Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress.
Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, beta-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H(2)O(2), as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell-matrix but not cell-cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.
Sáenz-Morales
David
D
Department of Pathology, Hospital Univ. Ramón y Cajal, Crta. de Colmenar, Km 9,1, 28034, Madrid, Spain.
Escribese
María M
MM
Stamatakis
Konstantinos
K
García-Martos
María
M
Alegre
Laura
L
Conde
Elisa
E
Pérez-Sala
Dolores
D
Mampaso
Francisco
F
García-Bermejo
M Laura
ML
D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000473:pathology; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D022001:Focal Adhesions; D066298:In Vitro Techniques; D007511:Ischemia / Q000378:metabolism* / Q000473:pathology*; D007668:Kidney / Q000098:blood supply* / Q000293:injuries; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000473:pathology*; D018384:Oxidative Stress; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D019108:Tight Junctions / Q000473:pathology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D017382:Reactive Oxygen Species; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein