Abstract
Ultrastructural analysis of the subpodocyte space (SPS) is essential to understanding podocyte injury in diabetic nephropathy, where SPS expansion represents an early marker of glomerular dysfunction. While traditionally assessed by transmission electron microscopy (TEM), we previously established a scanning electron microscopy (SEM) protocol enabling high-resolution SPS visualization in perfused kidney tissue. However, perfusion-based fixation, though effective in animal models, is not feasible in patients during the biopsy retrieval. To overcome these limitations, we developed a rapid SEM protocol based on secondary electron detection, specifically optimized for non-perfused samples. Using a paired design in healthy and diabetic (BTBR ob/ob) mice, we compared perfused and contralateral non-perfused kidneys, showing that SPS morphology and quantification remained accurate even in the absence of perfusion. The protocol was successfully applied to human kidney biopsies from controls and diabetic patients, allowing reliable SPS assessment and ultrastructural analysis across entire glomerular cross-sections. This optimized SEM protocol provides a reliable and versatile approach for SPS analysis in both non-perfused preclinical models and human kidney biopsies, thus broadening its applicability to clinically relevant settings.
Data availability
The data generated in the present article is available upon request to the corresponding author.
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Acknowledgements
We are indebted to Andrea Remuzzi for critical discussions and helpful suggestions. We also thank Daniela Corna, Domenico Cerullo and Daniela Rottoli for their technical support.
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S.C. performed the experiments and collected data; S.C. and S.T. developed the concept, analyzed data and wrote the manuscript; S.T., G.R., and A.B. supervised the experiments and approved the final results. All authors have read and agreed to the published version of the manuscript.
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The IRFMN adheres to the principles set out in the following laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law (D.Lgs 26/2014; Authorisation n. 35/2023-UT issued November 8, 2023 by Ministry of Health); Mario Negri Institutional Regulations and Policies providing internal authorisation for persons conducting animal experiments (Quality Management System Certificate – UNI EN ISO 9001:2015 – Reg. N° 6121; the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE). This study was approved by the Institutional Animal Care and Use Committees of Istituto di Ricerche Farmacologiche Mario Negri IRCCS and by the Italian Ministry of Health (approval number 712/2019-PR). All experimental protocols involving human subjects are carried out in accordance with the Declaration of Helsinki and good clinical guidelines. All patient information was anonymized and handled in accordance with institutional and ethical standards. As this study involved the use of de-identified archival biopsy material, informed consent was waived by the Territorial Ethics Committee Lombardia 6 of the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. The study was approved by Territorial Ethics Committee Lombardia 6 of the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy (procedure 2025 − 3.11/463).
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Conti, S., Benigni, A., Remuzzi, G. et al. A scanning electron microscopy—based approach to explore subpodocyte space remodeling in diabetic kidneys of mice and humans. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40816-9
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DOI: https://doi.org/10.1038/s41598-026-40816-9
