1999 — 2003 |
Atkinson, Simon J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Rho Gtpases and Actin Function in Renal Ischemia @ Indiana Univ-Purdue Univ At Indianapolis
Ischemic acute renal failure is a major cause of morbidity and mortality. Cells of the kidney proximal tubule are particularly vulnerable to ischemic injury, and this injury is characterized by breakdown of normal cellular architecture with consequent loss of polarized distribution of surface proteins. This disruption of cell structure and function correlates with rapid and reversible redistribution of components of the actin cytoskeleton. We propose that this abnormal distribution of actin filaments results from inactivation of rho family GTPases. Rho proteins are the branch of the ras superfamily of small GTPases that regulate cell morphology and motility, and their role in controlling actin function has been extensively studied in fibroblasts, but is less well characterized in epithelial cells. We propose three specific aims to test the involvement of rho proteins and their effectors in actin reorganization in a cell culture model of ischemia, using ATP depletion induced by substrate depletion and antimycin A treatment. We will use microinjection and transfection to introduce dominant active and dominant negative mutants of Rho, Rac and Cdc42 into LLC-PK cells to test the effect of blocking inactivation or activation of pathways through these proteins on the actin cytoskeletal response to ATP depletion or recovery. We will measure the activation state of rho family GTPases under conditions of ATP depletion and recovery by measuring the ratio of GTP:GDP bound to the GTPase, and analysing cellular localization of the GTPase using GFP-tagged proteins. Cytoplasmic isoforms of myosin II play an important role in function of the actin cytoskeleton in polarized epithelia, and are an important target of Rho regulation. We will examine myosin localization and light chain phosphorylation in control cells and in response to ATP depletion, and ezrin regulation, and we will determine the effect of regulators of myosin activity, Rho-kinase and myosin light chain kinase (MLCK), using dominant negative and constitutively active mutants. The data derived from the proposed studies will provide a basis for understanding the cellular mechanisms that underlie the abnormal response of the cytoskeleton to ischemia, and a mechanism for the known beneficial effects of growth factors. This will provide a basis for the development of improved therapeutic approaches to the management of human ischemic acute renal failure.
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0.924 |
2002 |
Atkinson, Simon J. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Imaging @ Indiana Univ-Purdue Univ At Indianapolis
The Imaging Core facility will service the program in the area of light microscope imaging and analysis. The core is built around the state-of- the-art facility available in the Nephrology Division of the Department of Medicine, and benefits from the experience of key core personnel in the application of imaging methodologies to the analysis of hematopoietic cell function. The facility is currently equipped with two confocal laser-scanning microscopes: a Zeiss LSM 510 capable of UV excitation of fluorescence; and a BioRad MRC-1024 with Ti: sapphire laser capable of 2-photon excitation as well as Kr/Ar laser for conventional confocal imaging. Two wide-field microscope stands equipped with cooled CCD cameras are available: an Applied Precision Deltavision system optimized for image deconvolution; and a second Nikon stand used for motility assays. Stage incubators with CO2 perfusion are available for maintaining and imaging live cells on the stage of the microscope for long periods. Computing resources available include workstations with software for image processing, image deconvolution, rendering of 3-D image volumes and quantitative analysis of image data. The aim of the imaging core is to provide 1) technology and expertise in imaging live hematopoietic cells by transmission light microscopy and fluorescence microscopy to facilitate real-time analysis of signaling events and Rac2 dependent processes; and 2) expertise and support for quantitative analysis of image data generated by fluorescence microscopy to complement biochemical and other analysis of hematopoietic cell function.
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0.924 |
2002 |
Atkinson, Simon J. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Rac2 in Reglation of Cytoskeletal Function @ Indiana Univ-Purdue Univ At Indianapolis
Rho GTPases are widely recognized as the critical integrators of signals from the cell surface that control cell morphology and motility in all eukaryotic cell types examined, including hematopoietic cells. The ultimate targets of these signals are alterations in the organization of the actin cytoskeleton. Signals from Rac, a rho family GTPase, result in actin polymerization at the cell periphery that is crucial for extension of the leading edge of crawling cells, for the phagocytosis of pathogens and for correct uptake and presentation of antigen by dendritic cells. The generation of a strain of mice deficient in the hematopoietic-specific GTPase Rac2 provides a unique animal model in which to study the role of signaling pathways utilizing this GTPase in their physiological context. Mice deficient in Rac2 exhibited multiple defects in neutrophil functions related to the actin cytoskeleton including chemotaxis, integrin- and selectin-mediated adhesion and phagocytosis. Underlying these defects appears to be a disturbance in actin dynamics at rest and in response to stimulus. We propose studies to determine the mechanisms by which Rac2 deficiency results in disturbed actin dynamics. We will characterize the phenotypes of Rac2 deficient macrophages, neutrophils and dendritic cells that relate to actin dynamics, and using retroviral transduction we will determine the requirement for Rac2-specific sequences for normal function in these cell types. The will determine the requirement for Rac2-specific sequences for normal function in these cell types. We will determine the mechanistic basis for disturbed actin dynamics in the absence of Rac2 by measuring the roles of de novo nucleation of actin filaments and barbed end uncapping, and the roles of components of signaling pathways that lie between Rac and components of the actin cytoskeleton. Studies described in this project will further understanding of the role of the cytoskeleton and the pathways that control it in innate and acquired immunity.
