Role of the actin cytoskeleton in ischemic injury.
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Actins↗
Biomedical subjects
B A Molitoris
Publications and source records attributed to B A Molitoris.
The potential role of ischemia in renal disease progression.
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Animals↗
Abnormal proximal tubule apical membrane protein composition in X-linked hypophosphatemic mice.
The hypophosphatemic (Hyp) mouse is characterized by an isolated X-linked defect in proximal tubular phosphate (Pi) reabsorption associated with a decreased maximum velocity (Vmax) and a normal affinity (Km). To directly investigate the underlying cellular defect proximal tubular brush-border membranes (BBM) from normal control (Con) and Hyp male littermates were examined for differences in cholesterol content, total and individual phospholipid composition, phospholipid incorporation rates, membrane fluidity, and by two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide protein electrophoresis. The cholesterol content, total and individual phospholipid profiles, phospholipid incorporation rates, and membrane fluidity of Con and Hyp BBM samples were comparable. However, the two-dimensional gel electrophoreses of Con and Hyp BBM proteins, run simultaneously under identical conditions, revealed a protein with an apparent abnormal isoelectric migration pattern in Hyp BBM samples. This protein had an apparent molecular weight 56,000 and an apparent pI of 7.2 and was consistently evident on Hyp gels (n = 3) but not on Con gels (n = 3). The appearance of this protein band was associated with a diminution in staining of a control protein of comparable apparent molecular weight but markedly lower apparent pI.
Animals↗
Myo-inositol uptake by rat cultured inner medullary collecting tubule cells: effect of osmolality.
Myo-inositol (MI) is involved in the adaptation to hyperosmolality. Its uptake by rat inner medullary collecting duct (RIMCD) cells has not been studied. Compared with cells grown in isotonic media, those grown in hyperosmolality display marked enhancement in [3H]MI uptake [counts/min (cpm).microgram protein-1.2 h-1] from 217 +/- 23 to 718 +/- 64, P less than 0.001. This is mimicked by the supplementation with 300 mM mannitol (638 +/- 59, P less than 0.001) but not by 300 mM urea. The increment in [3H]MI is observed at 37 degrees C but not at 4 degrees C. MI uptake is Na+ dependent in cells grown both in hyperosmolal or isotonic media. At least 12 h of hyperosmolality are needed to enhance MI uptake, and reexposure to isotonic media for at least 24 h is required for the enhancement to reverse. The effects of the microtubular inhibitor, nocodazole (10 micrograms/ml), and the protein synthesis inhibitor, cycloheximide (30 micrograms/ml), were studied. Cells grown with nocodazole show unimpaired enhancement of MI uptake. Cycloheximide exposure (16 h) does not affect MI uptake in isotonic media (182 +/- 23 vs. 191 +/- 15), but inhibited enhanced MI uptake in hyperosmolality (822 +/- 53 in the absence vs. 331 +/- 24 in the presence of cycloheximide, P less than 0.001). We conclude that hyperosmolality stimulates the synthesis of a protein, most likely an Na-MI cotransporter, that markedly enhances MI uptake. This process may be critical to the osmoregulation of RIMCD cells.
Animals↗
Ischemia-induced loss of epithelial polarity: potential role of the actin cytoskeleton.
Proximal tubule cells play a major role in the reabsorption of ions, water, and solutes from the glomerular filtrate. This is accomplished, in large part, by a surface membrane polarized into structurally, biochemically, and physiologically distinct apical and basolateral membrane domains separated by cellular junctional complexes. Establishment and maintenance of these unique membrane domains is essential for the normal functioning of proximal tubular cells and is dependent on cortical actin cytoskeletal-surface membrane interactions. Ischemia results in the duration-dependent loss of apical and basolateral surface membrane lipid and protein polarity. This loss of surface membrane polarity is associated with disruption of the cortical actin microfilament network and the opening of cellular tight junctions. Surface membrane lipids and proteins are then free to diffuse laterally within the membrane bilayer into the alternate membrane domain. Functionally, ischemia-induced loss of epithelial polarity is, in part, responsible for reduced sodium and glucose reabsorption. With recovery, proximal tubule cells undergo remodeling of the surface membrane such that the unique apical and basolateral membrane domains are reestablished allowing normal cellular function to return.
