Novel fluorescence techniques to quantitate renal cell biology

Abstract

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.

Intravital imaging reveals glomerular capillary distension and endothelial and immune cell activation early in Alport syndrome

“…Slides were mounted by using DAPI-containing mounting media (VectaShield, Vector Laboratories Inc., Burlingame, CA). Sections were examined with Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany) confocal/multiphoton laser scanning microscope systems as described previously (29)(30)(31). In addition, Periodic acid-Schiff (PAS) stained mouse kidney sections (2 µm thick) were imaged and analyzed using the Leica LAS EZ 3.4 software.…”

The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing

“…Under continuous anesthesia (Isoflurane 1-2% inhalant via nose-cone), mice were placed on the stage of the inverted microscope with the exposed kidney mounted in a coverslip-bottomed chamber bathed in normal saline as described previously [29,30]. Body temperature was maintained with a homeothermic blanket system (Harvard Apparatus).…”

A new view of macula densa cell protein synthesis

“…For O-propargyl-puromycin (OPP) labeling (Thermo Fisher Scientific), mice were injected intraperitoneally with 25 mL of 20 mM OPP dissolved in DMSO for 1 h before tissue harvest and staining as previously described (28,35). For OPP labeling, sections were developed using the Alexa Fluor 594 Click-iT OPP Protein Synthesis kit (Thermo Fisher Scientific) according to the manufacturer's instructions.…”

“…Although the morphological features of MD cells suggest the presence of robust protein synthesis machinery at the basal surface (1), the relative inaccessibility of MD cells and complex architecture of the JGA has limited our understanding of the protein synthesis and secretion capacity of MD cells using conventional histology techniques. The recent development of novel fluorescence techniques including an imaging-based, single cell-level global protein synthesis assay, and transgenic mouse models (25)(26)(27)(28) has made it possible to extensively characterize and quantify specific aspects of renal cell biology.…”