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1Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142; and 2The Water and Salt Research Center, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
Submitted 3 May 2004 ; accepted in final form 16 July 2004
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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kidney; sodium transporter; pressure diuresis; immunoelectron microscopy
This laboratory previously investigated the molecular mechanisms responsible for the decrease in PT sodium reabsorption during an experimental acute increase in BP and discovered there is a parallel retraction of NHE3 and NaPi2 from the apical microvilli (MV) to membranes of higher density enriched in intermicrovillar cleft (IMC) and endosomal markers, demonstrated by subcellular membrane fractionation (35, 41). Confocal microscopic evidence also supported that NHE3 is retracted from the apical MV in acute hypertension (20, 35). The destination of NHE3 and NaPi2 after retraction from the apical MV was, however, unclear. Like acute hypertension, in vivo parathyroid hormone (PTH) treatment inhibits both the sodium-phosphate-coupled transport and sodium/hydrogen exchange in the PT and also causes diuresis and natriuresis (1, 29). This laboratory observed a similar retraction of NHE3 and NaPi2 from the apical MV-enriched membrane fractions by density gradient centrifugation of renal cortex after in vivo treatment with PTH (43), which could contribute to the decrease in PT sodium and phosphate reabsorption. Immunocytochemical studies have already demonstrated that PTH causes NaPi2 internalization and degradation (22, 32). In contrast, the destination of NHE3 in response to PTH treatment in vivo has not been clarified.
There is evidence for regulated endocytosis of NHE3 in cultured cell lines. In Chinese hamster ovary cells, transfected NHE3 is localized to endosomal compartments in addition to the plasma membranes and the trafficking of NHE3 depends on dynamin and cytoskeleton (12, 15, 30). In opossum kidney (OK) cells, PTH or dopamine acutely stimulates the endocytosis of NHE3 via clathrin-coated vesicles (13, 17). Also, in OK cells, NHE3-mediated endosomal acidification is implicated in the endocytosis of albumin (16). However, PT brush border is very complex morphologically including tall and densely packed MV and well-defined IMC and coated pit regions, whereas in contrast, PT-derived OK cells have very sparse MV and no analogous IMC. Considering the pronounced difference in their respective morphologies, it is not obvious that results from studies conducted in cultured cells are applicable to the PT in situ (26).
The current study aimed to determine the routes of retraction of NHE3 vs. NaPi2 in vivo during these two distinct natriuretic stimuli, acute elevation of BP or PTH treatment, employing confocal and immunoelectron microscopy (immuno-EM) and dual labeling with markers of coated pits and endosomes. The results indicate that the retraced NHE3 and NaPi2 are routed to different membrane regions: NHE3 is redistributed to the base of the MV (not to endosomes), whereas NaPi2 is internalized to endosomes and perhaps lysosomes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Acute hypertension (high BP) protocol. Mean arterial pressure was increased 50–60 mmHg by constricting the superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery by tying silk ligatures around the vessels (35, 41). Endosomes were functionally labeled by horseradish peroxidase (HRP) uptake. Specifically, after 10-min acute hypertension, 25 mg of HRP in 1 ml of PBS were injected into the jugular vein, and then 10 min later kidneys were fixed in situ for 20 min while BP was recorded; thus the experimental time point was between 20 and 40 min.
PTH protocol. The synthetic bovine PTH fragment bPTH-(1–34) (Peninsula Lab, Belmont, CA) was dissolved in 4.0% BSA in 0.9% NaCl. PTH was infused intravenously in a bolus dose of 6.6 µg/kg followed by an infusion at 0.1 µg·kg–1·min–1 for 1 h. After 50 min, HRP was injected as described as in the high-BP protocol, and then 10 min after HRP injection kidneys were fixed in situ for 20 min (during which PTH was continuously infused at 0.1 µg·kg–1·min–1); thus the PTH infusion time point is between 60 and 80 min. In one series designed to examine early effects of PTH, kidneys were fixed starting 10 min after the PTH bolus and continuous infusion (for 20 min), without HRP injection; thus the PTH infusion time point is between 10 and 30 min.
