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Am J Physiol Renal Physiol 293: F1047-F1053, 2007. First published July 18, 2007; doi:10.1152/ajprenal.00074.2007
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Aminopeptidase N reduces basolateral Na+-K+-ATPase in proximal tubule cells

Kumar Kotlo,1,* Sagar Shukla,1,* Urmila Tawar,1 Randal A. Skidgel,3 and Robert S. Danziger1,2,3,4

Departments of 1Medicine, 2Physiology and Biophysics, and 3Pharmacology, University of Illinois at Chicago, Chicago; and 4Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois

Submitted 13 February 2007 ; accepted in final form 12 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na+-K+-ATPase, a preeminent basolateral Na+ transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na+-K+-ATPase activity and basolateral Na+-K+-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na+-K+-ATPase activity and basolateral abundance by ~50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na+-K+-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na+-K+-ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.

angiotensin IV; kidney

AMINOPEPTIDASE N (Anpep; EC 3.4.11.2 [EC] ), also known as CD13, gp150, microsomal aminopeptidase, and aminopeptidase M, is a homodimeric, membrane-bound, zinc-dependent aminopeptidase (for reviews, see Refs. 35, 47). In the kidney, it is concentrated in the brush-border membrane of proximal tubule (PT) cells (25, 37, 42). Anpep preferentially releases neutral amino acids from the NH2-terminal end of oligopeptides and has specificity similar to that of cytosolic leucine aminopeptidase. Reported substrates for Anpep include neuropeptides (e.g., Met- and Leu-enkephalins, neurokinin A, Met-lys-bradykinin, and endorphins) and vasoactive peptides (31, 44).

The natriuretic hexapeptide ANG IV (Val-Tyr-IIe-His-Pro-Phe) is generated by Anpep cleavage of the NH2-terminal Arg from angiotensin III (ANG III) and of des-Asp-ANG I (2–10) (which is further cleaved by angiotensin-converting enzyme) (37, 48). There is significant evidence that, in contrast to ANG II and ANG III, ANG IV both inhibits renal Na reabsorption and reduces renal vascular resistance (19, 21, 23). In rat PT cells, ANG IV decreases transcellular Na+ transport, as measured by PT O2 consumption rates (23), and in human kidney cells, ANG IV increases Na+ uptake, reflecting decreased Na+efflux (22).

The receptor for ANG IV (AGTRIV) has been identified as insulin-regulated membrane aminopeptidase, also referred to as cystinyl aminopeptidase (EC 3.4.11.3 [EC] ) (1–3, 6, 7, 15). AGTRIV is expressed in human PT, distal tubules, vascular smooth muscle, and endothelial cells (10, 21, 22).

Our laboratory, as well as others, reported data suggesting that renal Anpep may be important in salt handling. In the Goldblatt two-kidney, one-clip model of salt-sensitive hypertension, greater membrane-bound Anpep activity is present in the cortex of the nonclipped kidney (43). We found that Anpep transcript abundance, protein expression, and activity are greater in the kidneys of Dahl salt-resistant (SR/Jr) vs. Dahl salt-sensitive (SS/Jr) and Sprague-Dawley (SD) rat strains (16). The Anpep gene maps to a reported blood pressure quantitative trait locus in rats and has a functional polymorphism with promoter activity that is preferentially expressed in the Dahl SR/Jr strain vs. the SS/Jr and SD strains (26). However, a mechanistic link between renal tubule Anpep and Na transporters has not yet been established.

Normal adaptation to high salt occurs through a coordinated reduction in Na+ uptake by PT and distal nephrons (for reviews, see Refs. 4, 11). Na+-K+-ATPase is the preeminent basolateral Na+ transporter in PT. Moreover, adaptation to high salt has been directly linked to a reduction in basolateral Na+-K+-ATPase abundance via endocytosis or internalization of the membrane-associated transporter (36, 40, 41). The present study was performed to investigate Anpep regulation of basolateral Na+-K+-ATPase in PT.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The siRNAs to Anpep (5'-AATCGGGCACAGATCATTAAT-3') and AGTRIV (5'-AACCTGAGTCAGGACGTAAAT-3') were obtained from Qiagen. Rabbit anti-human polyclonal Anpep antibody (Ab) was from Santa Cruz Biotechnology, and monoclonal Na+-K+-ATPase Abs were from Upstate Biotechnology and Santa Cruz Biotechnology. Polyclonal AGTRIV Ab was kindly provided by Dr. P. Pilch, Boston University School of Medicine. Polyclonal NHE-3 Ab was provided by Dr. J. Liu, Medical College of Ohio. Monoclonal NHE-1 Ab was purchased from BD Biosciences. The control overexpression vector, pcDNA3.1D TOPO, and yellow fluorescent expression vector, pIRES-EYFP, were purchased from Invitrogen and Clontech, respectively. ANG IV was purchased from Phoenix Pharmaceuticals (Burlingame, CA). Porcine renal tubular epithelial cells (LLC-PK1 cells) were from ATCC.

