Premium
Ischemia leads to increases in tight junction mobility following FRAP in vivo
Author(s) -
Kolb Alexander,
Amsler Kurt,
Bacallao Robert
Publication year - 2017
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.31.1_supplement.703.11
Subject(s) - tight junction , occludin , in vivo , gap junction , ischemia , chemistry , biophysics , medicine , biology , biochemistry , intracellular , microbiology and biotechnology
Background After an injury to a cells tight junction it is hypothesized that these junctions become immobile and have a slow turnover rate contributing to leak and polarity changes leading to cell death. However, our in vivo data indicates ischemia increases the rate of tight junction turnover compared to pre ischemic tight junctions. Methods In vivo‐Adenoviral GFP occludin was hydrodynamically delivered through the renal vein. Four days later the kidney was exposed and labeled tight junctions were selected for FRAP. FRAP was conducted on both pre and post ischemic tight junctions and on non‐tight junction regions. In vitro‐GFP occludin transduced MDCK cells were serum starved overnight. Cells were treated with 0 μM H 2 O 2 , 55 μM H 2 O 2 or 110 μM H 2 O 2 for 2 hours at 37°C before FRAP. The tight junction region or a non‐tight junction region of the apical membrane was selected for FRAP. Results In vivo‐The immobility of pre ischemic tight junctions is 93% (SD 9%). The immobility of ischemic tight junctions is 34% (SD 24%). The average half‐life for recovery of ischemic tight junctions is 23.13 seconds (SD 1.07). The pre ischemic tight junctions show minimal recovery at 180 seconds post FRAP. In vitro: Control cells treated with 0 μM H 2 O 2 have an immobility of 43% (SD 14%;) with a half‐life of 68.2 seconds (SD 17.3 seconds). Cells treated with 55 μM H 2 O 2 have an immobility of 32% (SD 16%) with a half‐life of 88.5 seconds (SD 23.7 seconds). Cells treated with 110 μM H 2 O 2 have an immobility of 37% (SD 15%) with a half‐life of 82.3 seconds (SD 21.03 seconds). Results are statistically significant, p value <0.05 Conclusions In vitro tight junction turnover following FRAP occurs much faster in wild type cells then it does in injured cells. This data indicates that injury actually slows down tight junction turnover compared to control cells. However, these results are contradictory to the in vivo data that indicates ischemic injury increases tight junction turnover. Support or Funding Information VA Merit