Redistribution of Myosin VI from Top to Base of Proximal Tubule Microvilli during Acute Hypertension
Li E. Yang*,
Arvid B. Maunsbach,
Patrick K.K. Leong* and
Alicia A. McDonough*
* Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, Los Angeles California; and The Water and Salt Research Center, Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark
Address correspondence to: Dr. Alicia A. McDonough, Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, 1333 San Pablo Street, MMR 626, Los Angeles, CA 90089-9142. Phone: 323-442-1238; Fax: 323-442-2283; E-mail: mcdonoug{at}usc.edu
Received for publication April 6, 2005.
Accepted for publication July 15, 2005.
During acute hypertension, Na+/H+ exchangers (NHE3) retractfrom top to base of proximal tubule microvilli (MV) and Na+reabsorption decreases in proximal tubule. This study aimedto determine whether the actin-based motor myosin VI coordinatelyretracts with NHE3 in response to acute hypertension. BP wasraised approximately 50 mmHg in rats for 20 to 30 min or shamtreated, and kidneys were analyzed by subcellular fractionationor microscopy. During acute hypertension, myosin VI redistributedfrom low density apical MV-enriched membranes (from 23 ±2.4 to 11.4 ± 2.2%) into higher density membranes (from23.2 ± 0.7 to 36.9 ± 2.6%). By confocal microscopy,myosin VI was detected over the whole length of the MV in controls,then became completely focused at the base of MV during acutehypertension. For electron microscopic analysis using immunogoldlabeling, MV were divided into five zones from top (z1) to base(z5). In controls, myosin VI was evenly distributed throughthe five MV zones. In acute hypertension, myosin VI decreasedin z1 (from 20.6 ± 1.9 to 10.5 ± 2.3%) and z2(from 21.0 ± 2.0 to 13.2 ± 1.4%) and increasedin z5 (from 21.1 ± 3.3 to 38.6 ± 3.0%). Theseresults provide the first observation that acute hypertensioncauses myosin VI redistribution and support the idea that myosinVI may serve as the molecular motor for NHE3 retraction fromtop to base of MV during acute hypertension.
Na+/H+ exchange is the major route for apical sodium entry acrossthe proximal tubule (PT), and the Na+/H+ exchanger isoform 3(NHE3) is responsible for virtually all of the Na+/H+ exchangeactivity in this region (1,2). A rapid increase in BP acutelydecreases PT sodium reabsorption, which both increases NaClat the macula densa, a tubuloglomerular feedback signal to normalizerenal blood flow and GFR, and causes a pressure natriuresisthat reduces extracellular volume to counteract the hypertension(3–6). We previously determined (using subcellular fractionationand confocal and electron microscopy) that NHE3 is rapidly retractedto the base of the PT microvilli (MV) during acute hypertension(7–9) along with the suppression of PT Na+ reabsorption.During this response, NHE3 seems to migrate within the planeof the membrane from the top to the base of the MV, and themolecular mechanisms that could account for this movement havenot been determined. Relevant to this issue, the molecular motormyosin VI was shown recently to be highly enriched in the PTbrush border region (10,11), suggesting a candidate motor thatcould effect protein trafficking in this region.
Myosin VI, an unconventional myosin, moves toward the minusends of actin filaments, which are located at the base of theMV in the setting of the PT. Recent studies implicate this actin-basedmotor in forming clathrin-coated vesicles and in moving uncoatedvesicles through the actin mesh (12–15). Consistent witha role in endocytosis, Biemesderfer et al. (11) showed thatmyosin VI is highly enriched in the intermicrovillar-coatedpit region of the PT along with the clathrin adaptor proteinAP-2. These investigators also saw a significant amount of myosinVI in the apical MV of PT, where it presumably could move alongactin from the tops to the bases of the MV. The aim of thisstudy is to test the hypothesis that acute hypertension inducesredistribution of myosin VI along with NHE3 from the top tothe base of the MV. The results indicate coincident retractionof membrane-associated myosin VI and NHE3 from the top to thebase of the MV in response to rapid BP elevation, thus providingevidence that myosin VI may participate in driving the rapidtrafficking of Na+/H+ exchangers within the apical microvillardomain of the proximal tubule.
