F-actin disorganization in apoptotic cell death of cultured rat renal proximal tubular cells

Abstract

The mechanism of nephrotoxin-induced apoptosis was studied in rat renal proximal tubular cells (PTC) exposed to the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). After a 6-h incubation, DCVC caused a condensation of heterochromatin and a fragmentation of the nucleus in 84 and 16% of the cells, respectively, which is indicative of apoptosis. This was confirmed biochemically by agarose gel electrophoresis demonstrating the formation of DNA fragments with multiples of 200 bp. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented neither the fragmentation of the nucleus nor the formation of DNA fragments, but it did prevent lactate dehydrogenase release and bleb formation by DCVC. Apoptosis induced by DCVC was closely associated with F-actin disorganization: every cell with a fragmented nucleus displayed completely disorganized F-actin, while cells with a normal nucleus still possessed at least some intact F-actin also induced apoptosis in PTC. Similarly, dithiothreitol, which damages F-actin in PTC, caused apoptosis of PTC. These data suggest a causal relationship between F-actin disorganization and apoptosis of PTC.

Dephosphorylation of Focal Adhesion Kinase (FAK) and Loss of Focal Contacts Precede Caspase-mediated Cleavage of FAK during Apoptosis in Renal Epithelial Cells

“…Thereafter cells were treated with DCVC in EBSS in a final volume of 1, 2, or 10 ml, respectively, for 4 h. To study events that are related to apoptosis, cells were treated with DCVC in the presence of DPPD (20 M; 10 mM stock in dimethyl sulfoxide) which blocks necrotic cell death but allows the onset of apoptosis (58,65). The inhibitors zVAD-fmk (100 M; 100 mM stock in dimethyl sulfoxide) or Na 3 VO 4 (5, 10, 25, or 50 M; 100 mM stock in water) were added simultaneously with DCVC.…”

“…Since DCVC-induced apoptosis of RPTE is associated with impaired cell adhesion (57,58,65), and because FAK is cleaved by caspases in cell-free systems as well as in intact cells in vitro (12)(13)(14)(15), we determined whether DCVC-induced caspase activation is associated with FAK cleavage in RPTE. A major increase in the cleavage of FAK into two fragments with approximate sizes of 70 and 77 kDa was observed 6 h after DCVC treatment, but not before (Fig.…”

“…Therefore, we investigated the role of FAK phosphorylation and its cleavage during loss of focal adhesion integrity and apoptosis of primary cultures of rat RPTE using the well characterized nephrotoxi- (57, 58, 60 -65). DCVC is metabolized by a ␤-lyase to a reactive acylating metabolite that covalently modifies cellular macromolecules (60 -62); DCVC induces apoptosis of RPTE in culture (58,65). Cell death of primary cultured RPTE caused by DCVC treatment is preceded by disorganization of the F-actin cytoskeletal network and dissolution of focal adhesions (57,58).…”

“…DCVC is metabolized by a ␤-lyase to a reactive acylating metabolite that covalently modifies cellular macromolecules (60 -62); DCVC induces apoptosis of RPTE in culture (58,65). Cell death of primary cultured RPTE caused by DCVC treatment is preceded by disorganization of the F-actin cytoskeletal network and dissolution of focal adhesions (57,58). Thus, this compound is a useful agent to study the role of focal adhesion disturbance and cell detachment in apoptosis of primary cultures of kidney epithelial cells.…”

Reduction of trans-4,5-Dihydroxy-1,2-dithiane by Cellular Oxidoreductases Activates gadd153/chop andgrp78 Transcription and Induces Cellular Tolerance in Kidney Epithelial Cells

“…DTT and other classic endoplasmic reticulum stress inducers not only disrupt protein processing but also inhibit protein synthesis generally and can be quite toxic (64,65). Unlike DTT, DTTox does not inhibit protein synthesis, did not induce dome collapse in LLC-PK1 cells, as does DTT (22), nor did it cause lactate dehydrogenase release from LLC-PK1 cells, yet paradoxically, it activated grp78 expression.…”