Renal endosomes contain angiotensin peptides, converting enzyme, and AT1A receptors

John D. Imig1, Gabriel L. Navar2, Li-Xian Zou1, Katie C. O'Reilly2, Patricia L. Allen2, James H. Kaysen2, Timothy G. Hammond2, and L. Gabriel Navar1

1 Department of Physiology and Division of Nephrology, 2 Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kidney cortex and proximal tubular angiotensin II (ANG II) levels are greater than can be explained on the basis of circulating ANG II, suggesting intrarenal compartmentalization of these peptides. One possible site of intracellular accumulation is the endosomes. In the present study, we tested for endosomal ANG I, ANG II, angiotensin type 1A receptor (AT1A), and angiotensin converting enzyme (ACE) activity and determined whether these levels are regulated by salt intake. Male Sprague-Dawley rats were fed chow containing either high or low dietary sodium for 10-14 days. Blood and kidneys were harvested and processed for measurement of plasma, kidney, and renal intermicrovillar cleft and endosomal angiotensin levels. Kidney ANG I averaged 179 ± 20 fmol/g and ANG II averaged 258 ± 36 fmol/g in rats fed a high-sodium diet and were significantly higher, averaging 347 ± 58 fmol/g and 386 ± 55 fmol/g, respectively, in rats fed a low-salt diet. Renal intermicrovillar clefts and endosomes contained ANG I and ANG II. Intermicrovillar cleft ANG I and ANG II levels averaged 8.4 ± 2.6 and 74 ± 26 fmol/mg, respectively, in rats fed a high-salt diet and 7.6 ± 1.7 and 70 ± 25 fmol/mg in rats fed a low-salt diet. Endosomal ANG I and ANG II levels averaged 12.3 ± 4.4 and 43 ± 19 fmol/mg, respectively, in rats fed a high-salt diet, and these levels were similar to those observed in rats fed a low-salt diet. Renal endosomes from rats fed a low-salt diet demonstrated significantly more AT1A receptor binding compared with rats fed a high-salt diet. ACE activity was detectable in renal intermicrovillar clefts and was 2.5-fold higher than the levels observed in renal endosomes. Acute enalaprilat treatment decreased ACE activity in renal intermicrovillar clefts by 90% and in renal endosomes by 84%. Likewise, intermicrovillar cleft and endosomal ANG II levels decreased by 61% and 52%, respectively, in enalaprilat-treated animals. These data demonstrate the presence of intact angiotensin peptides and ACE activity in renal intermicrovillar clefts and endosomes, indicating that intact angiotensin peptides are formed and/or trafficked through intracellular endosomal compartments and are dependent on ACE activity.

kidney; fluorometry; rat; enalapril; salt diet; angiotensin AT1A receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTRARENAL renin-angiotensin system is an important paracrine system that regulates renal hemodynamics and tubular transport function (27). Angiotensin II in the kidney causes vasoconstriction (29) and stimulates proximal tubule sodium reabsorption (8, 17, 34) and acidification (24). Studies from several laboratories have provided evidence that the kidney renin-angiotensin system is selectively regulated and is sometimes dissociated from circulating renin and angiotensin II levels (6, 10, 26, 30). The kidney contains all the necessary components to generate angiotensin II from its precursor angiotensinogen (28, 36). Angiotensinogen has also been shown to be present in the proximal tubule segment by immunohistochemical methods (9, 20), and the mRNA for angiotensinogen is expressed in proximal tubule cells (11, 22, 37). Therefore, the proximal tubule may produce angiotensin II subsequent to angiotensinogen secretion, or angiotensin II may be formed intracellularly (27).

