Ischemia disrupts myosin Ibeta in renal tubules

Judy Boyd-White1, Anjaiah Srirangam1, Michael P. Goheen2, and Mark C. Wagner1

1 Renal Epithelial Biology Experimental Laboratories, Division of Nephrology, Department of Medicine, and 2 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In these studies we have examined rat kidneys biochemically and microscopically to determine where myosin Ibeta is located before, during, and after an acute ischemic injury. Myosin Ibeta is present in multiple tubule segments including the brush border (BB) of the proximal tubule cell (PTC). Its distribution is severely altered by a 15-min renal artery clamp. Myosin Ibeta is present in the urine during reflow and is found in the numerous cellular blebs arising from the damaged PTC and other tubules. Two hours of reflow result in a decrease in BB myosin Ibeta staining and an increase in its cytoplasmic staining. Interestingly, the return of the F-actin in the BB precedes the return of the myosin Ibeta , suggesting that this myosin I isoform may not play a role in rebuilding the microvilli after an ischemic injury. A nonstructural role for this myosin, such as transport or channel regulation, is supported by its presence in many tubule segments, all of which have transport and channel requirements but do not all contain microvilli.

kidney; cytoskeleton; motor; disease; actin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MYOSIN Ibeta is a member of the myosin superfamily that contains at least 15 distinct classes (33). All myosins contain an actin and ATP binding site in their globular head or motor region, while the myosin tail regions are quite diverse. Many myosin class members have been identified by molecular techniques in both lower and higher eukaryotes (23, 33, 40). In addition, multiple studies have identified multiple myosins in the same tissue and/or cells, suggesting that these molecular motors may coordinate their respective or redundant roles in cells. The myosin I class members have three functional domains (3, 12). At the amino terminus is a 70- to 80-kDa region that contains the ATP and actin binding site. The alpha -helical neck region contains one to six IQ (isoleucine-glutamine) motifs that interact with light chains that are calmodulin in vertebrates. The carboxy end domain, ~20 kDa, is enriched in basic amino acids that interact with acidic phospholipids. Numerous studies have proposed a role for myosin I's in cell motility, endocytosis, secretion, contractile function, and Golgi vesicle trafficking and in linking the actin bundles of microvilli to the plasma membrane. All these activities involve an interaction with membrane and actin filaments, a dual property for which myosin I's are well equipped.

There are at least four distinct classes of myosin I that are widespread in mammalian tissues, including kidney (12, 23, 33, 40). The four classes have been designated as Ialpha , Ibeta , IC, and Igamma , whereas in rat they are termed myr1, myr2, myr3, and myr4 (3, 33). The human cDNA for myosin Ibeta also has been characterized and is called Myo1c (33). Myosin Ibeta was the first mammalian myosin I purified (2). Western blots and immunofluorescence have shown this isoform to be widely distributed, being found in all rat tissues examined and present in active motile regions of cells, i.e., lamellipodia and ruffles (1, 12, 30, 36, 40). In addition, a punctate cytoplasmic pattern is observed in both cell and tissue immunolabeling. Recent studies suggest that myosin I may be involved in the regulation of mechanochemical transduction channels in the apical region of hair cell stereocilia in the inner ear (16, 18). We have previously localized myosin Ibeta to microvilli in a proximal tubule cell line, LLCPK, and have shown that it was also present in the brush border (BB) of proximal tubules (37).

Proximal tubule cells (PTC) have a specialized apical membrane that mediates the selective and efficient reabsorption of ions, water, and macromolecules from the glomerular filtrate (6, 24). Reabsorption requires microvilli and delivery of ion channels, receptors, and regulatory molecules to their correct destination, thus enabling the establishment and maintenance of surface membrane polarity. These processes depend, in part, on actin-surface membrane interactions and vesicular transport, both processes in which myosin I has been implicated. The apical region of the PTC is a very dynamic region composed of the terminal web and microvilli, each containing a high concentration of actin and lacking microtubules. The pit area at the base of the microvilli is a site of active endocytosis, and myosin Ibeta has been identified in an endosome fraction from this region (17). Importantly, membrane channels and other proteins and lipids are continuously delivered to the plasma membrane of all kidney tubules to replenish and maintain a cellular environment capable of executing urinary function. Ischemia in vivo or ATP depletion in vitro severely alters PTC actin cytoskeleton and inhibits ATP-dependent vectorial transport of organelles (4, 13, 24, 35). Data from numerous labs have documented that ischemia-reperfusion results in cellular changes throughout the kidney, including proximal and distal tubules (4, 13-15, 19, 21, 24, 25, 29). The alterations combined lead to cellular and organ-level dysfunction. Return to the normal physiological state requires cellular events involving both the actin cytoskeleton and vectorial transport.

