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【摘要】 The distal convoluted tubule (DCT) apical Na + -Cl - cotransporter (NCC) is responsible for the reabsorption of 5-10% of filtered NaCl and is the target for thiazide diuretics. NCC abundance is increased during dietary NaCl restriction and by aldosterone and decreased during a high-salt (HS) diet and mineralocorticoid blockade. This study tested the hypothesis that subcellular distribution of NCC is also regulated in response to changes in dietary salt. Six-week-old Sprague-Dawley rats were fed a normal-salt diet (NS; 0.4% NaCl) for 3 wk, then switched to a HS diet (4% NaCl) for 3 wk or a low-salt diet (LS; 0.07% NaCl) for 1 wk. Under anesthesia, kidneys were excised, renal cortex was dissected, and NCC was analyzed with specific antibodies after either 1 ) density gradient centrifugation followed by immunoblotting or 2 ) fixation followed by immunoelectron microscopy. The HS diet decreased NCC abundance to 0.50 ± 0.10 of levels in LS diet (1.00 ± 0.23). The HS diet also caused a redistribution of NCC from low to higher density membranes. Immunoelectron microscopy revealed that NCC resides predominantly in the apical membrane in rats fed the LS diet and increases in subapical vesicles in rats fed the HS diet. In conclusion, a HS diet provokes a rapid and persistent redistribution of NCC from apical to subapical membranes, a mechanism that would facilitate a homeostatic decrease in NaCl reabsorption in the DCT to compensate for increased dietary salt.
【关键词】 sodium transport distal convoluted tubule dietary salt thiazide receptor
THE NA + - CL - COTRANSPORTER (NCC) is expressed in the apical membrane of the distal convoluted tubule (DCT) and is responsible for the reabsorption of 5-10% of filtered Na + and Cl - ( 15 ). NCC is the target of the thiazide diuretics and is frequently used in the treatment of hypertension ( 2, 15 ). NCC abundance is increased by dietary NaCl restriction and decreased by a high-salt (HS) diet, during aldosterone escape, and by mineralocorticoid receptor blockade, implicating a homeostatic role of NCC abundance in adjusting renal Na + excretion to Na + intake ( 11, 14, 16, 19 ). This role of NCC in salt balance is supported by the study of the genetic disorder Gitelman?s syndrome, in which NCC is inactivated, resulting in salt wasting. Oocyte studies indicate that the Gitelman?s NCC is not processed properly or routed to the plasma membrane ( 4, 9 ).
Na + transport in the DCT could be regulated by changing the number or activity of NCC in the apical membrane. Although there is the evidence presented above that dietary salt and aldosterone levels can regulate total NCC abundance, there is scant information about whether the ratio of NCC between surface and intracellular pools is regulated in vivo. One relevant study is that of Verlander et al. ( 18 ), who reported that ovariectomy decreased both the complexity of the DCT apical surface and immunogold detection of apical NCC and that chronic estradiol replacement restored DCT ultrastructure and apical NCC density to normal. This study aimed to test the hypothesis that a low-salt (LS) diet favors expression of NCC in the apical membrane and a HS diet provokes redistribution of DCT NCC from apical to subapical membrane pools. Applying subcellular density gradient centrifugation and immunoelectron microscopy to animals placed on LS or HS diets, the results provide in vivo evidence for regulated redistribution of NCC between apical and subapical membranes.
