Methods in laboratory investigation: Noninvasive microscopic evaluation of the intact living nephrotic kidney

BACKGROUND: Tandem Scanning Confocal Microscopy (TSCM) is a new form of microscopy that allows one to noninvasively 'optically section' into intact structures and record microscopic images in real-time. In this study, we have evaluated the ability of this new technology to distinguish histopathologic changes in unstained living kidneys that occur coincident with the onset of puromycin aminonucleoside-induced nephrosis. EXPERIMENTAL DESIGN: The rats were anesthetized and a laparotomy was performed to expose the kidneys. Using a TSCM equipped with a 24x water-immersion objective, we viewed and recorded real-time images of subcapsular living uriniferous tubules and glomeruli of puromycin aminonucleoside-induced nephrosis rats exhibiting different levels of proteinuria. RESULTS: TSCM revealed a variety of histopathologic changes in the puromycin aminonucleoside-induced nephrosis uriniferous tubules including dilation of proximal tubular lumens, loss of microvillous brush border, cellular debris in the tubule lumens, and focal regions of extensive tubular necrosis. Using the fluorescent probe carboxyfluorescein, we were able to demonstrate significant heterogeneity in the movement of this fluorescent probe through the uriniferous tubules (e.g., some tubules were completely blocked), and irregular patterns in flow of carboxyfluorescein through nephrotic glomeruli. CONCLUSIONS: These observations indicate that TSCM is capable of revealing a variety of histopathologic changes in unstained, intact, living kidneys. In addition, many of the histopathologic features of the uriniferous tubules revealed by TSCM are difficult or impossible to distinguish in biopsied samples of renal tissue.