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.