MO429A NOVEL METHOD LINESCAN-DRIVEN FOR SNGFR MEASUREMENTS AS ALTERNATIVE TO HIGH FULL FRAME MULTIPHOTON MICROSCOPY ACQUISITION

Abstract

Abstract Background and Aims Renal micropuncture, which requires the direct access to the renal tubules, has been for long time the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models, but this approach is challenging by virtue of complex animal preparation and numerous careful steps. The introduction of intravital multiphoton microscopy (MPM) permitted to improve the study of renal functions exploiting the high laser penetration and the optical sectioning capacity. Previous MPM studies measuring in vivo the SNGFR relied on fast full frame acquisition during the filtration process obtainable with microscope resonant scanners, which represent optional expensive equipment able to reach very high acquisition speed. In this work we propose an innovative linescan-based MPM method to calculate SNGFR in rodents doable without using the fast acquisition rate offered by resonant scanners. Method An in vivo MPM approach was used to measure the SNGFR in control Munich Wistar Frömter rats (MWF) and to test the feasibility of the innovative linescan approach. In order to validate this method in conditions known for reduced and increased SNGFR, it was applied to ischemia reperfusion injury (IRI) and low-dose dopamine treated conditions, respectively. Results The glomeruli connected to S1 proximal tubules extending at least 100 μm from the exit of the Bowman’s space were chosen for the measurement. A linescan path starting from the urinary pole and crossing many times the tubular lumen orthogonally to the cellular wall was hand drawn. The linescan was acquired soon after a i.v. bolus of low-molecular weight fluorescent marker was injected. The tubular length, the mean diameter and the transit time of the fluorescent marker within two lines of interest (called cross1 and cross2) were measured to obtain the SNGFR. SNGFR measured in control rats was comparable with previous reported data both at MPM and micropuncture. Significantly higher values compared to control were obtained in 3 μg/kg/min dopamine-treated rats. In IRI-treated rats the SNGFR was reduced about 35% compared to the controls. Conclusion The results achieved with our linescan method were quite similar to those obtained with conventional micropuncture, suggesting that the two methods overlap for the normal, dopamine and IRI treatment. Our results show that linescan approach is a promising and cheap alternative to the fast full frame acquisition for the investigation of SNGFR in health and disease, offering results comparable to conventional micropuncture with unprecedent temporal resolution.