Homogenization and subcellular fractionation. The procedure for subcellular fractionation of renal cortex membranes has been described previously (41, 42). In brief, kidneys from control and treated animals were cooled in situ by flushing with cold PBS and then excised. The renal cortex was dissected, homogenized in isolation buffer (5% sorbitol, 0.5 mM disodium EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 mM histidine-imidazole buffer, pH 7.5), centrifuged at 2,000 g for 10 min twice to remove debris, the low-speed supernatants were pooled (So), loaded between two hyperbolic sorbitol gradients, and centrifuged at 100,000 g for 5 h. Twelve fractions were collected, pelleted, resuspended in isolation buffer, and stored at –80°C, pending assays.
Immunoblot analysis and antibodies. A 10-µl sample from each 1-ml gradient fraction was denatured in SDS-PAGE sample buffer for 30 min at 37°C, resolved on a 7.5% SDS polyacrylamide gel according to Laemmli (19), and transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P). Total sample protein loaded ranged from 1 µg (fraction 2) to 14 µg (fraction 7). For a typical high-BP experiment, blots were probed with either polyclonal NHE3-C00 (35) at 1:2,000 dilution or polyclonal anti-NaPi2 antibody provided by J. Biber (University of Zürich, Zürich, Switzerland) at 1:3,000 dilution (14), and then with Alexa 680-labeled goat-anti-rabbit secondary antibody. For detection of villin, the blots were probed with monoclonal anti-villin (Immunotech, Chicago, IL) at 1:1,000 dilution, and then with Alexa 680-labeled goat-anti-mouse secondary. All blots were detected with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) and accompanying software.
Indirect immunofluorescence. The left kidneys were fixed in situ by placing the isolated kidney in a small Plexiglas cup and bathing it in PLP fixative (2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4) for 20 min. The kidneys were then removed and cut in half on a midsagittal plane and postfixed in PLP for another 3–4 h. The fixed tissue was rinsed twice with PBS, cryoprotected by incubation overnight in 30% sucrose in PBS, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA), and frozen in liquid nitrogen. Cryosections (5 µM) were cut using a Microm Heidelberg (Mikron Instruments, San Marcos, CA) cryomicrotome and transferred to Fisher Superfrost Plus-charged glass slides and air dried. For immunofluorescence labeling, the sections were rehydrated in PBS 10 min, followed by 10-min washing with 50 mM NH4Cl in PBS, then with 1% SDS in PBS for 4 min for antigen retrieval (9). SDS was removed by two 5-min washes in PBS, and the sections were blocked with 1% BSA in PBS to reduce background. Dual labeling was performed by incubating with polyclonal antiserum NHE3-C00 at 1:100 dilution or anti-NaPi2 at 1:250 dilution and monoclonal antibody against the clathrin adaptor AP2 at 1:50 dilution or HRP (Sigma) at 1:100 dilution in 1% BSA/PBS for 1.5 h at room temperature. After being washed three times for 5 min in PBS, the sections were incubated with a mixture of FITC-conjugated goat-anti-rabbit (Cappel Research Products, Durham, NC) and Alexa 568-conjugated goat-anti-mouse (Molecular Probes, Eugene, OR) secondary antibodies diluted 1:100 in 1% BSA in PBS for 1 h, washed three times with PBS, mounted in Prolong Antifade (Molecular Probes), and dried overnight at room temperature. Slides were viewed with a Nikon PCM Quantitative Measuring High-Performance Confocal System equipped with filters for both FITC and TRITC fluorescence attached to a Nikon TE300 Quantum upright microscope. Images were acquired with Simple PCI C-Imaging Hardware and Quantitative Measuring Software and processed with Adobe PhotoDeluxe (Adobe Systems, Mountain View, CA).
Immuno-EM. Acute hypertension was induced for 20 min in four rats as described above. Four other rats served as controls and were treated the same way but without induction of high BP. None of the animals received HRP. In each group, two rats were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, and two rats were fixed by superfusion of the kidney surface with the fixative. BP was continuously monitored before and during fixation. Many surface-fixed tubules showed partial absence of lumen and some bulging of the apical cytoplasm into the tubule lumen. The immunolabeling pattern, described below, was the same with the two fixation methods. Tissue blocks were trimmed from the cortex and postfixed in the same fixative for 2 h, rinsed in buffer, infiltrated with 2.3 M sucrose, mounted on holders, and frozen in liquid nitrogen. Immunoelectron microscopy was performed either on thin (70 nm) cryosections prepared from the frozen tissue on a Reichert Ultracut S cryoultramicrotome (Leica) or on tissue that was cryosubstituted in a Reichert AFS freeze-substitution apparatus (Leica) and embedded in Lowicryl HM20 as previously described (25). Briefly, the samples were sequentially equilibrated over 3 days in methanol containing 0.5% uranyl acetate at temperatures gradually increasing from –90 to –70°C, rinsed in pure methanol, and infiltrated with Lowicryl HM20 at –45°C and, finally, UV-polymerization for 2 days at –45°C and 2 days at 0°C.
