These results are likely because activation and migration are not synchronous processes.. variants 1C10, is usually detected within tubular and interstitial cells as well as the glomerulus (indicated by black dashed square) in CD44+/+ mice. (J) higher magnification image of glomerulus in image I showing CD44 standard variant staining in PECs (black arrow heads). NIHMS837552-product-4.tif (25M) GUID:?3C7FCB3E-7C59-4485-842B-B925C57ADF48 5: Supplementary Figure 2 Inflammatory cells in glomeruli comparable in CD44+/+ and CD44?/? mice with FSGS Representative images of staining for (A) CD3 (activated T cell marker), (B) B220 (B cell marker), (C) F4/80 (macrophage marker) and (D) LY-6G (neutrophil marker) in diseased mice. White arrow heads show examples of positive staining in cells. The accompanying table shows quantification for 4′-Ethynyl-2′-deoxyadenosine the number of positive cells per glomerulus 4′-Ethynyl-2′-deoxyadenosine for each cell type . The number were extremely small, did not change much with disease (no significant glomerular influx) and showed no statistical differences between CD44+/+ and CD44?/? mice with this experimental model of FSGS. NIHMS837552-product-5.tif (24M) GUID:?8ABEC8DA-5AAB-40EF-A8F3-FD15354487E3 6: Supplementary Figure 3 Staining for hyaluronic acid binding protein is similar in CD44+/+ and CD44?/? mice with FSGS Representative images of staining for hyaluronic acid binding protein in (A) CD44+/+ mice and (B) CD44?/? mice. The accompanying table shows quantification for the percentage of glomeruli with positive HABP staining. There was no difference between CD44+/+ and CD44?/? mice at baseline. The percentage of glomeruli with positive HABP staining increased with disease, but there was no statistical difference between CD44+/+ and CD44?/? mice. NIHMS837552-product-6.tif (16M) GUID:?D26B978D-6D88-4DD3-BADC-62739580352D Abstract The glycoprotein CD44 is usually barely detected in normal mouse and human glomeruli, but is usually increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an and approach. Experimental FSGS 4′-Ethynyl-2′-deoxyadenosine was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells experienced lower migration from Bowmans capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, GATA3 overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two strategies were employed to show that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal 4′-Ethynyl-2′-deoxyadenosine epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is usually a regulator of CD44 expression and increased CD44 expression prospects to a pro-sclerotic and migratory parietal epithelial cells phenotype. expression of CD44 in PECs in glomerular diseases.8 CD44 is a family of trans-membrane glycoproteins consisting of different variants (CD44v) due to alternative splicing.9 CD44 is the main receptor for hyaluronic acid10 but binds other molecules, mostly components of extracellular matrix.11 Different biological functions have been 4′-Ethynyl-2′-deoxyadenosine explained and include proliferation,12 inflammation,13 tumor progression/ metastasis,14, 15 embryogenesis16 and cell migration.17, 18 CD44 is barely expressed in normal mouse and human glomeruli, being detected in only 0.8% of glomeruli in normal human biopsies.19 In contrast, CD44 is markedly increased in PECs in patients with FSGS, which might distinguish this podocyte disease from minimal change disease.20 CD44 is increased in PECs in mice with FSGS,1, 19C22 advanced age,4.
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