@article {70013, title = {Compartmentalization of the Endoplasmic Reticulum in Mouse Kidney Proximal Tubule Epithelial Cells}, journal = {Physiology}, volume = {40}, year = {2025}, month = {2025 May 12}, pages = {1280}, abstract = {

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 {\textmu}m3 of mouse kidney tissue, the mean volume of PT cells is 1980.25 {\textmu}m3 {\textpm} 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 {\textmu}m) 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.

}, doi = {10.1152/physiol.2025.40.S1.1280}, url = {https://doi.org/10.1152/physiol.2025.40.S1.1280}, author = {Reyna-Neyra, Andrea and Pandya, Raj D. and Lackner, Emily M. and Pang, Song and Li, Wei-Ping and Xu, C. Shan and Zugates, Christopher and Burdyniuk, Mariia and Pandya, Vraj D. and Weisz, Ora A. and Caplan, Michael J.} } @article {70147, title = {Illuminating Renal Proximal Tubule Architecture through High-Resolution Volume EM and Machine Learning Analysis.}, journal = {J Am Soc Nephrol}, year = {2025}, month = {2025 Oct 06}, abstract = {

BACKGROUND: Kidney epithelial cells perform complex vectorial fluid and solute transport at high volumes and rapid rates. Their structural organization both reflects and enables these sophisticated physiological functions. However, our understanding of the nanoscale spatial organization and intracellular ultrastructure that underlies these crucial cellular functions remains limited.

METHODS: To address this knowledge gap, we generated and reconstructed an extensive electron microscopic dataset of renal proximal tubule (PT) epithelial cells at isotropic resolutions down to 4nm. We employed artificial intelligence-based segmentation tools to identify, trace, and measure all major subcellular components. We complemented this analysis with immunofluorescence microscopy to connect subcellular architecture to biochemical function.

RESULTS: Our ultrastructural analysis revealed complex organization of membrane-bound compartments in proximal tubule cells. The apical endocytic system featured deep invaginations connected to an anastomosing meshwork of dense apical tubules, rather than discrete structures. The endoplasmic reticulum displayed distinct structural domains: fenestrated sheets in the basolateral region and smaller, disconnected clusters in the subapical region. We identified, quantified, and visualized membrane contact sites between endoplasmic reticulum, plasma membrane, mitochondria, and apical endocytic compartments. Immunofluorescence microscopy demonstrated distinct localization patterns for endoplasmic reticulum resident proteins at mitochondrial and plasma membrane interfaces.

CONCLUSIONS: This study provides novel insights into proximal tubule cell organization, revealing specialized compartmentalization and unexpected connections between membrane-bound organelles. We identified previously uncharacterized structures, including mitochondria-plasma membrane bridges and an interconnected endocytic meshwork, suggesting mechanisms for efficient energy distribution, cargo processing and structural support. Morphological differences between 4nm and 8nm datasets indicate subsegment-specific specializations within the proximal tubule. This comprehensive open-source dataset provides a foundation for understanding how subcellular architecture supports specialized epithelial function in health and disease.

}, issn = {1533-3450}, doi = {10.1681/ASN.0000000884}, url = {https://doi.org/10.1681/asn.0000000884}, author = {Pandya, Raj D and Lackner, Emily M and Xu, C Shan and Zugates, Christopher and Burdyniuk, Mariia and Reyna-Neyra, Andrea and Pandya, Vraj D and Li, Wei-Ping and Pang, Song and Weisz, Ora A and Caplan, Michael J} }