We previously documented the changes to myosin Ibeta in an established, reversible in vitro ischemic model, LLCPK ATP depleted/repleted (37). In that study, myosin Ibeta was shown for the first time to be present in microvilli, to undergo apparent cross-linking with actin after ATP depletion, and to return to a normal distribution after ATP repletion. Myosin Ibeta also was observed in the cytoplasm and at the lateral cell borders of cells. If this unconventional myosin participates in the structural stability of the microvilli, its presence in the BB should coincide with the return of the BB after ischemia. However, a return after BB formation would suggest that it is participating in some other cellular function in these cells. Consequently, the objective of these studies was to analyze the distribution of myosin Ibeta in the rat kidney under normal physiological conditions, during ischemia, and after reperfusion following ischemia. Interestingly, we found myosin Ibeta in many tubule segments. We found that myosin Ibeta is indeed concentrated in the BB of PTCs but also is present in a punctate pattern in the cytoplasm. Ischemia dramatically altered the BB morphology and resulted in a loss of myosin Ibeta from the BB and an apparent dissociation from F-actin. However, Western blot analysis of both kidney and urine samples from the injury and recovery process shows minimal loss of myosin Ibeta . This finding suggests that the changes we observed by immunofluorescence are the result of dramatic redistribution and/or reorganization of myosin Ibeta in the proximal tubule. Fifteen minutes of ischemia followed by 2 h of recovery resulted in structurally normal microvilli. However, myosin Ibeta , detected by immunofluorescence, had not returned to its predominant BB location in the proximal tubules but was found predominantly in the cytoplasm. Even after 24 h of recovery we observed more cytoplasmic and less BB staining of myosin Ibeta . These studies suggest that myosin Ibeta does not play a structural role in the early stages of microvilli rebuilding. In fact, its presence in all kidney tubules suggests a more ubiquitous role for myosin Ibeta , possibly in vesicular transport or other cellular functions requiring membrane-actin interactions.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies. The primary antibodies against myosin Ibeta have been characterized and used in multiple studies (1, 9, 36, 37). Briefly, highly purified adrenal myosin Ibeta was the antigen used, and the resulting mouse monoclonal antibodies react with a specific band in the rat kidney (Fig. 1). M2 and M3 have been mapped to a tail region specific for this myosin I isoform, and M4 and M5 have been mapped to a head domain region containing the actin and ATP binding sites (36, 37). All antibodies give similar immunolabeling results, while M2 and M3 have an apparent higher affinity for SDS-denatured myosin Ibeta and are routinely used for Western blots. The actin antibody was from Chemicon (no. MAB1501R; Temecula, CA).


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Fig. 1.   Myosin Ibeta is present throughout the kidney. A: specificity of the antibodies used. Three samples were blotted: rat kidney cortex (C), medulla (M), and partially purified brush-border membranes (BBM, or B). Ten micrograms of SDS-solubilized cortex (lanes 1 and 4), medulla (lanes 2 and 5), and BBM (lanes 3 and 6) were loaded. Lanes 1-3 were stained with Gel Code Silver Snap stain. Lanes 4-6 were Western blotted for myosin Ibeta and actin. M2 and M5 monoclonal antibodies against myosin Ibeta were used. Note the enrichment of the actin in the BBM sample. B and C: BBM, cortex, or medulla samples were extracted in <500 µl with 1% Triton X-100 (TX-100) plus (in mM) 250 sucrose, 6 PIPES, 2.5 HEPES, 0.2 EGTA, and 0.2 MgCl2, pH 6.9, on ice for 10 min. The solubilized proteins were separated from the insoluble by centrifugation at 48,000 g for 30 min. Equal amounts of cortex and medulla protein were extracted, and pellets were resuspended to volume. Equal volumes were then loaded on the gel. The BBM sample used for extraction contained approximately one-sixth the amount of protein. S, supernatant; P, pellet.

Western blot analysis. SDS-PAGE was carried out using Bio-Rad Criterion Tris-HCl gels (Hercules, CA) or Fisher PAGEr gold precast gels (Hanover Park, IL). Prestained molecular mass standards from Sigma (St. Louis, MO) and Bio-Rad were used. For immunoblotting, samples were resolved by SDS-PAGE and transferred by electroblotting to Millipore polyvinylidene difluoride (Bedford, MA). The chemiluminescence detection method used either the alkaline phosphatase substrate CSPD (Tropix) or Pierce's ECL kit according to the manufacturer's instructions. Gels were stained with either Gel-Code Blue (Pierce) or Gel-Code SilverSnap stain (Pierce).