The Lowicryl sections or ultrathin cryosection were first blocked by incubation in PBS containing 0.05 M glycine and either 0.1% skim milk powder or 1% BSA. The sections were then incubated for 1 h at room temperature with polyclonal antiserum NHE3-C00 at 1:100 dilution or anti-NaPi2 also at 1:100 dilution in PBS containing 0.1% skimmed milk powder. The primary antibodies were visualized using goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM1O, BioCell Research Laboratories, Cardiff, UK) diluted 1:50 in PBS with 0.1% skimmed milk powder and polyethyleneglycol (5 mg/ml). The Lowicryl sections were stained with uranyl acetate and the ultrathin cryosections with 0.3% uranyl acetate in 1.8% methylcellulose for 10 min before examination in a Morgagni electron microscope. Immunolabeling controls consisted of substitution of the specific primary antibodies with nonimmune rabbit IgG or incubation without primary antibody. All controls showed absence of labeling.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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-hexosaminidase and also contain megalin and the CCV adaptor protein AP2. In data collected from four density gradients, the NHE3, expressed as percent of total, is as follows: 24 ± 2% in WI, 63 ± 1% in WII, 13 ± 2% in WIII. NHE3 density distribution normalized to protein distribution provides an estimate of the concentration of NHE3 across the gradient, expressed as arbitrary density units/protein: 3.5 ± 1.1 in WI, 4.5 ± 0.9 in WII, 1.2 ± 0.2 in WIII. From this pattern, we conclude that WI and WII are both enriched in NHE3, and thus apical membranes, relative to WIII. Similar redistribution of NHE3 and NaPi2 by subcellular membrane fractionation. Our previous studies established that NHE3 and NaPi2 are acutely retracted from the PT MV during acute hypertension (high BP) or PTH treatment (35, 43), which accounts for, at least in part, the decrease in PT sodium and phosphate reabsorption during acute hypertension or PTH treatment. By subcellular membrane fractionation, NHE3 and NaPi2 undergo similar redistribution patterns out of light-density (WI) into heavier-density (WII and WIII) membranes in response to high BP or PTH corresponding to a retraction from the apical MV. Figure 1 contains typical immunoblots of density gradient fractions probed with antibodies to NHE3, NaPi2, and villin from control vs. 20 min high BP-challenged rats. Both NHE3 and NaPi2 transit from apical membrane-enriched fractions 3-5 (WI) to IMC-enriched fractions 6-8 (WII) and to IMC and coated pit and endosome-enriched fractions 9-11 (WIII). The redistribution responses of NHE3 and NaPi2 assessed by subcellular fractionation are indistinguishable. The actin-bundling protein villin broadly distributes between fractions 4-12, in a pattern unaltered by high BP or PTH treatment, indicating a translocation of the sodium transporters out of the apical MV rather than a change in the density of villin-associated membranes containing NHE3 and NaPi2.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Much of the recent physiological data describing the regulation of NHE3 in the PT has been derived from studies of stable epithelial cell lines such as OK and LLC-PK1. These studies built on our initial in vivo observation that NHE3 is regulated by trafficking between apical MV and an unidentified pool that was either at the base or below the MV (42). Some of the most convincing data supporting a role of membrane trafficking in the regulation of NHE3 comes from the Moe laboratory (13, 17, 18, 34). These studies in cultured cells used membrane-impermeant, cleavable biotinylation reagents to distinguish between plasma membrane and intracellular pools of NHE3 and demonstrated that inhibition of NHE3 activity by PTH or dopamine is accompanied by trafficking of NHE3 between the plasma membrane and an intracellular compartment. As predicted, these effects are dependent on the cells having an intact cytoskeleton, recently reviewed by Szaszi and co-workers (30, 31). Furthermore, also in OK cells, active NHE3 has been shown to facilitate the initial step of endocytosis of complexes of ligands bound to the scavenger receptor megalin (16), suggesting that a NHE3-megalin complex might be also important in receptor-mediated endocytosis.