Cell culture. For confocal imaging experiments, LLC-PK1 cells were cultured for passages 8–9 in our laboratory and then seeded in transwell plates at a density of 10,000 cells/cm2 and cultured for 4 days in DMEM 199 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. For transfection experiments, LLCPK1 cells were cultured in six-well plates at a density of 25,000 cells/cm2.

Transfections/plasmids. Anpep expression plasmid was generated by cloning PCR-amplified Anpep cDNA into pcDNA3.1 D His TOPO vector. Anpep cDNA was PCR amplified using primers (forward 5'-CACCATGGCCAAGGGTTTCTACATTTCC-3'; reverse 5'-CTAGCTGT TTTCTGTGAACCACTTGAG-3') and Anpep cDNA template (NM_001150 [GenBank] and gi: 4502094). Transfections with Anpep expression plasmid (2 µg) were performed using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions. In these experiments, a range of siRNA concentrations was tested and the optimal concentration, based on threshold for maximal suppression of basolateral Na+-K+-ATPase abundance, was 2 µg per transfection. Trans messenger transfect reagent (Qiagen) was employed for transfection of siRNA-Anpep (2 µg) and siRNA-AGTRIV (2 µg). Cells were harvested 48 h after transfection with siRNA or Anpep overexpression.

Na+-K+-ATPase activity assay. Na+-K+-ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain (13, 14, 32). To prepare membranes for Na+-K+-ATPase activity assay, cells from numbers 5–6 150-mm2 dishes were washed twice with 5 ml chilled phosphate-free buffer (2.36 M NaCl, 0.54 M NaHCO3, 0.4 M KCl, and 0.12 M MgCl2scraped in phosphate-free buffer) and centrifuged at 3,000 g for 8–10 min. The cells were then placed on ice and lysed by dounce homogenization in 2 ml of lysis buffer (1 mM NaHCO3, 2 mM CaCl2, and 5 mM MgCl2). Cellular lysates were centrifuged at 3,000 g for 1–2 min to remove intact cells, debris, and nuclei. The resulting supernatant was suspended in an equal volume of 1 M NaI, and the mixture was centrifuged at 48,000 g for 25 min. The pellet (membrane fraction) was washed one to two times and suspended in 10 mM Tris and 1 mM EDTA (pH 7.4). Protein concentrations were determined by the Bradford assay and adjusted to 1 mg/ml. The membranes were stored at –70°C until further use. To measure Na+-K+-ATPase activity, 100-µl aliquots of membrane fraction were added to an 800-µl reaction mixture (75 mM NaCl, 5 mM KCl, 5 mM MgCl2, 6 mM NaN2, 1 mM Na EGTA, 37.5 mM imidazole, 75 mM Tris·HCl, and 30 mM histidine; pH 7.4) with or without 1 mM ouabain (final volume = 1 ml) and preincubated for 5 min in a water bath at 37°C. Reactions were initiated by adding Tris ATP (4 mM) and were terminated after 15 min of incubation at 37°C by adding 50 µl of 50% TCA. For determination of ouabain-insensitive ATPase activity, NaCl and KCl were omitted from reaction mixtures containing ouabain. To quantify the amount of phosphate produced, 1 ml of coloring reagent (10% ammonium molybdate in 10 N sulfuric acid + ferrous sulfate) was added to the reaction mixture. The mixture was then combined thoroughly and centrifuged at 3,000 g for 10 min. Formation of phosphomolybdate was determined spectrophotometrically at 740 nm, against a standard curve prepared from K2HPO4. Na+-K+-ATPase activity was estimated as the difference between total ATPase activity and ouabain-insensitive ATPase activity and was expressed as nanomoles of phosphate produced per milligram of protein per minute.

Confocal microscopy. Cells were fixed in 2% paraformaldehyde and permeabilized with 0.08% saponin in phosphate-buffered saline for 10 min. Slides were then successively incubated with anti-Na+-K+-ATPase {alpha}-monoclonal primary Ab (1:2,000 dilution) for 1 h and with FITC-conjugated anti-mouse secondary antibody. Confocal images were obtained using Zeiss laser-scanning confocal microscope LSM 510 META.

