INTRODUCTION

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ANGIOTENSIN II is an octapeptide hormone that is the major effector molecule of the renin-angiotensin system. Biological effects of angiotensin II include vasoconstriction, aldosterone release, and cellular proliferation and growth. Angiotensin II acts as a circulating hormone as well as in a paracrine and/or autocrine fashion to modulate renal function. Angiotensin II has been shown to increase efferent arteriolar resistance and glomerular capillary hydraulic pressure and to decrease plasma flow rate, glomerular filtration rate, ultrafiltration coefficient (5, 7, 12, 22), and hydraulic conductivity (14) in the glomerulus.

Angiotensin II produces its diverse effects on glomerular function after its binding with specific cell surface receptors. Angiotensin II receptors have been demonstrated in murine and human glomerular preparations (1). The availability of non-peptide receptor ligands with variable affinity for angiotensin II receptor types has helped in distinguishing two main classes of angiotensin II receptors (7). The presence of angiotensin II receptor type 1 (AT₁) has been documented in mesangial cells (8) and proximal tubular epithelial cells (4). This class of angiotensin II receptors has been further divided into two subtypes, AT_1A and AT_1B. Both AT_1A and AT_1B subtypes have high affinity for biphenylimidazoles (e.g., losartan) and low affinity for tetrahydroimidazopyridines (e.g., PD-123,319). Both AT₁ subtypes are coupled to guanosine nucleotide binding proteins (G proteins), and their binding with angiotensin II can be inhibited by GTP analogs. These subtypes differ in the pattern of antagonist displacement by angiotensin II and inhibition by the GTP analog, guanosine 5'-O-(3-thiotriphosphate). Nearly all of the known physiological actions of angiotensin II are thought to be mediated by AT₁ receptors (5, 10, 25). Angiotensin II receptor type 2 (AT₂) has been documented in neuronal tissues and in fetal kidneys. AT₂ receptors selectively bind CGP-42112A, are highly sensitive to PD-123,319 and insensitive to losartan, and are not coupled to G proteins. The physiological action of AT₂ receptors has not been clearly established but may be involved in regulation of potassium channels. Both AT₁ and AT₂ receptors have been found to have seven transmembrane helices but have little homology in their amino acid sequences (10).

The filtration barrier of the glomerulus is composed of endothelial cells, the basement membrane, and glomerular epithelial cells. Angiotensin II may alter glomerular function directly and the filtration barrier indirectly through its effects on mesangial cells (1). It has been suggested that the structural features of glomerular epithelial cells make them an ideal candidate to respond to mechanical, endocrine, and paracrine signals through alterations in the slit-pore junction, and thus these cells may play an important part in regulation of filtration (6, 17). We have reported that angiotensin II caused an increase in intracellular cAMP and a rearrangement of actin cytoskeleton in cultured glomerular epithelial cells (23). These observations suggest a specific binding of angiotensin II with glomerular epithelial cells and thus a direct interaction of angiotensin II with one of the constituents of the glomerular filtration barrier.

In this study, we demonstrate the presence of angiotensin II receptors in cultured glomerular epithelial cells by immunoblotting, using antibodies specific for AT₁ and AT₂ receptor proteins. Because non-peptide receptor ligands show high selectivity for angiotensin II receptors, they can be employed to distinguish between receptor types. We used losartan and PD-123,319 to block AT₁ and AT₂ receptors, respectively. We measured angiotensin II-induced changes in the levels of cAMP as a marker for activation of cyclic nucleotide pathway of signal transduction.

	MATERIALS AND METHODS

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Establishment of glomerular epithelial cell cultures. A rat visceral glomerular epithelial cell line was obtained from the laboratory of Dr. William Couser (University of Washington, Seattle, WA; Ref. 9). Cells were grown for eight passages on collagen matrix in K-1/3T3-conditioned media and thereafter grown in K-1 medium supplemented with 2% Nu-Serum (Collaborative Research, Bedford, MA), insulin, transferrin, and selenium. At the 20th passage, cells were switched to plastic tissue culture dishes with a thin layer of bovine type I collagen and maintained in K-1/3T3-conditioned medium supplemented with 2% Nu-Serum, insulin, transferrin, and selenium. At confluency, the cells had a cobblestone appearance under light microscopy. The epithelial origin of the cells was verified by positive staining for the Fx1A antigen and negative for the Thy-1.1 antigen and factor VIII. These cells exhibit positive staining for podocalyxin, a podocyte marker, indicating visceral origin of these cells.

