Illuminating Mouse Renal Proximal Tubule Architecture through High-Resolution Volume EM and Machine Learning Analysis

Key Points High-resolution 3D imaging reveals new features of proximal tubule ultrastructure that suggested mechanisms for regulating kidney function. Our studies illuminate novel connections between membranes of the endoplasmic reticulum, plasma membrane, and apical endocytic compartments. The endoplasmic reticulum in proximal tubule cells has subdomains characterized by proteins involved in distinct biochemical functions. 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 physiologic 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 mouse renal proximal tubule epithelial cells at isotropic resolutions down to 4 nm. We used 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 (ER) 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 ER, plasma membrane, mitochondria, and apical endocytic compartments. Immunofluorescence microscopy demonstrated distinct localization patterns for ER 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. Morphologic differences between 4 and 8 nm 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.