Surface biotinylations. Biotinylations were performed with EZ-Link Sulfo-NHS-SS-Biotin (Pierce) in a sucrose-containing borate buffer (pH 9.0), as previously described (18). Cells were grown to 85–90% confluence in six-well transwell plates before transfection. Biotinylations were performed when the cells were completely confluent. The biotinylation buffer was placed in the bottom chamber of the transwell plates, and plates were incubated for 1.5 h at 4°C. Biotinylated proteins were immunoprecipitated from total cell extracts with streptavidin agarose, released by incubation in 10 mM dithiothreitol, reconstituted in Laemmli buffer, and subjected to SDS-PAGE and Western analysis.

Western analysis. Protein samples were prepared in lysis buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% NP-40, and protease inhibitor cocktail), and equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with the indicated primary Abs and incubated with appropriate horseradish peroxidase-conjugated secondary Ab. Chemiluminescence was detected using ECL Western blotting detection reagents (Amersham). Signal was normalized to total cell protein. The bands were quantified by NIH image analysis. Band saturation was avoided by using the shortest exposure time necessary to detect signals and comparing results with two different submaximal exposure times.

Statistical analysis. Comparisons were made by one-way ANOVA followed by the Tukey-Kramer multiple comparisons test. Data are expressed as means ± SD. P < 0.05 was accepted as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Anpep negatively regulates Na+-K+-ATPase activity in LLC-PK1 cells. To determine whether Anpep may regulate Na+-K+-ATPase, LLC-PK1 cells transiently transfected with full-length Anpep (Anpep OE). Transfected cells exhibited an approximate twofold increase in Anpep expression (Fig. 1A). Both Na+-K+-ATPase activity and basolateral Na+-K+-ATPase abundance, as measured by surface biotinylation, were reduced by ~70% with Anpep OE (P < 0.05; Fig. 1, B-C). Consistent with these results, surface staining for Na+-K+-ATPase {alpha}-subunit was reduced in confocal imaging (Fig. 2). In a complementary set of experiments, the effect of siRNA-mediated knockdown of Anpep on basolateral Na+-K+-ATPase abundance and Na+-K+-ATPase activity was examined. Endogenous Anpep protein levels were reduced by 50% in siRNA-transfected cells (Fig. 1). Conversely, both biotinylated Na+-K+-ATPase abundance and Na+-K+-ATPase activity approximately doubled (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Effect of aminopeptidase N/CD13 (Anpep) on the basolateral levels and activity of Na+-K+-ATPase in LLC-PK1 cells. To examine Anpep regulation of Na+-K+-ATPase, LLC-PK1 cells were transfected with Anpep expression plasmid (Anpep OE) or Anpep siRNA (siRNA-Anpep). Transfection with scrambled siRNA, pcDNA3.1 empty expression vector (vector OE), or yellow fluorescent protein expression vector (YFP vector) served as controls (see MATERIALS AND METHODS). A: to confirm Anpep overexpression or knockdown, whole cell lysates were subjected to Western blot analysis of Anpep. A representative Western blot is shown (n = 3). B: ouabain-inhibitable ATPase activity of membrane protein extracts from LLC-PK1 cells (n = 3). C: basolateral Na+-K+-ATPase abundance, as determined by surface biotinylation and Western analysis (n = 5). A representative Western blot of biotinylated Na+-K+-ATPase is shown. D: Na+-K+-ATPase abundance in whole cell extracts (n = 3). E: NHE-1 (n = 3). F: NHE-3 abundance (n = 3). *P < 0.01 vs. controls.

 

Figure 2
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  		Fig. 2. Anpep and ANG IV regulation of Na+-K+-ATPase distribution in LLC-PK1 cells. Cultures were transfected with expression plasmid (vector), Anpep expression plasmid (Anpep OE), ANG IV (10 nM 12 h) and cotransfected with ANG IV receptor (AGTRIV) siRNA + Anpep OE, and treated with ANG IV (see MATERIALS AND METHODS). Cells were immunostained for the {alpha}-subunit of Na+-K+-ATPase (green). All images were generated with a Zeiss LSM 410 laser-scanning confocal microscope. Images are representative of experiments repeated twice.

 
The abundance of whole cell Na+-K+-ATPase, the apical transporter NHE3, and basolateral transporter NHE1 (Fig. 1, D, E, and F, respectively) were also measured by Western analysis. The abundance of these proteins was similar with Anpep overexpression and Anpep siRNA, suggesting that NHE3 and NHE1 are not regulated, at least at the level of expression, by Anpep.

