Novel in vivo optogenetic tools reveal autonomous pacemaker control of renal hemodynamics by macula densa cells
Authors: Izuhara Audrey , Gyarmati Georgina , Becerra Calderon Alejandra , Trogen Greta , Deepak Sachin , Peti-Peterdi Janos 
Published: May 2024

Macula densa (MD) cells, the tubular component of the juxtaglomerular apparatus (JGA) are generally considered as sensor cells detecting tubular salt (NaCl) concentration and based on this information provide feedback control of glomerular hemodynamics (tubuloglomerular feedback, TGF). However, the neuron-like differentiation and activity as non-traditional MD functions are emerging. This study addressed the paradigm-shifting hypothesis that rather than being simply the sensory arm of TGF, MD cells function as the nephron’s autonomous pacemaker and the primary driver and oscillator of glomerular hemodynamics. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of MD-GT mice that selectively express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in MD cells. Optogenetic MD stimulation was induced in vivo by blue light (470nm) exposure of MD-Ai27 mouse kidneys that express the depolarizing channelrhodopsin ChR2 specifically in MD cells. Conversely, MD inhibition was induced by yellow light (560nm) exposure of MD-Ai39 mouse kidneys that selectively express the hyperpolarizing halorhodopsin NpHR in MD cells. Cell membrane expression of ChR2 (in MD-Ai27 mice) and NpHR (in MD-Ai39 mice) only in the MD among all renal cell types was confirmed using immunohistochemistry. Pacemaker-like regular Ca2+ oscillations (4-fold elevations in baseline Ca2+ and average frequency of 0.03 Hz) and their cell-to-cell propagation within the MD plaque via long cell processes were observed in MD-GT mice. These Ca2+ oscillations were MD cell autonomous, as they were preserved in freshly dissected single MD cells and in the in vitro dissected and microperfused JGA, and were exclusively confined to the MD area. Bulk and scRNA seq and MD transcriptome analysis identified the high MD expression of genes related to pacemaker function, cell membrane depolarization, voltage-dependent ion channels, Ca2+ signaling and synaptic transmission that was validated by immunohistochemistry. Blue light exposure of single glomeruli/JGAs using ROI scan in MD-Ai27 mouse kidneys that previously underwent superficial corticotomy (open loop nephrons) acutely and reversibly increased capillary and afferent (AA)/efferent arteriole (EA) blood flow by approx. 50% in the light-exposed glomerulus but not in adjacent glomeruli. In contrast, yellow light exposure of single glomeruli/JGAs in MD-Ai39 mouse kidneys acutely and reversibly decreased blood flow but increased glomerular capillary diameter (suggesting preferential EA vs AA vasoconstriction) in the exposed but not in adjacent single nephrons. In addition, yellow light stimulation of single glomeruli in MD-Ai39 mouse kidneys augmented the spontaneous glomerular blood flow oscillations in these open-loop nephrons (frequency of 0.03 Hz), but not in adjacent control nephrons. Altogether, these studies identified MD cells as the nephron’s central pacemaker and oscillator of single nephron blood flow, and emphasized the importance of MD cell membrane potential and Ca2+-dependent vasodilator release as a major driver of nephron function. DK064234 DK123564 DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.