Reassembly of the Permeability Barrier

Reutilization of existing junctional components

Synthesis of new junctional components

Synthesis of new junctional components

Nonpolarized renal epithelial cells

Cell death Apoptosis Necrosis

Polarized renal epithelial cells

Intact intercellular junctions

Nonpolarized renal epithelial cells

Compromised intercellular junctions

Nonpolarized renal epithelial cells

Damaged disassembled intercellular junctions

Cell death Apoptosis Necrosis t

Deplete ATP

Short-term ATP depletion 0-1 h

Replete ATP

Long-term ATP depletion 2.5-4 h

Replete ATP

Severe ATP depletion


Cell culture models of tight junction disruption and reassembly. The disruption of the permeability barrier, mediated by the tight junction, is a key lesion in the pathogenesis of tubular dysfunction after ischemia and reperfusion. Cell culture models employing ATP depletion and repletion protocols are a commonly used approach for understanding the molecular mechanisms underlying tight junction dysfunction in ischemia and how tight junction integrity recovers after the insult [6, 12, 42]. After short-term ATP depletion (1 hour or less) in Madin-Darby canine kidney cells, although some new synthesis probably occurs, by and large it appears that reassembly of the tight junction can proceed with existing (disassembled) components after ATP repletion. This model of short-term ATP depletion-repletion is probably most relevant to transient sublethal ischemic injury of renal tubule cells. However, in a model of longterm ATP depletion (2.5 to 4 hours), that probably is most relevant to prolonged ischemic (though still sublethal) insult to the renal tubule, it is likely that reestablishment of the permeability barrier (and thus of tubule function) depends on the production (message and protein) and bioassembly of new tight junction components. Many of these components (membrane proteins) are assembled in the endoplasmic reticulum.


Immunofluorescent localization of proteins of the tight junction after ATP depletion and repletion. The cytosolic protein zonula occludens 1 (ZO-1), and the transmembrane protein occludin are integral components of the tight junction that are intimately associated at the apical border of epithelial cells. This is demonstrated here by indirect immunofluorescent localization of these two proteins in normal kidney epithelial cells. After 1 hour of ATP depletion this association appears to change, occludin can be found in the cell interior, whereas ZO-1 remains at the apical border of the plasma membrane. Interestingly, the intracellular distribution of the actin-cytoskeletal-associated protein fodrin also changes after ATP depletion. Fodrin moves from a random, intracellular distribution and appears to become co-localized with ZO-1 at the apical border of the plasma membrane. These changes are completely reversible after ATP repletion. These findings suggest that disruption of the permeability barrier could be due, at least in part, to altered association of ZO-1 with occludin. In addition, the apparent co-localization of ZO-1 and fodrin at the level of the tight junction suggests that ZO-1 is becoming intimately associated with the cytoskeleton.


ATP depletion causes disruption of tight junctions. Diagram of the changes induced in tight junction structure by ATP depletion. ATP depletion causes the cytoplasmic tight junction proteins zonula occludens 1 (ZO-1) and ZO-2 to form large insoluble complexes, probably in association with the cytoskeletal protein fodrin [12], though aggregation may also be significant. Furthermore, occludin, the transmembrane protein of the tight junction, becomes localized to the cell interior, probably in membrane vesicles. These kinds of studies have begun to provide insight into the biochemical basis of tight junction disruption after ATP depletion, although how the tight junction reassembles during recovery of epithelial cells from ischemic injury remains unclear.

Low calcium (LC)

Low calcium (LC)

Calcium switch (NC)

Calcium switch (NC)


Madin-Darby canine kidney (MDCK) cell calcium switch. Insight into the molecular mechanisms involved in the assembly of tight junctions (that may be at least partly applicable to the ischemia-reperfusion setting) has been gained from the MDCK cell calcium switch model [43]. MDCK cells plated on a permeable support form a monolayer with all the characteristics of a tight, polarized transporting epithelium. Exposing such cell monolayers to conditions of low extracellular calcium (less than 5|xM) causes the cells to lose cell-cell contact and to "round up." Upon switching back to normal calcium media (1.8 mM), the cells reestablish cell-cell contact, intercellular junctions, and apical-basolateral polarity. These events are accompanied by profound changes in cell shape and reorganization of the actin cytoskeleton. (From Denker and Nigam [19]; with permission)

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