| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California
Submitted 22 January 2007 ; accepted in final form 18 July 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
hypertension; kidney; Na transporter; ACE inhibitor; triple therapy
Na+/H+ exchange (NHE) is the major route for apical sodium entry across the proximal tubule (PT), and the NHE3 isoform is responsible for virtually all the Na+/H+ exchange activity in this region (1, 2). We previously reported that during the development of hypertension in the SHR, there is a parallel redistribution of PT NHE3 and Na+-Pi cotransporter type 2 (NaPi2) from membranes of low density enriched in apical microvilli (MV) to membranes of higher density enriched in intermicrovillar cleft (IMC) and endosomal markers demonstrated by density gradient fractionation (22). This redistribution, which mimics the response observed in Sprague-Dawley (SD) rats in response to acute hypertension (37, 42), likely reflects a homeostatic compensation to the rising BP rather than a nonadaptive change that contributes to hypertension. The destination of NHE3 and NaPi2 after retraction from the low-density membranes was not determined in our prior SHR study; however, the acute redistribution in SD has been defined: NHE3 redistributes to the base of the MV while NaPi2 are endocytosed during acute hypertension or parathyroid hormone (PTH) treatment (38).
When BP is restored after a period of acute hypertension in the SD rats, the density distribution of NHE3 returns to the control pattern (42). However, when BP was acutely normalized in the chronically hypertensive adult SHR (with xylazine), there was no apparent normalization of NHE3 distribution (22). The aims of this study were to 1) define the subcellular location of NHE3 and NaPi2 in the PT of SHR, 2) determine the effect of chronic ACEI on Na+ transporter subcellular distribution and abundance, and 3) to test the hypothesis that chronically normalizing BP in SHR restores NHE3 and NaPi2 subcellular distribution to that seen in normotensive SD, that is, to the body of the MV. To determine whether changes in transporters’ distribution and/or abundance were due to BP normalization per se vs. chronic ACEI, two distinct methods were used to normalize BP: chronic ACEI or chronic triple therapy (hydralazine, reserpine, and hydrochlorothiazide). The results demonstrate that normalizing BP in SHR with either ACEI or triple therapy did not restore NHE3 or NaPi2 to the control distribution in the MV. Rather, ACEI provoked further redistribution of NHE3, NaPi2, NKCC, and NCC to higher-density membranes at base of MV or in intracellular pools and ACEI decreased NaPi2 abundance. Triple therapy normalized BP independent of changes in renal transporter distribution or abundance. These results suggest that antihypertensive therapy with ACEI depresses sodium transport all along the nephron by decreasing sodium transporter distribution and/or abundance in the apical plasma membrane, an effect that likely contributes to the BP-lowering effects of this drug.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Enalapril treatment of SHR. Male 8- to 9-wk-old SHR (Harlan) were provided with enalapril (Sigma; 30 mg·kg–1·day–1) in deionized drinking water for 4 wk or with untreated deionized water (n = 6 each). Untreated normotensive weight- and age-matched SD (Simonsen, n = 4) and Wistar-Kyoto (WKY; Charles River, n = 3) rats were included in some analyses.
Triple therapy treatment of SHR. Male 7- to 8-wk-old SHR were treated with 7.5 mg/day hydralazine, 0.15 mg/day reserpine, and 3 mg/day hydrochlorothiazide (Sigma) in deionized water for 7 wk or with untreated deionized water (n = 4 each).
In both treatment groups, drug concentrations in the drinking water were adjusted weekly to account for changes in rat body weight and water intake. At the end of the treatment, animals were anesthetized with inactin (Sigma; 125 mg/kg ip) and placed on a thermostatically controlled warming table to maintain body temperature at 37°C. A polyethylene catheter was placed into the carotid artery to monitor BP (Transbridge, World Precision Instruments). The jugular vein was cannulated to infuse 4.0% BSA in 0.9% NaCl at 50 µl/min to maintain euvolemia. The ureter was cannulated to collect urine. Glomerular filtration rate (GFR) was measured by FITC inulin clearance and urine flow rate was measured gravimetrically, both as previously described (18). After the in vivo measurements and kidney collection, the heart from each rat was excised, blotted dry, and weighed.
Homogenization and subcellular fractionation. As described in detail previously (42, 43), after the in vivo experiments, kidneys 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 g/ml aprotinin, and 5 mM histidine-imidazole buffer, pH 7.5), and 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 1 ml isolation buffer, and stored at –80°C, pending assays. Four animals per group were analyzed.