Anpep signaling requires AGTRIV. To investigate the ANG IV dependence of Anpep signaling, we examined the effect of AGTRIV siRNA on the basolateral abundance and activity of Na+-K+-ATPase. Forty-eight hours after transfection with AGTRIV siRNA, AGTRIV protein levels were reduced by 2.5-fold. This was accompanied by an approximate twofold increase in the basolateral Na+-K+-ATPase abundance (P < 0.05) and activity (P < 0.05; Fig. 3). In a second set of experiments, the effect of AGTRIV knockdown on regulation of Na+-K+-ATPase by Anpep was examined. Cells were first transfected with AGTRIV siRNA and 24 h later transfected again with Anpep expression vector. Inhibition of AGTRIV expression by siRNA was confirmed by Western analysis (Fig. 3B). AGTRIV knockdown relieved the negative effect of Anpep overexpression on Na+-K+-ATPase, as seen by approximately threefold greater basolateral Na+-K+-ATPase abundance and Na+-K+-ATPase activity in AGTRIV siRNA-Anpep OE cotransfected cells vs. scrambled siRNA-Anpep OE cotransfected counterparts (P < 0.05; Fig. 3). Thus, Anpep regulation of Na+-K+-ATPase requires AGTRIV expression.


Figure 3
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  		Fig. 3. Effect of siRNA-mediated AGTRIV knockdown on regulation of basolateral Na+-K+-ATPase by Anpep in LLC-PK1 cells. Cells were transfected with AGTRIV siRNA + Anpep expression plasmid (Anpep OE), scrambled siRNA + vector (OE vector; controls). A: Na+-K+-ATPase activity in membrane protein extracts (n = 4). B: biotinylated Na+-K+-ATPase in cotransfected LLC-PK1 cells; representative immunoblots for AGTRIV and actin in whole cell extracts (top 2 rows) and for biotinylated Na+-K+-ATPase (row 3; n = 4). *P < 0.05.

 
ANG IV negatively regulates Na+-K+-ATPase. Since ANG IV is formed by Anpep (37, 48), we examined the effect of ANG IV on Na+-K+-ATPase in LLC-PK1 cells. ANG IV treatment (10 nM, 30 min and 12 h) reduced Na+-K+-ATPase activity by approximately three- to fourfold (Fig. 4A, P < 0.05). Biotinylated Na+-K+-ATPase was reduced by approximately fourfold (P < 0.05) (Fig. 4B) and surface straining in confocal imaging decreased (Fig. 2, ANG IV). Whole cell (Fig. 4C) Na+-K+-ATPase abundance was not decreased.


Figure 4
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  		Fig. 4. Effect of ANG IV treatment on Na+-K+-ATPase. A: Na+-K+-ATPase activity in LLC-PK1 cells incubated with 10 nM ANG IV (10 nM, 30 min and 12 h; n = 4). Biotinylated (B) and whole cell (C) Na+-K+-ATPase in cells treated with ANG IV (10 nM, 12 h; n = 8, 4, respectively). Top rows: representative immunoblots. **P < 0.01 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of this study was to relate Anpep to regulation of Na+ handling in PT. We demonstrated that Anpep reduces basolateral Na+-K+-ATPase levels and activity of Na+-K+-ATPase. Immunohistochemical data suggest that these effects may be due to membrane trafficking of Na+-K+-ATPase. Anpep siRNA increased basolateral biotinylated Na+-K+-ATPase abundance and Na+-K+-ATPase activity in LLC-PK1 cells, suggesting that endogenous Anpep acts to downregulate Na+-K+-ATPase under basal conditions in these cells. Regulation by Anpep in cells has been postulated to be to reduced signaling by substrates, e.g., enkephalins, endorphins, cytokines, and somatostatin (31, 44) and the effect of Anpep inhibitors to reduced metabolism and increased signaling by these substrates. Our study is the first, to our knowledge, to link signaling by Anpep to a product, i.e., ANG IV, rather than substrate.

The reduction in basolateral Na+-K+-ATPase abundance and Na+-K+-ATPase activity associated with Anpep overexpression was inhibited by blocking AGTRIV expression with specific siRNA, suggesting signaling occurs through an ANG IV-dependent mechanism. This is the first time that this pluripotent signaling molecule has been implicated in intracellular signaling in LLC-PK1 cells. ANG IV has been reported to inhibit energy-dependent Na transport in PT cell lines (8) and also to inhibit Na+ uptake in a manner similar to ouabain (21). Our results suggest that inhibition of Na transport is due, at least in part, to reduced Na+-K+-ATPase.

