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.
Highlights
Interdigitating podocyte foot processes are an essential part of the kidney filtration barrier
Of all claudins annotated in the database, we found that Claudin-5 (CLDN5) transcripts clustered in the podocyte population whereas the other claudins were expressed more distally in the nephron (Figure 1A, Figure S1)
By using Podocyte Effacement Measurement Procedure (PEMP), we found that the Filtration slit density (FSD) in minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS) samples was statistically significantly reduced compared with healthy human nephrectomy samples (Figure 7D)
Summary
Interdigitating podocyte foot processes are an essential part of the kidney filtration barrier. In puromycin aminonucleoside nephropathy (PAN) rats, a well-established model to induce podocyte foot process effacement, these tight junction proteins were significantly up-regulated This suggested a basic mechanism of tight junction formation in nephrotic kidneys. Western blot analysis of isolated glomeruli revealed that CLDN5 is not up-regulated after PAN treatment This is contrastive to other SD-associated tight junction proteins like JAM-A, occludin and cingulin that were significantly regulated in glomerular disease.[8]. Since healthy podocyte foot processes are at the borderline of resolvability of conventional (fluorescence) light microscopes, which is limited to 200 nm in the lateral direction, electron microscopy has been the gold standard for its visualization for decades This technique is time-consuming, highly sophisticated and can only analyse a thin layer of 50-90 nm of a kidney section. We revealed by 3D-SIM that the podocyte-specific CLDN5 has a specific staining pattern in healthy and diseased kidneys that can be resolved by 3D-SIM, we suggest CLDN5 as a possible early effacement marker
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