Acute Hypertension Protocol
As described in detail previously (8,9,16), male Sprague-Dawleyrats (290 to 320 g body wt) were anesthetized intramuscularlywith ketamine (Fort Dodge Laboratories, Overland Park, KS) andxylazine (1:1, vol/vol; Miles, Shawnee Mission, KS) and placedon a thermostatically controlled table (37°C), BP was recordedfrom carotid artery, and 4.0% BSA in 0.9% NaCl was infused (50µl/min) to maintain euvolemia. Mean arterial pressurewas increased 50 to 60 mmHg over baseline by constricting thesuperior mesenteric artery, celiac artery, and abdominal aortabelow the renal artery with silk ligatures.
Subcellular Fractionation and Immunoblot
As described in detail previously (5,16), 20 min after BP waselevated (or sham treated), kidneys from control and acute hypertension-challengedanimal were cooled and excised; renal cortex was dissected andhomogenized; and a low-speed supernatant was isolated, loaded,and resolved on a sorbitol density gradient. Twelve fractionswere collected, pelleted, resuspended, and stored at –80°Cpending assays.
Ten-microliter aliquots of each gradient fraction or of pooledwindows (defined in Figure 1) were denatured in SDS-PAGE samplebuffer (30 min, 37°C), resolved on 7.5% SDS polyacrylamidegels (17), and transferred to polyvinylidene difluoride membranes(Immobilon-P; Millipore, Bedford, MA). Total sample proteinloaded ranged from 1 µg (fraction 2) to 14 µg (fraction7). Blots were probed with either polyclonal NHE3-C00 (8) oranti–myosin VI (T. Hasson, University of San Diego) (10)at 1:2000 dilution, then with Alexa 680–labeled goat anti-rabbitsecondary antibody. Villin was detected with a mAb (Immunotech,Chicago, IL) at 1:1000 dilution, then with Alexa 680–labeledgoat anti-mouse secondary. Signals were detected and quantifiedwith an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).
Figure 1. Myosin VI and Na+/H+ exchanger isoform 3 (NHE3) redistribute from low-density membranes to high-density membranes during acute hypertension (high BP). Renal cortices from the rat kidneys were removed and subjected to subcellular fractionation on sorbitol density gradients and collected as 12 fractions. A constant volume of sample from each gradient fraction was resolved by SDS-PAGE. Typical immunoblots of myosin VI, NHE3, and villin from control versus 20-min high BP rats are shown. The assayed volumes were adjusted to ensure that signals were within the linear range of detection. WI, window I, fractions 3 to 5 enriched in apical MV markers; WII, window II, fractions 6 to 8 enriched in MV, intermicrovillar cleft (IMC), and intermicrovillar coated pit (ICP) markers; WIII, window III, fractions 9 to 12 contains MV, IMC, ICP, and endosome/lysosome (endo, lyso) markers.
Indirect Immunofluorescence
As detailed previously (9), after BP was elevated for 10 min(or sham treated for same time), the left kidney was fixed insitu in a small Plexiglas cup in periodate-lysine-paraformaldehyde(PLP) fixative for another 20 min, during which time BP remainedelevated. Kidneys were removed, postfixed in PLP, rinsed, incubatedovernight in 30% sucrose/PBS, embedded, and frozen in liquidnitrogen. Cryosections (5 µm) were cut, transferred tocharged glass slides, air dried, rehydrated, incubated with1% SDS/PBS for 4 min (antigen retrieval) (18), washed, and blockedwith 1% BSA/PBS. Sections were dual labeled with either polyclonalanti–NHE3-C00 or anti–myosin VI (1:100) and eithermonoclonal anti-villin (1:100) or anti-AP2 (clathrin adaptor;1:50; Sigma, St. Louis, MO) in 1% BSA/PBS for 1.5 h at 25°C;then washed and incubated with a mixture of FITC-conjugatedgoat anti-rabbit (Cappel Research Products, Durham, NC) andAlexa 568–conjugated goat anti-mouse (Molecular Probes,Inc., Eugene, OR) secondaries (1:100) in 1% BSA/PBS for 1 h;and washed, mounted, and dried overnight, all as described (9).Slides were viewed with a Nikon PCM Quantitative Measuring High-PerformanceConfocal System equipped with filters for both FITC and TRITCfluorescence attached to a Nikon TE300 Quantum upright microscope.Images were acquired with Simple PCI C-Imaging Hardware andQuantitative Measuring Software (Compix Inc. Imaging Systems,Cranberry Township, PA).