Actins↗
Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells.
To further understand and investigate how ischemia affects the tight junction we have developed a 2-h model of rapidly reversible ATP depletion and cellular injury in confluent LLC-PK1 monolayers. ATP depletion was achieved utilizing substrate-free medium containing 0.1 microM antimycin A (AA). Cellular ATP levels dropped rapidly to less than 5% of control values, but recovery of ATP and cell morphology was possible even after 2 h of exposure to AA. Ruthenium red, an electron-dense marker of tight-junction integrity, was excluded from the tight junctions of control monolayers but penetrated cellular tight junctions during ATP depletion in a duration-dependent manner. Electrical resistance across the monolayers remained unchanged in control monolayers but decreased linearly during ATP depletion to 59% of control values. Transmonolayer movement of [3H]mannitol increased from a control level of 7 to 13.5% during ATP depletion. Recovery of tight-junction integrity was demonstrated by a slowing of [3H]mannitol transfer from the basolateral to the apical medium. The transfer rate in control monolayers was 0.0126%/min. During the initial 120 min of cellular recovery from 2 h of ATP depletion, the transfer rate was 0.0789%/min, but this decreased to 0.0045%/min between 2 and 4 h of recovery. In summary, physiology, biochemical, and morphological evidence indicates that reversible ATP depletion results in rapid opening of cellular tight junctions. After ATP-repletion physiological studies indicate a recovery of tight-junction integrity.
Adenosine Triphosphate↗
Dissociation and redistribution of Na+,K(+)-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion.
Establishment and maintenance of a polar distribution of Na+,K(+)-ATPase is essential for efficient Na+ reabsorption by proximal tubule cells and is dependent upon the formation of a metabolically stable, detergent-insoluble complex of Na+,K(+)-ATPase with the actin membrane cytoskeleton. The present studies show that cellular ATP depletion results in a rapid duration-dependent dissociation of Na+,K(+)-ATPase from the actin cytoskeleton and redistribution of Na+,K(+)-ATPase to the apical membrane. During ATP depletion, total cellular Na+,K(+)-ATPase activity was unaltered, but the Triton-X-100-insoluble fraction (cytoskeleton associated) of Na+,K(+)-ATPase activity decreased (P less than 0.01), with a corresponding increase in the detergent-soluble fraction of Na+,K(+)-ATPase (P less than 0.01). Indirect immunofluorescent studies of cells with depleted ATP revealed a redistribution of Na+,K(+)-ATPase from the basolateral membrane into the apical membrane and throughout the cytoplasm. ATP depletion also resulted in the redistribution of F-actin from a primarily cortical concentration to a perinuclear location. There was also a rapid, duration-dependent conversion of monomeric G-actin to F-actin starting during the first 5 min of ATP depletion. Taken together, these data suggest that ATP depletion causes profound alterations in cell polarity by inducing major changes in the actin cytoskeletal architecture.
Actins↗
New insights into the cell biology of ischemic acute renal failure.
Proximal tubule cells play an essential role in the reabsorption of ions, water, and solutes from the glomerular filtrate. This is accomplished, in large part, by having a surface membrane polarized into structurally, biochemically, and physiologically distinct apical and basolateral membrane domains separated by cellular junctional complexes. Establishment and maintenance of these unique membrane domains are essential for the normal functioning of the cell. Ischemia results in the duration-dependent loss of apical and basolateral surface membrane lipid and protein polarity. Loss of surface membrane polarity is preceded by disruption of the microfilament network and opening of cellular tight junctions. Surface membrane lipids and proteins are then free to diffuse laterally within the bilayer into the alternate membrane domain. Functionally, ischemia-induced loss of epithelial polarity has been shown to be responsible for reduced sodium and glucose reabsorption. Reduced Na+ reabsorption has been related to redistribution of Na+, K(+)-ATPase into the apical membrane. During recovery from ischemic injury, proximal tubule cells undergo remodeling of the surface membrane such that the unique apical and basolateral membrane domains are reestablished, allowing for the return of normal cellular function.
Actins↗
Mild ischemia predisposes the S3 segment to gentamicin toxicity.
