Kidney Int

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Kidney Int. were reversed with the help of exogenous recombinant ezrin. Recombinant crazy type ezrin significantly reduced the level of sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may improve mitochondrial function. In conclusion, the cytoskeletal linker protein SR3335 ezrin plays a significant part in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecules open construction (traditional linker features) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium level of sensitivity. for 30 min, washed, and resuspended in Weinbergs answer A (39). The tubules were homogenized in Sucrose HEPES buffer and centrifuged at 2500 RPM for 10 min. Mitochondria were harvested from supernatant by centrifugation at 11000g for 10 min at 4C. Specific Ezrin Binding to Mitochondria Mitochondria were fixed with 4% paraformaldehyde, clogged with 2% goat serum, and incubated with numerous concentrations (0.1C4.0 g) of rabbit polyclonal anti-ezrin antibody for 1 hour. After washing aside the unbound antibody (3x), the mitochondria were then incubated with FITC labeled goat anti-rabbit secondary antibody for 1 hour. After 3 washes with TBS, the fluorescence was measured by FLX800 microplate reader (bio-Tek, winoosk, VT) with excitation/emission filters arranged at 485/325. SR3335 In additional experiments, ezrin binding specificity was further tested by competition with exogenous recombinant mouse crazy type ezrin (produced in Dr. Manginos lab). Mitochondria were fixed with 4% paraformaldehyde and placed on poly-L-lysine coated slides. Then they were permeabilized (0.1% Triton X-100 and 0.1% citrate), blocked with 2% goat serum, and incubated with rabbit polyclonal anti-ezrin antibody for 1 hour followed by incubation with SR3335 Texas-red labeled goat anti- rabbit secondary antibody for 1 hour. After 3 washes in TBS, SR3335 the mitochondria were directly analyzed under a fluorescence microscope at 1000x magnification using an excitation wavelengths 596 nm and a detection wavelength of 620 nm. Control experiments were visualized without the primary antibody to measure non-specific fluorescence. The assay was carried out with and without the presence of recombinant rabbit crazy type ezrin (50 g/ml). Mitochondrial Permeability Transition Pore (mPTP) Mouse mitochondria were isolated as explained above for rabbit kidney tubules. The calcium sensitivity of the mPTP was measured by determining the threshold amount RGS7 of calcium required to open the pore as explained in detail (27). Briefly, mouse kidney mitochondria (125 g protein/ml) were added to 2 ml of reaction buffer comprising 20 mM Tris, 150 mM sucrose, 50 mM KCl, 2 mM KH2PO4, and 100 M Calcium Green dye (Sigma). The combination was softly stirred inside a cuvette at 30 C inside a Spectrofluorometer (Perkin Elmer Model LS55). Calcium chloride (0.5 M) was then administered to the cuvette incrementally in 10 l aliquots every minute and the calcium-induced fluorescence was monitored in real time. The calcium fluorescence SR3335 raises at each calcium pulse and does this inside a staircase fashion until the permeability transition pore opens and mitochondrial calcium rushes out to further drive the calcium signal upward. The time required for the mitochondria to lose the ability to maintain the fresh steady state of calcium is the point determined to be where the pore opens. This test is done for mitochondria isolated from new mouse kidneys and from kidneys chilly stored in UW answer for 24 hrs. For each mitochondrial preparation, the mPTP opening.