Immunoelectron Microscopy
BP was increased 50 to 60 mmHg over baseline as described inthe Acute Hypertension Protocol section or sham treated. After20 min of acute hypertension, two rats were perfusion-fixedwith 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH7.2), and the others were fixed by superfusion of the kidneysurface with the fixative. The immunolabeling pattern, describedbelow, was the same with the two fixation methods. As detailedpreviously (9), tissue blocks were trimmed from the cortex,postfixed in the same fixative for 2 h, rinsed in buffer, infiltratedwith 2.3 M sucrose, mounted on holders, and frozen in liquidnitrogen. Immunoelectron microscopy was performed either onthin (70 nm) cryosections prepared on a Reichert Ultracut Scryoultramicrotome (Leica, Germany) or on tissue that was cryosubstitutedin a Reichert AFS freeze-substitution apparatus (Leica, Germany)and embedded in Lowicryl HM20 as described previously (19).
The Lowicryl sections or ultrathin cryosection were blocked(PBS that contained 0.05 M glycine and either 0.1% skim milkpowder or 1% BSA) and incubated with polyclonal anti–myosinVI (1:100, 1 h) in PBS/0.1% skim milk powder. Anti–myosinVI was visualized with goat anti-rabbit IgG conjugated to 10nm of colloidal gold particles (GAR.EM1O; BioCell Research Laboratories,Cardiff, UK; 1:50 in PBS/0.1% skim milk powder and 5 mg/ml polyethyleneglycol).Lowicryl sections were stained with uranyl acetate, and theultrathin cryosections were stained with 0.3% uranyl acetatein 1.8% methylcellulose for 10 min. Immunolabeling controlswere incubated with nonimmune rabbit IgG or without primaryantibody. All controls showed absence of labeling. Electronmicrographs were taken at x16,000 in a Morgagni FEI electronmicroscope (Hillsboro, OR) from brush border areas where themicrovilli were sectioned longitudinally. Thus, the micrographsrepresent random samples of microvilli in PT segments S1 andS2.
Quantitative Analysis of Myosin VI Distribution
The distribution of myosin VI, demonstrated by immunogold labeling,was determined in cryosections from six controls and five animalswith high BP over three areas: The MV, where the MV were closeto parallel with the section; the intermicrovillar zone (IMZ);and the apical cytoplasmic zone (ACZ) as defined in Figure 2.The MV was divided in five zones, counted from top to base ofthe microvilli: Zone 1 from 0 to 20% of the length of a givenmicrovillus, zone 2 from 20 to 40%, etc. Zone 5 thus representsthe very base of the MV and does not include the membrane betweenthe MV or the endocytic invaginations. The IMZ begins wherethe MV end and is arbitrarily defined to extend to a (measured)depth of 0.5 µm into the apical part of the cell. It containsthe plasma membrane between the MV, the coated invaginations,small coated endocytic vacuoles, dense apical tubules, and sometimeslarge endocytic vacuoles. The ACZ is arbitrarily defined asthe next 0.5-µm-deep zone into the apical cytoplasm. Themeasured location of individual gold particles, representinglabeled myosin VI, was assigned to one of the five zones alongthe microvillus or to IMZ or ACZ. The number of gold particleswithin each zone was expressed as percentage of total gold particlescounted in each animal. The numbers for each zone are the accumulatednumbers from several sections from each animal. Mean valuesfor each zone in controls and in animals with high BP were comparedwith ANOVA.
