which involve different renal insults and signaling pathways. The renoprotective properties of sEH inhibition will be studied in both a unilateral ureter ligation model and in ischemia/reperfusion. Finally, the third aim will directly test the ability of EETs to protect against drug- or ischemia-induced renal cell injury, using cultured renal epithelial cells. The relative contribution of vascular versus tubular formed fatty acid epoxides in renoprotection will also be tested, using mouse strains with tissue specific overexpression or disruption of CYP epoxygenases and sEH. A combination of chemical and genetic tools to modulate sEH activity and EET production provide the critical framework to advance our preliminary observation of renoprotection associated with sEH inhibition. A long term goal of these studies is to develop strategies for the therapeutic modulation of sEH for the prevention and treatment of acute kidney injury. The general nature of the anti-inflammatory and anti-apoptotic effects of sEH inhibition will make our findings more broadly relevant to diseases affecting other organs as well. PUBLIC HEALTH RELEVANCE: Acute kidney injury is a complex syndrome occurring in 20% to 30% of critically ill patients, and is associated with increased mortality, hospitalization, use of healthcare resources, and costs. Despite decades of research in animal models, effective strategies for prevention of acute kidney injury have yet to make it to the clinic. The studies proposed in this application will explore a novel pathway for protection against acute kidney injury which exploits an abundant renal fatty acid epoxide with established roles in inflammation and apoptosis.

DESCRIPTION (provided by applicant): The Pharmaceutical Sciences and Pharmacogenomics Graduate Program (PSPG) is a cross-disciplinary program that consists of scientists at the University of California, San Francisco (UCSF) working in pharmaceutical sciences, computation and contemporary genetics. This Program encompasses the areas of molecular pharmacology related to drug development including: drug action, drug transport, drug metabolism and drug/gene delivery. The Program also includes the areas of pharmacokinetic/dynamic modeling, pharmacogenomics and bioinformatics applied to genetics/genomics. The Program offers training in these research areas through a core curriculum that encourages a breadth of knowledge about these fields and an elective curriculum that provides in-depth education on specific topics or techniques. Students participate in an ethical conduct in science course. The program immerses young scientists in the culture of science through a seminar program in pharmacogenomics, an annual retreat and seminars from industrial scientists. Students are recruited into the program with undergraduate degrees in the basic sciences and from Doctor of Pharmacy programs. There are 50 students in the program and approximately 10% of the students come from under represented groups. The program capacity was increased in 2000 from 35 students to 60 students and we anticipate reaching the 60 student capacity in 2012. There are 49 training faculty. Our program is one of the very few in the World with faculty expertise that spans the fields of pharmaceutics, genetics and computation;hence the training program is ideally situated to educate future scientific leaders in the emerging research areas at the intersection of these disciplines. The integrative approach has resulted in Ph.D. graduates who have the insights and quantitative scientific tools required for success in the rapidly advancing fields of drug development and pharmacogenomics;consequently graduates from the program are in high demand in academia, government and industry.

DESCRIPTION (provided by applicant): Project Summary/Abstract The Pharmaceutical Sciences and Pharmacogenomics (PSPG) Graduate Program is a cross-disciplinary program that represents the merger at the University of California San Francisco of scientists working in pharmaceutical sciences, drug development and contemporary genetics. The graduate program reflects the exciting scientific developments in the area of genomics, quantitative and systems biology, and computation that have far-reaching implications to the pharmaceutical and pharmacological sciences. The goal of the PSPG Graduate Program is to educate students to address the major questions in the pharmaceutical sciences, teach students the basic sciences needed to address these questions, and create an environment where the students can develop into independent and creative scientific problem solvers. The program brings together 50 faculty members spanning 18 departments with well-funded research programs. This multidisciplinary graduate program has a dual focus: 1) pharmaceutical sciences and drug development, including molecular and systems pharmacology, drug delivery and therapeutic bioengineering, drug metabolism and transport, and pharmacokinetics/pharmacodynamics; and 2) pharmacogenomics, which includes clinical, functional and computational genomics related to drug disposition and response. The training program includes a series of core courses providing an in- depth understanding of the principles of pharmaceutical sciences and pharmacogenomics, complemented by core and general electives covering advanced drug delivery and pharmacokinetic principles, principles of genetics and cell biology, bioinformatics, tissue and organ biology, and advanced statistics. Students also participate in laboratory rotations that expose them to the diversity of potential projects available for their dissertation research and a university-wide course on responsible conduct of research. The program immerses young scientists in the culture of science through a seminar program in pharmaceutical sciences and pharmacogenomics which brings in leading academic and industrial scientists, student research presentations, and an annual retreat. The program goal is to recruit 12 outstanding students per year for a total training program of 60 students. Underrepresented students are actively recruited through a number of faculty activities and represent approximately 10% of our students. Our program is one of the very few in the world with faculty expertise that spans the fields of pharmaceutics, genetics and computation; hence the training program is ideally situated to educate future scientific leaders in the emerging research areas at the intersection of these disciplines. The integrative approach has resulted in Ph.D. graduates who have the insight and quantitative scientific tools required for success in the rapidly advancing fields of drug development and pharmacogenomics; consequently graduates from the program are in high demand in academia, government and industry.