There are striking differences in the phenotypes of renal cell lines contrasted with renal PT cells (26). The brush border of the PT is very dense and consists of two distinct microdomains: the MV and the intermicrovillar domain, which can be subdivided into IMC and the intermicrovillar coated pits (ICP). Cultured PT cells have sparse MV and the ICP microdomain of the PT is all but lacking in cell lines. Although there is evidence for substantial intracellular pools of NHE3 in cultured cells, reviewed above, evidence for a significant pool of intracellular NHE3 in vivo at baseline, analogous to the pool of water channels seen in collecting duct cells at baseline, is all but lacking. Our previous studies demonstrate the appearance of a putative endosomal pool by subcellular fractionation after acute hypertension or PTH treatment: NHE3 shifts to high-density membranes enriched in endosomal markers and, by confocal microscopy, from MV (where NHE3 overlaps with villin staining) to a region just below villin staining (20, 35). However, the results of this current study contradict our previous interpretation that NHE3 was actually endocytosed. Confocal and electron microscopy results from the present study showed that NHE3 is redistributed between a pool in the MV to a pool in the intermicrovillar region, that is, redistribution within the apical membrane without endocytosis.
The presence of a recruitable pool of NHE3 at the base of the MV is physiologically relevant. This laboratory previously established that redistribution of NHE3 in response to acute hypertension is a reversible response: when BP is restored, NHE3 returns to its original density distribution pattern and sodium transport in the PT is restored (41). We recently reported that in hypertension induced by renal injury/sympathetic nervous system activation, NHE3 and NaPi2 abundance in MV-enriched low-density membranes increases, whereas transporter abundance in the high-density membranes enriched in IMC, ICP, and endosomes decreases (36, 37), a response that may contribute to the generation and maintenance of hypertension. Besse-Eschmann et al. (4) demonstrated a similar mechanism of NHE3 regulation in puromycin aminonucleoside-induced nephrotic syndrome; that is, NHE3 is shifted from an inactive pool to an active pool in the apical brush border, which would contribute to the sodium retention observed in nephritic syndrome.
The factors that constrain the reserve compartment of NHE3 to the base of MV remain to be defined. The current results indicate there is little if any NHE3 colocalization with AP2 or HRP during acute hypertension or PTH treatment. Immuno-EM results showed that NHE3 did not (or only to a very small extent) move into the components of the endocytic apparatus including clathrin-coated endocytic invaginations/pits, dense apical tubules, small and large endocytic vacuoles, or lysosomes. The EM results are consistent with the confocal results that localized NHE3 above AP2 during PTH or high-BP treatment (Fig. 3). In contrast, the results of Yip et al. (38) studying the one-clip Goldblatt hypertensive model, also using confocal microscopy, suggested that NHE3 colocalizes with AP2. This difference may be due to differences in the animal models, kidney fixation protocols, or the resolution by light microscopy.
Can the retraction of NHE3 observed during acute BP elevation cause the decrease in sodium entry into the PT? Yip et al. (39) measured Na+/H+ exchanger activity in vivo after loading cells with the intracellular pH indicator BCECF and determined that 20-min acute hypertension caused a 50% reduction in Na+/H+ exchanger activity. Studies from this laboratory, using the acridine orange quench method to assess Na+/H+ exchanger transport activity in membrane fractions from control vs. acute hypertension renal cortex, concluded that there was no change in Na+/H+ exchanger activity/transporter when NHE3 retracted to the intermicrovillar domain. In a related analysis, Biemesderfer et al. (5) fractionated unstimulated rabbit renal brush-border membranes, assayed Na+/H+ exchange activity using the same acridine orange quench method, and concluded that NHE3 exists in two oligomeric states: an active 9.6S form present in brush border MV and an inactive 21S megalin-associated NHE3 in dense vesicles containing markers of the IMCs. The authors postulated that the 21S form could serve as a reservoir of NHE3 for its rapid regulation. There are clear differences between the membrane preparation methods used in the two studies, and it remains to be determined whether key regulatory elements were lost in our fractionation procedure. Taken together, these studies are consistent with the hypothesis that during hypertension, NHE3 is rapidly retracted from the MV to the IMC where transporters form complexes with regulatory proteins that reversibly inactivate activity and decrease PT Na+ reabsorption, without NHE3 internalization.
