Super‐resolved local recruitment of CLDN5 to filtration slits implicates a direct relationship with podocyte foot process effacement

Abstract

Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron‐dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell‐cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super‐resolution 3D‐structured illumination microscopy, we observed a spatially restricted up‐regulation of the tight junction protein claudin‐5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA‐salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS‐treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up‐regulation in injured foot processes. Moreover, CLDN5 up‐regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up‐regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement.

scoMorphoFISH: A Deep-Learning enabled toolbox for single-cell single-mRNA quantification and correlative (ultra-)morphometry

“…We deliberately established scoMorphoFISH on these rather challenging archived clinical samples with a higher degree of heterogeneity compared to standardized material generated in a basic-research context. Thus, the functionality of this method is not species-limited and readily usable in rodent models for which we have already shown the performance of PEMP 4 . Due to high sensitivity and specificity, this technique poses excellent functionality in routinely generated and archived formalinfixed paraffin-embedded (FFPE) material with the potential to correlate spatial transcription and morphometric data with corresponding clinical data and disease history.…”

“…Recently, several methods have been established to improve the analysis depth of formalin-fixed paraffin-embedded (FFPE) kidney biopsies: The filtration barrier can be morphometrically analyzed by 3D structured illumination microscopy (3D-SIM) [1][2][3] . The determination of the filtration slit density by PEMP (podocyte exact morphology measurement procedure) emerged as a tool that can be combined with co-staining of multiple proteins 4 . However, antibody-based quantification of local protein abundance has limitations as it depends on antibody availability and performance.…”

ScoMorphoFISH: A deep learning enabled toolbox for single‐cell single‐mRNA quantification and correlative (ultra‐)morphometry

“…Thus, the functionality of this method is not species‐limited and readily usable in rodent models for which we have already shown the performance of PEMP. 4 Due to high sensitivity and specificity, this technique poses excellent functionality in routinely generated and archived formalin‐fixed paraffin‐embedded (FFPE) material with the potential to correlate spatial transcription and morphometric data with corresponding clinical data and disease history. Therefore, scoMorphoFISH enables hypothesis testing in sample repositories with archived medical records.…”

“… 1 , 2 , 3 Since the filtration barrier could be morphometrically analysed by 3D‐structured illumination microscopy (3D‐SIM), 3 the determination of the filtration slit density by PEMP ( podocyte exact morphology measurement procedure ) emerged as a tool that can be combined with co‐staining of multiple proteins. 4 , 5 However, antibody‐based quantification of spatial protein abundance has limitations as it depends on individual antibody availability and performance. Additionally, locally secreted factors are typically not captured by immunofluorescence techniques.…”

The Pathology Lesion Patterns of Podocytopathies: How and why?

“…This technique permits imaging of large tissue areas and to resolve fine structural details at once. Therefore, we can visualize the slit diaphragm 3D, giving direct evidence of structural changes or podocyte loss ( Figure 2 ) ( Artelt et al, 2018 ; Tesch et al, 2021 ).…”