Locally produced angiotensin II may act as an important regulator of proximal tubular transport function. Angiotensin converting enzyme (ACE) is in abundance on the brush-border membrane of proximal tubule cells and may generate intraluminal angiotensin II (21, 33, 42). Once generated, intraluminal angiotensin II could exert effects on proximal tubule transport upon activation of apical AT1 receptors. Endocytosis of the angiotensin II-AT1 receptor complex has been demonstrated to be important for the full expression of physiological responses to angiotensin II (5, 23). In proximal tubule cells, binding of angiotensin II and endocytosis of the AT1 receptor-angiotensin II complex is coupled to the activation of signal transduction pathways and sodium transport (4, 31, 32, 38). Angiotensin II produced intracellularly may also bind to AT1 receptors and have an autocrine effect on proximal tubular transport function. A recent study demonstrated that microinjection of angiotensin II directly into the cytosol of vascular smooth muscle cells causes an increase in intracellular calcium (12). The effect of intracellular angiotensin II on vascular smooth muscle calcium was an AT1 receptor-mediated event, since concomitant injection of an AT1 receptor blocker prevented this response (12). Thus intracellular trafficking of angiotensin II may be important for directing angiotensin II to certain cellular locations for full expression of its biological response.

The objective of the present study was to determine whether renal endosomes contain angiotensin peptides and whether these peptides are regulated by chronic changes in dietary salt intake and acute enalapril treatment. Kidney and plasma samples were obtained for measurement of angiotensin peptides. Endosomes and intermicrovillar clefts were obtained from the other kidney by fractionation and were assayed for angiotensin peptides and ACE activity. Studies were performed utilizing flow cytometry to determine whether renal endosomal AT1A receptor levels are regulated by dietary salt intake. Additionally, we determined whether an acute blockade of the renin-angiotensin system with an ACE inhibitor would affect angiotensin II levels in the intracellular organelles. The results of this study demonstrate that angiotensin I and angiotensin II levels and ACE activity are detectable in renal endosomes and intermicrovillar clefts and are influenced by acute alterations of the renin-angiotensin system, but not by chronic changes in salt intake.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Design

Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed in wire cages and maintained in a temperature- and light-controlled room. Animals had free access to tap water and were fed semisynthetic diets (Harlan Teklad, Madison, WI) containing low (0.003 meq/g) or high (1.34 meq/g) sodium content for 10 days. The chloride contents of the low- and high-sodium diets were 0.28 and 1.34 meq/g, respectively. Before collection of blood and kidney samples, each rat was placed in a metabolic cage and urine was collected for 24 h. All experimental procedures were approved by the Tulane University Animal Care and Use Committee.

Protocol 1: Renal Endosomal Angiotensin Levels in Rats Fed a High- or Low-Salt Diet

Rats received either a high- or low-sodium diet, and tissue samples were harvested 10-14 days following the start of the salt diet treatment. On the day samples were collected, the rats were decapitated between 9:00 AM and 11:00 AM. Trunk blood was collected for measurement of plasma renin activity (PRA) and angiotensin I and angiotensin II concentrations. Kidneys were immediately harvested, and one kidney was homogenized in methanol for measurement of tissue angiotensin I and angiotensin II levels. The time delay between decapitation and homogenization of the kidney was ~60 s. Renal endosomes for measurement of angiotensin levels were obtained from the other kidney by fractionation. A heavy endosome fraction was prepared as previously described (15, 16) and is characterized by homogeneity for entrapped markers that colocalize with apically derived enzymes and glycoproteins, suggesting that this is an apically derived membrane fraction. This heavy endosome fraction will be referred to as "intermicrovillar clefts," on the basis of heavy enrichment with clathrin, actin, gp280, gp330, and megalin (16). During the preparation of heavy endosomes, a thick layer of basolateral membranes forms about one-eighth of the way down the 16% Percoll gradient. Above the basolateral membranes is a faint white band of vesicles that is homogeneous for entrapped endosomal markers, and this vesicle fraction will be referred to as "endosomes." This light endosomal fraction lacks clathrin or clathrin-associated proteins, and the paucity of cathepsin B confirms that this fraction is prelysosomal and a true intracellular compartment (15).

Protocol 2: Flow Cytometry Analysis of Endosomal Purity and AT1A Receptor Antibody Binding

Kidneys were harvested from rats fed either high- or low-salt diet. To quantitate the endosomal expression of AT1A receptor in endosomes from rats fed low-salt or high-salt diet, endosomes were prepared with labeled fluorescein dextran (13, 14) and antibody binding curves were performed (18). Immediately prior to homogenization, 0.3 mg/ml 10S fluorescein-dextran was added to the homogenate at room temperature (14, 18). Ice-cold buffer was added to the membranes, and the membranes were homogenized on ice with six passes of a tight-fitting glass-Teflon motor-driven homogenizer. Light endosomes were prepared as above. Aliquots of membrane vesicles were labeled with an antibody to the cytosolic tail of the AT1A receptor. This is a rabbit polyclonal antibody we prepared to the hemocyanin-conjugated peptide sequence, CLSTKMSTLSYRPSDNM, and affinity purified.

