Intravital Multiphoton Microscopy as a Tool for Studying Renal Physiology, Pathophysiology and Therapeutics

Abstract

Intravital multiphoton microscopy has empowered investigators to study dynamic cell and subcellular processes in vivo within normal and disease organs. Advances in hardware, software, optics, transgenics and fluorescent probe design and development have enabled new quantitative approaches to create a disruptive technology pioneering advances in understanding of normal biology, disease pathophysiology and therapies. Offering superior spatial and temporal resolution with high sensitivity, investigators can follow multiple processes simultaneously and observe complex interactions between different cell types, intracellular organelles, proteins and track molecules for cellular uptake, intracellular trafficking, and metabolism in a cell specific fashion. The technique has been utilized in the kidney to quantify multiple dynamic processes including capillary flow, permeability, glomerular function, proximal tubule processes and determine the effects of diseases and therapeutic mechanisms. Limitations include the depth of tissue penetration with loss of sensitivity and resolution due to scattered emitted light. Tissue clearing technology has virtually eliminated penetration issues for fixed tissue studies. Use of multiphoton microscopy in preclinical animal models offers distinct advantages resulting in new insights into physiologic processes and the pathophysiology and treatment of diseases.

A unique subset of pericystic endothelium associates with aberrant microvascular remodelling and impaired blood perfusion early in polycystic kidney disease

“…It would be prudent to apply such techniques to future clinical trials of ADPKD, monitoring the response of intrinsic renal blood flow to pharmacological agents. To mitigate the limitations of MRI, such as partial volume effect due to averaging ‘zero’ flow regions of epithelial cysts within renal blood flow measurements, alternative and emerging techniques, such as photoacoustic imaging (27) or multiphoton microscopy (95), could be used to non-invasively assess kidney vasculature paired with novel 3D analysis approaches to assess vascular patterning described in this paper. Our findings also have therapeutic implications.…”

Lrpap1 (RAP) Inhibits Proximal Tubule Clathrin Mediated and Clathrin Independent Endocytosis, Ameliorating Renal Aminoglycoside Nephrotoxicity

“…Notably, no S3 PTs extend to the outer cortex accessible to the two-photon imaging performed here, which was limited to approximately 50 μ m deep for quantitation. 19 , 20 In addition, identification of different tubule types is also possible due to the endogenous autofluorescence and apical brush border membrane present in the PTs but lacking from collecting ducts or distal tubules (dt). 20 , 21 To examine any reduction in the uptake of the markers because of RAP administration, regions containing glomeruli and S1 segments were selected for study and marked, with images obtained at time points of 15, 30, and 60 minutes after bolus infusion of the two markers.…”

“… 19 , 20 In addition, identification of different tubule types is also possible due to the endogenous autofluorescence and apical brush border membrane present in the PTs but lacking from collecting ducts or distal tubules (dt). 20 , 21 To examine any reduction in the uptake of the markers because of RAP administration, regions containing glomeruli and S1 segments were selected for study and marked, with images obtained at time points of 15, 30, and 60 minutes after bolus infusion of the two markers. Fluorescence from the images was quantified and normalized to the highest average value in the time series.…”