Intravital Imaging of the Mouse Kidney Reveals Axial Differences in Calcium Signaling along the Nephron
Authors: Martins Joana Raquel Delgado , Haenni Dominik , Bugarski Milica , Hall Andrew M. 
Published: June 2021

Ca2+ is an important ion involved in various physiological processes and signaling pathways in the kidney. Intracellular levels are tightly regulated and changes in Ca2+ signaling are associated with kidney diseases. Intravital multiphoton (MP) imaging is a powerful tool in providing novel insights into renal physiology and pathophysiology. Although various cellular readouts can be assessed, effective visualization of Ca2+ signaling remains challenging due to a lack of adequate loading or expression of Ca2+ sensitive probes in the mouse kidney.To overcome these challenges, we generated a double transgenic mouse line expressing the Ca2+ sensitive protein GCamp6s and generalized Cre in all tissues. This resulted in very high expression of the sensor in tubular cells along the nephron and collecting duct system, suitable for intravital MP imaging.In the intact kidney cortex we observed unsynchronized spontaneous Ca2+ oscillations in neighboring cells along the proximal tubule (PT). We developed new image processing algorithms to perform automated analysis of their temporal correlation and combined it with network clustering analysis which revealed that oscillations were markedly different between early (S1) and later (S2) cortical PT segments. Oscillations were not detected in the distal convoluted tubule (DCT), where Ca2+ absorption is entirely transcellular, nor in the collecting duct system. The signals were modulated by blocking of purinergic signaling, changes in luminal flow and when axial distribution of solute load was altered. In addition, increased Ca2+ was observed in PT cells in response to physiological ligands, such as Angiotensin II.To further investigate these mechanisms we prepared fresh ex vivo kidney slices. Oscillations with comparable kinetics were still present, although in much fewer tubules, implying that luminal flow is important for their resting activity. Oscillations in the PT could be restored by purinergic stimulation with ATP and blocked by the P2 antagonist Suramin. Spontaneous oscillations persisted in PT cells upon acute removal of extracellular Ca2+, suggesting that they were sustained by intracellular stores. Oscillations were also observed in the thick ascending limb (TAL), a segment where calcium absorption occurs through a combination of transcellular and paracellular routes.In summary, using a new transgenic mouse model and automated analysis, we report for the first time the detection of spontaneous, ligand‐induced Ca2+ signals in PTs in vivo. Moreover, we have found evidence of striking axial differences along the nephron, even within the PT, most likely related to specialized functions of tubular sub‐segments. Oscillations were present in the PT and TAL, segments where paracellular Ca2+ transport occurs, while they were markedly absent from the DCT, where Ca2+ is solely transported transcellularly. Since previous in vitro studies suggest that flow triggered local nucleotide release in renal epithelia leads to changes in ion transport and apical endocytosis, we speculate that purinergic dependent Ca2+ oscillations in the PT and TAL might serve to locally regulate transport capacity in response to acute changes in demand, for example due to variations in glomerular filtration rate.Support or Funding InformationJRM is a IKPP2 fellow funded by the European Union's Seventh Framework Programme for research, technological development and demonstration under the grant agreement no 608847This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.