METHODS
Animal protocol. Experiments were performed using 6-wk-old Sprague-Dawley rats (Harlan, San Diego, CA) fed a pelleted diet with 0.4% NaCl (normal salt; NS) for 3 wk, then switched to either a pelleted 4% NaCl (HS) diet for 3 wk or a gelled 0.07% NaCl (LS) diet for 1 wk. All diets were from Dyets, Bethlehem, PA. The gelled diet was prepared from sodium-free powdered rat chow by the addition of 0.07% NaCl (wt/wt), 25 ml deionized water/15 g rat chow, and 0.5% agar. Food intake was controlled in the LS group so that each rat received 0.2 meq of Na + ·200 g body wt -1 ·day -1. Animals had free access to water in all protocols. At the end of each protocol, urine was collected for 5 h in a metabolic cage. Plasma was collected from the tail vein in anesthetized rats (ketamine im, Fort Dodge Laboratories, Overland Park, KS; xylazine, 1:1, vol/vol, Miles, Shawnee Mission, KS). Urine and plasma Na + and K + were measured using a flame photometer (Radiometer FLM3, Copenhagen, Denmark). Plasma aldosterone levels were measured in rats fed the LS or HS diets for 1 wk ( n = 6 each) in plasma collected via heart puncture after anesthesia (ketamine/xylazine, 1:1 vol/vol) using a Coat-A-Count solid phase 125 I radioimmunoassay (Diagnostic Products, Los Angeles, CA). All animal experiments were approved by the University of Southern California Keck School of Medicine Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Density gradient fractionation of NCC. To assess density distribution of NCC in response to dietary salt, kidneys from rats fed LS and HS diet regimens ( n = 5 each) were subjected to subcellular fractionation on sorbitol density gradients. The procedure for subcellular fractionation has been described in detail previously ( 24 ). In brief, after anesthesia (ketamine im; xylazine, 1:1, vol/vol) the kidneys were cooled in situ by flushing with cold PBS to block membrane trafficking, excised, renal cortexes were dissected, homogenized (in 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) with a Tissuemizer (Tekmar Instruments), and centrifuged at 2,000 g for 10 min (supernatant retained); the pellet was rehomogenized and centrifuged, and the two low-speed supernatants (S 0 ) were pooled. A 4-ml aliquot of the S 0 sample was mixed with 6 ml 87.4% sorbitol and equilibrated on ice for 1 h. Gradients were constructed by the delivery of sorbitol to the bottom of the centrifuge tube in the following order: 1 ml of 5% sorbitol isolation buffer, a first hyperbolic gradient of 35-55% sorbitol generated by adding 18 ml of 55% sorbitol to a constant-volume mixing chamber initially loaded with 8.9 ml of 35% sorbitol, a second hyperbolic gradient of 55-70% sorbitol generated by mixing 8.2 ml of 70% sorbitol with 6.3 ml of 55% sorbitol, and, finally, 1 ml of 80% sorbitol. Equilibrated S 0 was loaded between the two hyperbolic sorbitol gradients, centrifuged at 100,000 g for 5 h, 12 fractions were collected, and the density of each was measured using a DA-100M Density/Specific Gravity meter (Mettler Toledo). Fractions were diluted and pelleted at 250,000 g for 75 min, resuspended in 1 ml isolation buffer, and stored at -80°C pending assay.
To assess the density distribution of a protein in the gradient, a constant volume of each of the 12 fractions (8 µl) was assayed by immunoblotting, and results are expressed as the percentage of the total on the gradient (signal in all 12 fractions = 100%). To assess the total pool size of NCC in crude membranes, a constant amount (50 µg) of S o protein was analyzed. In all immunoblot assays, one-half the volume or one-half the protein was also assayed in parallel to verify linearity of the detection system. Samples were denatured in SDS-PAGE sample buffer for 30 min at 37°C, resolved on 7.5% SDS polyacrylamide gels ( 10 ), and transferred to polyvinylidene diflouride membranes (Millipore Immobilon-P). Blots were incubated with polyclonal antiserum against NCC (TSC; D. Ellison, Oregon Health and Science Univ., Portland, OR) at 1:500 dilution or polyclonal antiserum against aquaporin-2 (AQP2) at 1:1,000 dilution (M. A. Knepper, National Institutes of Health, Bethesda, MD) and then incubated with Alexa 680-labeled goat anti-rabbit (Molecular Probes, Eugene, OR) and detected and quantitated with the Odyssey Infrared Imaging System (Li-COR, Lincoln, NB) and accompanying LI-COR software.
Immunoelectron microscopy. To analyze NCC subcellular distribution at the ultrastructural level, kidneys from five animals each from LS and HS regimes were analyzed. After anesthesia, kidneys were excised, tissue blocks were trimmed from the cortex and fixed in 4% paraformaldehyde solution containing 0.1 M Na + cacodylate, pH 7.2, for 3 h, rinsed in PBS, and infiltrated with 2.3 M sucrose, mounted on holders, and frozen in liquid nitrogen. Immunoelectron microscopy was performed on thin (70 nm) cryosections prepared from the frozen tissue on a Reichert Ultracut S cryoultramicrotome (Leica) as previously described ( 12 ). Briefly, the cryosections 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 or overnight at 4°C with polyclonal antiserum against NCC (TSC ) at 1:1,600 dilution in PBS containing 0.1% skim milk powder. The primary antibody was 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% skim milk powder and polyethylene glycol (5 mg/ml). The cryosections were stained with 0.3% uranyl acetate in 1.8% methylcellulose for 10 min before examination in a FEI Morgagni electron microscope (Eindhoven, The Netherlands). Immunolabeling controls consisted of substitution of the specific primary antibody with nonimmune rabbit IgG or incubation without primary antibody.
