Myristoylated Protein Kinase C Epsilon Peptide Inhibitor (Myr-PKC ε-) mitigates renal ischemia-reperfusion injury and PKCε translocation to epithelial cell membranes in vivo

Philadelphia, PA

Introduction:

Delayed graft function (DGF), a post-transplant acute kidney injury, occurs in one-third (~7000) of the total number of kidney transplant recipients (~20,000) in the U.S. annually. It is characterized by prolonged ischemia (~20 mins) and subsequent restoration of blood flow to the previously ischemic renal tissue. Collectively, this is described as renal ischemia-reperfusion (I/R) injury. During reperfusion of blood to the previously ischemic renal tissue, generation of reactive oxygen species (ROS) from mitochondrial and uncoupled endothelial nitric oxide synthase (eNOS) is known to contribute to increased ROS-induced infarct size during reperfusion in myocardial I/R. Previously, Myr-PKCε- when given i.v. at the onset of reperfusion, decreased infarct size in both in vivo and ex vivo myocardial I/R models. Myr-PKCε- also decreased serum H2O2 in vivo in both renal extracorporeal shock wave lithotripsy and in a hindlimb I/R model. Myr-PKCε-(N-myr-EAVSLKPT) is known to have a protective effect by inhibiting superoxide production from uncoupled eNOS and mitochondrial ATP-sensitive K+ channels. We hypothesized that Myr-PKCε- would mitigate murine renal I (19min bilateral)/R(96 hrs.) injury and PKCε expression in renal tubular epithelium when given i.v. at the onset of reperfusion compared to scrambled control peptide (N-myr-LSETKPAV; Myr-PKCε-scram).

Methods:

Renal pedicles of anesthetized male C57BL/6J mice (25–30g) were clamped bilaterally for 19 mins. One minute before unclamping, 1.6 mg/kg (~20 µM serum concentration) Myr-PKCε- (n=6) or Myr-PKCε-scram (n=7) was administered by tail vein injection. Serum creatinine (Cr) (mg/dL) was measured at baseline, 24hrs, 72hrs, and 96hrs post-injury. Glomerular filtration rate (GFR) (µl/min) was measured via fluorescein-isothiocyanate (FITC)-Sinistrin. At the conclusion of the experiment, mice kidneys were removed, sectioned, formalin fixed and paraffin embedded. Immunohistochemistry (IHC) was performed using a PKCε antibody to evaluate PKCε localization and samples were analyzed using Aperio ImageScope.

Results:

Myr-PKCε- (n=6) significantly improved both GFR and Cr throughout reperfusion compared to Myr-PKCε-scram control (n=7, p<0.05). Myr-PKCε- significantly improved both GFR and Cr throughout the 96 hrs reperfusion period compared to myr-PKCε-scram control. Myr-PKCε- restored final GFR and Cr to 52% and 54% vs. myr-PKCε-scram 29% and 18% respectively, compared to initial baseline values. IHC staining of kidney sections following I/R, diaminobenzidine chromogen reaction resulted in a brown precipitate indicating detection of PKCε and was defined to be a positive signal. All other signals were defined to be negative. Myr-PKCε- (1.77x108 ± 3.14x107) resulted in a significant decrease in the number of positive signals, in whole-kidney samples compared to Myr-PKCε-scram (3.58x10-1±5.03x10-2) (p < 0.05). There was no difference in the number of negative signals or total number of signals between Myr-PKCε- and Myr-PKCε-scram.

Discussion:

Results suggest that Myr-PKCε- improved post-reperfused kidney function following bilateral renal I(19min)/R(96 hrs) ischemia and attenuated PKCε localization in tubular epithelium compared to Myr-PKCε-scram. In future studies, PKCε IHC staining and analysis will be performed on pig heart samples that have undergone I/R injury.