Protective effects of ezrin on cold storage preservation injury in the pig kidney proximal tubular epithelial cell line (LLC-PK1).
BACKGROUND
Renal damage caused by cold preservation and warm reperfusion has been well documented and involves tissue edema, cell swelling, ATP depletion, calcium toxicity, and oxidative stress. However, more common proximal mechanisms have not been identified, which may limit the development of effective clinical treatment strategies. Previous work indicates that many cytoskeletal structures are affected by cold preservation and reperfusion, including membrane-rich ezrin-associated complexes. The aim of this study was to investigate whether the sublamellar cytoskeletal protein ezrin is causally involved in cold preservation injury in renal tubule epithelial cells.


METHODS
We created a stably transfected cell line (LLC-EZ) using the pig kidney proximal tubular epithelial cell line (LLC-PK1), which constitutively overexpresses wild-type ezrin. These cells were cold stored in University of Wisconsin Solution and reperfused in vitro to model renal tubule preservation injury, which was assessed by biochemical, metabolic, functional, and structural endpoints.


RESULTS
Overexpression of ezrin increased cell viability (lactate dehydrogenase release), mitochondrial activity (ATP synthesis, dehydrogenase activity, and inner mitochondrial membrane potential), and protected the structure of cell membrane microvilli and mitochondria after cold storage preservation injury. Reperfusion-induced apoptosis was also significantly reduced in LLC-EZ cells overexpressing ezrin.


CONCLUSIONS
Enhanced ezrin expression protects tubule epithelial cells from cold storage preservation injury, possibly by membrane or mitochondrial mechanisms.
Tian
Tao
T
Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, USA.
Lindell
Susanne L
SL
Henderson
Scott C
SC
Mangino
Martin J
MJ
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D003080:Cold Temperature; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000627:therapeutic use*; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology*; D007668:Kidney; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D051379:Mice; D008928:Mitochondria / Q000502:physiology / Q000648:ultrastructure; D015427:Reperfusion Injury / Q000517:prevention & control*; D013552:Swine; D014021:Tissue Preservation / Q000379:methods*; D014162:Transfection; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D000099149:Ezrin