Quantitation and statistical analysis. Data are expressed as means ± SD. Differences were regarded significant at P < 0.05. In density gradient assays, two-way ANOVA was applied to determine whether there was a significant effect of treatment on the overall density distribution pattern. After significance was established, the location of the difference in the pattern was assessed by unpaired a two-tailed Student?s t -test assuming equal variance with Bonferroni adjustments for multiple comparisons. The effect of treatment on total NCC abundance was assessed by a two-tailed Student?s t -test assuming equal variance for unpaired samples (LS vs. HS).
Quantitation of immunogold labeling was performed on electron micrographs (final magnification 48,000) of DCT cells from five LS and five HS rats. From each rat 4-7 cells were analyzed from sections oriented approximately at right angles to the apical cell membrane and showing negligible background over mitochondria and nuclei. Gold particles were ascribed to the cell membrane when located on its outer surface, above it, or within 30 nm from its inner surface. Gold particles over the apical cytoplasm were counted below the analyzed stretch of cell membrane. The percentage of particles associated with the cell membrane, compared with all counted particles, was determined for each cell. The mean percentage was then determined for each animal, and the means for HS and LS rats were compared by two-tailed Student?s t -test for unpaired samples.
RESULTS AND DISCUSSION
Effect of dietary NaCl on physiological parameters. As summarized in Table 1, neither body weight, kidney weight, urine output, nor plasma Na + or K + concentration was affected by changes in dietary NaCl. Compared with the LS diet, the HS diet increased urinary Na + excretion 100-fold and decreased plasma aldosterone to undetectable levels. The HS diet had a tendency to decrease urinary K + excretion, which is consistent with the fall in plasma aldosterone.
Table 1. Effect of dietary salt on body weight, plasma, and urine electrolytes
Effect of dietary NaCl on the abundance of NCC. NCC abundance has been reported to increase with salt restriction and to decrease after 28 days of an elevated (2.59 %) NaCl diet ( 11, 16 ). In this study, NCC abundance was measured in kidney cortex homogenates following low-speed centrifugation to remove debris (S o fraction). As shown at the top of Fig. 1, NCC abundance decreased 50% in rats fed the HS diet for 3 wk (0.50 ± 0.10) compared with NCC abundance in rats exposed to the LS diet (1.00 ± 0.23), confirming the previous studies. Whether NCC abundance decreases during HS due to increased degradation or decreased synthesis or a combination of both was not determined. However, because NCC mRNA is unaffected by a variety of stimuli, including changes in dietary NaCl, furosemide treatment, spironolactone treatment, and aldosterone escape ( 1, 11, 14, 19 ), we would postulate the decrease is due to increased degradation or decreased mRNA translatability.
Fig. 1. Effect of dietary salt on Na-Cl - cotransporter (NCC) in cortical homogenate and aquaporin-2 (AQP2) abundance in medullary homogenate. Rats were fed either a low-salt [0.07% NaCl (LS), n = 6] or 3 wk of a high-salt diet [4% NaCl (HS), n = 6]. Protein aliquots (50 and 25 µg) of each sample were analyzed by immunoblotting for NCC, and 40- and 20-µg protein aliquots of each sample were analyzed by immunoblotting for AQP2. Shown are representative blots of 4 samples from each group run at 50 µg protein for NCC and 40 µg for AQP2. Relative abundance of all the samples, normalized to the LS group = 1.00 and is indicated below the blots. * P < 0.05, assessed by unpaired Student?s t -test compared with LS values.
Vasopressin infusion has been shown to increase NCC abundance ( 5 ) and would be suppressed in rats with increased water intake. Samples were also probed for medullary AQP2 abundance, an indicator of vasopressin levels ( 17 ). As shown at the bottom of Fig. 1, AQP2 abundance increased significantly during HS feeding, as demonstrated previously ( 16 ). Because urine output was similar in the LS group (fed a gelled diet) and HS group (where salt stimulates thirst) ( Table 1 ), the change in AQP2 appears to be secondary in the HS diet rather than water intake per se.