How does NHE3 move within the plane of the MV? It is well established that brush-border MV are filled with bundled actin filaments, and evidence suggests that NHE3 can be tethered to the actin via the PDZ domain protein NHE RF and ezrin (40). Recently, the unconventional myosin VI, which moves toward the pointed ends of actin filaments, was, in fact, localized in the PT, mainly to the base of the MV (6). Together, these findings suggest myosin VI is a good candidate for moving cargo proteins such as NHE3 and/or NaPi2 along the MV down to the IMC/ICP region. Evidence from subcellular fractionation in our laboratory showed that myosin VI redistributes with NHE3 during acute BP elevation (in preparation).
The difference in the retraction patterns between NHE3 and NaPi2 was clearly demonstrated by their tandem analysis using both confocal and immuno-EM. During acute hypertension, there is an apparent internalization of NaPi2 to early endosomes evident by colocalizing with HRP (Fig. 4, middle) consistent with the subcellular fractionation results previously published by this laboratory (43). Fifteen minutes after injecting 100 µg of PTH in rats, Traebert et al. (32) detected significant colocalization of PTH with HRP. After 2-h high-phosphate-diet feeding, NaPi2 was colocalized with Golgi and lysosomal markers (21). During 1-h PTH treatment, we observed a pronounced internalization of NaPi2 to intracellular vesicles including perinuclear compartments (Fig. 4, bottom) and no NaPi2 colocalization with HRP, suggesting that by 1 h NaPi2 has already passed through the early endosomes en route to lysosomes. During acute hypertension or 10 min after PTH infusion, NaPi2 appears as the punctate staining either colocalizing with AP2 or right below AP2 staining region (Fig. 5, B and C), consistent with the findings from Traebert et al. (32) and indicating that clathrin-coated pits may contribute to the endocytosis of NaPi2. The beaded appearance of NaPi2 staining right below the clathrin staining domain was also observed in a NHERF1 knockout mouse that has a problem inserting NaPi2 to the brush border (33).
The immuno-EM results from the current study indicate that during acute hypertension, NaPi2 enters the normal endocytic pathway, which transports proteins from the tubule lumen into the cell (23). Thus NaPi2 is present in the small coated endocytic vacuoles, as well as in the uncoated large endocytic vacuoles and the dense apical tubules, which are located around the vacuoles. The apical tubules have been shown to connect directly to the vacuoles and may return membranes from the endocytic vacuoles to the apical cell membrane in a recycling mechanism (24). When the dense apical tubules move away from the endocytic vacuoles, their membranes evidently include NaPi2 but not the clathrin coats and most of the endocytosed protein is left behind. Therefore, in double immunofluorescence, there is overlap between NaPi2 and AP2 in many coated endocytic vacuoles, but the surrounding cytoplasmic regions contain many apical tubules labeled only with NaPi2. This is probably the case in Fig. 5, B and C, and explains why some NaPi2 staining is present below the AP2 staining zone.
In conclusion, the parallel study of NHE3 and NaPi2 redistribution provides direct in vivo evidence that NHE3 and NaPi2 are regulated via distinct trafficking pathways. Although both are retracted from the top of the MV and the mechanism for the initial retraction may be shared, after that point the retraction pathways appear distinct. There is no evidence for a significant pool of intracellular NHE3 in vivo at either baseline or the presence of natriuretic stimuli such as increased BP or PTH. The well-defined intermicrovillar domain in the PT, not observed in cultured cells, appears to serve as a storage pool for NHE3. Thus caution must be applied in studies using cultured renal cell lines to define molecular mechanisms of sodium transport regulation. The NHE3 pool at the base of the MV and IMC is likely important for the rapid retraction and insertion of NHE3, necessary to change PT Na+ transport to generate the autoregulatory signal relayed to the macula densa during changes in BP or GFR. In contrast, NaPi2 is not retracted to a pool at the base of the MV in the apical surface but internalized, likely through clathrin-coated pits, and destined to endosomes/lysosomes. It remains to be determined whether internalized NaPi2 is degraded, as is evident after PTH treatment (22), and/or recycled back to the cell surface via the dense apical tubules like internalized insulin receptors (28).
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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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.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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