Membrane vesicles were first preincubated in 50% normal goat serum for 2 h to reduce nonspecific binding of secondary antisera raised in goat. Following washing of membrane vesicles, serial log dilutions of antisera were added to each aliquot, and the membrane vesicles incubated at 4°C overnight. After further washing, 1:40 of goat anti-rabbit affinity-purified rat preabsorbed phycoerythrin-conjugated secondary antiserum was added, and aliquots were incubated for 4 h at room temperature. Prior to flow cytometry the membrane vesicles were washed and resuspended in 200 mM mannitol, 100 mM KCl, and 10 mM HEPES, pH 8.0 with Tris. This pH ensures optimal fluorescence of the highly pH-dependent fluorescein-dextran emission. Fluorescein-dextran and antibody staining tagged by phycoerythrin were analyzed and colocalized on a vesicle-by-vesicle basis by flow cytometry.

Flow cytometry analysis was performed on a Becton-Dickinson FACSVantage flow cytometer using a dedicated Power Mac computer (13, 14, 18). Excitation was at 488 nm using a Coherent 5-W argon-ion laser. For each particle, emission was measured using photomultipliers at 530 ± 30 and 585 ± 26 nm. Data were collected as 2,000 event list mode files and were analyzed using Cell Quest software.

Protocol 3: Effects of ACE Inhibition on Renal Endosomal Angiotensin II

Rats were fed chow containing low sodium content and on the day of tissue harvesting either enalaprilat (10 mg ip) or saline vehicle was administered. One hour following the injection of enalaprilat or vehicle, rats were decapitated, and blood and kidneys were immediately harvested. Trunk blood was collected for determination of PRA and angiotensin I and angiotensin II levels. Renal intermicrovillar clefts and endosomes were obtained from the kidneys for measurement of ACE activity and angiotensin II levels.

Analytical Procedures

Angiotensin peptide assays. Angiotensin I and angiotensin II levels were measured by radioimmunoassays as previously reported (10). For assessment of plasma angiotensin levels, trunk blood was collected into chilled tubes containing a mixed inhibitor solution (5 mM EDTA, 10 µM pepstatin, 20 µM enalaprilat, and 1.25 mM 1,10-phenanthroline). Blood samples were immediately centrifuged at 4°C for 10 min at 1,000 g to minimize in vitro generation of the peptides. Plasma was separated and immediately extracted by absorption to and elution with 100% methanol from a phenyl-bonded solid-phase extraction column (Bond-Elut, Varian) for measurement of angiotensin peptides. The eluates were collected and stored at -20°C. Before radioimmunoassay, the eluates were evaporated to dryness under vacuum and reconstituted in assay diluent.

For analysis of renal angiotensin peptide levels, the kidney was weighed, immersed in cold methanol (100%), and homogenized with a glass homogenizer immediately after harvesting. The kidney supernatants were dried overnight in a vacuum centrifuge. The dried residue was reconstituted in 4 ml of 50 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA, 0.25 mM thimerosal, and 0.25% heat-inactivated bovine serum albumin. These samples were purified and stored as described above for plasma. Aliquots of renal intermicrovillar clefts and endosomes (200 µl) were transferred to a tube containing 1 ml of methanol and stored at -20°C for a maximum of 2 days. A separate aliquot was taken for determination of endosomal protein concentration using the method of Lowry et al. (25). These samples were evaporated to dryness in a vacuum centrifuge, reconstituted in assay diluent, and assayed directly.