Effect of changes in dietary NaCl on density distribution of NCC. Previous studies from our laboratory have demonstrated a shift in density distribution of renal cortical type 3 Na + /H + exchanger and type 2 Na + -P i cotransporter to higher densities in response to hypertension or PTH treatment coinciding with subcellular redistribution, determined by microscopy, of transporters to the base of the microvilli (type 3 Na + /H + exchanger) or to endosomes (type 2 Na + -P i cotransporter) ( 23 ). Membrane marker characteristics of the sorbitol density gradients used in these studies indicate that plasma membranes have a lower density than endosomes or lysosomes ( 22, 24 ). Stimulated by the usefulness of these gradients as one approach to examine Na + transporter trafficking, we used the same sorbitol density gradients to determine whether there was evidence for redistribution of NCC in response to changes in dietary salt. Figure 2 A illustrates that the density distribution profile of the sorbitol gradients is highly reproducible between samples. In LS diet-fed rats, NCC distributed in a major peak between fractions 4 and 7 and a broad shoulder between fractions 7 and 10 ( Fig. 2 B ). The 3-wk HS diet provoked a significant shift of NCC from the lower density peak to the higher density shoulder ( Fig. 2 B ). As discussed in METHODS, the NCC density distribution results are normalized to the sum of the NCC signal in all 12 fractions of that 1 gradient (=100%); thus the total decrease in NCC in HS group is not evident in this analysis. These results, suggestive of a change in the ratio of NCC between the apical surface and intracellular membranes, provided the impetus to look for evidence of subcellular redistribution in these groups with microscopy.
Fig. 2. Distribution of NCC in density gradient fractions of renal cortex from Sprague-Dawley rats on 1 wk LS or 3 wk on HS diet. A : density distribution profile of samples from LS and HS gradients demonstrates reproducibility. B : HS diet provoked a significant redistribution of NCC out of lower density membranes into higher density membranes compared with rats fed a LS diet. Data are expressed as means ± SD; n = 5. * P < 0.05 in HS vs. LS, assessed by ANOVA ( P < 0.000) followed by unpaired Student?s t -test with Bonferroni adjustment.
Immunocytochemistry of NCC distribution with changes in dietary NaCl. Immunoelectron microscopy was employed to determine whether the shift in density distribution of NCC with dietary salt represented a change in subcellular distribution. In DCT of rats fed the LS diet, immunogold labeling of NCC was pronounced over the entire apical plasma membrane surface, both over the small microvilli and between the microvilli ( Fig. 3 ). This labeling pattern was observed in both cells with many or few microvilli. There was also NCC labeling associated with a small population of vesicles located in the most apical region of the cytoplasm. Most NCC-labeled vesicles were 0.05-0.15 µm in diameter and appeared devoid of a clathrin-like cytoplasmic coat. In axially sectioned cells, most vesicles were located <1.0 µm from the plasma membrane, and no label was observed deeper in the cytoplasm. Overall, the gold labeling of NCC in LS diet-fed rats was much greater over the apical plasma membrane than over the subapical vesicles ( Fig. 3 ). In contrast, rats on the HS diet exhibited a significant increase in the frequency of labeled subapical vesicles in DCT cells and an apparent relative decrease in plasma membrane labeling ( Fig. 4 ). Quantitative analysis showed that 70.2 ± 3.2% of the immunogold particles were associated with the cell membrane in LS rats compared with 38.5 ± 3.9% in HS rats ( P << 0.05). Adjacent cells in proximal and cortical collecting tubules in both groups were completely devoid of label, as were intercalated cells occasionally observed immediately adjacent to labeled DCT cells.
Fig. 3. Immunoelectron microscopic localization of NCC in distal convoluted tubule (DCT) cells from 2 different rats on 1 wk of LS diet. There is abundant gold labeling of the apical plasma membrane (arrows), in both cells with few ( top cell) and many ( bottom cell) microvilli. Much of the label is associated with the inner surface of the plasma membrane and a stained layer on the inside of the membrane. Some label is also present over small vesicles in the apical cytoplasm (arrowheads). N, nucleus; M, mitochondrion. Scale bar = 0.5 µm.
Fig. 4. DCT cells from 2 different rats on HS diet showing increased label-associated apical vesicles (arrowheads) and a relative decrease in label over the plasma membrane (arrows). Bar = 0.5 µm.
