(963.4) Myristoylated Protein Kinase C Beta II Inhibitor Attenuates Renal Injury in Mice Subjected to Severe Bilateral Ischemia-Reperfusion

Poster Board Number: E644

Lisa Shah (Philadelphia College of Osteopathic Medicine), Devani Johnson (Philadelphia College of Osteopathic Medicine), Alexis Verwoert (Philadelphia College of Osteopathic Medicine), Sunit Singh (Philadelphia College of Osteopathic Medicine), Tameka Dean (Philadelphia College of Osteopathic Medicine), Qian Chen (Philadelphia College of Osteopathic Medicine), Robert Barsotti (Philadelphia College of Osteopathic Medicine), Yanlin Jiang (University of Alabama at Birmingham School of Medicine), James George (University of Alabama at Birmingham School of Medicine), Anupam Agarwal (University of Alabama at Birmingham School of Medicine), Lindon Young (Philadelphia College of Osteopathic Medicine, Young Therapeutics, LLC)

Acute kidney injury (AKI) due to ischemia-reperfusion (I/R) insult involves oxidative stress and inflammation leading to rapid renal decline and remains a significant cause of post-operative mortality. Myristoylated protein kinase C beta II peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-) is known to attenuate myocardial I/R injury in ex vivo rat hearts. We hypothesized that myr-PKCβII- would attenuate severe renal I/R injury that is characterized by elevated serum creatinine (Cr) and a decrease in glomerular filtration rate (GFR). We predict that treatment with myr-PKCβII- will improve these indices of kidney function compared to a scrambled control peptide (N-myr-WNPESLNTE; myr-PKCβII-scram).

Renal pedicles of anesthetized male C57BL/6J mice (25–30g) 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 i.v. into the tail vein. Cr (mg/dL) was measured at baseline, 24h, 72h, and 96h post-injury. GFR (µl/min) was determined with fluorescein-isothiocyanate (FITC)-Sinistrin renal clearance. Data were evaluated by unpaired Student’s t-test.

Following 20-min renal ischemia (Fig 1.), myr-PKCβII- (n=9) significantly reduced Cr at 24h and 72h post-injury compared to myr-PKCβII-scram control (n=8, plt;0.05). However, there were three fatalities prior to 96h. In 19-min ischemia, there were no fatalities up to 96h post-injury. However, Cr levels of the myr-PKCβII-scram control (n=8) in 19-min I/R were significantly lower than 20-min I/R at all time points post-injury (all plt;0.01): 24h (0.54 ± 0.10 vs 1.59 ± 0.06 mg/dL), 72h (0.24 ± 0.06 vs 1.28 ± 0.25 mg/dL), and 96h (0.17 ± 0.03 vs 0.72 ± 0.15 mg/dL). Myr-PKCβII- did not significantly reduce Cr (Fig. 1) nor improve GFR (Fig. 2) following 19-min I/R.

Results suggest that 20-min renal ischemia was more severe, which was indicated by a 5-fold increase of peak Cr levels at 72h post-injury compared to 19-min ischemia and the unanticipated fatalities of three mice. Myr-PKCβII- attenuated renal injury following 20-min renal ischemia, but not at the milder 19-min model in which peak Cr levels were too low to detect therapeutic benefit. The large difference in injury severity between 20-min and 19-min renal ischemia emphasizes the temporal relationship between renal function and ischemic duration.

Future studies will characterize myr-PKCβII- protection against AKI in 19-min bilateral renal ischemia based on biomarkers indicating proximal renal tubule damage (e.g. NGAL and Kim-1). A more optimal ischemic duration between 19 min and 20 min will be investigated for future experiments.

This research was supported by the Center of Chronic Disorders of Aging and the Division of Research at Philadelphia College of Osteopathic Medicine, and Young Therapeutics, LLC. NIH funding was provided by the National Institute of Diabetes and Digestive and Kidney Diseases Grant #1R43DK121626-01A1.





Creatinine Timecourse of 19 min and 20 min Myr-PKCβII Inhibitor vs Myr-PKCβII Scram Control in a Murine Bilateral Renal I/R Model. Serum creatinine was measured from retro-orbital blood samples taken at baseline, 24h, 72h, and 96h post-injury. * p<0.05, 20-min Myr-PKCβII- vs Myr-PKCβII Scram Control; # p<0.05, 20-min Myr-PKCβII Scram Control vs 19-min Myr-PKCβII Scram Control.; Glomerular filtration rate (GFR) Timecourse of 19 min Myr-PKCβII Inhibitor vs Myr-PKCβII Scram Control in a Murine Bilateral Renal I/R Model. GFR levels were calculated using FITC-Sinistrin renal clearance. The sample size for Myr-PKCβII Scram Control was n=7 at 96h, due to malfunctioning transdermal monitors.