Imaging of renal medullary interstitial cells in situ by confocal fluorescence microscopy

Abstract

 Renal medullary interstitial cells are a prevalent and characteristic feature of the inner medulla of the kidney, but the physiological significance of this is unclear. We have developed a method for imaging renal medullary interstitial cells in situ by loading the cells with fluorescent dyes and monitoring their distribution using confocal microscopy. The pH-sensitive probe 2’7’-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester was used as a marker of cytoplasmic volume and therefore of cell morphology. Nile Red was used to demonstrate the presence of renal medullary interstitial cell lipid droplets. Papillae were excised from 100 g Sprague-Dawley rats and loaded with the appropriate dye. The papillae were then examined using a Leica TCS 4D confocal microscope and oil immersion lenses. Fluorescence was excited (488 nm) using an argon laser and emission wavelengths above 515 nm collected using a long pass filter. Images of papillae loaded with 2’7’-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester clearly demonstrate a ladder-like arrangement of renal medullary interstitial cells. More detailed examination revealed the presence of cytoplasmic extensions that appear to make close contact with adjacent loops of Henle. Three-dimensional reconstructions of serial sections revealed spiral arrangements in some ladders of renal medullary interstitial cells. Nile Red-labelled lipid droplets of 0.5–1.0 µm diameter were located throughout the cytoplasm of renal medullary interstitial cells and especially within the cytoplasmic extensions. These experiments highlight the ability of confocal microscopy to allow investigation of renal medullary interstitial cells in situ.