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Increased differentiating kidney epithelial cell density induced by changes in fluid flow velocity (1180.12)
Author(s) -
Seetharaman Vikram,
Resnick Andrew
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1180.12
Subject(s) - biology , cell cycle , chemistry , cyclin dependent kinase 1 , microbiology and biotechnology , cell , biochemistry
Mouse cortical collecting duct cells were allowed to differentiate under conditions of no fluid flow (vf = 0; negative control) or constant rapid flow (vf = max; positive control) on orbital shakers. Cells were subjected to Δvf at 3, 5, 10, 20 days post‐differentiation, from vf = 0 to vf = max, or vice versa. At various times post‐Δvf, cells were harvested, nuclei were DAPI stained for cell counting, and RNA/protein extracted. Some cells were fixed and immunostained for the presence of the markers of MCCD cell differentiation E‐cadherin (E‐cad), important in adherens junction formation, and polycystin 1 (PC1), important in cellular mechanosensation. If MCCD’s exposed to Δvf do indeed de‐differentiate, then the relative expression of these 2 proteins should decrease. Although PC1 was absent from all cells sampled before 10 days post‐differentiation and unchanged in cells exposed to 5 days of vf = max followed by 5 days of vf = 0 compared to cell exposed to 10 days of vf = max only, E‐cad expression appeared slightly lower in cells exposed to 5 days of vf = max followed by 4 hours of vf = 0 compared to cells exposed to both 5 days of vf = 0 only and 5 days of vf = max only. Quantitative real time‐polymerase chain reaction and Western blot analyses will subsequently be used to determine concentrations of several mRNA transcripts [cyclin dependent kinase 1 (cdk1)/c‐Myc/cyclin D] and phosphorylated proteins [phospho‐Protein Kinase B (PKB, also known as Akt)/phospho‐mammalian Target of Rapamycin (mTOR)/phospho‐Mitogen Activated Protein Kinase (MAPK, also known as ERK1&2)], respectively, considered markers of cell proliferation. If MCCD’s exposed to Δvf do indeed divide, then all of these mRNA transcripts and phospho‐proteins should increase in concentration except for phospho‐PKB, which should decrease in concentration. Grant Funding Source : Supported by the National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716