Compartmentalization of the endoplasmic reticulum in mouse kidney proximal tubule epithelial cells

The renal proximal tubule plays a critical role in water and solute reabsorption. Recently we generated a high resolution 3D, quantifiable volume microscopic identification of the ultrastructure of kidney Proximal Tubule (PT) cells using enhanced Focused Ion Beam Scanning Electron Microscopy (eFIB-SEM) and machine learning-based segmentation approaches. This analysis revealed that, in a volume of 70x60x177 µm3 of mouse kidney tissue, the mean volume of PT cells is 1980.25 µm3 ± 491.28 μm3. In an analysis of 25 PT cells, mitochondria (MITO) and endoplasmic reticulum (ER) accounted for an average of 26.4% and 6.3% of cell volume, respectively. Importantly, 91% of the total ER volume appeared to be comprised of a single contiguous ER structure as determined by tracing the ER surface. Using semi-thin sections (0.5 µm) of mouse kidney and antibodies directed against ER proteins we assessed the functional compartmentalization of the ER in PT cells by immunofluorescence microscopy. We find that ER proteins that participate in maintaining ER structure and lipid exchange, such as CLIMP-63 and VAP-A, localize to regions of the ER that are in close apposition to the basolateral plasma membrane (BL PM) of the PT cell. This distribution is confirmed by co-staining with an antibody directed against the Na, K-ATPase, a marker of the BL PM. In contrast, regions of the ER that are involved in calcium ion storage, as detected by staining for the SERCA calcium ATPase, are distributed broadly through the cytoplasm in the area of the cell that is rich in MITO. Staining for mitofilin, a MITO outer membrane protein, confirmed the abundance and distribution of the MITO in all of the PT cells. PDI, a protein that regulates proper folding and maturation of newly synthesized proteins in the lumen of the ER, resides primarily in portions of the ER that surround the nucleus and extend into the apical regions of the cell. PDI is mostly absent from the BL portions of the PT cells. Interestingly, calreticulin, which participates both in ER calcium storage and newly synthesized protein folding and quality control processes, is heavily concentrated in the subapical region of the cell. Using the machine learning algorithm to segment the lumen of the seemingly continuous ER structure demonstrates that, within the limit of resolution of this technique, continuity of ER lumens is limited to discrete patches. The defined distributions of these ER markers demonstrates that the extensive ER network in proximal tubule cells is divided into subdomains with distinct functional capacities and properties. NIH-RC2 DK120534. RDP and EML conributed equally. OAW and MJC contributed equally. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.