? DESCRIPTION (provided by applicant): Cancer chemotherapy can significantly improve patient outcomes but is often limited by toxicity. The long term goal of the proposed pharmacogenomic studies is to identify genetic predictors of drug toxicity that can be used to achieve maximal therapeutic benefit with minimal toxicity. This proposal is focused on microtubule targeting agent-induced sensory peripheral neuropathy, a dose-limiting toxicity associated with these widely used chemotherapy agents. We have recently identified several common genetic variants associated with paclitaxel-induced sensory peripheral neuropathy through a genome-wide association study. The studies proposed here will extend these findings and test the central hypothesis that both common and rare variants contribute to interindividual variability in microtubule targeting agent toxicity. The studies in aim 1 will identify rare varians in novel genes associated with microtubule targeting agent-induced sensory peripheral neuropathy. Sequencing of exomes and selected regulatory regions of 622 DNA samples from subjects enrolled in a Phase III trial of microtubule targeting agents in advanced breast cancer (CALGB 40502) will be performed and novel genes identified that are associated with drug-induced sensory peripheral neuropathy. The results from aim 1 and from our earlier genome-wide association study will be replicated in aim 2. Replication samples will come from three clinical trials testing paclitaxel in the treatment of early stage and advanced breast cancer and from the BioVU resource at Vanderbilt University. Finally, aim 3 will be functional genomics studies to characterize the effect of novel genes identified in our earlier genome-wide association study on neuronal and Schwann cell function. Specifically, neurite extension assays, cell shape and cellular signaling will be used to screen for the most biologically relevant genes identified in our genomic studies. Identification and validation of genetic predictors of this toxiity will advance the long term goal of these studies to use biomarkers for the identification of patients at increased risk of chemotherapy-induced sensory peripheral neuropathy and to develop targeted therapies to prevent or treat this toxicity.

Project Summary The modern era of discovery and clinical use of small molecule therapeutics to treat infectious diseases is threatened by rapidly spreading drug resistance. Drug resistance affects all major groups of infectious diseases but is particularly troubling among causative agents of such devastating infections as diarrheal and respiratory diseases, malaria and tuberculosis. Concerted efforts among basic, translational and drug discovery researchers are urgently needed to prevent further spread of resistance and to develop effective approaches to anticipate and to counter present and future drug resistant pathogens. This proposal requests partial support for the Gordon Research Conference (GRC) and Gordon Research Seminar (GRS) on Multidrug Efflux Systems to be held in Barga, Italy, April 27-May 3, 2019. This conference will fill a major gap in addressing critical issues and novel approaches to achieve the goals of effective reversal of persistent multidrug resistance worldwide and the development of therapeutics with optimal efficacy and reduced toxicity. The goal of this conference and seminar is to provide a stimulating and collaborative environment for researchers to openly discuss unpublished work, push the boundaries of science, and foster scientific interactions between young investigators, leaders in the field, and colleagues working in academia and the pharmaceutical/biotechnology industry. A major goal is to bring together researchers with expertise in diverse disciplines who traditionally have limited interaction to share progress and engage in discussion of challenges and opportunities in the field. The participation of researchers studying multidrug resistance pumps in microorganisms and those studying mammalian efflux pumps is designed to introduce technological advances and transformative approaches to each field. The goals of this GRC will be accomplished by the internationally recognized keynote speakers, the eight scientific sessions with over 30 invited speakers and additional discussion leaders, and the unique format and setting that facilitates fruitful interactions between participants at all career stages. Speakers in each session will present cutting-edge, unpublished data and innovative ideas on issues that are critical to the success of targeting multidrug efflux pumps for the reversal of resistance. The interactive nature of the GRC scientific and poster sessions and the participation of researchers from diverse research sectors is an optimal forum for graduate students and postdoctoral fellows to expand their scientific breadth, interact with leaders in the field, establish future collaborative relationships and consider various career paths. Submitted abstracts from trainees and underrepresented minorities will be targeted for oral presentations in each session. Through participation in the GRS prior to the main conference, trainees will have additional opportunities for oral and poster presentations and to develop relationships with their peers in a nurturing environment. The 2019 GRC and GRS on Multidrug Efflux Systems will further stimulate cutting-edge basic and translational research on the important biomedical problem of multidrug resistance.