If Anpep synthesizes ANG IV in cultured LLC-PK1 cells, this suggests of that either local renin-angiotensin system (RAS) signaling is present in renal epithelial cells or that the culture media, including fetal bovine serum, provides substrates. The existence of principal components of RAS, including ANG II, is well established in a variety of tissues (27, 39, 45). Substrates for ANG IV formation include ANG III and ANG I (R-V-Y-I-H-P-F-H-L; Angiotensin 2–10) which is converted to ANG IV by Anpep and carboxypeptidase or angiotensin. The in vivo utilization of these substrates is not known. However, our results suggest that if signaling is through ANG IV formation that the process is not substrate limited since Anpep overexpression increases signaling.

Although AGTRIV has been identified as a receptor for ANG IV, its role has remained controversial since ANG IV also has a low affinity for AT1, AT2, and ANG-(1-7) (5, 17, 30, 49) receptors. The finding that the effect of ANG IV on Na+-K+-ATPase is blocked by AGTRIV siRNA provides unique supportive evidence that this signaling occurs via AGTRIV, but does not exclude simultaneous effects on other receptors. A rigorous investigation of the mechanism(s) underlying selectivity for specific receptors under different conditions will require measurements of local ANG IV concentration and receptors. There is also the possibility that expression of AGTRIV is regulated and plays an independent role in Anpep signaling. For instance, it has been shown that AGTRIV receptor binding is greater in the renal outer medulla of Wistar-Kyoto rats than of spontaneously hypertensive rats (19).

The reduction in biotinylated Na+-K+-ATPase induced by Anpep is not accompanied by a decrease in whole cell Na+-K+-ATPase abundance. This is consistent with the report by Choukroun et al. (9) in which surface expression, also measured by biotinylation, of Na+-K+-ATPase in cultured renal epithelial cells was reduced by phospholipase A2while total cell Na+-K+-ATPase was unaffected. This was interpreted as an indication that trafficking of Na+-K+-ATPase was primarily being regulated in this setting. Determination of regulation of intracellular Na+-K+-ATPase transport and synthesis by Anpep will further understanding of regulation of basolateral Na+-K+-ATPase by Anpep.

The results also raise the intriguing possibility that two zinc-dependent aminopeptidases, AGTRIV and Anpep, may regulate Na+-K+-ATPase in cells. Although both aminopeptidases metabolize ANG III and ANG IV and are inhibited by ANG IV, they are distinct enzymes that likely serve nonoverlapping functions. AGTRIV is insulin regulated and is present in GLUT4-containing vesicles. AGTRIV, but not Anpep, has been linked to activation of Erk1/2 (29), which may mediate endocytosis of basolateral Na+-K+-ATPase. AGTRIV also can activate NF-{kappa}B and related proinflammatory genes in vascular smooth muscle cells (15). AGTRIV is the only membrane aminopeptidase reported to cleave vasopressin (24). The results also highlight the possibility that Anpep may not only signal through regulation of metabolism of substrates but also through the synthesis of products, e.g., ANG IV.

There is the possibility that other apical and basolateral transporters may be regulated directly or indirectly by Anpep and ANG IV. Some agents, e.g., PTH, which redistributes apical NHE3 and NaPi2 transporters while inhibiting basolateral Na+-K+-ATPase (50), and captopril, which redistributes multiple membrane-associated proteins (28), regulate more than one PT transporter. Some changes in transporters may be secondary to others, e.g., basolateral Na+-K+-ATPase is known to regulate the expression and activity apical transporters, including NHE3 (38) and, vice versa, it is possible that apical transporters modulate basolateral transporters. To more fully understand regulation by Anpep of Na+-K+-ATPase will require insight into effects on apical and other basolateral transporters. As a first step to addressing this, we measured NHE3 and NHE1 protein abundance. A significant change abundance of neither was detected with Anpep overexpression nor siRNA to Anpep, suggesting that these transporters are not regulated by Anpep. Clearly, further studies are needed to critically investigate regulation of Na transport by Anpep.

