Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells.
Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.
Ashworth
S L
SL
Renal Epithelial Biology Experimental Laboratories, Indiana University Department of Medicine, and Roudebush Veterans Administration Medical Center, Indianapolis, IN 46202-5116, USA.
Sandoval
R M
RM
Hosford
M
M
Bamburg
J R
JR
Molitoris
B A
BA
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000818:Animals; D051345:Destrin; D005455:Fluorescent Antibody Technique; D007511:Ischemia / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000098:blood supply / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008566:Membranes / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism* / Q000652:urine; D008871:Microvilli / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D051339:Actin Depolymerizing Factors; D000199:Actins; D051345:Destrin; D008840:Microfilament Proteins