ER and Mitochondria Regulate Intracellular Ca2+ and ENaC in Mouse Renal CCD Principal Cells
Authors: Li Linda Wenyi , Galarza‐Paez Laura , Zhou Li , Linck Valerie A , Duke Billie Jeanne , Al‐Khalili Otor , Thai Tiffany L , Eaton Douglas Charles 
Published: November 2023

Cortical collecting duct (CCD) principal cells fine tune total body salt and water balance. Part of this regulation is via intracellular Ca2+; in particular, intracellular Ca2+ regulates renal epithelial sodium channels (ENaC). We have shown that mitochondria in principal cells sequester intracellular Ca2+ to form microdomains within cells with different Ca2+ concentrations in different areas of the cell, thereby allowing differential regulation of ENaC in different parts of the cell. Failure to establish the microdomains produces disorders of Ca2+ signaling including hypertension and polycystic kidney disease. Therefore, determining how mitochondria take up Ca2+ to form these domains is crucial to understanding pathophysiology. Mitochondria in CCD principal cells interact with the ER via voltage‐dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane and IP3 receptor subtype 3 (IP3RIII) on the ER membrane to form a signaling complex known as mitochondrial‐associated ER membranes (MAMs). We hypothesize that MAMs are important for mitochondrial Ca2+ sequestration by allowing the rapid release of Ca2+ from ER into the cytosol to produce a local increase in intracellular Ca2+. At the same time the MAM complex activates mitochondrial VDAC1 to take up Ca2+ and prevent a persistent or global increase in Ca2+. Subsequently, mitochondria slowly return sequestered Ca2+ to the ER to restore ER Ca2+ stores. To determine whether the absence of VDAC1 disrupts MAMs, we perfused the kidneys of VDAC1 knockout mice and their littermate wild‐type mice with glutaraldehyde. TEM imaging indicates that although MAMs in the CCD principal cells were intact in both VDAC1 knockout and wild type mice, the mitochondria of the VDAC1 knockouts were larger than those of their wild‐type counterparts. To examine whether MAMs affect Ca2+ signaling domains, we imaged mpkCCD cells (n = 5) transfected with green Lck‐GCaMP2 (AddGene), a membrane‐associated Ca2+ sensor, and treated the cells with MDIVI‐1, an agent that selectively inhibits MAMs by blocking the dynamin‐dependent processes required for MAM formation. We applied UTP apically to increase intracellular Ca2+ beneath the apical membrane and found that Ca2+ increased strongly near the apical membrane, but only weakly in other parts of the cell. In cells treated with MDIVI‐1, Ca2+ increased less but was more uniformly distributed in all parts of the cell. To test whether VDAC1 influences ENaC activity in vivo, since ENaC currents increase as intracellular Ca2+ decreases, we examined ENaC regulation in renal tubules from VDAC1 knockout mice compared to wildtype mice on diets with different amounts of salt. We dissected and split open the cortical collecting duct, and then applied single‐channel patch clamp to the CCD principal cells. The activity of ENaC in wildtype mice decreased with increased sodium in the diet. ENaC activity in VDAC1 was significantly higher than wild type mice and activity did not depend upon dietary sodium. We conclude that inhibition of the associations between mitochondria and ER interferes with local Ca2+ signaling and therefore disrupts the ability of Ca2+ to regulate ENaC.

Support or Funding Information

Supported by APS UGSRF and Emory's Robert P. Apkarian Integrated Electron Microscopy Core. VDAC1 knockout mice were obtained from Dr. William Craigen at Baylor University.