In vivo evaluation of renal function using intravital multiphoton microscopy on rats and mice with induced or spontaneous renal injury

Abstract

Multiphoton microscopy is a powerful tool for the in vivo imaging of renal processes thanks to the improved tissue penetration and the high spatial and temporal resolution. Intravital imaging permits to evaluate dynamic processes and pathophysiological parameters in real time using non-invasive methods. This project aims to use existing multiphoton microscopy approaches to evaluate in vivo many renal parameters in our animal models and, at the same time, to develop an original application for the assessment of single nephron glomerular filtration rate. In addition, a method for detection and quantification of renal fibrosis ex vivo was developed coupling multiphoton microscopy and machine learning-based segmentation software. The linescan-based innovative methodology offered a great improvement in terms of temporal resolution and provided reliable values of single nephron glomerular filtration rate comparable with previous methods. The validation of this approach was carried out measuring renal filtration in low-dose dopamine and ischemic treated rats, which showed significantly higher and lower values of single nephron glomerular filtration rate, respectively, compared to the control group. The dynamic process of renal glucose reabsorption was elucidated in GLUT2 cKO mice mimicking the Fanconi Bickel Syndrome. Intravital multiphoton microscopy of the renal tubules after the continuous infusion of fluorescent 2-NBDG demonstrated an impaired utilization of glucose in these mice compared to the control. We also developed a novel approach based on second harmonic generation tool provided by the multiphoton microscopy and a machine learning-based segmentation software to detect and quantify renal fibrosis in kidney slices. These multiphoton applications highlighted the great potential of intravital microscopy to elucidate in real time the mechanisms involved in renal pathophysiology at cellular and subcellular resolution