The purpose of these studies was to determine if a functionally insignificant ischemic insult, occurring prior to gentamicin administration, enhanced gentamicin nephrotoxicity. Bilateral renal pedicle clamp studies demonstrated that 15 minutes of ischemia did not increase the plasma creatinine yet markedly enhanced gentamicin nephrotoxicity. Further studies, in uninephrectomized rats, demonstrated that following fifteen minutes of renal ischemia and four hours of reperfusion inulin clearance, FENa+ and cellular morphology were normal. This model, therefore, was used in all subsequent studies. While the plasma creatinine concentrations were normal 24 hours following 15 minutes of ischemia and only slightly increased following gentamicin administration (100 mg/kg, i.p.) gentamicin administered four hours following 15 minutes of renal ischemia resulted in significantly increased 24-hour plasma creatinine values. Light microscopic quantitation of tissue injury, performed 24 hours following experimental manipulation, was notable for S3 segment damage in the ischemia plus gentamicin group. This was not observed in either the ischemia group or the sham operated gentamicin group. Cortical gentamicin levels were elevated in the ischemia plus gentamicin group, despite similar plasma gentamicin half-lives. However, the elevation in cortical gentamicin levels was dissociated from the enhanced nephrotoxicity seen following mild ischemic injury. Taken together these data indicate that mild renal ischemia, occurring prior to gentamicin administration, greatly enhanced gentamicin nephrotoxicity with the greatest damage occurring to S3 cells.
Acute Kidney Injury↗
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)
Actin Cytoskeleton↗
Alterations in the establishment and maintenance of epithelial cell polarity as a basis for disease processes.
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Animals↗
Loss of epithelial polarity: a novel hypothesis for reduced proximal tubule Na+ transport following ischemic injury.
Ischemia results in the marked reduction of renal proximal tubule function which is manifested by decreased Na+ and H2O reabsorption. In the present studies the possibility that altered Na+ and H2O reabsorption were due to ischemia-induced loss of surface membrane polarity was investigated. Following 15 min of renal ischemia and 2 hr of reperfusion, proximal tubule cellular ultrastructure was normal. However, abnormal redistribution of NaK-ATPase to the apical membrane domain was observed and large alterations in apical membrane lipid composition consistent with loss of surface membrane polarity were noted. These changes were associated with large decreases in Na+ (37.4 vs. 23.0%, P less than 0.01) and H2O (48.6 vs. 36.9%, P less than 0.01) reabsorption at a time when cellular morphology, apical Na+ permeability, Na+-coupled cotransport, intracellular pH and single nephron filtration rates were normal. We propose that the abnormal redistribution of NaK-ATPase to the apical membrane domain is in part responsible for reduced Na+ and H2O reabsorption following ischemic injury.
Adenosine Triphosphate↗
Markedly increased clearance of vancomycin during hemodialysis using polysulfone dialyzers.
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Biocompatible Materials↗
Citrate: a major factor in the toxicity of orally administered aluminum compounds.
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Aluminum↗
Site and mechanism of enhanced gastrointestinal absorption of aluminum by citrate.
Clinical and experimental studies have shown that citrate markedly enhances the intestinal absorption of aluminum (Al), but the site and mechanism of enhanced absorption are unknown. To determine where in the gastrointestinal tract aluminum citrate (Alcitr) was absorbed. Alcitr was gavaged with D-[1-3H] glucose in male Sprague-Dawley rats. Plasma Al levels increased rapidly and simultaneously peaked with D-[1-3G] glucose, suggesting early proximal bowel absorption. In in vitro duodenal and jejunal everted gut preparations, Alcitr incubation resulted in increased tissue Al levels and markedly enhanced transmural transport of Al and citr. Unlike citr, the transmural movement of Al was independent of temperature (37 degrees C vs. 4 degrees C). On the other hand, Al lactate (al Lac) increased tissue associated Al levels but had no effect on transmural Al movement. To determine if this large flux of Al following Alcitr administration was due to paracellular movement, ruthenium red and Ussing chamber studies were used to evaluate the morphologic and functional integrity of cellular tight junctions. Alcitr, as opposed to AlCl3, markedly increased ruthenium red deposits in intercellular spaces, especially around goblet cells, and induced a prolonged significant reduction in transmural resistance. Alcitr also resulted in rapid and nearly complete (99.7%) chelation of free calcium, an event known to disrupt cellular tight junction integrity. Taken together, these data suggest that enhanced Al absorption following administration of Alcitr occurs in the proximal bowel via the paracellular pathway due to the opening of cellular tight junctions.