The present findings demonstrate that Anpep regulates basolateral Na+-K+-ATPase abundance and activity in LLC-PK1 cells via a novel signaling pathway that requires AGTRIV. Because renal Anpep expression is greater in the Dahl SR/Jr rat than in the Dahl SS/Jr rat, Anpep may contribute to reduction of basolateral Na+-K+-ATPase in the SR/Jr strain and adaptation to high salt (16, 26, 33, 34). Inappropriately low levels of renal tubule Anpep may thus contribute to salt sensitivity. The in vivo relationship between Anpep and mediators of salt adaptation such as nitric oxide (20), dopamine (12, 33), natriuretic peptides (46), and marinobufagenin (41) will be necessary to understand the role of Anpep and ANG IV/AGTRIV in blood pressure regulation. Nevertheless, the present results raise the possibility that Anpep and ANG IV/AGTRIV agonists are novel therapeutic targets and agents, respectively, for salt-dependent hypertension.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grant R21-DK-065628 (R. S. Danziger), American Heart Association Grant-in-Aid (R. S. Danziger), and Phillip Morris (R. S. Danziger)


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. S. Danziger, Dept. of Medicine, Univ. of Illinois at Chicago, 840 S. Wood St., Chicago, IL 60612 (e-mail: rdanziger{at}aol.com)

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.