The reconstituted samples were incubated with rabbit anti-angiotensin I or anti-angiotensin II antisera and 125I-labeled angiotensin I or angiotensin II for 48 h at 4°C. Bound and free angiotensin peptides were separated by dextran-coated charcoal, and the supernatants were counted by a gamma counter for 3 min. Results are presented as femtomoles per gram kidney weight, femtomoles per milligram endosomal protein content, or femtomoles per milliliter plasma. The sensitivities of the angiotensin I and angiotensin II assays were 2.1 ± 0.5 and 1.4 ± 0.4 fmol, respectively, during 90% of maximal binding. For the angiotensin I and angiotensin II assays, the specific binding was 38.4 ± 0.9% and 37.1 ± 1.2%, respectively, with a nonspecific binding of 1.6 ± 0.3% and 1.0 ± 0.1%, respectively. As previously reported, cross-reactivity for antisera demonstrated that angiotensin-(1-10) and angiotensin-(2-10) were equipotent for the angiotensin I antisera, whereas angiotensin-(1-8) and its COOH-terminal fragments were more than 10,000-fold less effective in displacing 125I-angiotensin I. Angiotensin-(1-8) and angiotensin-(2-8) exhibit similar potencies for the angiotensin II antisera. Angiotensin-(3-8) was also detected by the angiotensin II antisera, but the displacement of 125I-angiotensin II was not parallel to that elicited by angiotensin II, and angiotensin-(1-10), angiotensin-(2-10), and short COOH-terminal fragments of angiotensin II were only detected at concentrations more than 100-fold higher that those for angiotensin II. The recoveries of angiotensin peptides through the extraction and purification procedures were determined by directly adding 125I-angiotensin I and 125I-angiotensin II into the plasma samples immediately before application to the solid-phase extraction columns and into the homogenization solutions immediately before collection of the tissues. The recoveries of angiotensin I and angiotensin II from plasma were 90-95% and from tissues were both 80%.

Other analysis. Trunk blood was collected in chilled tubes containing EDTA (5 mM) and was centrifuged at 4°C for 10 min at 1,000 g for determination of PRA. Plasma was separated, immediately frozen, and stored at -20°C until assayed. PRA was measured by the radioimmunoassay of angiotensin I generation with a standard commercial kit (Incstar) as described previously (10). ACE activity was determined by a fluorometric assay as previously described (7). Intermicrovillar cleft and endosomal samples were added to a substrate solution in 0.05 Tris · HCl, pH 8.0, containing 0.1 M NaCl. ACE activity in the samples was determined by fluorometric measurement of the enzymatic cleavage of 2-aminobenzoylglycine from 2-aminobenzoglyl-p-nitrophenylalanine. Intermicrovillar cleft and endosome ACE activity is expressed as fluorescence units per milligram of protein. Plasma and urinary sodium levels were quantified by flame photometry.

Statistical Analysis

All data are presented as means ± SE. An unpaired two-tailed t-test was applied to compare the angiotensin peptide levels, PRA, and ACE activity. Flow cytometry data was analyzed by two-way analysis of variance followed by Bonferroni test. The significance level was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Dietary Sodium on Excretory Function and PRA

Body weight was not significantly different between the two groups, averaging 358 ± 5 and 356 ± 6 g for rats placed on a low- and high-sodium diet, respectively. Urinary volume and sodium excretion averaged 17.8 ± 2.0 ml/day and 0.01 ± 0.002 µmol/min, respectively, in rats fed chow containing low dietary sodium. As expected, urinary volume (71.0 ± 8.8 ml/day) and sodium excretion (15.1 ± 1.7 µmol/min) were significantly higher in the group receiving high dietary sodium. Plasma sodium concentrations were not different between the groups, averaging 143 ± 7 and 147 ± 5 mM for rats placed on a low- and high-sodium diet, respectively. As evidence of upregulation of the renin-angiotensin system, animals placed on low dietary sodium had a fourfold elevation of PRA compared with those given high dietary sodium (16.8 ± 3.7 vs. 5.4 ± 1.3 ng ANG I · ml-1 · h-1).

Effect of Dietary Sodium on Circulating, Kidney, and Endosomal Angiotensin Peptide Levels

Plasma angiotensin I and angiotensin II levels were elevated in animals receiving chow containing low sodium content (Fig. 1). Likewise, rats placed on a low-sodium diet had elevated kidney angiotensin I and angiotensin II levels (Fig. 1). Plasma and kidney angiotensin II levels were elevated to a similar degree in the low dietary sodium rats. Renal endosomes and intermicrovillar clefts contained measurable amounts of angiotensin I and angiotensin II (Fig. 2). Of the two angiotensin peptides measured, endosomal angiotensin II comprised 87% and 78% of the total content in animals fed low and high dietary sodium, respectively. Angiotensin II comprised 90% of the total intermicrovillar cleft angiotensin peptide content in rats fed chow containing either low or high dietary sodium content. In contrast to the activation of the plasma and kidney angiotensin system in low-salt diet animals, intermicrovillar cleft and endosomal angiotensin peptide levels were not different between the high and low dietary sodium groups.


