#2518 The decrease of the filtration slit density, not proteinuria, is the early indication of the filtration barrier dysfunction

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Abstract Background and Aims Intact podocyte foot processes (FP) play a crucial role in ensuring proper selective filtration in the kidney. Changes in FP morphology are thought to be the initial trigger for proteinuria, but definitive proof is still missing. As FP structures are below the optical resolution of light microscopes, changes are investigated by electron microscopy (EM) on ultrathin sections. In this study, the correlation between morphology and proteinuria was investigated using the Podocyte Exact Morphology Measurement Procedure (PEMP), a 3D-SIM-based technique. Our investigation was focused on podocyte damage caused by treatment with nephrotoxic serum (NTS) as a model for focal segmental glomerulosclerosis. Method The mice were treated with nephrotoxic serum (NTS), and kidneys were taken at 24, 48, and 72 hours after injection. Kidney sections (3 µm thick) were prepared for 3D-SIM analysis after staining with podocin and integrin and then fixed in formalin and embedded in paraffin. Filtration slit density (FSD) was quantified using the Podocyte Exact Morphology Measurement Procedure (PEMP) as described earlier (Siegerist et al. 2017). In addition, EM was performed on glutaraldehyde-fixed tissue samples using standard protocols. The albumin to creatinine ratio was also determined. Results We observed that NTS-treated mice developed moderate proteinuria after 48 hours. Using the super-resolution microscopy-based 3D-SIM technique to quantify podocyte PF morphology by calculating the filtrations slit density (FSD) revealed a significant reduction of the FSD in NTS-treated mice already after 24 hours. This structural change was confirmed by transmission electron microscopy analysis, showing that the effacement of podocytes FPs is the first sign of a change in the filtration barrier function before proteinuria is measurable. Conclusion PEMP analysis of standard histological sections from NTS-injected and control mice demonstrated that the first event in the functional loss of the size selectivity of the filtration barrier is the effacement of FP morphology, followed by detectable proteinuria. Therefore, the assessment of the FSD is a promising strategy for early and accurate detection of the loss of filtration barrier integrity, providing opportunities for faster and more precise diagnostics.