Document Detail


Ezrin functionality and hypothermic preservation injury in LLC-PK1 cells.
MedLine Citation:
PMID:  22554620     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Renal epithelial cells from donor kidneys are susceptible to hypothermic preservation injury, which is attenuated when they over express the cytoskeletal linker protein ezrin. This study was designed to characterize the mechanisms of this protection. Renal epithelial cell lines were created from LLC-PK1 cells, which expressed mutant forms of ezrin with site directed alterations in membrane binding functionality. The study used cells expressing wild type ezrin, T567A, and T567D ezrin point mutants. The A and D mutants have constitutively inactive and active membrane binding conformations, respectively. Cells were cold stored (4 °C) for 6-24 h and reperfused for 1h to simulate transplant preservation injury. Preservation injury was assessed by mitochondrial activity (WST-1) and LDH release. Cells expressing the active ezrin mutant (T567D) showed significantly less preservation injury compared to wild type or the inactive mutant (T567A), while ezrin-specific siRNA knockdown and the inactive mutant potentiated preservation injury. Ezrin was extracted and identified from purified mitochondria. Furthermore, isolated mitochondria specifically bound anti-ezrin antibodies, which were reversed with the addition of exogenous recombinant ezrin. Recombinant wild type ezrin significantly reduced the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may modify mitochondrial function. In conclusion, the cytoskeletal linker protein ezrin plays a significant role in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecule's open configuration (traditional linker functionality) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium sensitivity.
Authors:
Tao Tian; Susanne L Lindell; Melody Lam; Martin J Mangino
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-04-23
Journal Detail:
Title:  Cryobiology     Volume:  65     ISSN:  1090-2392     ISO Abbreviation:  Cryobiology     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-06-04     Completed Date:  2012-12-19     Revised Date:  2013-08-14    
Medline Journal Info:
Nlm Unique ID:  0006252     Medline TA:  Cryobiology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  60-7     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
Affiliation:
Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Membrane Permeability
Cell Survival / physiology
Cells, Cultured
Cold Temperature
Cytoskeletal Proteins / genetics,  metabolism,  physiology*
Epithelial Cells / cytology,  metabolism,  physiology*
Gene Knockdown Techniques
Hypothermia, Induced / methods*
LLC-PK1 Cells
Mitochondria / metabolism
Mitochondrial Membrane Transport Proteins / metabolism*
Organ Preservation / methods
Point Mutation
Reperfusion Injury / prevention & control*
Swine
Grant Support
ID/Acronym/Agency:
R01 DK087737/DK/NIDDK NIH HHS; R01-DK-087737/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Cytoskeletal Proteins; 0/Mitochondrial Membrane Transport Proteins; 0/ezrin; 0/mitochondrial permeability transition pore
Comments/Corrections
Comment In:
Cryobiology. 2013 Apr;66(2):93   [PMID:  23261414 ]
Cryobiology. 2013 Apr;66(2):94   [PMID:  23274401 ]

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