Microfilament disruption occurs very early in ischemic proximal tubule cell injury

Abstract

Experimental ischemic acute renal failure results in disruption of proximal tubule apical membranes. Previous work utilizing immunofluorescence with an anti-actin antibody has demonstrated that the apical cytoskeleton of proximal tubule cells is disrupted during ischemic injury. In this study, using rhodamine-phalloidin which stains only filamentous actin, we demonstrate that graded durations of ischemia resulted in progressive disruption of proximal tubule apical microfilaments. Quantification using spectrofluorometry showed that 5, 15 and 50 minutes of ischemia resulted in 32.8 +/- 4%, 48.8 +/- 2.5%, and 58.4 +/- 2.6% decreases in apical F-actin relative to controls. Ischemia did not qualitatively affect either glomerular or distal tubule F-actin structure, though there were nonprogressive increases in glomerular fluorescence. In summary, rhodamine-phalloidin staining can be used to qualitatively and quantitatively assess proximal tubule microfilaments in vivo. We conclude that ischemia results in very early loss of proximal tubule apical microfilaments, with the majority of F-actin loss occurring within five minutes.

Exogenous adenosine triphosphate (ATP) preserves proximal tubule microfilament structure and function in vivo in a maleic acid model of ATP depletion.

“…Photographs Spectrofluorometry. The technique of spectrofluorometry is well established ( 1 1, 12) and has been previously performed with this fluor by us and others (10,13). Apical fluorescence of rhodamine-phalloidin, which is linearly related to F-actin concentration ( 14), was measured with a MPV compact microscope photometer (E. Leitz, Inc.) mounted on the fluorescence microscope.…”

“…F-actin in outer cortical renal tissue was stained as previously described ( 10). Outer cortical tissue slices were quickly frozen in liquid nitrogen, embedded in the cryofixative Tissue-Tek OCT (Miles, Inc., Elkhart, IN), and 5-,um sections cut on a cryotome (2800 Frigocut; Reichert Scientific Instruments, Buffalo, NY).…”

“…Using spectrofluorometry of rhodamine-phalloidin-stained sections of outer cortical proximal tubules, we have recently demonstrated that the majority of apical filamentous (F-) actin loss occurs during the first 5 min of experimental ischemic injury (10). Since both ATP depletion and apical microfilament disruption are very early events during ischemia, and the binding of ATP to F-actin has been postulated to be necessary for optimal filamentous actin growth, we hypothesize that microfilament structure and cellular location in vivo are dependent on cellular ATP levels.…”

Inducible Nitric-oxide Synthase Is an Important Contributor to Prolonged Protective Effects of Ischemic Preconditioning in the Mouse Kidney

“…Renal ischemia/reperfusion results in disruption of the actin cytoskeleton (6,34). We evaluated the effect of ischemic preconditioning on postischemic actin cytoskeleton changes using immunocytochemistry techniques.…”

Rehmannia glutinose Ameliorates Renal Function in the Ischemia/Reperfusion-Induced Acute Renal Failure Rats

“…21,22) Na,K-ATPase is an oligomeric enzyme composed of stoichiometric amounts of two major polypeptides, the a 1 and b 1 subunit. The a 1 subunit is a multispanning membrane protein that is responsible for the catalytic properties of the enzyme and b 1 subunit is a polypeptide that is essential for the normal activity.…”