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Fig. 1.   Effect of dietary salt on plasma and kidney angiotensin I (A) and angiotensin II (B) levels. * Significant difference between high- (n = 11) and low-salt (n = 12) diet groups (P < 0.05).



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Fig. 2.   Effect of dietary salt on renal endosomal (A) and intermicrovillar cleft (B) angiotensin I and angiotensin II levels (n = 11 high salt diet group; and n = 12 low-salt diet group).

Effect of Dietary Sodium on Endosomal AT1A Receptors

Classic serial log dilution antibody curves were determined to examine the protein expression of AT1A receptors in renal endosomes. There was significantly more AT1A receptor antibody binding in the low-salt rats at the 1:1,000 antibody dilution (Fig. 3A). Comparison of frequency histograms of the binding in 2,000 individual endosomes demonstrates that this is a shift in the entire population, not an effect in a subpopulation of endosomes (Fig. 3B).


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Fig. 3.   Entrapped endosomal fluorescein dextran and AT1A receptor antibody binding in endosomes prepared from rats maintained on a low- or high-salt diet. A and B: quantification of AT1A receptor antibody binding to endosomes from rats maintained on low- or high-salt diet. A: binding of anti-AT1A antisera detected by the fluorescence of a phycoerythrin-tagged secondary antibody in endosomes prepared from rats maintained on low-salt diet compared with high-salt diet rats (n = 6). *Significant difference between high- and low-salt diet groups (P < 0.05). B: overlaid flow cytometry histograms of the individual values in 2,000 endosomes show that the low-salt diet effect is a shift in the entire population of endosomes, not a subpopulation effect (representative of n = 6). C: purity of endosomal fractions assayed by entrapped fluorescein dextran; overlaid flow cytometry histograms of the individual values in 2,000 endosomes show that the pH quenching of fluorescein dextran is a shift in almost the entire population of endosomes, not a subpopulation effect (representative of n = 6). D-F: colocalization of entrapped fluorescein and AT1A receptor antibody binding. Ability of flow cytometry to make simultaneous measurements of entrapped fluorescein dextran as an endosomal marker and antibody binding allows construction of 3-dimensional frequency histograms displaying entrapped fluorescein dextran fluorescence against antibody binding on horizontal axes and number of vesicles in each channel up out of the page. D: a control without fluorescein entrapped; minimum antibody binding defines the level of autofluorescence. E: colocalization of anti-AT1A binding with entrapped fluorescein dextran in rats maintained on a high-salt diet (representative of n = 6). F: colocalization of anti-AT1A binding with entrapped fluorescein dextran in rats maintained on a low-salt diet (representative of n = 6). Arrow points to unique population of endosomes that demonstrate colocalization of anti-AT1A binding with entrapped fluorescein dextran.

To confirm and demonstrate the purity of the endosomal fractions, we quenched FITC-fluorescence by acidification and used this distribution of fluorescence to set a gate to assay FITC-dextran uptake in the labeled fractions. This is a severe standard, as it uses only the pH-dependent component of the fluorescence signal. Under these conditions, more than 95% of the endosomes were positive for entrapped markers, and this was confirmed in replicates. An overlaid flow cytometry histogram of values demonstrate that pH quenching of fluorescein dextran shifts the entire population of endosomes and is not a subpopulation effect (Fig. 3C).

The ability of flow cytometry to make simultaneous measurements of entrapped fluorescein dextran as an endosomal marker and antibody binding allows construction of three-dimensional frequency histograms displaying entrapped fluorescein dextran fluorescence against antibody binding on horizontal axes and number of vesicles in each channel up out of the page (Fig. 3, D-F). A control without fluorescein entrapped, and minimum value of secondary antibody binding defines the level of background autofluorescence (Fig. 3D). Colocalization of AT1A antibody binding with entrapped fluorescein is represented by the hills in Figs. 3, E and F. High-salt diet rats showed a single endosomal population exhibiting colocalization of anti-AT1A receptor binding (Fig. 3E), whereas two distinct endosomal populations contained anti-AT1A receptor binding when animals were maintained on a low-sodium diet (Fig. 3F).

