Early recovery of the actin cytoskeleton during renal ischemic injury in vivo.

The actin cytoskeleton of proximal tubule cells is important for both the maintenance of membrane domains and attachment to neighboring cells and underlying substrata. Adenosine triphosphate (ATP) depletion during ischemic injury causes early alterations in the actin cytoskeleton, resulting in loss of membrane domains and cellular attachment. We examined the actin cytoskeleton during recovery from ischemic injury. As shown previously in cell culture studies, ATP depletion to 14% of control values from in vivo ischemia resulted in decreases in G-actin consistent with net polymerization of the cytoskeleton. After 20 minutes of recovery restored ATP levels to 24% of control values, percent G-actin increased back to control values, yet cytoplasmic actin polymerized with little evidence of apical recovery. After 120 minutes of recovery, ATP levels had increased to 48% of control values with little qualitative or quantitative change in actin polymerization from 20 minutes of recovery. When ATP levels recovered to 65% of control values at 360 minutes after ischemia, movement of F-actin back toward the apical surface was observed. These data, along with prior data using maleic acid, suggest that thresholds of cellular ATP may cause differing effects on distinct cellular actin pools. We conclude that actin cytoskeletal recovery occurs very early and may be necessary for reestablishment of polarity essential for normal reabsorptive functions.

Cellular and metabolic consequences of chronic ischemia on kidney function.

Approximately 15% of end-stage renal disease is attributable to chronic ischemic nephropathy from renovascular disease, representing significant patient morbidity and sizable medical costs. Although the pathophysiology of both ischemic acute renal failure and renovascular hypertension are under intense study, there have been little data obtained on the pathophysiology of chronic ischemic injury to the kidney. Data from studies of renovascular hypertension demonstrate the primary dependence of the stenotic kidney on angiotensin II in maintenance of glomerular filtration rate, although other vascular regulators, such as endothelium-derived nitric oxide and endothelin, may also play a role. Clues to the pathophysiology of cellular injury in chronic ischemic nephropathy can be found in acute models of ischemic injury to the tubules, toxic models of chronic decreased blood flow such as cyclosporine, and from recent pathological studies showing immunologic alterations. Because there are very little data on the cellular mechanisms of chronic ischemic injury to the kidney, this is an important area for laboratory investigation, particularly because the techniques developed both in studies of acute renal ischemia and chronic renovascular hypertension are readily available. Further understanding of the cellular mechanisms of chronic renal ischemia may eventually lead to medical interventions for patients with ischemic nephropathy too ill to undergo major abdominal surgery.

Perioperative care of the renal patient.

Due to the hormonal and hemodynamic alterations inherent in the surgical experience, acute renal failure is common during the perioperative period. Acute renal failure occurs in 5% of hospital admissions, and the surgical setting is the second most common cause of inpatient acute renal failure. Because this setting has the highest mortality for acute renal failure, recognition of high-risk patients is essential for careful monitoring and prophylactic measures. Patients with chronic renal insufficiency, elderly patients, jaundiced patients, diabetics, and those undergoing cardiac or aortic surgery are at greatest risk for perioperative acute renal failure. Patients with severe chronic renal failure or end-stage renal disease are at significant risk for development of complications during the perioperative period, due both to renal and nonrenal reasons. Hyperkalemia, infections, arrhythmias, and bleeding commonly occur in these patients during the perioperative period. This population has a reasonable surgical mortality for both general and cardiac surgery, but the extremely high morbidity warrants careful perioperative monitoring and care.

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

The hallmark of ischemic acute renal failure is a rapid and early decline in proximal tubule ATP. Since we have previously shown that over half of apical microfilament losses occur within the first 5 min of experimental ischemic injury, we postulated that microfilament (F-actin) structure and cellular location are dependent on cellular ATP levels. To test this hypothesis, we used maleic acid to selectively inhibit renal cortical ATP production in vivo. Maleic acid significantly decreased tissue ATP and apical F-actin in a dose-dependent manner relative to equimolar sodium chloride controls, yet higher doses of maleic acid quantitatively resulted in net actin polymerization, primarily in the cytoplasm. Functionally, maleic acid decreased glomerular filtration rate (GFR) and tubular reabsorption of sodium (TRNa) in a dose-dependent manner relative to sodium chloride controls. Administration of exogenous ATP resulted in significant increases in tissue ATP, net actin depolymerization, and relocation of F-actin from the cytoplasm back to the apical surface coinciding with increases in GFR and TRNa. Thus, ATP depletion induced by maleic acid resulted in significant cytoskeletal and functional alterations that were ameliorated by exogenous ATP. We therefore conclude that the structure and cellular location of F-actin necessary for normal functioning of proximal tubule cells in vivo is dependent on tissue ATP levels.

Microfilament disruption occurs very early in ischemic proximal tubule cell injury.

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.

Role of microfilaments in maintenance of proximal tubule structural and functional integrity.

To determine the selective effect of microfilament disruption on both cellular structure and function, microfilament-specific doses of cytochalasin D (10 microM) were used in an isolated perfused kidney system. Structurally, cytochalasin D resulted in extensive disruption of the apical surface with blebbing, vacuolization, and patchy loss and fusion of microvilli. Functionally, cytochalasin D resulted in an initial decrease in glomerular filtration rate (300.8 +/- 29.9 vs. 541.6 +/- 51 microliters.min-1.g-1, P less than 0.05) with subsequent stabilization throughout the duration of the perfusion. In contrast, the tubular reabsorption of sodium decreased significantly in a linear fashion from 97.1 +/- 0.7 to 64.3 +/- 7.0% over the duration of the perfusion. Similarly, the tubular reabsorption of lithium decreased linearly from 74.8 +/- 2.6%, before the addition of cytochalasin, to 33.6 +/- 6.8% by the end of the perfusion. Correlation of the decrements in percent tubular reabsorption of sodium and lithium for individual kidneys was 0.87 (P less than 0.01), suggesting the effect of microfilament disruption on tubular reabsorption of sodium was localized primarily to the proximal tubule. Because ischemic injury is characterized by time-dependent structural alterations in the apical membrane of proximal tubule cells, we set out to determine whether microfilament disruption occurs during ischemic acute renal failure. Utilizing indirect immunofluorescence with an anti-actin antibody, control kidneys demonstrated intact circumferential apical immunofluorescence representing brush-border and terminal web actin staining. Fifteen minutes of ischemia resulted in multiple large gaps in the terminal web, and 50 min of ischemia caused diffuse redistribution of actin immunofluorescence throughout the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)