Northern blot analysis. At the specified time of ischemia and/or reperfusion, the kidneys were briefly (<20 s) perfused with PBS, and the cortex, medulla, and papilla were separated and rapidly frozen in liquid nitrogen. Frozen tissue was homogenized with the Bio-Pulverizer (Biospec Products, Bartlesville, OK) in liquid nitrogen, and powdered tissue was then poured into a tube containing the TRI reagent (Molecular Research Center, Cincinnati, OH). Total RNA was prepared according to the manufacturer's protocol. For Northern blots, total RNA was electrophoresed in 1% agarose gels containing 1.85% formaldehyde. Equal loading was assessed by optical density reading of samples before loading and by analysis of 28S RNA bands after the gel run. The RNA was transferred onto nylon membranes using the Schleicher and Schuell Turbo blotter (Keene, NH). The membranes were prehybridized for 15 min and hybridized for 2 h at 65°C using Rapid-hyb buffer (Amersham). The probe was a 700-bp myosin Ibeta cDNA tail fragment labeled using the Rediprime DNA labeling system (Amersham). The filter was washed twice with 2× sodium saline citrate (SSC)-0.1% SDS at room temperature for 5 min each time, once for 15 min with 0.5× SSC-0.1% SDS at 65°C, and once for 15 min with 0.2× SSC-0.1% SDS at 65°C. Analysis and quantitation were conducted with a Molecular Dynamics PhosphorImager. All analyses compared the clamped kidney with the contralateral unclamped kidney. RNA levels of the paired kidneys were normalized using a glyceraldehyde-3-phosphate dehydrogenase probe. Data are presented as means ± SE. Statistical analysis was performed with the ANOVA and Tukey's honestly significant difference analysis, and a P value <0.05 was considered statistically significant.

Immunofluorescence of rat kidney sections. Rat kidneys were perfusion-fixed according to the procedure of Maunsbach and Afzelius (22). Briefly, the animals were anesthetized with an intraperitoneal injection of ketamine-xylazine and atropine. The abdominal aorta and viscera were exposed via a midline incision, and sutures were placed superior and inferior to the renal blood vessels to confine the perfusate flow primarily to the kidneys. A catheter was secured in the abdominal aorta in the retrograde direction for delivery of a prefixation rinse of 20 ml of warmed and filtered 0.85% saline containing 3 U/ml of heparin, followed by the fixation solution containing filtered 2% paraformaldehyde in PBS, pH 7.4, warmed to 37°C. The kidneys were perfusion-fixed for 5 min at a flow rate of 50 ml/min at an average pressure of 140 mmHg. After perfusion fixation, the kidneys were removed from the animal, the capsule and perirenal fat were dissected away, and the tissue was trimmed for immersion fixation at 4°C. Larger blocks were sectioned on a Vibratome for immunofluorescence, and smaller blocks ~1 mm3 were processed for electron microscopy (EM). Sections 50-100 µm thick were obtained with the use of the Vibratome, and the sections were placed in 0.20% paraformaldehyde until labeled. For immunolabeling, the sections were rinsed with PBS and placed into 1% SDS or 1% Triton X-100 for 5 min (5). After detergent treatment, the tissue sections were rinsed in PBS and placed in PBS-50 mM EGTA containing the primary antibody (10 µg/ml). Incubation was at room temperature overnight, with gentle agitation. The sections were extensively (minimum 6× 30 min) washed with PBS at room temperature, followed by an overnight incubation with the secondary antibody (goat anti-mouse IgG conjugated to Cy5, Jackson ImmunoResearch, or Alexa 568, Molecular Probes, Eugene, OR) 5 µg/ml in 3% BSA in PBS. F-actin was labeled by including Alexa 488 or Oregon Green phalloidin (Molecular Probes) diluted 1:200 with the secondary antibody. FITC-labeled lectins were used at a concentration of 10 µg/ml. The sections were again rinsed with PBS and mounted on coverslips using ProLong (Molecular Probes). Immunofluorescence controls included omission of the primary antibody, use of multiple secondary antibodies that gave the same pattern for myosin Ibeta , and use of several additional irrelevant monoclonal antibodies at the same concentration as the myosin Ibeta antibodies. Omission of both the primary and irrelevant antibodies resulted in negligible staining.

