Cellular osmotic homeostasis is a fundamental requirement for life. All cells are exposed to osmotic challenges brought about by changes in intracellular solute flux and/or perturbations in extracellular osmolality. Most mammalian cells are protected from extracellular osmotic challenges by the kidney, which tightly regulates blood ionic and osmotic concentrations. Renal medullary cells are an important exception to this generalization and are subjected normally to extreme osmotic stress by the renal concentrating mechanism. Cells maintain osmotic homeostasis by the tightly regulated gain and loss of salt and organic solutes termed organic osmolytes, and by detecting and repairing osmotic stress induced damage. The transport and metabolic pathways that mediate animal cell osmoregulatory solute fluxes are well described. However, little is known about the signaling mechanisms by which animal cells detect osmotic perturbations, about the types of cellular and molecular damage induced by osmotic stress, and about how this damage is detected, repaired and prevented. DK61168 supported studies developed the nematode C. elegans as a novel genetically tractable model system for defining fundamental mechanisms of animal cell osmosensing and osmotic homeostasis. During the previous funding period, we made the novel observation that disruption of protein synthesis activates expression of genes required for organic osmolyte accumulation. We also demonstrated for the first time that hypertonicity causes rapid and extensive protein damage in vivo and that genes required for protein degradation are essential for survival during hypertonic stress. The current proposal builds on these new findings and addresses three questions with broad biological and pathophysiological significance. How does disruption of protein synthesis activate osmosensitive gene expression? What are the quality control mechanisms utilized by cells to detect, degrade and repair proteins damaged by hypertonic stress? What are the mechanisms by which acclimation to hypertonic stress suppresses hypertonicity induced protein damage? We will utilize a combination of cell biological, molecular and biochemical approaches to provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. We will also exploit the genetic tractability of C. elegans and begin to define the signals and signaling pathways that regulate expression of genes required for survival in hypertonic environments. Our work will provide novel insights into cellular osmosensing and signal transduction and into the mechanisms that protect hypertonically stressed cells from protein damage and associated injury and death. Detailed understanding of hypertonicity induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases. Public Health Relevance: Cellular osmotic homeostasis is a fundamental requirement for life. Studies described in this application will define the signals and signaling pathways that regulate expression of genes required for survival of cells in hypertonic environments and will provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. Detailed understanding of osmotic stress induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases.

Public Health Relevance

Cellular osmotic homeostasis is a fundamental requirement for life. Studies described in this application will define the signals and signaling pathways that regulate expression of genes required for survival of cells in hypertonic environments and will provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. Detailed understanding of osmotic stress induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK061168-13
Application #
8420529
Study Section
Special Emphasis Panel (ZRG1-DKUS-B (02))
Program Officer
Ketchum, Christian J
Project Start
2001-09-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
13
Fiscal Year
2013
Total Cost
$352,065
Indirect Cost
$153,830
Name
Mount Desert Island Biological Lab
Department
Type
DUNS #
077470003
City
Salsbury Cove
State
ME
Country
United States
Zip Code
04672
Burkewitz, Kristopher; Choe, Keith P; Lee, Elaine Choung-Hee et al. (2012) Characterization of the proteostasis roles of glycerol accumulation, protein degradation and protein synthesis during osmotic stress in C. elegans. PLoS One 7:e34153
Burkewitz, Kris; Choe, Keith; Strange, Kevin (2011) Hypertonic stress induces rapid and widespread protein damage in C. elegans. Am J Physiol Cell Physiol 301:C566-76
Przybysz, Aaron J; Choe, Keith P; Roberts, L Jackson et al. (2009) Increased age reduces DAF-16 and SKN-1 signaling and the hormetic response of Caenorhabditis elegans to the xenobiotic juglone. Mech Ageing Dev 130:357-69
Choe, Keith P; Przybysz, Aaron J; Strange, Kevin (2009) The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans. Mol Cell Biol 29:2704-15
Falin, Rebecca A; Morrison, Rebecca; Ham, Amy-Joan L et al. (2009) Identification of regulatory phosphorylation sites in a cell volume- and Ste20 kinase-dependent ClC anion channel. J Gen Physiol 133:29-42
Choe, Keith P; Strange, Kevin (2008) Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response. Am J Physiol Cell Physiol 295:C1488-98
Strange, Kevin (2008) Authorship: why not just toss a coin? Am J Physiol Cell Physiol 295:C567-75
Strange, Kevin (2007) Revisiting the Krogh Principle in the post-genome era: Caenorhabditis elegans as a model system for integrative physiology research. J Exp Biol 210:1622-31
Strange, Kevin; Christensen, Michael; Morrison, Rebecca (2007) Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies. Nat Protoc 2:1003-12
Choe, Keith P; Strange, Kevin (2007) Molecular and genetic characterization of osmosensing and signal transduction in the nematode Caenorhabditis elegans. FEBS J 274:5782-9

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