Abstract
Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.
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Acknowledgments
The authors thank Ms. Kanako Oda for her skillful assistance of transplantation of fertilized eggs and Dr. Kosei Takeuchi for helpful discussion and helping transfection of claudins to COS7 cells. This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology (No. 21591021 to E.Y.).
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R. Koda and L. Zhao contributed equally to this work.
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Supplementary Fig. S1
Claudin mRNA levels in rat isolated glomeruli quantified by real-time PCR using another primer sets. Quantification was performed with vectors containing each PCR product as a standard. Results are shown as the ratio of claudin to GAPDH, and expressed as the mean +/- SD (n = 4).
Supplementary Fig. S2
Light microscopy (a-d) and electron microscopy (e, f) of glomeruli of neonatal animals from claudin-5 deficient mice (KO) (a, c, e) and wild mice (wild) (b, d, f). Sections for light microscopy were stained with haematoxylin and periodic acid-Schiff reagent (a-d). No morphological difference was found between deficient and wild mice. Pod: podocyte, *: glomerular capillary lumen (GIF 530 kb)
Supplementary Fig. S3
Double-labeled immunofluorescence photomicrographs of frozen kidney sections of neonatal animals from claudin-5 deficient mice (KO) (a, a’) and wild mice (wild) (b, b’) incubated with antibodies against ZO-1 (red; a, b) and podocin (green; a’, b’) (GIF 94 kb)
Supplementary Fig. S4
Double-labeled immunofluorescence photomicrographs of frozen sections of rat neonatal kidneys incubated with antibodies against claudin-5 (green; a, b) and podocin (red; a’) or laminin (red; b). The neonatal kidney displays a developmental gradient with immature glomeruli located toward the kidney surface and more mature glomeruli toward the corticomedullary junction. Staining for claudin-5 is observed in the area closer to the kidney surface but not for podocin (a, a’). Both claudin-5 and podocin are detected in deeper glomeruli of the capillary-loop stage. Double labeling with anti-laminin antibody shows claudin-5 expression at the vesicle stage (b). Dotted lines: kidney surface, v: renal vesicle (GIF 127 kb)
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Koda, R., Zhao, L., Yaoita, E. et al. Novel expression of claudin-5 in glomerular podocytes. Cell Tissue Res 343, 637–648 (2011). https://doi.org/10.1007/s00441-010-1117-y
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DOI: https://doi.org/10.1007/s00441-010-1117-y