Protective effects of ezrin on cold storage preservation injury in the pig kidney proximal tubular epithelial cell line (LLC-PK1)

Abstract

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.

Figure 1

LLC-PK Cells transfected with the plasmid pEX Ezrin-YFP express YFP and ezrin. The transfected cell line LLC-EZ expressing YFP under fluorescence microscopy (A) and sham transfected LLC-PK1 cells showing only background autofluorescence (B). A representative Western blot shows the expression of Ezrin protein after cold storage and warm reperfusion (C). There is strong expression of Ezrin protein in fresh LLC-EZ cells. The expression of ezrin decreased to 15.3% of the baseline after 24 hours of cold preservation and recovered to 27.2% of the baseline after 1 hour of warm reperfusion. Comparatively little expression of Ezrin protein was observed in any of the LLC-PK cells.

Figure 2

Progressive cold storage and reperfusion caused a storage time dependent loss of cell viability, which was further dependent on the quality of the organ preservation solution used (UW Vs DMEM). Over expression of ezrin (LLC-EZ cell line) significantly increased cell viability as assessed by LDH release after reperfusion (A), by WST-1 dye conversion (B), and by intracellular ATP regeneration after reperfusion (C). Values are mean ± SD from 4 independent experiments. * P< 0.05 between the LLC-PK1 controls and the corresponding values from the LLC-EZ cell line.

Figure 3

Over expression of Ezrin protein protects cells from apoptosis during cold preservation and warm reperfusion. Apoptosis rates of LLC-PK and LLC-EZ cells before storage (fresh), after 24 h of cold storage, and after 1 h of warm reperfusion (A, 120x). Positive control LLC-PK1 cells treated with Staurosporin (B, 480x). TUNEL positive labeling shows red color with blue DAPI staining of nuclei as a counter stain.

Figure 4

MitoTracker Red dye (M7512, red) and YFP (green) fluorescence of fresh epithelial cells (A) and epithelial cells after 24 hrs of cold storage and 1 hr of reperfusion (B). The LLC-EZ cells show strong MitoTracker Red staining after cold storage and warm reperfusion, whereas the non-transfected cells show almost no signal. The co-localization and proportionality of the MitoTracker Red and the YFP fluorescent signals in the cold stored LLC-EZ cells (B-Left, arrows) suggests an ezrin-induced protection of the inner mitochondrial potential required for mitochondrial function and ATP synthesis.

Figure 5

Transmission electron microscopy (TEM) of fresh LLC-PK cells before cold storage (left), LLC-PK cells after 8 hrs of cold storage (middle), and TEM of ezrin over expressing LLC-EZ cells after 8 hrs of cold storage preservation (right). Microvilli details of the plasma membrane are featured in panel-A and show loss of brush border after cold storage in LLC-PK cells that is largely prevented in cells that over express ezrin. Arrows show cell microvilli. Similarly, mitochondria (panel-B) are shrunken, electron dense, and some show signs of cristalysis after 8 hrs of cold storage in control LLC-PK1 cells (B, middle). However, the mitochondrial changes are much milder in the ezrin over expressing LLC-EZ cells after cold storage (B, right). Many of their mitochondria appear normal with fewer electron dense structures. The mitochondrial structural preservation associated with ezrin over expression is consistent with the changes observed in mitochondrial function in these cells. Arrows show mitochondria.