Immunoblot analysis and antibodies. To determine the density distribution pattern of sodium transporters and associated proteins, 10 µl of each fraction were denatured in SDS-PAGE sample buffer for 30 min at 37°C, resolved on a 7.5% SDS-polyacrylamide gel according to Laemmli (16), and transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P). Total sample protein loaded ranged from 1 µg (10 µl fraction 2) to 14 µg (10 µl fraction 7). The density distribution of a protein is expressed as the percentage of the total signal in each fraction where the sum total signal in 12 fractions = 100%, thus the density pattern is independent of the total amount of protein loaded on the gradient. A change in the density distribution pattern of a protein, assessed by ANOVA, implies a change in the density of the membrane-associated protein which could be due to either trafficking of proteins between membranes of distinct densities, or redistribution between domains in the same planar membrane with distinct domains.
To assess the total pool size of transporters or associated proteins, a constant amount of the So protein (the 2,000-g supernatant of the homogenates) from each cortex or medulla was analyzed. Polyclonal anti-NHE3 [NHE3-C00 (37)] and anti-myosin VI (T. Hasson, Univ. of California at San Diego) were used at 1:2,000 dilution. Polyclonal anti-NaPi2 (McNaPi2; McDonough lab) and anti-dipeptidyl peptidase IV (DPPIV, M. Farquhar, UC San Diego) were used at 1:1,000 dilution. Polyclonal anti-NCC (Tsc; D. Ellison, Oregon Health and Science Univ.) and anti-ENaC 
, 
, 
(L. Palmer, Cornell Univ.) were used at 1:500. Polyclonal anti-NHERF1 (R-1046; E. Weinman, Univ. of Maryland) and monoclonal anti-NKCC (T4; C. Lytle, UC Riverside) were used at 1:3,000 dilution. Polyclonal anti-ACE (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:200 dilution. All blots were incubated with Alexa 680 labeled goat-anti-rabbit, goat-anti-mouse, or donkey-anti-goat secondary antibody (Molecular Probes, Eugene, OR), detected with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NB) and accompanying software.
Confocal microscopy. As described previously (37), kidneys from age-matched SD, WKY, SHR treated with or without enalapril (n = 2–3 each) were fixed in situ by bathing in PLP fixative (2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4) for 20 min and then postfixed in PLP for another 2–3 h. The fixed tissue was 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 and transferred to Fisher Superfrost Plus-charged glass slides and air dried. For immunofluorescence labeling, the sections were rehydrated in PBS, followed by 10-min wash in 50 mM NH4Cl in PBS, then with 1% SDS in PBS for 4 min for antigen retrieval (3). Dual labeling was performed by incubating with polyclonal antiserum NHE3-C00 at 1:100 dilution and monoclonal antibody against villin (Immuntech, Chicago, IL) at 1:100 or with antiserum McNaPi2 at 1:50 dilution and monoclonal antibody against clathrin adaptor AP2 (Sigma, St. Louis, MO) at 1:50 dilution, for 1.5 h. The sections were then incubated with a mixture of FITC-conjugated goat-anti-rabbit (Cappel Research Products, Durham, NC) and Alexa 568-conjugated goat-anti-mouse (Molecular Probes) secondary antibodies diluted 1:100 for 1 h, mounted in Prolong Antifade (Molecular Probes). 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. For NCC localization, surface sections were labeled with polyclonal anti-NCC antibody and then FITC-conjugated goat-anti-rabbit secondary antibody. Confocal immunofluorescence and differential interference contrast analysis of the DCT cells were performed and recorded using a Leica TCS SP2 AOBS MP confocal microscope (Leica Microsystems, Heidelberg, Germany).