* K. Kotlo and S. Shukla contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Albiston AL, Fernando R, Ye S, Peck GR, Chai SY. Alzheimer's, angiotensin IV and an aminopeptidase. Biol Pharm Bull 27: 765–767, 2004.[CrossRef][Web of Science][Medline]
  2. Albiston AL, McDowall SG, Matsacos D, Sim P, Clune E, Mustafa T, Lee J, Mendelsohn FA, Simpson RJ, Connolly LM, Chai SY. Evidence that the angiotensin IV [AT(4)] receptor is the enzyme insulin-regulated aminopeptidase. J Biol Chem 276: 48623–48626, 2001.[Abstract/Free Full Text]
  3. Albiston AL, Mustafa T, McDowall SG, Mendelsohn FA, Lee J, Chai SY. AT4 receptor is insulin-regulated membrane aminopeptidase: potential mechanisms of memory enhancement. Trends Endocrinol Metab 14: 72–77, 2003.[CrossRef][Web of Science][Medline]
  4. Aperia A. Regulation of sodium/potassium ATPase activity: impact on salt balance and vascular contractility. Curr Hypertens Rep 3: 165–171, 2001.[Medline]
  5. Bernier SG, Servant G, Boudreau M, Fournier A, Guillemette G. Characterization of a binding site for angiotensin IV on bovine aortic endothelial cells. Eur J Pharmacol 291: 191–200, 1995.[CrossRef][Web of Science][Medline]
  6. Braszko JJ. Involvement of D1 dopamine receptors in the cognitive effects of angiotensin IV and des-Phe6 angiotensin IV. Peptides 25: 1195–1203, 2004.[CrossRef][Web of Science][Medline]
  7. Chai SY, Fernando R, Peck G, Ye SY, Mendelsohn FA, Jenkins TA, Albiston AL. The angiotensin IV/AT4 receptor. Cell Mol Life Sci 61: 2728–2737, 2004.[CrossRef][Web of Science][Medline]
  8. Chen JK, Zimpelmann J, Harris RC, Burns KD. Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells. Am J Physiol Renal Physiol 280: F980–F988, 2001.[Abstract/Free Full Text]
  9. Choukroun GJ, Marshansky V, Gustafson CE, McKee M, Hajjar RJ, Rosenzweig A, Brown D, Bonventre JV. Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins. J Clin Invest 106: 983–993, 2000.[Web of Science][Medline]
  10. Czekalski S, Chansel D, Vandermeersch S, Ronco P, Ardaillou R. Evidence for angiotensin IV receptors in human collecting duct cells. Kidney Int 50: 1125–1131, 1996.[Web of Science][Medline]
  11. Dantzler WH. Regulation of renal proximal and distal tubule transport: sodium, chloride and organic anions. Comp Biochem Physiol A Mol Integr Physiol 136: 453–478, 2003.[CrossRef][Medline]
  12. Efendiev R, Yudowski GA, Zwiller J, Leibiger B, Katz AI, Berggren PO, Pedemonte CH, Leibiger IB, Bertorello AM. Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis. J Biol Chem 277: 44108–44114, 2002.[Abstract/Free Full Text]
  13. Ellis DZ, Nathanson JA, Sweadner KJ. Carbachol inhibits Na+-K+-ATPase activity in choroid plexus via stimulation of the NO/cGMP pathway. Am J Physiol Cell Physiol 279: C1685–C1693, 2000.[Abstract/Free Full Text]
  14. Esmann M. ATPase and phosphatase activity of Na+,K+-ATPase: molar and specific activity, protein determination. Methods Enzymol 156: 105–115, 1988.[Web of Science][Medline]
  15. Esteban V, Ruperez M, Sanchez-Lopez E, Rodriguez-Vita J, Lorenzo O, Demaegdt H, Vanderheyden P, Egido J, Ruiz-Ortega M. Angiotensin IV activates the nuclear transcription factor-{kappa}B and related proinflammatory genes in vascular smooth muscle cells. Circ Res 96: 965–973, 2005.[Abstract/Free Full Text]
  16. Farjah M, Roxas B, Danziger RS. Dietary NaCl regulates in renal APN transcript/protein abundance and activity: relevance to hypertension in the Dahl rat. Hypertension 43: 282–285, 2004.[Abstract/Free Full Text]
  17. Faure S, Javellaud J, Achard JM, Oudart N. Vasoconstrictive effect of angiotensin IV in isolated rat basilar artery independent of AT1 and AT2 receptors. J Vasc Res 43: 19–26, 2006.[CrossRef][Web of Science][Medline]
  18. Gottardi CJ, Dunbar LA, Caplan MJ. Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol Renal Fluid Electrolyte Physiol 268: F285–F295, 1995.[Abstract/Free Full Text]
  19. Grove KL, Deschepper CF. High salt intake differentially regulates kidney angiotensin IV AT4 receptors in Wistar-Kyoto and spontaneously hypertensive rats. Life Sci 64: 1811–1818, 1999.[CrossRef][Web of Science][Medline]
  20. Hakam AC, Hussain T. Angiotensin II AT2 receptors inhibit proximal tubular Na+-K+-ATPase activity via a NO/cGMP-dependent pathway. Am J Physiol Renal Physiol 290: F1430–F1436, 2006.[Abstract/Free Full Text]
  21. Hamilton TA, Handa RK, Harding JW, Wright JW. A role for the angiotensin IV/AT4 system in mediating natriuresis in the rat. Peptides 22: 935–944, 2001.[CrossRef][Web of Science][Medline]
  22. Handa RK. Characterization and signaling of the AT(4) receptor in human proximal tubule epithelial (HK-2) cells. J Am Soc Nephrol 12: 440–449, 2001.[Abstract/Free Full Text]
  23. Handa RK, Krebs LT, Harding JW, Handa SE. Angiotensin IV AT4-receptor system in the rat kidney. Am J Physiol Renal Physiol 274: F290–F299, 1998.[Abstract/Free Full Text]
  24. Herbst JJ, Ross SA, Scott HM, Bobin SA, Morris NJ, Lienhard GE, Keller SR. Insulin stimulates cell surface aminopeptidase activity toward vasopressin in adipocytes. Am J Physiol Endocrinol Metab 272: E600–E606, 1997.[Abstract/Free Full Text]
  25. Kenny AJ, Maroux S. Topology of microvillar membrance hydrolases of kidney and intestine. Physiol Rev 62: 91–128, 1982.[Free Full Text]