Figure 2. Schematic drawing and subdivision of apical surface of proximal tubule (PT) cell for immunogold analysis of myosin VI distribution. The locations of gold particles were determined over three areas of PT cells: Microvilli (MV), intermicrovillar zone (IMZ), and apical cytoplasmic zone (ACZ). The IMZ is arbitrarily defined to extend 0.5 µm into the apical part of the cell. It contains the plasma membrane between the MV, the coated invaginations, coated or uncoated endocytic vacuoles, and dense apical tubules (striated). The ACZ is defined as an adjacent 0.5-µm-wide zone deeper in the apical cytoplasm. The location of individual gold particles, representing labeled myosin VI, was measured from the tip of each microvillus and related to the measured length of the same microvillus. Each gold particle then was referred to one of five equal zones of the MV: Zone 1 from 0 (tip of MV) to 20% of the length of a given microvillus, zone 2 from 20 to 40%, etc. Zone 5 thus contains the very base of the MV. The percentage of colloidal gold particles in each zone was determined for each animal.
Coincident Redistribution of Myosin VI and NHE3 by Subcellular Membrane Fractionation
Membrane marker characteristics of the fractions collected fromthe sorbitol density gradients have been reported previously(5,8). In brief, fractions 3 to 5 (window I [WI]) contain plasmamembrane markers, including apical markers alkaline phosphatase,dipeptidyl-peptidase IV, NHE3, and basolateral Na,K-ATPase;fractions 6 to 8 (window II [WII]) also contain apical membranemarkers as well as most of the intermicrovillar cleft (IMC)marker megalin and the clathrin-coated pit adaptor protein AP2;fractions 9 to 11 (window III [WIII]) are enriched in the endosomalmarker rab 5a and the lysosomal marker -hexosaminidase as wellas some apical membrane and intermicrovillar cleft markers.We established that NHE3 is acutely retracted from the top tothe base of the proximal tubule MV during acute hypertension(high BP) (9). Figure 1 demonstrates typical density gradientpatterns in renal cortex from control versus 20-min high BPchallenged rats probed with antibodies to myosin VI, NHE3, andvillin. Myosin VI and NHE3 transit in concert out of light density(WI) into higher density (WII and WIII) membranes in responseto high BP. The broad distribution pattern of the actin-bundlingprotein villin (between fractions 4 and 12) is unaltered byhigh BP, demonstrating that there is not an overall change indensity of the apical membranes. For quantification of the densityshift, the density gradient fractions were pooled into the threewindows as defined in Figure 1, and percentage of total in eachwindow was calculated. Twenty-minute high BP provoked coincidentredistribution of myosin VI (Figure 3A) and NHE3 (Figure 3B)out of low-density WI membranes: Myosin VI decreased from 23± 4.8% (control) to 11.4 ± 4.4% (high BP), andNHE3 decreased from 23.7 ± 4.8% (control) to 8.1 ±3.9% (high BP). Both proteins redistributed to higher densityWIII membranes: Myosin VI increased from 23.2 ± 1.4%(control) to 36.9 ± 5.2% (high BP), and NHE3 increasedfrom 13.5 ± 3.4% (control) to 22.1 ± 4.8% (highBP). Villin distribution was not changed (Figure 3C) and totalprotein distribution changed only minimally (Figure 3D) by highBP, indicating that the redistribution of myosin VI and NHE3are specific.
Figure 3. Summary of myosin VI, NHE3, villin, and protein distribution in three windows, expressed as the percentage of the total signal in all three windows. On the basis of previous marker analyses (8), membrane fractions were pooled into three windows and subjected to Western blot analysis: Fractions 3 to 5 were pooled into WI, fractions 6 to 8 were pooled into WII, and fractions 9 to 11 were pooled into WIII. A constant volume of sample from each window was resolved by SDS-PAGE. Values are means ± SD; n = 4 in each group. *P < 0.05 versus control, assessed by ANOVA and followed by paired t test.
Immunofluorescence Evidence for Myosin VI Redistribution
Double labeling with antibodies to myosin VI versus markersof brush border MV or clathrin coated-pits was performed onkidneys that were harvested from control and acute high BP challengedrats. Kidneys were surface rather than perfusion fixed in thisseries. Villin was stained to indicate position of MV; its distributionis not altered by acute hypertension (7). At baseline pressure(Figure 4A, left), there is a significant amount of myosin VIboth in the proximal tubule MV (arrow) and at the base of theMV (arrowhead) as previously reported (11). During high BP (Figure 4A,right), there is a shift in myosin VI distribution to thebase of the MV (green, arrowhead) leaving the red villin stainingin the MV.