Quantitation and statistical analyses. Data are expressed as means ± SD. Differences were regarded significant as P < 0.05. To assess whether there was a significant effect of treatment on density distribution pattern, two-way ANOVA was applied. After significance was established, the location of the difference in the pattern was assessed by unpaired two-tailed Student's t-test assuming equal variance with Bonferroni adjustments for multiple comparisons.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
-hexosaminidase as well as some apical membrane and IMC markers. As hypertension develops in SHR, NHE3 has been shown to redistribute out of the low-density fractions enriched in apical membrane markers to higher-density membranes (22). This response in SHR mimics the responses of normotensive SD rats to acute hypertension and likely serves as homeostatic adaptation to chronically elevated BP (21). In this current study, we aimed to determine the effect of chronically normalizing BP in SHR with ACEI and tested the hypothesis that NHE3 returns to apical membrane-enriched low-density fractions when BP is chronically normalized. Distribution of NHE3, expressed as percent of the total signal in the gradient, is shown in Fig. 1A. In SD rats, 42% of total NHE3 is located in low-density apical plasma membranes (WI), 
47% in WII, and 10% in high-density WIII enriched in endosomes and lysosomes. In untreated SHR, only 22% of total NHE3 is located in apical enriched WI, 65% in WII, and 12% in WIII. In enalapril-treated SHR, only 5% remains in WI, 78% is in WII, and 17% in WIII. These results demonstrate that ACEI treatment, which lowers BP to normal levels, does not normalize NHE3 distribution to that observed in nonhypertensive rats or that observed before hypertension develops (22). Rather, NHE3 is further retracted out of the apical enriched low-density membranes to higher-density membranes by ACEI treatment. This response would, theoretically, contribute to the antihypertensive action of ACEI by depressing PT Na+ reabsorption.
|
|
28% of total NaPi2 is located in WI, 47% in WII, and 20% in WIII. In untreated SHR, NaPi2 in WI is reduced to 16% of total and NaPi2 in WII is increased to 53%, and in WIII to 25% of total. In SHR treated with enalapril, NaPi2 in WI is reduced to 8% of total and increased in WIII to 31% of total. Thus, there is evidence for further net removal of NaPi2 from the MV when BP is reduced to control levels with ACEI. Immunofluorescence analysis was used to determine the subcellular distribution of NaPi2 in SD, WKY, untreated SHR, and SHR treated with enalapril. To establish whether NaPi2 is internalized, double labeling with antibodies to NaPi2 vs. the clathrin-coated vesicle adaptor protein AP2 was performed on cryosections of kidneys (Fig. 3). In both age-matched SD and WKY rats, the majority of NaPi2 is located in the brush border above AP2 staining. In untreated SHR, there is a detectable punctuate labeling of NaPi2 below AP2-stained coated pits but most of the NaPi2 is in the brush border above the border of AP2 staining. In SHR treated with enalapril, there is a reduced staining of NaPi2 in the MV and more pronounced NaPi2 staining in intracellular compartments, occasionally overlapping with AP2 staining. These findings demonstrate that the redistribution of NaPi2 to higher-density membranes, especially to endosome/lysosome-enriched WIII, corresponds to a redistribution of NaPi2 from the apical MV to subapical membrane vesicles. These results indicate that NaPi2 distribution is not returned to the apical MV when BP is normalized with ACEI, rather, further internalized into endosomes or lysosomes.
|
Distribution of DPPIV in untreated vs. enalapril-treated SHR is shown in Fig. 4A. Enalapril provoked a significant 12% shift of the total DPPIV out of WI into WII (3% increase) and WIII (8% increase). There was an analogous shift in myosin VI distribution (Fig. 4B) of 14% of total myosin VI out of WI into WII (8% increase) and WIII (6% increase). The distributions of the NHE3-associated protein NHERF1 and of ACE were not significantly altered by ACEI (Fig. 4, C–D).
|
10% of the NCC in fractions 3–4 shifts into fractions 5–6 (10% increase; Fig. 4E). Confocal immunofluorescence analysis of NCC in treated vs. untreated SHR, shown in Fig. 5, is not able to detect a significant spatial redistribution. The region of staining is quite narrow due to the paucity and short height of MV in the DCT. Recently, Sandberg et al. (28) demonstrated that 20-min acute captopril infusion in SD rats causes similar redistribution of NCC from low- to high-density fractions on density gradients. Using immunoelectron microscopy, they demonstrated a quantifiable and significant redistribution of 15% of the total NCC from apical plasma membranes to subapical cytoplasmic vesicles. Extrapolating from this study from our group, we postulate that chronic ACEI causes a similar redistribution of NCC from apical plasma membranes to subapical cytoplasmic vesicles. The current study also shows that 56% of cortical thick ascending limb NKCC in untreated SHR is located in low-density fractions 4–6 and that enalapril treatment provoked a redistribution of 15% of total NKCC from fractions 4–6 into fractions 8–10 (Fig. 4F). We have not examined the corresponding distribution of NKCC by microscopy but postulate that, like NHE3, NaPi2, and NCC, the apical membranes have a lower density than the intracellular membranes. Taken together, these results demonstrate that long-term ACEI provokes a coordinated redistribution of PT NHE3, NaPi2, DPPIV, and myosin VI, thick ascending limb NKCC, and distal tubule NCC from lower-density membranes to higher-density membranes which is postulated to contribute to BP lowering by blunting Na+ reabsorption.