A Bio-Rad (Hercules, CA) MRC 1024 laser scanning confocal microscope was used to acquire images. Focal sections were obtained in 0.5- or 1-µm steps with Kalman averaging. Each black-and-white image presented is a single focal plane unless stated otherwise. Three-dimensional (3-D) reconstruction of a series of images was performed with the use of MetaMorph's (Universal Imaging, West Chester, PA) stack 3-D reconstruction function. All digital images were imported into MetaMorph, and image planes were selected and transferred to PowerPoint for labeling and scaling. Color encoding was performed in MetaMorph by assigning the red and blue channels to the myosin Ibeta signal and the green channel to the F-actin signal. The resulting images show myosin Ibeta in purple and F-actin in green, and colocalization is shown as white.

Induction of renal ischemia and urine collection. In Sprague-Dawley rats utilized for microscopy and RNA isolation, renal ischemia was induced by clamping of the renal pedicle of the left kidney, with the contralateral kidney serving as the paired control (26). The rats used for urine and total protein changes were subjected to a bilateral renal clamp. We found no difference in the morphological changes and distribution of myosin Ibeta and F-actin between the 15-min unilateral and bilateral clamp models. Rats were acclimated to metabolic cages for 2-3 days before the bilateral clamp was performed. Urine was collected 24 h before surgery and after 2 and 24 h of reperfusion following the 15-min clamp. Urine volume was measured and monitored using Bayer Multistix for urinalysis. The urine was centrifuged for 15 min at 17,000 g, and the supernatant was assayed. Kidneys were removed at 2 or 24 h after reperfusion and separated into cortex and medulla (including papilla and pelvis). Tissue was solubilized with SDS. These protein and urine samples were used for semiquantitative analysis of myosin Ibeta changes after the ischemic injury (see Fig. 4).

Other methods. Tissue for EM analysis was obtained as described in Immunofluorescence of rat kidney sections, with the tissue blocks processed according to the procedure of Maunsbach and Afzelius (22). Briefly, blocks were rinsed in PBS twice for 30 min to remove fixative, postfixed in 1% osmium tetraoxide for 1 h at 4°C, rinsed in PBS twice for 30 min, dehydrated through an ethanol series, and embedded in Spurrs resin. Thick sections (0.5 µm) were cut on an RMC (Research Manufacturing Company, Tucson, AZ) microtome and stained with 1% toluidine blue for light microscopy. Thin sections (90-120 nm) were cut and stained with uranyl acetate followed by lead citrate and were viewed with a Philips CM 10 electron microscope. Protein concentration was determined using Pierce's BCA Protein Assay Reagent or Bio-Rad's Protein Assay.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myosin Ibeta is widely distributed in the rat kidney. We previously showed that myosin Ibeta was present in the microvilli of LLCPK1 PTCs and that its distribution was altered by ATP depletion (37). The primary objective in these studies was to determine the distribution of myosin Ibeta in the rat kidney under normal physiological conditions, after an ischemic insult, and during reperfusion. Figure 1A shows the specificity of the antibodies used in these studies. Western blot analysis of rat kidney cortex, medulla, and BB membranes (BBM) is shown.

Most myosin Ibeta in rat kidneys is Triton X-100 insoluble in contrast to that in LLCPK cells, which under control conditions is close to 90% soluble (37). Figure 1, B and C, shows the results from a Triton X-100 extraction of BBM, cortex, and medulla. After extraction on ice, the sample was centrifuged, and the supernatant and pellet were analyzed by silver stain (Fig. 1B) and Western blot (Fig. 1C) for myosin Ibeta . Note that the majority of myosin Ibeta is present in the F-actin-containing pellet in all samples. Over 90% of myosin Ibeta was found in the supernatant when ATP was added before centrifugation (data not shown). This finding agrees with our data from the in vitro ATP-depletion model. We observed no significant change in the Triton X-100 solubility of myosin Ibeta after ischemia or reperfusion in rat kidney (data not shown). This is likely due to the highly organized and developed cytoskeleton in vivo compared with the in vitro model.