  26. Kotlo K, Hughes DE, Herrera VL, Ruiz-Opazo N, Costa RH, Robey RB, Danziger RS. Functional polymorphism of the Anpepgene increases promoter activity in the Dahl salt-resistant rat promoter. Hypertension 49: 1–6, 2007.[Free Full Text]
  27. Lavoie JL, Sigmund CD. Minireview: overview of the renin-angiotensin system–an endocrine and paracrine system. Endocrinology 144: 2179–2183, 2003.[Abstract/Free Full Text]
  28. Leong PK, Devillez A, Sandberg MB, Yang LE, Yip DK, Klein JB, McDonough AA. Effects of ACE inhibition on proximal tubule sodium transport. Am J Physiol Renal Physiol 290: F854–F863, 2006.[Abstract/Free Full Text]
  29. Li YD, Block ER, Patel JM. Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation. Am J Physiol Lung Cell Mol Physiol 283: L707–L716, 2002.[Abstract/Free Full Text]
  30. Lochard N, Thibault G, Silversides DW, Touyz RM, Reudelhuber TL. Chronic production of angiotensin IV in the brain leads to hypertension that is reversible with an angiotensin II AT1 receptor antagonist. Circ Res 94: 1451–1457, 2004.[Abstract/Free Full Text]
  31. Matsas R, Stephenson SL, Hryszko J, Kenny AJ, Turner AJ. The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase. N Biochem J 231: 445–449, 1985.
  32. Newaz MA, Ranganna K, Oyekan AO. Relationship between PPARalpha activation and NO on proximal tubular Na+ transport in the rat. BMC Pharmacol 4: 1, 2004.[CrossRef][Medline]
  33. Nishi A, Bertorello AM, Aperia A. High salt diet downregulates proximal tubule Na+,K+-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats: evidence of defective dopamine regulation. Acta Physiol Scand 144: 263–267, 1992.[Web of Science][Medline]
  34. Nishi A, Celsi G, Aperia A. High-salt diet upregulates activity and mRNA of renal Na+-K+-ATPase in Dahl salt-sensitive rats. Am J Physiol Renal Fluid Electrolyte Physiol 264: F448–F452, 1993.[Abstract/Free Full Text]
  35. Noren O, Sjostrom H, Olsen J. Aminopeptidase N. In: Cell-Surface Peptidases in Health and Disease, edited by Kenny AJ and Boustead CM. Oxford: BIOS Scientific, 1997, p. 175–191.
  36. Ogimoto G, Yudowski GA, Barker CJ, Kohler M, Katz AI, Feraille E, Pedemonte CH, Berggren PO, Bertorello AM. G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin. Proc Natl Acad Sci USA 97: 3242–3247, 2000.[Abstract/Free Full Text]
  37. Olsen J, Kokholm K, Noren O, Sjostrom H. Structure and expression of aminopeptidase N. Adv Exp Med Biol 421: 47–57, 1997.[Web of Science][Medline]
  38. Oweis S, Wu L, Kiela PR, Zhao H, Malhotra D, Ghishan FK, Xie Z, Shapiro JI, Liu J. Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK1 cells. Am J Physiol Renal Physiol 290: F997–F1008, 2006.[Abstract/Free Full Text]
  39. Paul M, Poyan MA, Kreutz R. Physiology of local renin-angiotensin systems. Physiol Rev 86: 747–803, 2006.[Abstract/Free Full Text]
  40. Pedemonte CH, Efendiev R, Bertorello AM. Inhibition of Na,K-ATPase by dopamine in proximal tubule epithelial cells. Semin Nephrol 25: 322–327, 2005.[CrossRef][Web of Science][Medline]
  41. Periyasamy SM, Liu J, Tanta F, Kabak B, Wakefield B, Malhotra D, Kennedy DJ, Nadoor A, Fedorova OV, Gunning W, Xie Z, Bagrov AY, Shapiro JI. Salt loading induces redistribution of the plasmalemmal Na+-K+-ATPase in proximal tubule cells. Kidney Int 67: 1868–1877, 2005.[CrossRef][Web of Science][Medline]
  42. Poumarat JS, Houillier P, Rismondo C, Roques B, Lazar G, Paillard M, Blanchard A. The luminal membrane of rat thick limb expresses AT1 receptor and aminopeptidase activities. Kidney Int 62: 434–445, 2002.[CrossRef][Web of Science][Medline]
  43. Prieto I, Hermoso F, Gasparo M, Vargas F, Alba F, Segarra AB, Banegas I, Ramirez M. Angiotensinase activities in the kidney of renovascular hypertensive rats. Peptides 24: 755–760, 2003.[CrossRef][Web of Science][Medline]
  44. Riemann D, Kehlen A, Langner J. CD13–not just a marker in leukemia typing. Immunol Today 20: 83–88, 1999.[CrossRef][Web of Science][Medline]
  45. Sakai K, Sigmund CD. Molecular evidence of tissue renin-angiotensin systems: a focus on the brain. Curr Hypertens Rep 7: 135–140, 2005.[Web of Science][Medline]
  46. Scavone C, Scanlon C, McKee M, Nathanson JA. Atrial natriuretic peptide modulates sodium and potassium-activated adenosine triphosphatase through a mechanism involving cyclic GMP and cyclic GMP-dependent protein kinase. J Pharmacol Exp Ther 272: 1036–1043, 1995.[Abstract/Free Full Text]
  47. Sjostrom H, Noren O, Olsen J. Structure and function of aminopeptidase N. Adv Exp Med Biol 477: 25–34, 2000.[Web of Science][Medline]
  48. Ward PE, Benter IF, Dick L, Wilk S. Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M. Biochem Pharmacol 40: 1725–1732, 1990.[CrossRef][Web of Science][Medline]
  49. Wright JW, Krebs LT, Stobb JW, Harding JW. The angiotensin IV system: functional implications. Front Neuroendocrinol 16: 23–52, 1995.[CrossRef][Web of Science][Medline]
  50. Zhang Y, Norian JM, Magyar CE, Holstein-Rathlou NH, Mircheff AK, McDonough AA. In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition. Am J Physiol Renal Physiol 276: F711–F719, 1999.[Abstract/Free Full Text]



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