|
-subunit abundance increased 17%. Enalapril treatment had no significant effect on immunoreactivity of NHE3, the NHE3-associated proteins DPPIV or NHERF1, or the abundance of DCT NCC or TAL NKCC in SHR.
|
|
seen after enalapril were due to ACEI or BP lowering, total pool size of the Na+ transporters and related proteins was assessed after 7-wk treatment of triple therapy. Unlike ACEI, triple therapy did not significantly alter total abundance of these proteins, although there were marginal changes in cortical myosin VI and medullary ENaC subunits (Fig. 7D, Table 4), indicating that the changes seen with enalapril were primarily due to ACEI rather than BP lowering per se.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Inhibitors of ANG II production or action are commonly used to treat hypertension even in cases where elevated renin-angiotensin system (RAS) components do not appear to account for the hypertension. For example, in SHR the RAS is not believed to play a major role in maintaining the high BP because aldosterone levels are not changed in young and adult SHR rats compared with age-matched WKY. In the current study, we treated SHR with enalapril for 4 wk, a well-established protocol to correct the hypertension in the SHR (14, 34), and found that the treatment corrected both hypertension and cardiac hypertrophy. Related to these findings, a recent study by Crowley et al. (6) established that the effects of ANG II infusion in normotensive mice, specifically hypertension and cardiac hypertrophy, were due to ANG II actions in the kidney. The study employed a kidney cross-transplantation model to produce animals that had AT1 receptors only in the kidney or everywhere but the kidney and then infused them with ANG II. When AT1 receptors were eliminated from the kidney, the residual repertoire of systemic, extrarenal AT1 receptors was not sufficient to induce hypertension or cardiac hypertrophy. Their findings demonstrate the critical role of the kidney in the pathogenesis of hypertension and its cardiovascular complications. Furthermore, the results suggest that the major mechanism of action of ACE inhibitors to lower BP is attenuation of ANG II effects in the kidney.
When comparing hypertension and control models, it is not always obvious which differences contribute to hypertension and which are adaptive responses to the hypertension. By both confocal and cell fractionation strategies, the McDonough and Yip labs (22, 40) showed that in 3- to 4-wk-old SHR, before hypertension is evident, NHE3 distribution was the same as in aged-matched normotensive SD and then by 12 wk of age, NHE3 and NaPi2 moved out of the body of the MV. The confocal microscopy images in the current study provided clear evidence for distinct trafficking patterns of NHE3 vs. NaPi2 in adult SHR, patterns that were not resolved by fractionation (22): NHE3 is partially retracted from the body to the base of the MV while NaPi2 is partially internalized into endocytic vacuoles during development of hypertension. As we discussed before, this redistribution likely reflects a homeostatic compensation to the rising pressure, rather than a change that contributes to hypertension.
In comparing SHR to normotensive SD or WKY rats, there are renal differences that could potentially account for the development of hypertension including a failure to respond to dopamine (9, 20) and enhanced TGF (5). Both of these differences may be secondary to the altered distribution of sodium transporters. The defect in dopamine-mediated natriuresis in SHR could be secondary to redistribution of NHE3 to a domain not accessible to dopamine regulation. Previously, by electron microscopy, we defined, in SD rats, a distinct domain at the base of the MV which is the apparent storage pool of NHE3 after its retraction during acute hypertension or PTH treatment. The NHE3 pool at the base of the MV is likely an important component of the machinery for rapid retraction and insertion of NHE3, necessary to change PT Na+ transport rate to generate the autoregulatory signal relayed to the macula densa during changes in BP or GFR. Thus this retraction of NHE3 to the base of the MV would amplify NaCl delivery to the macula densa which could contribute to enhanced TGF seen in SHR.