Figure 4. Indirect immunofluorescence microscopy of myosin VI redistribution during acute hypertension. Kidneys from control and 10-min acute hypertension challenged rats were fixed in situ with PLP for 20 min, followed by in vitro fixing for another 3 to 4 h. (A) Kidney surface sections were double labeled with polyclonal anti–myosin VI, then FITC-conjugated goat anti-rabbit secondary antibody (green) and with monoclonal anti-villin antibody, then with Alexa 568–conjugated goat anti-mouse secondary antibody (red). Villin was labeled to indicate where the MV are. At baseline pressure, there is a significant pool of myosin VI in the proximal tubule MV (arrow) and an enriched myosin VI staining at the base of the MV (arrowhead). During acute high BP, myosin VI is retracted from the MV to the base of MV (arrowhead). (B) Sections were double labeled with polyclonal anti–myosin VI then FITC-conjugated goat anti-rabbit secondary antibody (green), and with monoclonal anti–AP-2 antibody, then Alexa 568–conjugated goat anti-mouse secondary antibody (red). In controls, myosin VI is both located in the MV above AP-2 staining (arrow) and partially co-localized with AP-2 at the base of the MV (arrowhead). After high BP, myosin VI completely concentrated at the base of the MV overlapping with AP-2 staining (yellow, arrowhead). Myosin VI staining does not retract to below AP-2–stained domain in acute hypertension. Bar = 10 µm.
To define further the destination of the retracted myosin VIduring high BP, we performed dual labeling of myosin VI andthe clathrin adaptor protein AP-2. In controls (Figure 4B, left),myosin VI is partially co-localized with AP-2 at the base ofthe MV (arrowhead), with a significant amount of myosin VI locatedin the MV above AP-2 staining (arrow). After high BP (Figure 4B,right), myosin VI is concentrated at the base of the MVevident either right above or overlapping with AP-2 staining(yellow, arrowhead). However, there is no obvious myosin VIinternal to AP-2 staining. These results indicate that myosinVI is retracted from the top to the base of the MV during highBP rather than to pools below the MV.
Immunoelectron Microscopic Analysis of Myosin VI Redistribution
In Figure 5, myosin VI labeling is observed along the lengthof the MV in both controls and animals with high BP. However,in controls, labeling is evenly distributed along the MV (Figure 5A),whereas after acute hypertension, gold particles appearmore frequent over the inner half of the MV (Figure 5B). Thisdifference in labeling of myosin VI between controls and animalswith high BP was documented in a quantitative analysis of myosinVI distribution as revealed by the colloidal gold particles,which eliminates cell-to-cell variability (Figure 6A): MyosinVI–associated particle density decreased significantlyin the top of the MV (zones 1 and 2) and doubled at the baseof the MV (zone 5). In both controls and animals with high BP,myosin VI was also present in the IMZ and in the ACZ fartherinto the cytoplasm. The quantitative analysis showed that myosinVI distribution, as percentage of total in the slice, was notaltered by high BP in MV (approximately 50% of total myosinVI), IMC (approximately 30%), and ACZ (approximately 20%; Figure 6B).That is, there was significant redistribution within theMV during high BP but not between MV, IMC, and ACZ zones asdefined.
Figure 5. Immunoelectron microscopic analysis of the myosin VI redistribution during acute hypertension. In control rats (A), gold particles labeling myosin VI are present along the whole length of the MV (arrows), whereas in high BP rats (B), there are more gold particles over the inner half of the MV than over the outer half. There is equal labeling of myosin VI (arrowheads) in IMZ and ACZ in control and high BP rats. In high BP rats, immunogold labeling of villin (C) shows gold particles along the whole length of the MV. Bar = 0.5 µm.
