Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Abstract

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman's space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman's space and all segments of the nephron within the imaging depth limit, about 100 µm below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman's space. We expect this technique to have wide applicability in renal physiologic investigation.

Multicore-fiber microendoscopy for functional cellular in-organ imaging

“…6). To investigate organs such as the kidney it is usually required to either explant and stabilize the kidney [26,45] or to use techniques that cannot resolve single cells [39,[46][47][48][49], whereas the microendoscope enables flexible and cellular-resolution imaging of the kidney in-situ.…”

“…Currently, longitudinal studies rely on solid structures, such as the skull, to mount a camera system such as the Miniscope [56] or they rely on non-invasive techniques that lack cellular resolution [47,49,57]. Alternatively, a population of animals is imaged at different time points, but each animal only once [45]. Investigating for example the progression of kidney injury, kidney disease or the effect of heart disease on the renin-angiotensin-aldosterone-system at the cellular level could lead to new insights and disease models, while reducing the number of animals required for such studies.…”

A quantitative 3D intravital look at the juxtaglomerular renin-cell-niche reveals an individual intra/extraglomerular feedback system

“…Either a chemoattractive gradient within the glomerulus towards the JGA, a cell junction mediated interconnective signaling mechanism, or an intraglomerular release of (death) molecules into Bowman space with a following interaction with the macula densa mimicking a real glomerular-tubulo-extraglomerular feedback mechanism may regulate the RCN and its responses. The model system presented here can be combined with new sophisticated technologies such as two-photon guided glomerular/tubular micropuncture and liquid chromatography/mass spectrometry of nanoliter range samples from recovered filtrates from Bowman space and segments of the nephron to answer the above questions ( Matsushita et al, 2018 ). Subsequent studies utilizing the pluripotent ( Pippin et al, 2015 ) potential of RC or further controlled intraglomerular (micro-)injection experiments ( Ikeda et al, 2017 ; Saxena et al, 2021 ) will extend the presented research of RC-mediated repair and RCN replenishment regulation via neogenesis.…”

Renal injury in cardiorenal syndrome type 1 is mediated by albumin

“…Following CA/CPR or sham procedure, general anesthesia and the jugular catheter were maintained, and mice were subjected to in vivo microscopic imaging as described previously (Matsushita et al, 2018 ). A small skin incision was made on the left flank, and the kidney was partly extruded through the muscle and subcutaneous tissue.…”