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.