This study demonstrated that normalizing BP with ACEI or HRH did not return sodium transporter distribution to that seen in normotensive rats. Rather, NHE3 is further retracted from the body to the base of the MV, and NaPi2 is further internalized from the MV in SHR as BP was normalized with ACEI (Figs. 1–3). This further trafficking of sodium transporters out of the MV driven by ACEI likely contributes to the correction of hypertension and is consistent with the effects of endogenous ANG II on apical NHE3 regulation. Our lab recently discovered that acute infusion of the ACE inhibitor captopril (12 µg/min for 20 min) in SD rats causes the similar redistribution of NHE3 from the body to the base of the MV (17). Apparently, this ACEI effect is still dominant in NHE3 distribution over long-term treatment and suggests that basal levels of ANG II are required for targeting of NHE3 and NaPi2 to the body of the MV. The molecular details concerning NHE3 activity vs. distribution along the brush-border MV remain to be determined. This current study revealed that DPPIV is also retracted from the body of the MV during enalapril treatment. This may be important to sodium transport regulation as Girardi and colleagues (10, 11) reported that DPPIV associates with NHE3 in the MV and DPPIV activity increases NHE3 activity. Taken together, these studies are consistent with the hypothesis that during BP elevation or ACEI, NHE3 is retracted from the apical MV to the base of the MV where transporters form complexes with regulatory proteins that reversibly inactivate transporters and decrease PT Na+ reabsorption, without NHE3 internalization. An alternative hypothesis is that the "effective" Na+ reabsorption area on the apical surface is reduced when most NHE3 moved to the bottom of the MV.
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 NHERF and ezrin (41). Interestingly, the unconventional myosin VI, which moves toward the pointed ends of actin filaments, is found localized in the PT. Our quantitative immuno-EM analysis determined that 50% of the myosin VI was localized to the MV, 30% to the intermicrovillar zone (IMZ), and 20% to the apical cytoplasmic zone (39). Supporting a functional role for this motor in moving NHE3 in the MV, myosin VI redistributes with NHE3 from the top to the base of the MV during acute BP elevation in SD (39). In this study ACEI also provoked a redistribution of myosin VI to higher-density membranes. The current study also demonstrated a 49% decrease in PT myosin VI abundance. 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 IMZ and/or internalization of the proteins to endocytic compartments (12). It has been shown that NHERF1 interacts with NaPi2 and that the interaction is necessary for maintaining NaPi2 in the brush border (29, 30). In a NHERF1 knockout mouse, there is problem inserting NaPi2 into the brush border (33). Recently, Déliot et al. (7) demonstrated that PTH-induced NaPi2 endocytosis was accompanied by the dissociation of NaPi2 and NHERF1: NaPi2 is internalized while NHERF1 stays in the brush border. The finding in our current study showed that long-term ACEI treatment internalized NaPi2 but did not change NHERF1 distribution, supporting the notion that the dissociation of NaPi2 and NHERF1 may be critical for the NaPi2 internalization during ACEI.
In this study, we also measured a 33% decrease in ACE expression in renal cortex after 4-wk ACEI. There is now abundant evidence that the antihypertensive ACE inhibitors have renal protective effects and ameliorate the progression of chronic renal disease (31). Largo et al. (16a) demonstrated in the PTs of rats with intense proteinuria that ACE expression, not AT1 receptor expression, is upregulated, indicating that increased intrarenal RAS may be responsible for the tubulointerstitial lesions in this model. In diabetes, intrarenal ANG II production is elevated despite suppressed systemic RAS (4). Nagai et al. (24) found that AT1 blocker or ACE inhibitor at the prediabetic stage suppressed proteinuria and attenuated the development of renal injury in type 2 diabetes, suggesting that elevated intrarenal RAS may be important in renal injury. Kobori and colleagues (14) recently demonstrated that intrarenal angiotensinogen and ANG II are increased in SHR even though intrarenal renin levels are normal. The increased angiotensinogen and renal injury in SHR are both prevented by AT1 receptor blocker treatment. Therefore, Kobori and colleagues concluded that this enhanced intrarenal RAS may contribute to the early renal injury in SHR. In addition, their results, as well as those of Ingelfinger et al. (13), support the notion of positive feedback effect of ANG II on its precursor, angiotensinogen, i.e., elevated ANG II stimulates angiotensinogen generation. Interestingly, we found a similar relationship in the opposite direction in this study: inhibition of ANG II production by ACEI lowers ACE expression in the kidney. This could potentially decrease the intrarenal RAS and contribute to the correction of BP and prevention of renal injury in SHR. It remains to be determined whether there is an upregulation of intrarenal ACE during the development of hypertension in SHR compared with SD rats, and whether the decrease in ACE by ACEI treatment is renal specific.