Effect of ACE Inhibition on Renal Endosomal Angiotensin II

Administration of enalaprilat for 1 h to conscious rats fed a low-sodium diet resulted in a twofold increase in PRA (Fig. 4A). ACE inhibition resulted in a fourfold increase in plasma angiotensin I levels from 240 ± 65 to 883 ± 74 fmol/ml, but plasma angiotensin II levels decreased by 64% (Fig. 4B). Renal endosomes and intermicrovillar clefts had substantial amounts of ACE activity in rats fed a diet containing low sodium content (Figs. 5 and 6). Renal intermicrovillar cleft and endosomal ACE activity was inhibited by greater than 80% by the enalaprilat treatment. Renal intermicrovillar cleft angiotensin II levels were decreased by 61% from 98 ± 16 to 38 ± 8 fmol/mg (Fig. 5). In addition, acute inhibition of ACE activity decreased renal endosomal angiotensin II levels by 52% from 68 ± 14 to 32 ± 8 fmol/mg (Fig. 6).


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Fig. 4.   Effect of enalaprilat treatment on plasma renin activity (PRA, A) and plasma angiotensin II (B) levels in rats fed a diet containing low sodium. * Significant difference between control (n = 12) and enalaprilat-treated (n = 12) groups (P < 0.05).



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Fig. 5.   Effect of enalaprilat treatment on intermicrovillar cleft angiotensin converting enzyme (ACE) activity (A) and angiotensin II (B) levels in rats fed a diet containing low sodium. * Significant difference between control (n = 12) and enalaprilat-treated (n = 12) groups (P < 0.05).



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Fig. 6.   Effect of enalaprilat treatment on renal endosomal ACE activity (A) and angiotensin II (B) levels in rats fed a diet containing low sodium. * Significant difference between control (n = 12) and enalaprilat-treated (n = 12) groups (P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Several studies (2, 5, 19, 39, 40) have demonstrated that angiotensin II is internalized into cells via an AT1 receptor-mediated process, but the amount of angiotensin II in intracellular compartments and regulation of intracellular angiotensin II levels have not been established. The present study was performed to determine whether renal endosomes contain detectable amounts of angiotensin peptides and whether these levels are altered by dietary salt intake or ACE inhibition. We demonstrated the presence of angiotensin I, angiotensin II, ACE activity, and AT1A receptors in renal intermicrovillar clefts and endosomes. Renal endosomes harvested from animals fed a diet containing low dietary sodium exhibited significantly greater AT1A receptor binding compared with rats fed a high-salt diet. In addition, ACE activity was inhibited by enalapril treatment and was associated with a decrease in intermicrovillar cleft and endosomal angiotensin II levels.

The importance of tissue renin-angiotensin systems and the paracrine actions of angiotensin II are being extensively investigated. Several studies have shown that renal content (10, 28) and kidney interstitial (35) angiotensin II concentrations greatly exceed the circulating levels, suggesting that intrarenal angiotensin II is greater than can be explained from the circulating levels and may be an important regulator of renal function. There is substantial evidence that the components of the renin-angiotensin system necessary for the formation of angiotensin II are present in the proximal tubules of the kidney (28, 36). The protein and mRNA for the angiotensin II substrate, angiotensinogen, have been localized to the proximal tubules (9, 11, 20, 22, 37). Additionally, ACE is present in abundance on the brush-border membranes of proximal tubule cells (9, 11, 22). The present study demonstrates the presence of ACE activity in intermicrovillar clefts and in endosomes. Even though previous studies have shown that the kidney and proximal tubule ratio of angiotensin I to angiotensin II is close to one (1, 10, 28), the ratios of angiotensin I to angiotensin II are less than 0.2 in the intermicrovillar clefts and in the endosomes. These results provide further evidence that angiotensin II is generated intraluminally from angiotensin I at the brush-border membrane and actively transported to the endosomal compartment.

