Myristic Acid-Trans-Activator of Transcription Dual Conjugation Enhances Intracellular delivery of protein kinase C beta II peptide inhibitor for concentration-dependent attenuation of superoxide release in isolated rat polymorphonuclear leukocytes

Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previously, myristic acid conjugated (or myristoylated) PKCβII inhibitor (myr-PKCβII-) significantly attenuated PMN SO release when dual conjugated to myristic acid and Trans-activator of transcription (myr-Tat-PKCβII-; N-myr-Tat-CC-SLNPEWNET) compared to myr-conjugation alone. However, the optimal concentration of myr-Tat-PKCβII- has yet to be determined. We hypothesized that myr-Tat conjugation would enhance the intracellular delivery of PKCβII- cargo and attenuate SO release in a concentration-dependent manner while retaining greater cell viability at lower concentrations. This study tested the concentration-dependent effects of myr-Tat-PKCβII- on SO release and cell viability compared to myr-Tat-PKCβII scrambled (myr-Tat-PKCβII- scram; N-myr-Tat-CC-WNPESLNTE), unconjugated PKCβII-, and untreated control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII- (20μM), myr-Tat-PKCβII- (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr-Tat-PKCβII-scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after phorbol-12-myristate-13 acetate (PMA) stimulation (100nM). Intracellular delivery was evaluated by the magnitude of PMA-induced PMN SO release attenuation with the PKCβII- cargo. Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis. Myr-Tat toxicity occurs at concentrations higher than 2 μM. Myr-Tat PKCβII- significantly decreased SO release compared to control while retaining similar cell viability at 5 μM (n=15, 0.384±0.03) and 7.5μM (n=11, 0.391±0.05) concentrations. Myr-Tat PKCβII-scram 5μM (n=7, 0.44±0.07) and 7.5μM (n=5, 0.409±0.11) were not different from control. Results suggest that Myr-Tat-PKCβII- 5μM significantly attenuates PMA-induced PMA SO release while retaining cell viability at \u3e 80%. Future studies will assess the effects of myr- or myr-Tat-PKCβII- peptides on PKCβII- translocation activity

Renal function in a mouse model of acute kidney injury is improved by myristoylated protein kinase c epsilon inhibitor

Introduction: Delayed graft function (DGF) is a post-transplant acute kidney injury that is caused by prolonged ischemia resulting in oxidative stress-mediated damage during reperfusion. Previously, myr-PKCε- reduced infarct size and serum H2O2 when administered upon reperfusion in an ex vivo rat heart ischemia-reperfusion (I/R) model and in vivo hindlimb I/R model, respectively. Myristoylated protein kinase C epsilon peptide inhibitor (N-myr-EAVSLKPT; myr-PKCε-) is known to confer protection by inhibiting superoxide production from uncoupled endothelial nitric oxide synthase and mitochondrial ATP-sensitive K+ channels. We hypothesized myr-PKCε- would attenuate renal injury, characterized by elevated serum creatinine (Cr) and decreased glomerular filtration rate (GFR), compared to a scrambled control peptide (N-myr-LSETKPAV; myr-PKCε-scram). Methods: Renal pedicles of male C57BL/6J mice (25–30g) were clamped bilaterally for 19 min. Myr-PKCε- or myr-PKCε-scram (1.6 mg/kg; 20 µM blood) were administered into the tail vein 1 min before unclamping. Cr was measured at baseline, 24h, 72h, and 96h post-injury. GFR was determined with fluorescein-isothiocyanate (FITC)-Sinistrin renal clearance. Data were evaluated by unpaired Student’s t-test. Results: This bilateral 19 min renal ischemia resulted in significant GFR reduction (Fig. 1) and Cr elevation (Fig. 2) throughout the post-ischemic time-course. Myr-PKCε- (n=6) significantly improved both GFR and Cr at 72h and 96h compared to myr-PKCε-scram control (n=4, p\u3c0.05). Conclusions: Results support the hypothesis that Myr-PKCε- improves kidney function after 19-min warm ischemic injury. Immunolabeling of PKCε, Kim-1, and NGAL biomarkers will be used to further evaluate PKCε localization and the extent of Myr-PKCε- protection against renal tubular damage

Myristoylated Protein Kinase C Beta II Inhibitor Attenuates Severe Acute Kidney Injury Induced by Ischemia-Reperfusion

INTRODUCTION: Acute kidney injury (AKI) due to ischemia-reperfusion (I/R) insult involves oxidative stress and inflammation leading to rapid renal decline. In this study, we test a novel myristoylated protein kinase C beta II peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-), known to attenuate ex vivo rat myocardial I/R injury in a murine model of renal ischemia. We aim to demonstrate the attenuation of renal I/R injury by myr-PKCβII- in comparison to scrambled control peptide (N-myr-WNPESLNTE; myr-PKCβII-scram) by quantifying serum creatinine (Cr) and glomerular filtration rate (GFR). METHODS: Renal pedicles of anesthetized male C57BL/6J mice were clamped bilaterally for 20-min or 19-min. Five minutes before unclamping, 2.0 mg/kg (20 µM serum) myr-PKCβII- or myr-PKCβII-scram were given IV into the tail vein. Cr was measured at baseline, 24h, 72h, and 96h post-injury. GFR was determined with fluorescein-isothiocyanate (FITC)-Sinistrin renal clearance. Data were evaluated by unpaired Student’s t-test. RESULTS: Following 20-min renal ischemia, Myr-PKCβII- (n=9) significantly reduced Cr at 24h and 72h post-injury compared to myr-PKCβII-scram (n=8; p\u3c0.05). Three unexpected fatalities followed 20-min ischemia, but not 19-min ischemia. Serum Cr levels modestly increased following 19-min renal I/R and were similar for Myr-PKCβII-scram control and Myr-PKCβII- treated groups. Compared to myr-PKCβII-scram control, Myr-PKCβII- demonstrated a trend towards improved GFR at 72hr (130 ± 28 vs 96 ± 44 µl/ml; p=0.065) and 96 hr (133 ± 33 vs 113 ± 50 µl/ml; p=0.095) post-I/R injury. DISCUSSION: Results suggest 20-min renal ischemia was more severe, indicated by a 5-fold increase in Cr at 72h post-injury compared to 19-min ischemia and unanticipated fatalities of three mice. Myr-PKCβII- attenuated renal injury following 20-min renal ischemia, but not after 19-min in which Cr levels were too low to detect therapeutic benefit. The difference in injury severity between 20-min and 19-min renal ischemia emphasizes the temporal relationship between renal function and ischemic duration

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

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\u3c0.05). 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 \u3c 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