Real-time Imaging of Ca-handling in Intact Renal Glomeruli Using Confocal Microscopy.

Glomeruli are filtering units in the kidneys. Being multicellular and complex in structure, many aspects of glomerular function are yet to be elucidated. Most studies use glomerular cells in culture, which may exhibit altered physiology compared to native cells. Confocal microscopy has opened new avenues in exploring in situ glomerular function and physiology. In this report, we propose experimenting with glomerular cells in renal cortical slices and isolated intact glomeruli for Ca(2+)-handling studies. Cortical slices (100 μm thick) were obtained from mice while intact glomeruli were isolated from rats using the sieving method. These were loaded with fluo-4 and then placed in a confocal microscope. Fluo-4 was excited using a 488 nm photodiode laser and images were collected at 1 frame/sec. Changes in average fluorescence intensity (AFI) were interpreted as changes in [Ca(2+)](i). AFI increased to 37.1 ± 6.7% and 84.3 ± 20.9% with Ang II (0.01 and 0.1 μM respectively). Norepinephrine (10 μM), arginine vasopressin (0.1 μM) and K(+) (30 mM) also elevated AFI by 26.5 ± 6.8%, 22.3 ± 1.0% and 39.8 ± 10.3% respectively in the glomerular cells. Likewise in isolated glomeruli, Ang II (0.1-10 μM), K(+) (30-90 mM) and endothelin-1 (0.01-1 μM), all showed elevation in [Ca(2+)](i). These results give an impetus for future studies examining Ca(2+)-handling by confocal microscopy in glomerular cells using renal cortical slices and isolated intact glomeruli. The results support the utility of this system for study of glomerular physiology and pharmacology.