Immunofluorescence images in Figs. 2 and 3 show that myosin Ibeta is widely distributed in the rat kidney. In Fig. 2, three rat kidney Vibratome sections double-labeled for myosin Ibeta (A, C, and E) and F-actin (B, D, and F) are shown. Figure 2, A and B, represent single planes, while Fig. 2, C-F, are reconstructions from multiple confocal planes. Note the prominent myosin Ibeta BB labeling in the proximal tubules. The intense labeling of F-actin with phalloidin easily identifies proximal tubules. We often observed a zone of reduced myosin Ibeta labeling under the BB that may correspond to the terminal web. There is also distinct cytoplasmic myosin Ibeta staining that appears rather punctate in many of the tubules. In Fig. 3, sections were double-labeled for myosin Ibeta (A and C) and peanut lectin (B and D) to address the presence of myosin Ibeta in non-PTCs. Peanut lectin labels primarily the intercalated cells in the rat kidney, though some staining has been observed in the inner medulla (7, 20). Myosin Ibeta is clearly present throughout the cytoplasm of collecting duct cells and is often concentrated near the apical surface of these cells. This distribution, along with its presence in the BB of PTCs, is consistent with a role for myosin Ibeta that involves regulating the dynamic actin network found near the plasma membrane in these cells (6).


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Fig. 2.   Myosin Ibeta is concentrated in the brush border (BB) of proximal tubule cells (PTC). Vibratome sections of rat kidney were double-labeled for myosin Ibeta and F-actin. A, C, and E show low- and high-power views of myosin Ibeta labeling in primarily proximal tubules (PT), whereas B, D, and F show F-actin labeling in the same tubule field. C and D are reconstructions from 24 0.5-µm planes, and E and F are reconstructions from 19 0.5-µm planes. Note the prominent staining of myosin Ibeta in the BB, the frequent weak staining zone under the BB, and consistent but weaker cytoplasmic staining. Bars = 10 µm.



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Fig. 3.   Presence of myosin Ibeta in nonproximal tubules. Vibratome sections of rat kidney were immunolabeled for myosin Ibeta and peanut lectin. A and C show the myosin Ibeta signal, whereas B and D show the peanut lectin labeling in the same tubule field. Note the presence of myosin Ibeta in the non-PTC, i.e., peanut lectin-positive tubules in addition to PTC. Bars = 10 µm.

Changes induced by ischemia. Ischemia has been shown to alter many cellular proteins and functions and has been most extensively studied in the PTCs (4, 24, 35). Our objective in these studies was to assess changes to myosin Ibeta caused by ischemia and to determine when myosin Ibeta returns to normal. We chose to use a 15-min ischemic clamp because structural alterations to the proximal tubules have been documented to follow this period of ischemia (26, 38, 39). Furthermore, this brief ischemic injury will result in less cell death and enable a quicker return to normal cell structure and function.

The first analysis addressed whether ischemia-reperfusion altered levels of myosin Ibeta mRNA. Quantitation of Northern blots, shown in Fig. 4A, showed changes to myosin Ibeta expression in both cortex and medulla after a 15-min clamp and different times of recovery. Note that after 2 and 4 h of reperfusion, there was an increase in expression of myosin Ibeta mRNA in both the cortex and medulla. After 24 h there was a significant decrease in myosin Ibeta expression in the medulla that did not return to normal levels till after 3 days of reperfusion. Myosin Ibeta mRNA in the cortex returned to normal levels within 3 days of reperfusion. These data suggest that myosin Ibeta mRNA expression is regulated differently in cortex and medullary cells and that its recovery from an ischemic injury occurs more slowly in the medullary cells. An increase in mRNA expression could be caused by the need to replace myosin Ibeta that has been lost. Western blot analysis of kidney proteins (Fig. 4B) was conducted to determine whether myosin Ibeta protein levels change after an ischemic injury. Quantitation using the UN-SCAN-IT program (Silk Scientific, Orem, Utah) on scanned blots showed that changes between control and recovery time points varied <10%. In addition, urine was collected and analyzed for its protein content to further evaluate myosin Ibeta release. Figure 4C shows the changes in urine protein before and after the ischemic clamp. Note that the urine output (ml/h) is similar under all conditions. To evaluate the urine protein content of the control and 15-min ischemia-24-h reperfusion collections, they were concentrated fivefold. The 15-min ischemia-2-h reperfusion sample was not concentrated. The most notable difference besides the increase in albumin after ischemia was the minor increase in the release of myosin Ibeta and actin at the 15-min ischemia-2-h reperfusion collection.


