title: Fluorescence imaging of intracellular calcium signals in intact kidney tissue.
abstract: BACKGROUND
  Intracellular calcium (Ca(2+)) plays an important role in normal renal physiology and in the pathogenesis of various kidney diseases; however, the study of Ca(2+) signals in intact tissue has been limited by technical difficulties, including achieving adequate loading of Ca(2+)-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
  Ca(2+)-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 Ca(2+) signals was recorded by confocal microscopy. Kidney slices were also obtained from transgenic mice expressing the GCaMP2 Ca(2+)-sensor in their endothelial cells and real time Ca(2+) transients stimulated by physiological stimuli.
  
  
  RESULTS
  Wide spread loading of Ca(2+) indicators was achieved in the tubular and vascular structures of both the medulla and cortex. Real time Ca(2+) 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 Ca(2+) transients were similarly recorded in kidney slices taken from the transgenic mouse expressing the GCaMP2 Ca(2+)-sensor.
  
  
  CONCLUSIONS
  We present new approaches that can be adopted to image cytosolic and mitochondrial Ca(2+) 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 Ca(2+) homeostasis in renal health and disease.
authors.affiliation: Medway School of Pharmacy, The Universities of Kent and Greenwich at Medway, Chatham Maritime, UK. C.M.Peppiatt @ kent.ac.uk
authors.forename: C M
authors.initials: CM
authors.lastname: Peppiatt-Wildman
authors.forename: C
authors.initials: C
authors.lastname: Crawford
authors.forename: A M
authors.initials: AM
authors.lastname: Hall
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D007668:Kidney / Q000502:physiology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002118:Calcium