Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI75447.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI72517.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI73174.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI70313.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI71702.
Intracellular calcium ([Ca2+]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca2+]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca2+]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca2+]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca2+]i around the injury site and promoted cell-to-cell propagating podocyte [Ca2+]i waves along capillary loops. [Ca2+]i wave propagation was ameliorated by inhibitors of purinergic [Ca2+]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca2+]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca2+]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca2+]i in glomerular pathology and suggest that purinergic [Ca2+]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI72674.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI71194.
Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI73787.
Citation Information: J Clin Invest. 2014. https://doi.org/10.1172/JCI72126.