Abstract Tight junctions are intercellular adhesion complexes that connect epithelial cells and thus prevent leakage between cells. They are made of tight junction proteins such as occludin, E-cadherin, Zonnula occludins, and Nidogen-1. Meprins are metalloproteinases that are abundantly expressed in the brush border membrane of the proximal kidney tubules. Ischemia reperfusion (IR) causes injury to the kidneys. Disruption of the meprin B gene or pre-treatment with the meprin inhibitor, actinonin, has been shown to protect mice from IR induced renal injury. This suggests that meprins play a role in the pathology of IR induced renal injury. The mechanism by which meprins enhance kidney injury is not fully understood. Meprin beta has been shown to degrade the tight junction proteins, E-cadherin and occludin in vitro. The goal of the current study was to determine whether hypoxia activates meprin B leading to degradation of the tight junction proteins. Meprin β transfected Madin-Darby canine kidney (MDCK) cells were depleted of oxygen by treatment with cobalt chloride (CoCl 2 ) for 0, 0.5, 1, 2 and 3 hours. Non-transfected MDCK cells were used as controls. Proteins were then extracted from the cells, and Western blot analysis used to quantify the levels and fragmentation of occludin and E-cadherin. Degradation of both E-cadherin and occludin was observed in the cobalt chloride treated meprin β transfected cells but not in non-transfected control cells. This suggests that hypoxia activates meprin B, leading to degradation of tight junction proteins. This may be partly responsible for the IR induced renal injury observed in vivo. Western Blot Analysis Proteins were electrophoretically separated on 10% acrylamide gels and transferred to nitrocellulose membrane. Non-specific binding sites were blocked by incubation in 8% fat-free milk for 1h at room temperature. Bands for E-cadherin and occludin were detected by incubation in the following primary antibodies: Anti-E. Cadherin (Abcam, Cambridge, MA), Rabbit Polyclonal, diluted 1:1000 Anti-Occludin (Origene, Rockville, MD), rabbit polyclonal, diluted 1:1000 Actin and tubulin antibodies were used for loading controls: Anti-Tubulin and ant-actin (Abcam, Cambridge, MA), mouse monoclonal, diluted 1:1000 Rabbit IgG and Mouse IgG (Bio-Rad), diluted 1:10,000 were used as secondary antibodies To detect bands, ECL substrate was added to the membranes, and bands developed on X-ray film Results Meprin B degradation of tight junction proteins in kidney cells subjected to hypoxia Jasmine George, Elimelda Ongeri, PhD Department of Biology, North Carolina Agricultural and Technical State University, Greensboro, NC Summary The intensity of the bands for E-cadherin and occludin in meprin ß transfected MDCK cells treated with CoCl 2 decreased in a time- dependent manner. This decrease was not observed in non- transfected control cells exposed to CoCl 2. - This suggests that both hypoxia and presence of meprins play a role in reducing the amount of occludin and E-cadherin in the MDCK cells Protein levels for actin and tubulin, which were used as loading controls, did not change, suggesting that the decrease was protein specific. Acknowledgements iBLEND NSF Grant No Literature J. Bylander Qing Li, Ganesan Ramesh, W. Brian Reeves and Judith S. Bond(2008) Targeted disruption of the meprin beta gene results in decreased renal ischemia/reperfusion injury in mice. The FASEB Journal 294(3) Naoki Ichikawa-Tomikawa, Kotaro Sugimoto, Seiro Satohisa, Keisuke Nishiura, and Hideki Chiba (2007). Transmembrane proteins of tight junctions. BBA Journal Lee SY, Shin JA, Kwon HM, Weiner ID, Han KH(2011). Renal ischemia-reperfusion injury causes intercalated cell- specific disruption of occludin in the collecting duct. Histochemistry and Cell Biology 136(6) LIU Shu-xin,CHEN Xiang-mei,SUN Xue-feng (2006). The change of E-cadherin expression in renal ischemia reperfusion injury. Chinese Journal of Practical Internal Medicine. E. Steed, Balda MS, Matter K(2009). Dynamics and Functions of Tight junction Proteins. Trends in Cell Biology 20(3) Yura RE, Bradley SG, Ramesh G, Reeves WB, Bond JS(2009). Meprin A metalloproteases enhance renal damage and bladder inflammation after LPS challenge. American Journal of Physiology Renal Physiology 296(1) Discussion/Conclusion Tight junction complexes help maintain a functional barrier for epithelial cells. Degradation of these proteins will compromise the integrity of the epithelial cells barrier. Meprins have previously been shown to cleave both occludin and E-cadherin in vitro. Our data suggests that hypoxia activates meprin B, leading to the degradation of both occludin and E-cadherin. This may be partly responsible for the kidney damage observed in IR, a condition in which kidney tissue is subjected to hypoxia in vivo. More studies will be done to determine if meprin activity is increased under hypoxia conditions. Methods Cell Culture and Oxygen depletion MDCK cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) to 90% confluence. They were serum-starved by overnight culture in serum-free media with 0.1% BSA. The cells were treated with 125 µM Cobalt Chloride (CoCl 2 ) to deplete oxygen and thus induce hypoxia conditions for 0, 0.5, 1, 2 and 3h Proteins were extracted from the cells and fractionated into a soluble-, nuclear-, and membrane-enriched fractions. Western blot analysis was used to quantify tight junction proteins (E-cadherin and occludin) in the membrane-enriched fraction. Figure 2. Representative immunoblot for E-cadherin in MDCK cells treated with CoCl 2. MDCK cells were treated with CoCl 2 as described in figure 1 above. Western Blot analysis was used to detect the levels of E-cadherin. Tubulin was used as a loading control. The levels of E-cadherin were decreased in meprin β transfected cells exposed to CoCl 2 for 1, 2 and 3 h. E-Cadherin Tubulin hours post CoCl 2 exposure Meprin β transfected cells Non-transfected control cells Figure 1. Exposure to CoCl 2 decreased occludin levels in meprin β transfected MDCK cells. Meprin transfected MDCK cells were treated with 125 µM CoCl 2 for 0-3 h. Proteins were then extracted and electrophoretically separated on 10% acrylamide gels. Western blot analysis was used to probe for occludin. Actin was used as a loading control. The levels of occludin in meprin transfected cells started decreasing at 1h post-CoCl 2 treatment. Figure 4.1 Replication of western blot analysis was performed for the tight junction protein E. Cadherin in HEK293 cells transfected with both meprin A and meprin B. Non-transfected HEK293 cells were used as controls. The levels of E. Cadherin decreased after 1h in the HEK293 cells transfected with meprin B. There was no observed change in the non transfected cells or the cell lines transfected with meprin A. Figure 4.2. Under hypoxia conditions induced by treatment with COCl 2, occludin decreased at at time dependent manner. Western Blot analysis showed a decrease after 1h and continued at 2 and 3h time points. Statistical analysis suggested a decrease consistent with the one viewed in figure 3 above.