The overall mortality rate for patients with acute renal failure (ARF) has remained 40-50% for the past 30 years. The associated annual cost of ARF is over $12 billion. Ischemia is the leading cause of ARF and alterations in the actin cytoskeleton of renal tubular cells during ischemia and recovery have important consequences for kidney function. The long-term goal of this project is to understand how myosin Il (MII) and the Rho GTPases interact to regulate protective and reparative alterations in the actin cytoskeleton of renal tubular cells during ischemia and recovery. The general strategy is to study ATP depletion as a reversible cell culture model of ischemia that has been shown to recapitulate alterations in the actin cytoskeleton observed from in vivo studies of ischemia. Mutant proteins will be used as molecular probes to conduct studies not possible in animals or humans. The proposed experiments will test the hypothesis that MII is an important distal effector of the Rho regulatory cascade and plays a critical role in regulating the actin cytoskeleton in renal tubular cells during ischemia and recovery. Grant funding and extended training in cell biology is a major factor in the PI's immediate goal of attaining research independence and the long-term career goal of becoming a successful and productive investigator in an academic institute.
The specific aims of this present study are as follows: 1) Determine the effects of altering MII activation on the actin cytoskeleton and cell adhesion during cellular ATP depletion and recovery. Mutant forms of myosin Iight chain kinase (MLCK) will be expressed in MDCK cells to alter the activation state of myosin II. In all specific aims, immunofluorescence confocal microscopy will be utilized to examine actin and MII distribution as well as structures important for cell-matrix adhesion. Standard assays will be used to examine MII activation and cell adhesion. 2) Determine the effects on the actin cytoskeleton of altering MII activation while inversely altering Rho signaling during cellular ATP depletion and recovery. Mutant forms of MLCK and Rho proteins will be co-expressed in MDCK cells to alter cellular signaling. 3) Determine the effects on the actin cytoskeleton of altering Rho-kinase (ROCK) signaling while inversely altering Rho signaling during cellular ATP depletion and recovery. Mutant forms of ROCK and Rho proteins will be co-expressed in MDCK cells to alter cellular signaling. 4) Determine the effects on the actin cytoskeleton of altering MII activation while inversely altering ROCK signaling during cellular ATP depletion and recovery. Mutant forms of MLCK and ROCK proteins will be co-expressed in MDCK cells to alter cellular signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK060621-05
Application #
7008237
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
Project Start
2002-04-15
Project End
2007-11-30
Budget Start
2005-12-01
Budget End
2007-11-30
Support Year
5
Fiscal Year
2006
Total Cost
$125,676
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Horbelt, M; Wotzlaw, C; Sutton, T A et al. (2007) Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy. Kidney Int 72:422-9
Sutton, Timothy A; Kelly, K J; Mang, Henry E et al. (2005) Minocycline reduces renal microvascular leakage in a rat model of ischemic renal injury. Am J Physiol Renal Physiol 288:F91-7
Molitoris, Bruce A; Sutton, Timothy A (2004) Endothelial injury and dysfunction: role in the extension phase of acute renal failure. Kidney Int 66:496-9
Sutton, Timothy A; Mang, Henry E; Campos, Silvia B et al. (2003) Injury of the renal microvascular endothelium alters barrier function after ischemia. Am J Physiol Renal Physiol 285:F191-8
Sutton, Timothy A; Fisher, Charles J; Molitoris, Bruce A (2002) Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int 62:1539-49