Figure 6. Quantification of myosin VI redistribution by immunoelectron microscopy during acute hypertension. (A) Distribution of immunogold labels for myosin VI along the length of the MV in high BP versus control. The distribution of gold label is uniform along the MV in the control group, but in the high BP group, there is a significant decrease in myosin VI labeling at the tip and outer part of the MV and a significant increase in myosin VI labeling in the innermost zone of the MV. (B) Both in the high BP group and in the control group, approximately 50% of the myosin VI is associated with the brush border, 30% with the IMZ, and 20% with the ACZ. There are no statistical differences between the groups with respect to the frequency of gold labels over these zones. This suggests that there is no movement of myosin VI into or out of the brush border, relative to the cell, after acute increase in BP. Values are means ± SEM; n = 6 in control and 5 in high BP. *P < 0.05 versus control, assessed by ANOVA.
Comparison of villin labeling of the MV in controls and animalswith high BP revealed no difference in villin distribution.Specifically, there was no evidence of decreased labeling inthe top of the MV (Figure 5C).
This laboratory previously established that in response to eitheracute hypertension or parathyroid hormone treatment, NHE3 isretracted from the top to the base of the proximal tubule MVassociated with decreased PT Na+ reabsorption; that is, NHE3is retracted within the apical membrane plane without endocytosis(7,9). This study addresses how NHE3 moves within the planeof the MV membrane. The trafficking of the membrane proteinsin epithelial cells often utilizes actin as a track or relieson the interaction of the membrane proteins with actin and itsmolecular motors. Recently, Wagner et al. (20) showed that expressionof myosin I (Myo1c), an actin-based unconventional myosin inM1 collecting duct cells, participates in the regulation ofthe Na+ channel after ADH stimulation. It is well establishedthat the brush border MV are dense and filled with bundled actinfilaments. The unconventional myosin VI is highly enriched inthe PT brush border region (10,11). Myosin VI moves toward thepointed end of actin filaments (21), which are located at thebase of the MV in the setting of the PT, suggesting that itcould move cargo proteins such as NHE3 along the MV down tothe base of the MV. The findings from subcellular fractionation(Figures 1 and 3) and confocal (Figure 4) and electron microscopy(Figures 5 and 6) provide evidence for movement of myosin VIfrom the top to the base of the MV in response to acute hypertension,coincident with redistribution of NHE3 within the plane of membranealong the microvillus. Myosin VI staining did not retract tobelow AP-2–stained domain (Figure 4B), suggesting thatthere is no detectable myosin VI–involved endocytosisin acute high BP. At the electron microscopic level, quantitativeanalysis of the gold particles that label myosin VI indicatesthat at baseline pressure, approximately half of the myosinVI protein is located in the MV, the other half split betweenthe IMZ and the ACZ. High BP did not cause redistribution ofmyosin VI among these three defined regions; rather, it provokedsignificant retraction of myosin VI from the tops to the basesof the MV. These results, taken together, support the hypothesisthat myosin VI may play a role in the regulated decrease inproximal tubule sodium transport that occurs during acute hypertension(4,6,22), specifically by driving the retraction of NHE3 withinthe MV. Considering the results from another angle, they providethe first evidence for regulation of renal myosin VI distribution,establishing this motor as a candidate that could regulate anumber of PT functions. We have evidence (not shown) that theredistribution of myosin VI is not isolated to high BP treatmentas there is also a coordinate redistribution of myosin VI andNHE3 from low to higher density membranes in rats infused withthe angiotensin-converting enzyme inhibitor captopril, whichoccurs in the absence of a change in BP (manuscript submitted).
This study also highlights the notion of rapid regulation ofmembrane transporters and membrane-associated proteins in vivobetween subdomains of the plasma membrane (without internalization)associated with a change in Na+ transport. NHE3 apparently movesto a reservoir pool from which it could be rapidly recruitedwhen a need for increased Na+ reabsorption occurs. Recently,Inoue et al. (23) demonstrated that in potassium deficiency,NaPi IIa is increasingly partitioned in the low-density microdomainsof the apical membranes enriched in cholesterol, sphingomyelin,and glycosphingolipid, characterized as "lipid raft" fractionson a detergent-free density gradient. By using fluorescencecorrelation spectroscopy, NaPi IIa lateral diffusion was decreasedand NaPi IIa aggregation/clustering increased, associated withdecreased Na/Pi co-transport activity in K+ deficiency. Chaet al. (24) recently expressed chimera NHE3–enhanced greenfluorescent protein (EGFP) in renal epithelial opossum kidney(OK) cells and demonstrated that the lateral mobility of NHE3on the apical membrane of OK cells is dependent on an intactactin cytoskeleton determined by fluorescence recovery afterphotobleaching and confocal microscopy.