Angiotensin content of the kidneys, intermicrovillar clefts, and endosomes was determined by harvesting the tissue, quickly processing it to prevent enzymatic action, and measuring the amount of angiotensin peptides present. Because angiotensin peptides are continuously being formed and degraded, this dynamic regulation precludes precise regional and compartmental comparisons of angiotensin peptide levels. In addition, the yield of endosomes compared with the original tissue mass is complicated by the classic tradeoff of yield versus purity, and in the present study we opted for purity. However, based on the intermicrovillar cleft and endosomal protein concentrations and amount of kidney sample used for their collection, we can estimate the percent of the total kidney angiotensin peptide levels present in these compartments. In the low dietary salt group, average weight of both kidneys was 3.1 ± 0.1 g, the total amount of angiotensin II averaged 1,197 fmol, and angiotensin I was 1,076 fmol, which are within the ranges previously reported (10, 43, 44). Kidneys contained 4.1 ± 0.2 mg intermicrovillar cleft protein, and this compartment can account for 288 fmol of the total angiotensin II and 31 fmol of the total angiotensin I. The endosomal compartment contained 1.7 ± 0.1 mg protein, 88 fmol of the total angiotensin II, and 13 fmol of the total angiotensin I. Thus, on the basis of the measurements from intermicrovillar clefts and endosomes, these organelles can account for an estimated 31% of the total angiotensin II and 4% of the total angiotensin I contained in the kidneys. These findings of high levels of angiotensin II versus angiotensin I in these compartments support the concept that internalization of angiotensin II is importantly involved in the physiological actions of this peptide.

Experiments were performed to determine the effects of dietary salt on renal intermicrovillar cleft and endosomal angiotensin levels. Although animals maintained on low dietary salt had elevated circulating and kidney angiotensin content, renal intermicrovillar cleft and endosomal levels of angiotensin I and angiotensin II were not different between the high and low dietary salt treatment groups. This was an unexpected finding since low-salt diet has been demonstrated to increase plasma, kidney, and renal interstitial (35) angiotensin II levels to maintain fluid and electrolyte balance. One possibility is that in the low dietary salt group there was an increase in intermicrovillar cleft and endosomal angiotensin II levels that occurred early in the treatment period. Likewise, an increase in sensitivity to angiotensin II, upregulation of the AT1 receptor, or increased degradation of angiotensin II could have resulted in unchanged renal intermicrovillar cleft and endosomal levels. A previous study demonstrated upregulation of AT1 mRNA levels in the kidney and adrenal gland in response to low dietary sodium (41). Thus an increase in AT1 receptors may be responsible for the actions of angiotensin II to increase sodium reabsorption, whereas an increase in intermicrovillar cleft and endosomal levels may not be necessary for long-term regulation of sodium reabsorption.

We also performed flow cytometric studies to quantitate the endosomal expression of AT1A receptors from rats fed high- or low-salt diet. The results of these studies demonstrated that AT1A receptors were present in renal endosomes and that dietary sodium intake affected these receptors. AT1A receptor antibody binding was increased in rats fed a low-salt diet compared with those maintained on high dietary sodium. Interestingly, a unique AT1A receptor-containing population of endosomal vesicles was apparent in the low-salt diet group that was not observed in animals fed high dietary sodium. Thus renal endosomal AT1 receptors are increased in animals maintained on a low-sodium diet. The identification of a distinct endosomal population with higher AT1A receptor antibody binding in the endosomal fractions isolated from animals on a low-salt diet demonstrates a minor subpopulation of endosomes with different protein content and properties. This suggests that the AT1A receptor traffics in a highly select population of endosomes during regulated transport, separate from bulk flow in other compartments.

Previous studies have demonstrated that internalization of angiotensin II occurs and is necessary for activation of certain signal transduction pathways. We previously demonstrated that chronic infusion of angiotensin II leads to increases in renal tissue levels of angiotensin II (43). This accumulation of angiotensin II is dependent on binding to the AT1 receptor, since accumulation was inhibited by losartan administration (44). In a recent study, van Kats et al. (40) demonstrated the accumulation of radiolabeled angiotensin II but not angiotensin I by a number of tissues, with the greatest accumulation in the adrenal glands and kidneys. The accumulation of angiotensin II was AT1 receptor mediated, since it was blocked by the nonpeptide AT1 receptor blocker, L-158,809 (40). This process appears to be selective for the angiotensin II-AT1 receptor complex, since internalization of fluorescein-labeled angiotensin II was observed with the AT1 receptor but not the AT2 receptor (19).

