Supplementary Material for: Fluorescence Imaging of Intracellular Calcium Signals in Intact Kidney Tissue

Background: Intracellular calcium (Ca2+)
plays an important role in normal renal physiology and in the
pathogenesis of various kidney diseases; however, the study of Ca2+ signals in intact tissue has been limited by technical difficulties, including achieving adequate loading of Ca2+-sensitive
fluorescent dyes. The kidney slice preparation represents a model
whereby three-dimensional tissue architecture is preserved and
structures in both the cortex and medulla can be imaged using confocal
or multiphoton microscopy. Methods: Ca2+-sensitive
dyes Rhod-2, Fura-red and Fluo-4 were loaded into tubular and vascular
cells in rat kidney slices using a re-circulating perfusion system and
real-time imaging of Ca2+ signals was recorded by confocal microscopy. Kidney slices were also obtained from transgenic mice expressing the GCaMP2 Ca2+-sensor in their endothelial cells and real time Ca2+ transients stimulated by physiological stimuli. Results: Wide spread loading of Ca2+ indicators was achieved in the tubular and vascular structures of both the medulla and cortex. Real time Ca2+
signals were successfully recorded in different intracellular
compartments of both rat and mouse cortical and medullary tubules in
response to physiological stimuli (ATP and angiotensin II). Glomerular
Ca2+ transients were similarly recorded in kidney slices taken from the transgenic mouse expressing the GCaMP2 Ca2+-sensor. Conclusion: We present new approaches that can be adopted to image cytosolic and mitochondrial Ca2+
signals within various cell types in intact kidney tissue. Moreover,
techniques described in this study can be used to facilitate future
detailed investigations of intracellular Ca2+ homeostasis in renal health and disease.