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Fig. 4.   Ischemia causes minor alterations to kidney myosin Ibeta mRNA and protein levels. A: Northern analysis of myosin Ibeta mRNA levels in both cortex and medulla is presented at selected time points of ischemia-reperfusion. All analyses compared the clamped kidney with the contralateral unclamped kidney. For each time point, a minimum of 3 samples was analyzed. Data are presented as means ± SE. Cortex and medulla values were combined to perform statistical analysis using ANOVA and the Tukey's honestly significant difference test. The variable tested was recovery time. *Significant differences (P < 0.05) were found between 2 and 4 h of recovery compared with the 24-h recovery time point. B: a Gel-Code Blue-stained gel and Western blot of 2 identical gels that contained kidney cortex and medulla samples under control conditions, after 15-min ischemia and 2-h recovery (15-2 h), or after 15-min ischemia and 24-h recovery (15-24 h). Total protein (50 µg) was loaded in each lane. The gel for Western blotting was cut and probed for myosin Ibeta or actin (A), and their respective bands are shown below the Gel-Code Blue-stained gel. C: a Gel-Code Blue-stained gel and Western blot of rat urine samples (lanes 1-8) under control (before clamping), after 15-min ischemia and 2-h recovery (15-2 h), or after 15-min ischemia and 24-h recovery (15-24 h). Results from identical gels blotted for myosin Ibeta and actin are presented below the stained gel. The control and 15-24 h urine samples were concentrated 5-fold because of their low protein levels. Concentrated urine (5 µl) was loaded for the control and 15-24 h samples, and 5 µl of collected urine was loaded for the 15-2 h collection.

Figure 5 shows two rat kidney sections double-labeled for myosin Ibeta (A and C) and F-actin (B and D) after 15 min of ischemia. Note the presence of myosin Ibeta closer to the apical surface and its presence in cellular blebs (Fig. 5C, arrows). These alterations were observed in tubules containing distinct F-actin labeling, i.e., proximal tubules, and also in those without distinct F-actin labeling, i.e., nonproximal tubules (Fig. 5A, star). The changes in location of myosin Ibeta are not restricted to proximal tubules and suggest that recovery will require reorganization and retargeting of this protein.


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Fig. 5.   Cellular redistribution of myosin Ibeta following ischemia. Vibratome sections from ischemic kidneys were double-labeled for myosin Ibeta (A and C) and F-actin (B and D). Note the presence of myosin Ibeta in cellular blebs from both PTC (C, arrows) and non-PTC (A, star). Myosin Ibeta was also found in the tubule lumens. C and D are single confocal planes, and A and B are reconstructions from 14 1-µm confocal planes. Bars = 10 µm.

Recovery from ischemia: Does myosin Ibeta participate in rebuilding the microvilli? Having established that myosin Ibeta undergoes dramatic changes with ischemia, our next objective was to establish the structural relationship between the reappearance of microvilli and the return of myosin Ibeta to the BB. Figure 6 shows the overall cell structure of primarily proximal tubules at both the light and EM level. While there are clearly some areas lacking complete microvilli that can be found after only 2 h of reperfusion (Fig. 6F, arrow), the majority of the proximal tubules appear to have reformed normal BB with microvilli.


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Fig. 6.   Effect of ischemia-reperfusion on PT morphology. The overall cell structure of PTs is shown at both the light level, stained with toluidine blue (Tol. Blue; A-C), and at the electron microscopy (EM) level (D-F) under control conditions (A and D), after 15 min of ischemia (B and E), and after 15 min of ischemia followed by 2 h of reperfusion (C and F). Asterisks identify PT lumens. Note the prominent BB and microvilli in A and D, their dramatic change in B and E, and their structural return by 2 h of reperfusion in C and F. The arrow in F identifies a PT with incomplete recovery of its microvilli. Bars = 10 µm.

Figure 7 highlights the major observations reported for myosin Ibeta under control (A) and ischemic (B) conditions and shows the location of myosin Ibeta after 15 min of ischemia and 2 (C-F) or 24 h (H and I) of reperfusion. Color images present myosin Ibeta in purple and F-actin in green. Colocalization between myosin Ibeta and F-actin is indicated as white. Note the prominent BB labeling for both F-actin and myosin Ibeta in the control state (Fig. 7A), where significant white is indicated, and the dramatic disruption to both myosin Ibeta and F-actin after ischemia (Fig. 7B). With 2 h of reperfusion, a modest increase in myosin Ibeta mRNA was found (Fig. 4A) with a similar decrease in myosin Ibeta protein occurring (Fig. 4B), and it also was found in the urine (Fig. 4C). Immunolocalization showed a more dramatic decrease in myosin Ibeta staining (Fig. 7, C, E, and F) after 2 h of reperfusion. Although the magnitude of the myosin Ibeta decrease was variable, there was a significant reorganization of this protein after ischemia and reperfusion (compare the color image of the control in Fig. 7A with the 2-h recovery state in Fig. 7F). This was most evident by the reduced staining of the BB for myosin Ibeta . In addition, the intensity of myosin Ibeta labeling was often increased in nonproximal tubular cells at 2 h. These results suggest that myosin Ibeta does not participate in the early rebuilding of the microvilli that occurs after reperfusion. By 24 h of reperfusion (Fig. 7, G-I) there were clearly regions of proximal tubules that had myosin Ibeta in the BB (Fig. 7H, arrows), although there was still a higher percentage of cytoplasmic labeling than observed in the normal physiological state, and many proximal tubules had little myosin Ibeta in the BB (Fig. H, triangles). In addition, we often observed an increase in myosin Ibeta staining in nonproximal tubules at 24 h of recovery. Interestingly, myosin Ibeta was also found in the urine from the 2- to 24-h reperfusion collection. The results combined suggest that a dramatic cytoskeletal and membrane reorganization of kidney cells has taken place, after 15-min clamp, that does not cause any significant release of protein but requires the cells to retarget and reorganize multiple cellular components, one of which is myosin Ibeta . Normal proximal tubule staining for myosin Ibeta returned by 3 days of reperfusion (data not shown). To obtain a better understanding of myosin Ibeta changes in other tubule segments, changes to specific tubular markers during ischemia-reperfusion must be followed. These studies, along with studies aimed at better defining the recovery of myosin Ibeta in the PTC, are needed to provide a clearer picture of the role of myosin Ibeta in kidney tubule function and recovery from ischemia.