We know that during acute hypertension, NHE3 is retracted tothe base of the MV, and we know that PT Na+ reabsorption isinhibited by the high BP, but we do not know whether retractioncauses or is simply associated with inhibition of NHE3 transportactivity. Recently, Kocinsky et al. (25) demonstrated that thereis a distinct pattern of NHE3 phosphorylation in the MV:NHE3phosphorylated at serine 552 localized to the coated pit regionof the brush border membrane, whereas total NHE3 was found throughoutthe brush border membrane. Whether NHE3 phosphorylation at serine552 is increased during high BP and whether it is critical forPT Na+ transport inhibition remain to be determined.
The factors that constrain the Na+ transporter and myosin VIat the base of the MV during hypertension remain to be defined.Evidence suggests that NHE3 can be tethered to the actin cytoskeletonvia the PDZ domain protein NHERF and ezrin (26). Biemesderferet al. (27) provided evidence that NHE3 in the PT exists intwo oligomeric states: An active 9.6S form present in brushborder MV and an inactive 21S megalin-associated NHE3 in densevesicles that contain markers of the IMZ (28). The same groupalso demonstrated that the adaptor protein Dab2 co-localizeswith both myosin VI and NHE3/megalin complex at the coated pitregion of the brush border. It is interesting that Swiatecka-Urbanet al. (15) demonstrated that in polarized human airway epithelialcells (Calu-3), myosin VI forms a complex with Dab2 and cysticfibrosis transmembrane conductance regulator and facilitatescystic fibrosis transmembrane conductance regulator endocytosisby a mechanism that requires actin filaments. It remains tobe determined whether there is an increased association of myosinVI, Dab2, and NHE3/megalin during high BP.
Acknowledgments
This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases grant DK-34316 (A.A.M.) and DanishMedical Research Council and the Water and Salt Research Centerestablished and supported by the Danish National Research Foundation(Grundforskningsfonden; A.B.M.). L.E.Y. was supported by anAmerican Heart Association Postdoctoral Fellowship award. Confocalmicroscopy was supported by Core Center grant DK-48522.
Components of this project were presented in abstract form (JAm Soc Nephrol14: 318A, 2003; J Am Soc Nephrol 15: 196A, 2004).
We thank Else-Merete Locke, Tina Drejer, and Karen Thomsen forexpert technical assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication April 6, 2005.
Accepted for publication July 15, 2005.
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Top
Abstract
Introduction
-hexosaminidase as well as some apical membrane and intermicrovillar cleft markers. We established that NHE3 is acutely retracted from the top to the base of the proximal tubule MV during acute hypertension (high BP) (9). Figure 1 demonstrates typical density gradient patterns in renal cortex from control versus 20-min high BP challenged rats probed with antibodies to myosin VI, NHE3, and villin. Myosin VI and NHE3 transit in concert out of light density (WI) into higher density (WII and WIII) membranes in response to high BP. The broad distribution pattern of the actin-bundling protein villin (between fractions 4 and 12) is unaltered by high BP, demonstrating that there is not an overall change in density of the apical membranes. For quantification of the density shift, the density gradient fractions were pooled into the three windows as defined in Figure 1, and percentage of total in each window was calculated. Twenty-minute high BP provoked coincident redistribution of myosin VI (Figure 3A) and NHE3 (Figure 3B) out of low-density WI membranes: Myosin VI decreased from 23 ± 4.8% (control) to 11.4 ± 4.4% (high BP), and NHE3 decreased from 23.7 ± 4.8% (control) to 8.1 ± 3.9% (high BP). Both proteins redistributed to higher density WIII membranes: Myosin VI increased from 23.2 ± 1.4% (control) to 36.9 ± 5.2% (high BP), and NHE3 increased from 13.5 ± 3.4% (control) to 22.1 ± 4.8% (high BP). Villin distribution was not changed (Figure 3C) and total protein distribution changed only minimally (Figure 3D) by high BP, indicating that the redistribution of myosin VI and NHE3 are specific. 
