Conventional and Confocal Epi-Reflection and Fluorescence Microscopy of the Rat Kidney in vivo

Abstract

To visualize superficial and accessible renal tubule cells functioning in situ and to relate what we can ‘see’ to what we know of their function from more invasive in vivo or less direct in vitro studies means applying and adapting recent advances in epifluorescence and confocal microscopy to improve image resolution and to combine this with the use of fluorescent labels to monitor the handling of specific molecules by the proximal and distal renal tubule cells in vivo. Doing this in living tissue is novel, especially in the kidney. Application of confocal microscopy to the imaging of living tissue, as opposed to isolated cells, has not been widely reported. The kidney surface has been imaged before using the confocal microscope and in preliminary studies we have extended this by using a different confocal system with and without fluorescence. While the studies published up to now have been morphological, comparing standard renal (structural) histology of surface glomeruli and renal tubules with the corresponding in vivo confocal images, more dynamic, real-time studies have been limited. Individual red blood cells can be seen flowing around the peritubule capillary network and nucleated white blood cells can also be distinguished. Tubule cells, endothelial cells, the proximal tubule cell brush border and cell mitochondria can be visualized. Filtration and secretion can be observed, and the early and late parts of the proximal tubule distinguished, and the distal tubule recognized. Localization of fluorescently labeled insulin to the luminal brush border and progressive uptake of label and distribution within proximal tubule cells toward the basolateral (blood side) membrane can be demonstrated. The possibility of monitoring hemodynamic changes and tracking the filtration, uptake, secretion and absorption of fluorescently tagged molecules, as well as intracellular fluorescence, e.g. calcium or pH, is an exciting prospect and is ripe for detailed exploration.

Molecular Aspects of Renal Anionic Drug Transport

“…Intracellular concentrations of three to five times the medium concentration were determined (142). Fluorescein accumulation in proximal tubules during secretory transport has been confirmed in rat kidney in vivo (143). Confocal microscopic images of rat proximal tubule cells showed that fluorescein was compartmentalized within subcellular organelles, predominantly in mitochondria.…”

The use of microcomputed tomography to study microvasculature in small rodents

“…In the kidney, the capillary network of the glomerulus (50) and its circulation (70) have been studied. Boyde et al (15) were able to visualize individual red blood cells and white blood cells as they flowed through the peritubular capillary circulation near the surface of living kidneys. However, the maximum depth of focus for all of these studies is Ͻ1 mm and, consequently, in a whole kidney that has greater tissue depth, continuous measurement from the arterial trunk to the downstream capillaries has not been possible.…”

In vivo Confocal Laser Scanning Microscopy and Micropuncture in Intact Rat

“…Second, organ movements caused by microcirculation and respiration may create problems, especially at higher magnifications. Few organs have been visualized by this technique: brain [8], spleen [9], skin [10], cornea [11], lung [12], and kidney [13]and most of them for studies of morphological changes and hemodynamics in the intact organ. Recently, Dunn et al [14]successfully studied the dynamics of receptor-mediated endocytosis in intact kidney by two-photon laser scanning microscopy (TPLSM) demonstrating individual endosomes containing fluorescence-labeled dextran and gentamicin.…”