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Fig. 7.   Cellular location of myosin Ibeta and F-actin after 15 min of ischemia and 2 or 24 h of reperfusion. Control tissue and ischemic tissue are presented in A and B, respectively, as color composites of images in Fig. 2, E and F, and Fig. 5, C and D. All color overlays show myosin Ibeta in purple and F-actin in green, and colocalization is indicated as white. D-F: reconstructions of 15 0.5-µm planes that show myosin Ibeta , F-actin, and the color composite from 15 min of ischemia and 2 h of reperfusion. C: myosin Ibeta staining from a kidney treated with 15 min of ischemia and 2 h of reperfusion; black-triangle indicates the PT. G-I: images of myosin Ibeta (H and I)- and F-actin-labeled (G) kidney sections after 15 min of ischemia and 24 h of reperfusion. Even after 24 h of recovery, we still found most PTs lacking the normal staining pattern for myosin Ibeta . Arrows identify PTs with some normal myosin Ibeta staining, and triangles identify PTs lacking any significant myosin Ibeta staining in the BB. Bars = 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ischemia triggers many cellular alterations along the nephron (4, 24, 35, 39). Reduced blood flow results in low cellular ATP levels, effectively inhibiting many ATP-dependent processes such as phosphorylation, ion regulation, and vesicle trafficking. These alterations contribute to the cell injury observed and lead to tubule and kidney dysfunction. The best-studied ischemic changes occur in proximal tubules, where the apical cytoskeleton undergoes dramatic rearrangement after cessation of blood flow in vivo. Actin, the major protein component of the microvilli, is redistributed in the apical cytoplasm. Its regulation and associated proteins, including ezrin and actin-depolymerizing factor (ADF), are also altered under ischemic conditions (10, 11, 31). It has become increasingly apparent that ischemia disrupts the normal cellular morphology and physiology of the distal segments of the nephron as well (14, 15, 19, 21, 29). Return to the normal physiological state requires retargeting of misdirected cellular components and rebuilding of the cytoskeleton.

Myosin Ibeta is an actin-activated ATPase with both actin and membrane binding domains (3). It has three calmodulin light chain binding domains that bind calmodulin strongest in the absence of calcium. Its widespread distribution argues for involvement in a fundamental cell process shared by multiple cell types. In these studies, we analyzed its tubular distribution and addressed its potential structural role in PTC microvilli. It has previously been suggested that myosin I tethers actin to the plasma membrane of the microvillus (27). Under control conditions, myosin Ibeta was found to be ~90% Triton insoluble, probably due to its interaction with F-actin. Immunofluorescence labeling of myosin Ibeta and F-actin in normal kidney tissue sections showed extensive colocalization of these two proteins in the BBM. There is strikingly no colocalization of myosin Ibeta with actin in the stress fibers of these cells. There appears to be a zone of minimal myosin Ibeta labeling beneath the BB, which may correspond to the proximal tubule terminal web region. This zone is often followed by a punctate distribution of myosin Ibeta in the cytoplasm. The myosin just beneath the BB may be associated with vesicles that are to be transported along actin filaments to the microvillar membrane. Multiple studies have implicated myosin I's as playing a role in endocytosis and intracellular trafficking (23, 33, 40). Movement into and throughout the BB region, a region lacking microtubules, may utilize an actin-based motor such as myosin Ibeta . Alternatively, myosin Ibeta in the subapical region could be simply waiting for transport to the BB via another myosin motor. Distinguishing whether this myosin is actively involved in transport to the BB or a non-transport function would be aided by a better understanding of what this myosin interacts with and how these interactions are regulated. Interactions between multiple myosin Ibeta proteins have been suggested as a prerequisite for this motor to perform work, and the subapical punctate distribution may represent a small cluster of myosin Ibeta molecules (28). It is widely accepted that the actin cytoskeleton plays a role in the regulation of multiple channels; thus it is possible that myosin I actin-based motors contribute to these regulatory processes (8, 32, 34, 38). Myosin I's may also help shuttle membrane vesicles containing channels to and from the plasma membrane and/or may affect the dynamics of the actin cytoskeleton, thus indirectly contributing to channel regulation.