Aluminum↗
Beta-2-microglobulin-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome.
The clinical manifestations of beta-2-microglobulin (beta 2M)-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome from a medical center hospital are presented. The predominant morbidity of beta 2M-amyloid was musculoskeletal, with deposits identified in surgical or biopsy specimens from trigger fingers, carpal tunnels, fractures, and radiolucent bone lesions. Lucent bone lesions were the characteristic radiologic finding of beta 2M-amyloidosis and were most commonly found in carpal bones, humeral heads, and femoral heads. Carpal tunnel syndrome occurred in greater than 20% of our chronic hemodialysis patients. The longer the period of time on chronic hemodialysis the greater the morbidity from beta 2M-amyloid. Although significant amounts of beta 2M-amyloid were detected in the perivascular regions of viscera, clinical compromise of internal organs from this type of amyloid was not documented. In acute studies, beta 2M clearance during hemodialysis was markedly increased using the Fresenius polysulfone dialyzers compared to cuprophane dialyzers. In summary, beta 2M-amyloid is common and causes significant morbidity in chronic hemodialysis patients. Long-term dialysis with highly permeable membranes effects greater beta 2M clearance which may result in less tissue deposition of beta 2M-amyloid, and therefore, fewer clinical complications.
Amyloid↗
Epithelial polarity following ischemia: a requirement for normal cell function.
Ischemic injury results in proximal tubule (PT) dysfunction and loss of surface membrane (SM) polarity. Since epithelial vectorial transport requires SM polarity, we set out to determine if correction of renal cortical PT dysfunction following ischemia was dependent on the reestablishment of SM polarity. Acute renal failure was induced using a bilateral 50-min pedicle clamp. Serum creatinine and fractional sodium excretion were maximal on day 1, remained elevated on day 3, and returned toward base line by day 8. PT cellular ultrastructure was normal by day 3. Despite rapid morphological recovery, ischemia resulted in a prolonged defect in glucose reabsorption. The delayed recovery of normal glucose handling closely paralleled the slow normalization of apical membrane lipid polarity. Na+-K+-ATPase polarity was also lost secondary to ischemia as demonstrated cytochemically and biochemically by the redistribution of Na+-K+-ATPase to the apical membrane. The time required to reestablish normal Na+-K+-ATPase polarity (8 days) paralleled the recovery of normal PT Na+ reabsorption (8 days), as assessed by fractional lithium clearances. This finding supports the hypothesis that apical Na+-K+-ATPase is in part responsible for reduced Na+ reabsorption following ischemic injury. In summary, these data suggest that functional recovery of PT glucose and Na+ reabsorption following a reversible ischemic insult requires not only morphological recovery, but also the reestablishment of surface membrane lipid and protein polarity.
Animals↗
Ischemia-induced loss of epithelial polarity. Role of the tight junction.
In proximal tubular cells ischemia is known to result in the redistribution of apical and basolateral domain-specific lipids and proteins into the alternate surface membrane domain. Since tight junctions are required for the maintenance of surface membrane polarity, the effect of ischemia on tight junction functional integrity was investigated. In vivo microperfusion of early loops of proximal tubules with ruthenium red (0.2%) in glutaraldehyde (2%) was used to gain selective access to and outline the apical surface membrane. Under control situations ruthenium red penetrated less than 10% of the tight junctions. After 5, 15, and 30 min of ischemia, however, there was a successive stepwise increase in tight junction penetration by ruthenium red to 29, 50, and 62%, respectively. This was associated with the rapid duration-dependent redistribution of basolateral membrane domain-specific lipids and NaK-ATPase into the apical membrane domain. Taken together, these data indicate that during ischemia proximal tubule tight junctions open, which in turn leads to the lateral intramembranous diffusion of membrane components into the alternate surface membrane domain.
Animals↗