In this study, we also discovered that long-term enalapril provoked significant differences in overall distribution patterns of both NKCC and NCC, specifically, from low-density to higher-density membranes. Related to these studies, Kwon et al. (15) demonstrated that ANG II treatment of rats for 7 days increased medullary thick ascending limb total NKCC abundance which could contribute to enhanced renal Na+ reabsorption in response to ANG II. Our density gradient results are consistent with movement of NKCC out of apical membranes to intracellular membranes during ACEI, although we do not have direct microscopic evidence for internalization of NKCC. We do have immuno-EM evidence for the redistribution of DCT NCC from low- to higher-density membranes consistent with trafficking of NCC from apical membranes to subapical vesicles (27, 28). Specifically, we discovered that in SD rats fed high-salt diet or infused with captopril (12 µg/min) for 20 min there is a redistribution of NCC from DCT apical plasma membranes to the subapical cytoplasmic vesicles. Related to this, an earlier study by Wang and Giebisch (32) demonstrated that ANG II stimulates sodium, bicarbonate, and volume reabsorption in the DCT. The results from the current study provide evidence consistent with chronic redistribution of NCC out of the apical membrane into subapical vesicles during ACEI, suggesting a previously unreported mechanism of action of ACE inhibitors.
Regarding the ENaC regulation by diuretics, Na et al. (23) previously demonstrated that chronic thiazide infusion (3.75 mg/day hydrochlorothiazide) through osmotic minipumps in SD rats increased ENaC in the cortex and ENaC and in the outer medulla, suggesting a mechanism for diuretic tolerance. In our current study, HRH given in drinking water (including 3 mg/day hydrochlorothiazide) caused slight increases in three subunits of medullary ENaC but these changes did not reach statistical significance. The difference between our study and that of Na et al. could potentially be explained by the higher dose and direct infusion used in their study. It is likely that the osmotic minipumps deliver more thiazide to the site of action in the DCT than administering the thiazide in the drinking water and, in addition, the dose used in our study was 20% lower. Interestingly, 4-wk enalapril induced a small but significant increase in medullary ENaC abundance, which may also indicate a tolerance mechanism in long-term ACEI treatment.
In conclusion, the redistribution of Na+ transporters, including NHE3, NaPi2, NCC, and NKCC, during long-term enalapril treatment appears to be a direct consequence of ACEI rather than BP lowering because lowering BP with the triple therapy treatment did not significantly alter transporter density distribution. This study provides evidence for previously unreported actions of ACEI in the kidney that likely contribute to the BP-lowering effects of these drugs. The results indicate that long-term enalapril may reduce Na+ retention and BP by reducing the abundance of Na+ transporters and their associated regulators in apical membranes along the nephron, as well as lowering renal ACE abundance itself.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. D. M. Riquier, D. H. Lee, and A. A. McDonough Renal NHE3 and NaPi2 partition into distinct membrane domains Am J Physiol Cell Physiol, April 1, 2009; 296(4): C900 - C910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. H. Lee, A. D. M. Riquier, L. E. Yang, P. K. K. Leong, A. B. Maunsbach, and A. A. McDonough Acute hypertension provokes acute trafficking of distal tubule Na-Cl cotransporter (NCC) to subapical cytoplasmic vesicles Am J Physiol Renal Physiol, April 1, 2009; 296(4): F810 - F818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Kastner, M. Pohl, M. Sendeski, G. Stange, C. A. Wagner, B. Jensen, A. Patzak, S. Bachmann, and F. Theilig Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis Am J Physiol Renal Physiol, April 1, 2009; 296(4): F902 - F911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Yang, M. B. Sandberg, A. D. Can, K. Pihakaski-Maunsbach, and A. A. McDonough Effects of dietary salt on renal Na+ transporter subcellular distribution, abundance, and phosphorylation status Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1003 - F1016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. K. Madala Halagappa, S. Tiwari, S. Riazi, X. Hu, and C. M. Ecelbarger Chronic candesartan alters expression and activity of NKCC2, NCC, and ENaC in the obese Zucker rat Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1222 - F1231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. C. Girardi, L. E. Fukuda, L. V. Rossoni, G. Malnic, and N. A. Reboucas Dipeptidyl peptidase IV inhibition downregulates Na+-H+ exchanger NHE3 in rat renal proximal tubule Am J Physiol Renal Physiol, February 1, 2008; 294(2): F414 - F422. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
, n = 4), untreated spontaneously hypertensive rats (SHR; 
, n = 4), and SHR treated with enalapril (30 mg·kg–1·day–1 for 4 wk; 
, n = 4). Typical immunoblots of NHE3 and NaPi2 from a constant sample volume from each gradient fraction are shown. Immunoreactivity is expressed as the percentage of the total signal in all 12 fractions. Values are means ± SD. *P < 0.05 vs. SD. #P < 0.05 vs. untreated SHR group, assessed by ANOVA and followed by paired Student's t-test.
