Imaging of Glomerular Endothelial Cell Calcium Dynamics in vivo Identifies Endothelial Progenitor Cell Subpopulation
Authors: Schiessl Ina Maria , Desposito Dorinne , Izuhara Audrey , Gyarmati Georgina , Peti‐Peterdi Janos 
Published: June 2021

Glomerular endothelial cells (GEnC) are important components of the glomerular filtration barrier and are often the first site of injury in many renal diseases. It is therefore important to understand how GEnC regenerate. Due to the lack of specific endothelial progenitor cell (EPC) markers, it is unknown if certain GEnC can proliferate or weather a local EPC population exists. Intracellular calcium [Ca] is a key modulator for cell proliferation. Here we aimed to identify and characterize local EPCs based on GEnC calcium dynamics and proliferation patterns, by performing genetic lineage tracing and [Ca] measurements using serial intravital microscopy in three different injury models.GEnC [Ca] was evaluated using inducible Cdh5‐GCaMP5 mice, which express the calcium‐sensitive fluorescent protein GCaMP5G specifically in endothelial cells. To study GEnC remodeling, we used inducible homozygous Cdh5‐Confetti mice, which identify individual endothelial cells by the expression of one out of 10 color combinations and EPC proliferation by clonal cell division over time.Baseline imaging of Cdh5‐GCaMP5 kidneys revealed generally stable GEnC [Ca] levels. High‐speed time‐lapse recording did not detect [Ca] elevations or myogenic oscillations in afferent (AA) and efferent arterioles (EA, n=10).We next determined GEnC [Ca] levels in Streptozotocin‐induced type 1 diabetes. On the first day of hyperglycemia (day 0) we found distinct calcium elevations in the EA. At day 14, scattered GEnC showed 3.4x higher [Ca] levels compared to control, which was accompanied by enlarged glomeruli. Serial imaging of the same glomeruli in Cdh5‐Confetti kidneys at days 0 and 14 revealed clonal expansion of single GEnC at the vascular pole.Similarly, we found distinct calcium elevations in EA and scattered GEnC at the vascular pole early in ureter obstructed kidneys (6d after UUO). Lineage tracing of Cdh5‐Confetti kidneys suggested vascular pole‐derived clonal expansion of EPCs.In a third model, we applied laser injury to ablate single GEnC along the glomerular tuft. This immediately induced the cell‐to‐cell propagation of a calcium wave including in the EA. Consistently with the involvement of purinergic calcium signaling, acute infusion of the non‐selective P2‐inhibitor suramin (150 mg/kg/h) reduced wave propagation velocity (7.5 vs. 0.6 μm/s, p<.0001, n=13 each) and GEnC [Ca] elevation (6.3‐ vs. 2.4‐fold increase, p<.0001, n=13 each). One day after laser‐induced cell ablation, we detected a 5.2‐fold increase in calcium within only a few GEnC at the vascular pole. Even though GEnC ablation was not performed in close vicinity to the vascular pole, serial imaging of Cdh5‐Confetti kidneys suggested clonal expansion of single EPCs at the vascular pole within 3–7 days.In summary, serial imaging of Cdh5‐GCaMP5 and Confetti kidneys in different states of disease suggested the presence of a novel EPC subpopulation localized at the glomerular vascular pole. EPC activation and proliferation was associated with robust [Ca] changes and may be used to functionally identify EPC and to further study mechanisms of their activation in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.