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0.924 |
2007 — 2012 |
Atkinson, Simon J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Rho Gtpases and Actin Cytoskeletal Function in Renal Ischemia @ Indiana Univ-Purdue Univ At Indianapolis
DESCRIPTION (provided by applicant): The persistence of ischemic acute renal failure as a major cause of morbidity and mortality in hospitalized patients and the relative lack of improvement in outcomes for these patients challenges us the better understand the cellular mechanisms underlying this injury. Disruption of the actin cytoskeletal organization that normally underpins the structure and function of proximal tubule epithelial cells is well-established as an early and critical aspect of injury, with direct consequences for renal function and cell survival. In the previous years of this award we have shown that Rho GTPases play critical roles that may mediate the cytoskeletal alterations observed with ischemia. Moreover, we showed that activities of RhoA, Rac1 and Cdc42 are sensitive to ATP depletion, but that the activity of each protein is differentially affected by depletion. We proposed that alterations in Rho GTPase activity and consequent cytoskeletal disruption are the result of signaling through the AMPK-TSC1/2 pathway resulting in inactivation of the type II (rapamycin insensitive) mTor complex 2, which has recently been shown to regulate the cytoskeleton through Rho GTPases or via novel mTOR independent pathways downstream of TSC1/2. We propose studies to test and elucidate our proposed mechanism using complementary studies in cell culture and in animal models of renal ischemia. We propose four specific aims to address our central hypothesis: 1) Investigate the activation of AMPK by ATP depletion in vitro and ischemia in vivo; 2) Investigate the role of TSC1/2 and Rheb GTPase signaling on Rho GTPase activity and cytoskeletal organization in vitro and in vivo; 3) Determine the effect of AMPK activation on the type II mTOR complex; 4) Determine the effect of altered AMPK, TSC1/2, Rheb and mTOR signaling on actin cytoskeletal organization and proximal tubule cell function in animal models of ischemic renal injury. These studies will determine the key mechanism linking energy depletion to cytoskeletal alterations, and afford an opportunity to identify targets for novel therapies. Narrative Ischemia (loss of blood flow) is a major cause of acute kidney failure. Currently there is a poor understanding of the way that kidney cells respond to ischemia, which makes it difficult to design therapies or drugs. The studies proposed will test a possible mechanism leading to cell injury that could be a target for drug therapy.
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0.924 |
2011 — 2014 |
Atkinson, Simon J. Molitoris, Bruce A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Utilizing Sirna to Minimize Nephrotoxic Injury @ Indiana Univ-Purdue Univ At Indianapolis
DESCRIPTION (provided by applicant): Nephrotoxins remain a major cause of acute kidney injury (AKI). RNA interference holds great promise as a novel and specific therapeutic gene-silencing technology for a wide range of diseases. Oligonucleotides, like many other small macromolecules, are present in the ultrafiltrate formed at the renal corpuscle, and therefore are presented to the tubular epithelial cells lining the nephron at relatively high concentrations. These cells, particularly those in the proximal tubule segments, avidly take up oligonucleotides, so that the kidney has by far the greatest accumulation of systemically delivered oligonucleotides. This makes siRNA a promising technology for treatment of kidney disease, especially given the particular sensitivity of proximal tubule cells to toxic or ischemic injury. Given the clinical and biological importance of these observations, the handling of siRNA by renal cell types needs to be better characterized. We propose that siRNA is preferentially accumulated in proximal tubule epithelial cells because of the specialization of these cells for rapid endocytosis of macromolecules delivered to the filtrate. New technologies in fluorescence microscopy, particularly the application of multi-photon imaging to intravital microscopy, allow the cellular and subcellular distribution of labeled oligonucleotides to be analyzed in real time in live animals. We propose to use this methodology in four specific aims to better understand the mechanism of uptake of siRNA by proximal tubule cells and to establish the feasibility of using siRNA to prevent toxic acute kidney injury (AKI). We propose: 1) to determine the kinetics of cellular and intracellular accumulation, catabolism and biological effects of siRNA in the normal kidney;2) to determine the effect of chronic kidney disease on siRNA handling by the kidney;3) to determine the effect of endocytic blockade on siRNA uptake;and 4) to test the ability of siRNA to prevent aminoglycoside uptake by proximal tubule cells and hence limit renal toxicity. PUBLIC HEALTH RELEVANCE: RNA interference is a method that can be used to stop the harmful effects of normal or mutant proteins, and may be an important and specific therapy for diseases in the future. The RNA molecules used in this technology are concentrated in the kidney, and the studies in this proposal will examine the mechanisms and consequences of this accumulation, the way that kidney disease can affect this process, and the possibility of using RNA to protect the kidney against toxins.
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0.924 |