Evidence is accumulating that the internalization of the AT1 receptor and angiotensin II does not occur for the sole purpose of trafficking angiotensin II to the lysosomes for degradation and recycling of the receptor. Rather, this process may be vital for some of the proximal tubular actions of angiotensin II. Utilizing cultured rat proximal tubule epithelial cells, Schelling and Linas and colleagues demonstrated that endocytosis of apical AT1 receptors was coupled to increases in phospholipase C and decreases in adenylate cyclase (31, 38) and angiotensin II-induced sodium flux (32, 38). Becker et al. (3, 4) showed in a tubule cell line expressing rabbit AT1 receptors (LLC-PK-AT1R) that apical membrane AT1 receptor-mediated endocytosis of angiotensin II stimulates phospholipase A2, which is necessary for sodium flux and recycling of the AT1 receptor. These investigators also demonstrated that compared with the rapid internalization of the apical membrane AT1 receptor complex, the basolateral membrane internalization of the AT1 receptor was a slow process and was not linked to phospholipase A2 (3). In the present study, we demonstrated an increase in endosomal AT1A receptors and the appearance of a unique AT1A receptor-containing endosomal population in rats fed low dietary sodium, but the physiological consequences of these changes are unknown.

One possibility to explain the unaltered renal intermicrovillar cleft and endosomal angiotensin II levels was that the radioimmunoassay was not sufficiently sensitive or the variability was too large to detect subtle changes. Therefore, we performed an additional set of studies to determine whether ACE inhibition could alter renal intermicrovillar cleft and endosomal angiotensin II levels. We were able to detect ACE activity in the intermicrovillar clefts and endosomes of rats fed a low-salt diet. The ACE activity was 2.5-fold higher in the intermicrovillar clefts, in agreement with previous studies demonstrating an abundance of ACE along the brush-border membrane of the proximal tubule (21, 33, 42). Acute treatment with the ACE inhibitor, enalapril, increased PRA and plasma angiotensin I levels and decreased plasma angiotensin II levels in animals fed a low-salt diet. In addition, ACE activity of the intermicrovillar clefts and endosomes was greatly diminished. The decrease in ACE activity was associated with a substantial decrease in the angiotensin II levels of the intermicrovillar clefts and endosomes. These results demonstrate that intermicrovillar cleft and endosomal angiotensin II levels can be influenced and that the sensitivity of the radioimmunoassay is sufficient to detect changes of this magnitude. The physiological consequences of alterations in intracellular endosomal angiotensin II levels remain to be determined.

In summary, the present study demonstrated that intact angiotensin I and II could be detected by radioimmunoassay techniques in renal intermicrovillar clefts and endosomes. The intermicrovillar clefts and endosomes contained primarily angiotensin II. Additionally, animals maintained on low dietary sodium had an increase in AT1A receptors associated with endosomes. These findings support the concept that angiotensin I is converted to angiotensin II at the proximal tubule brush-border membrane and that angiotensin II is then internalized into the cell complexed to the AT1 receptor. We also demonstrated that acute treatment with enalapril decreased ACE activity and angiotensin II levels in renal intermicrovillar clefts and endosomes. Thus angiotensin II is either formed or trafficked through renal intracellular endosomal compartments, and these intracellular angiotensin II levels are influenced by ACE activity.


    ACKNOWLEDGEMENTS

We thank Ginny Primrose, Wendy Campbell, and Paul Deichmann for excellent technical assistance. This work was supported by National Institutes of Health Grants HL-26371 (to L. G. Navar) and DK-46117 (to T. G. Hammond), National Aeronautics and Space Administration Grant 9-811 Basic (to T. G. Hammond), a Veterans Affairs Research Associate Career Development Award (to T. G. Hammond), and an American Heart Association Grant-in-Aid (to J. D. Imig).


    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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. D. Imig, Dept. of Physiology, SL39, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: jdimig{at}mailhost.tcs.tulane.edu).

Received 8 October 1998; accepted in final form 29 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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