Abstract 478: Development Of A Novel Technique To Visualize In Vivo Renin Activity And Assess Its Use In The Study Of The Pathogenesis Of Diabetic Nephropathy
Introduction and aims: Although systemic renin activity is suppressed in diabetes, a renin-angiotensin system (RAS) inhibitor exerts renoprotective effects. Activation of local/tissue RAS, therefore, appears to be involved in the pathogenesis of diabetic nephropathy. We attempted to develop a technique to visualize tissue renin activity in vivo using the fluorescence resonance energy transfer (FRET) system and multiphoton laser microscopy (MP-LM). In the FRET peptide, the fluorescence of 5-fluorescein amidite (FAM) is intramolecularly quenched. After cleavage into two fragments by renin, the fluorescence of 5-FAM is recovered and can be visualized at excitation/emission wavelengths of 490/520 nm.
Methods and results: Diabetes was induced with streptozotocin (STZ) in C57B6/ mice, followed by aliskiren (Alis: 25 mg/kg/day with osmotic pump) or valsartan (Val: 15 mg/kg/day by lavage) treatment for 4 weeks starting at 6 weeks after STZ injection. Diabetic mice (DM) showed higher urinary albumin excretion (UAE; control: 85±15 μg/day, DM: 220±25 μg/day, P<0.05). Alis or Val treatment significantly reduced UAE (Alis: 120±21 μg/day, Val: 135±18 μg/day). Renin activity in the kidney assessed by the FRET system was markedly enhanced in the glomeruli in DM (1.7±0.2-fold that in controls, P<0.05). Alis reduced plasma and renal renin activity to baseline levels. Production of reactive oxygen species (ROS) and nitric oxide (NO) was evaluated by MP-LM using dichlorofluorescin and diaminorhodamine-4M acetoxymethyl ester. Increased ROS production and diminished bioavailable NO were noted in the glomeruli in DM, but were significantly ameliorated with Alis or Val treatment. Glomerular permeability of macromolecules was visualized in vivo using MP-LM with 70-kDa dextran. Glomerular permeability was significantly greater in DM than in the controls and was ameliorated by Alis or Val treatment.
Conclusions: We developed an in vivo imaging technique to visualize renin activity in living animals. Alis inhibited both plasma and tissue renin activity, ameliorated tissue oxidative stress, and attenuated UAE in a diabetes model. The in vivo imaging technique with the FRET technique will provide a novel approach to study the pathophysiology of kidney diseases.
Author Disclosures: M. Satoh: B. Research Grant (includes principal investigator, collaborator, or consultant and pending grants as well as grants already received); Modest; Grant-in-Aid for Challenging Exploratory Research. C. Other Research Support (includes receipt of drugs, supplies, equipment or other in-kind support); Significant; Novartis Pharmaceuticals Japan. K. Kidokoro: B. Research Grant (includes principal investigator, collaborator, or consultant and pending grants as well as grants already received); Modest; Kawasaki Foundation for Medical Science and Medical Welfare. N. Kashihara: B. Research Grant (includes principal investigator, collaborator, or consultant and pending grants as well as grants already received); Modest; Uehara Memorial Foundation. B. Research Grant (includes principal investigator, collaborator, or consultant and pending grants as well as grants already received); Significant; Grant-in-Aid for Challenging Exploratory Research. D. Speaker (includes speakers bureau, symposia, and expert witness); Significant; Novartis Pharma, Daiichi-sankyo, Takeda Pharmaceutical, MSD.
- © 2014 by American Heart Association, Inc.