Under normal physiological conditions, myosin Ibeta and actin are concentrated in the BB of the proximal tubule. With ischemic injury, the actin cytoskeleton is disrupted, microvilli shorten and fuse, ATP levels fall, and intracellular calcium levels rise. Low levels of ATP favor formation of a rigor complex between myosin and actin. Interestingly, after 15 min of ischemia, myosin Ibeta is observed in cellular blebs in the proximal tubule lumens without F-actin. This early separation of myosin Ibeta and F-actin may result from the rapid degradation of microvillar F-actin by ADF, which is present in these blebs, and the calcium activation of F-actin severing proteins, such as gelsolin (6, 24, 31). What is clear is that the microvillar F-actin undergoes dramatic alterations within 15 min of ischemia that result in apical enrichment of myosin Ibeta and G-actin in this area (31, 35). A possible scenario to explain what happens to myosin Ibeta follows: increased intracellular calcium releases calmodulin from myosin Ibeta , causing an increase in ATPase activity and membrane binding. This enables the myosin to move quickly up the collapsing microvillar F-actin, where it associates with the membrane as F-actin breaks down. The polarity of F-actin (barbed end apical) is such that myosin Ibeta would move to the barbed end. Myosin Ibeta could then associate with a select group of acidic phospholipids that collect in the blebs or bind to a receptor protein that has accumulated in the blebs. We are currently examining some of these possibilities by analyzing BBM vesicles from normal and ischemic proximal tubules.

While the precise sequence of these cellular ischemic events is uncertain, it is well established that return of physiological cell and organ function requires reestablishment of the normal cell architecture. This will involve, in part, rebuilding the apical microvilli and retargeting membrane and proteins back to their correct location. We showed in these studies that normal F-actin structure is obtained after 2 h of reperfusion following a 15-min clamp. Surprisingly, myosin Ibeta normal staining pattern is not returned to normal. In fact, myosin Ibeta is present throughout the cytoplasm in both proximal and distal tubules and often appears in larger punctate structures than in the control tissue. This suggests that either the cellular process(es) in which this motor protein participates has not recovered or it may be actively assisting in the retargeting and/or reorganization of actin cytoskeleton and membrane components. Even after 24 h of reperfusion there are regions of PTC that do not appear normal for myosin Ibeta . However, at 2 and 24 h of recovery, we have shown that there are only minor changes in both protein and mRNA levels for myosin Ibeta . This finding suggests that the changes we observe by immunofluorescence are the result of dramatic cytoskeletal and membrane reorganization that occurs after an ischemic insult. Apparently, instead of simply making more of the myosin Ibeta , the cells have to retarget and reestablish this protein's normal cellular interactions, which our data suggest is not complete even after 24 h of recovery. We believe that interactions of myosin Ibeta interactions with membrane phospholipids, F-actin, and possibly membrane proteins undergo significant reorganization during reperfusion that ultimately contributes to the return of functioning kidneys. During this reorganization, a reduced staining pattern for myosin Ibeta is observed in the BB. Our results suggest that this is caused by a redistribution of this myosin away from the BB. Analysis of distinct subcellular fractions, i.e., BBM, will help to explain these changes. We are presently examining the recovery time points following the ischemic block to better define the physiological changes that may coincide with the return of myosin Ibeta . The major conclusion from these studies is that myosin Ibeta is present throughout the kidney tubules, its distribution is severely altered by ischemia, and its return follows the apparent structural return of the BB.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54923.


    FOOTNOTES

Address for reprint requests and other correspondence: M. C. Wagner, Indiana Univ. School of Medicine, 1120 South Dr., FH 115, Indianapolis, IN 46202-5116 (E-mail: wagnerm{at}iupui.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 October 2000; accepted in final form 25 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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