Glomerular Endothelial Cell Calcium Dynamics Visualized in vivo
Authors: Schiessl Ina Maria , Gyarmati Georgina , Peti‐Peterdi Janos 
Published: June 2021

Glomerular endothelial cells (GEnC) are a critically important component of the glomerular filtration barrier and are subject to continually oscillating hemodynamics‐based high mechanical forces. Several in vitro studies suggest a key role of calcium in endothelial cell functions, such as the production of nitric oxide and cell proliferation. However, little is known about GEnC calcium dynamics in vivo. Here, we generated a new genetic mouse model to study calcium levels in GEnCs during physiological and several pathophysiological conditions.

Endothelial cell calcium reporter mice (Cdh5‐Cre × PC‐G5‐tdT) were developed by crossing Cdh5 (VeCadherin)‐CreERT2 and PC‐G5‐tdT mice. After tamoxifen‐induction, these mice co‐express tdTomato and the calcium sensitive fluorescent protein GCaMP5G in endothelial cells, which allows the study of intracellular calcium changes in a semi‐ratiometric fashion. Endothelial cell‐specificity of GCaMP5G expression in the kidney was confirmed by co‐localization of GCaMP5G with the endothelial marker CD31 using immunohistochemistry.

Intravital 2‐photon imaging of fully induced Cdh5‐Cre × PC‐G5‐tdT kidneys at baseline revealed generally stable intracellular calcium levels in GEnCs, which were occasionally disrupted by spontaneous calcium elevations in single cells. Consistent with regular oscillations in myogenic contractions of the afferent arteriole, time‐lapse imaging of endothelial cells in the afferent arteriole depicted calcium fluctuations.

Acute injection of sphingosine 1 phosphate (4 ug/g BW), which is an important factor of endothelial cell migration and proliferation, induced a 3‐fold increase of GEnC calcium levels. Furthermore, laser‐induced ablation of a single macula densa cell stimulated a pronounced calcium elevation in the afferent arteriole endothelium of the same nephron, suggesting a paracrine communication between macula densa and endothelial cells within the juxtaglomerular apparatus.

We next analyzed GEnC calcium levels during pathologic alterations in glomerular hemodynamics. Acute bolus injection of L‐NAME (10 mg/kg BW) caused a 2‐fold increase of afferent endothelial cell calcium levels and increased GEnC calcium values 1.3‐fold. Laser‐induced formation of a local glomerular thrombus provoked a 1.7‐fold elevation of GCaMP5G/tdTomato ratio in affected GEnCs. Furthermore, GEnC calcium levels were assessed 1 week after unilateral ureteral obstruction (UUO), and found to be 2‐fold elevated compared to GEnCs in the contralateral kidneys.

In summary, GEnC calcium levels fluctuate in response to physiological and pathophysiological hemodynamic changes. Intravital 2‐photon microscopy of newly generated Cdh5‐Cre × PC‐G5‐tdT kidneys is a promising new tool to improve our understanding of GEnC calcium dynamics in vivo and to further study its role in endothelial cell physiology and regeneration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.