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

Abstract

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.

Figure 1. Emitted and transmitted images showing different areas in a glomerulus

The glomerulus is captured in a mouse renal cortical slice (100 μm thick) surrounded by renal tubules (see the transmitted image). The slice was loaded with a Ca²⁺ indicator dye (fluo-4, 7 μM), then visualized under a 40× oil-immersion objective of a confocal laser scanning microscope (white scale bar = 50 μm).

Figure 2. Angiotensin II (Ang II) elevated [Ca²⁺]_i in renal glomerular cells

A representative tracing showing a glomerular cell, A without and B with Ang II (0.01 μM) treatment that results in an increase in average fluorescence intensity (AFI) or [Ca²⁺]_i in response to the agonist. C shows a line graph of the increased AFI in response to Ang II (0.01 μM).

Figure 3. Increase in fluorescence intensity by different agonists

Tracings showing change in average fluorescence intensity evoked by norepinephrine (NE), arginine vasopressin (AVP) and K⁺ [concentrations indicated] in fluo-4 loaded murine whole kidney slices via confocal microscopy.

Figure 4. Pooled data for effect of different agonists on [Ca²⁺]_i

Mean changes in average fluorescence intensity (AFI) evoked by angiotensin II (n=6), norepinephrine (n=5), arginine vasopressin (n=4) and K⁺ (n=5) in fluo-4 loaded murine whole kidney cortical slices recorded via confocal microscopy (values shown are mean ± SEM; *P < 0.05, Student’s t-test).

Figure 5. Images showing an isolated rat glomerulus

A shows a comparison of the emitted (fluorescent) and transmitted (laser) images of an isolated decapsulated rat glomerulus at rest. The glomerulus was loaded with a Ca²⁺ indicator dye (fluo-4, 7 μM), then visualized under a 40× oil-immersion objective of a confocal laser scanning microscope. B shows the same glomerulus but after application of endothelin-1 (1 μM). Note the appearance of multiple bright spots (arrows) in the structure indicating increase in [Ca²⁺]_i in those glomerular cells (white scale bar = 50 μm).

Figure 6. Pooled data for stimulatory effect of different agonists on [Ca²⁺]_i

Mean (± SEM; one-way ANOVA) increases in AFI in response to A angiotensin II (n=3–9), B K⁺ (n=5–10) and C endothelin-1 (n=3–4) in isolated fluo-4 loaded rat renal glomeruli recorded via confocal microscopy.