In general, the activity of a transporter can be regulated by covalent modification of transporters in the membrane or by changing the number of transporters in the apical membrane. The number of transporters in the apical membrane can be changed by regulating the total pool size or by varying trafficking of the transporter between the apical membranes and subapical pools. This study provides evidence not only of regulation of the total pool size of NCC but also of regulated redistribution between the plasma membrane and subapical pools in response to changes in dietary NaCl intake. Redistribution of NCC to subapical vesicles would significantly amplify the effect of decreased NCC pool size with a high-salt diet, and redistribution of NCC to the plasma membrane during salt restriction could contribute to increasing NaCl reabsorption. In vivo microperfusion studies of DCT demonstrated increased capacity of the DCT to transport Na + and Cl - during salt restriction and furosemide treatment ( 6, 7 ).
The results in this study demonstrate that the redistribution of NCC from low- to higher-density membranes coincides with a cellular redistribution from the plasma membrane to the subapical membranes. This important region of the nephron has been very difficult to study because it is short compared with other regions of the nephron found in the cortex, such as the proximal tubule and cortical collecting duct. However, the localization of NCC specifically to the DCT as well as the specificity of the antibody allows one to analyze the NCC distribution both biochemically on density gradients and structurally by microscopy. The density gradients, which can enrich for NCC on the plasma membrane vs. NCC in subapical vesicles, have the potential of providing useful starting material for biochemical analyses of associated proteins localized with NCC to these domains.
The importance of NCC in control of blood pressure has been widely studied. Loss-of-function mutations of NCC have been linked to Gitelman?s syndrome, an autosomal recessive disease characterized by salt wasting, low blood pressure, hypokalemia, and secondary hyperaldosteronism ( 3, 13 ). In contrast, in pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder with the opposite clinical phenotype of hypertension and hyperkalemia, mutations in WNK [ w ith n o lysine ( K )] kinases WNK1 and WNK4 have been linked to increased NCC activity, presumably due to a loss of inhibitory influence on NCC ( 20, 21 ). There is evidence for genetic defects in NCC trafficking. When NCC with the Gitelman mutations was expressed in oocytes, metolazone (an inhibitor of NCC)-sensitive 22 Na + uptake was either significantly lower than wild-type or completely abolished ( 4 ). Immunocytochemistry in the oocytes showed that, unlike wild-type, the mutant NCC was localized in the cytoplasm and below the plasma membrane, explaining the lack of metolazone-sensitive Na + uptake. The lack of plasma membrane NCC may be attributed to reduced or lack of NCC glycosylation in the mutants: mutants lacking glycosylation localized only to the cytoplasm below the plasma membrane, whereas NCC mutants with partial glycosylation localized to both the plasma membrane and to a pool just below the plasma membrane ( 4, 9 ). Regarding PHAII, in which NCC activity is increased, presumably secondary to mutations in WNK 4, there is also evidence that WNK4 plays a role in NCC trafficking. Coexpression of wild-type WNK4 in oocytes reduces plasma membrane expression of NCC by 85% compared with what is observed in oocytes expressing NCC alone, and coexpression of both WNK1 and WNK4 with NCC increases plasma membrane expression of NCC to levels found in cells not expressing WNKs ( 20, 21 ). These results suggest the possibility that the redistribution of NCC during a HS diet may involve WNK4 activity. Another candidate regulator is aldosterone, which is a key regulator of NCC abundance ( 8 ).
In conclusion, this study provides in vivo evidence for NCC redistribution in response to changes in dietary NaCl. In animals fed a moderately LS to NS diet, NCC was distributed primarily in the apical plasma membrane and to a lesser extent in small vesicles found close to the apical membrane. In comparison, a HS diet provoked both a decrease in NCC abundance as well as redistribution of NCC from the apical surface to the small subapical vesicles.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34316 (A. A. McDonough) and the Danish Medical Research Council and the Water and Salt Research Center established and supported by the Danish National Research Foundation (Grundforskningsfonden; A. B. Maunsbach). M. Sandberg was partially supported by a predoctoral fellowship from the American Heart Association, Western States Affiliate.
ACKNOWLEDGMENTS
We thank Else-Merete Løcke and Karen Thomsen for expert technical assistance.
Components of this project were presented in abstract form ( J Am Soc Nephrol 16: 346A, 2005).
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作者单位：1 Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California; and 2 The Water and Salt Research Center, Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus C, Denmark