Autonomous Calcium Signaling in Human and Zebrafish Podocytes Controls Kidney Filtration Barrier Morphogenesis
Authors: Djenoune Lydia , Tomar Ritu , Dorison Aude , Ghobrial Irene , Schenk Heiko , Hegermann Jan , Beverly-Staggs Lynne , Hidalgo-Gonzalez Alejandro , Little Melissa H. , Drummond Iain A. 
Published: April 2021

Significance Statement
Podocytes are critical to maintaining the kidney glomerular filtration barrier. Mutations in genes associated with development of nephrotic syndrome lead to elevated cytoplasmic calcium in podocytes and cause disruption of filtration barrier function. Whether calcium signaling plays a role in the initial formation of the filtration barrier is not known. Using live calcium imaging in two models, larval zebrafish and human kidney organoids, the authors demonstrate that podocyte calcium signaling is active during podocyte differentiation, is podocyte-cell autonomous, occurs independently of neighboring cell types, and is required for foot process and slit diaphragm formation. Their findings also show that developmental calcium signaling occurs by a different mechanism than disease-associated calcium perturbations, and represents a critical regulatory signal for podocyte morphogenesis and filtration barrier formation.

Background
Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown.

Methods
We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids.

Results
Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis.

Conclusions
These findings establish podocyte cell–autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.