Preparation of membrane fraction from cultured glomerular epithelial cells. Confluent cultures of glomerular epithelial cells were washed three times with 10 mM potassium phosphate buffer (pH 7.7) containing 250 mM sucrose, 1 mM EDTA (disodium salt), 10 mM magnesium chloride, and 0.1 mM of phenylmethylsulfonyl fluoride (PMSF). Cells were scraped using disposable cell scrapers and homogenized in two volumes of 10 mM potassium phosphate buffer (pH 7.7) by passing at least five times through a 25-gauge needle. The homogenate was centrifuged at 5,000 g for 15 min at 4°C, and the supernatant was further ultracentrifuged at 100,000 g for 45 min. The final pellet was resuspended in 100 µl of 100 mM potassium phosphate (pH 7.7) buffer containing 30% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.1 mM of PMSF. The membrane preparation was stored at 80°C. Protein concentration was measured by Bradford's method (3), using the Coomassie brilliant blue reagent (Bio-Rad, Hercules, CA).

Receptor protein antibodies. A rabbit anti-rat AT₁ polyclonal antibody was obtained from Chemicon International (Temecula, CA). This antibody was equally immunoreactive against AT_1A and AT_1B receptors, according to the supplier. Anti-AT₂ receptor antibodies were developed in the laboratory. Briefly, amino acid sequences of high antigenicity were selected from the published amino acid sequence of AT₂ receptor protein (19), using the GCG computer software (Genetics Computer Group, Madison, WI) and were used to synthesize peptides with help from the Protein and Nucleic Acid Core Facility (Medical College of Wisconsin, Milwaukee, WI). Synthesized peptides were purified by reverse-phase high-pressure liquid chromatography and conjugated to ovalbumin by 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) method (Imject Immunogen EDC Conjugation Kit; Pierce Chemical, Rockford, IL). The conjugate was purified by gel filtration and mixed (1:1) with Freund's complete adjuvant and injected intradermally into New Zealand White rabbits. Animals were boosted 21 days later with a similar mixture but using Freund's incomplete adjuvant. Preimmunization serum was obtained prior to the initial immunization, and subsequent blood collection was carried out 3 wk after the booster injection. Blood was withdrawn from the ear vein, and the serum was separated and stored frozen. Antigen recognition of the antiserum was carried out by testing a series of diluted serum samples (1:1,000, 1:2,000, 1:10,000) against several concentrations of conjugated peptide (0.6, 0.3, 0.15, 0.075 mg/blot) on a nitrocellulose membrane. Specificity of AT₂ antiserum was tested by a competitive binding assay, using the peptide conjugate and the native AT₂ receptor protein on a Western blot, followed by the quantitation of the unbound native AT₂ receptor protein. The specificity of AT₁ and AT₂ receptor antisera was also demonstrated by their binding with their respective antigens, using total adrenal protein as a source of the AT₁ and AT₂ receptor protein.

Documentation of receptor proteins using immunoblot analysis. SDS-PAGE of the membranes was performed using the Mini-Protean II apparatus (Bio-Rad). Separation of proteins was accomplished using 10% resolving gels and 5% stacking gels. Gels were placed in a chamber and submerged in electrophoresis running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS). Twenty micrograms of sample protein in 20 µl running buffer and 5 µl of 5× sample buffer were mixed and heated at 100°C for 10 min. Solutions were vortexed and spun down for 1 s. One marker lane was added with a commercially produced mixture of molecular weight standards (Bio-Rad). Gels were run at a constant voltage of 200 volts for 45 min and transferred to a nitrocellulose membrane using a Mini Transblot (Bio-Rad) at 100 volts for 60 min. The membrane was blocked overnight in 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBS-T), followed by incubation with immune serum and antibody in 2% nonfat dry milk in TBS-T at a concentration of 1:1,000 (0.15 ml/cm²). The membrane was incubated at room temperature on a rocking platform for 90 min and washed three times with TBS-T for 5 min and sealed in a plastic bag with 1:1,000 secondary anti-rabbit antibody conjugated with horseradish peroxidase (Bio-Rad) and incubated on a rocking platform at room temperature for 60 min. The membrane was washed three times for 5 min each in TBS-T, followed by a wash with ECL enhanced chemiluminescence (Amersham, Arlington Heights, IL) for 1 min to activate a chemiluminescent reaction, and exposed to X-ray.

Measurement of cAMP in glomerular epithelial cells and inhibition of cAMP accumulation by non-peptide receptor ligands. Glomerular epithelial cells were grown in RPMI 1640 (GIBCO-BRL; Life Technologies, Grand Island, NY) with 10% fetal calf serum. Confluent cells were washed twice with control medium (RPMI 1640 without fetal calf serum). Washed cells were incubated in the control medium or in control medium containing angiotensin II (10¹¹, 10⁹, 10⁷, or 10⁷ M) for 2 h at 37°C. Cells were washed with PBS (pH 7.4) and treated with 1 ml of 80% methanol. The extract in methanol was used for the measurement of cAMP levels, using RIANEN cAMP ¹²⁵I radioimmunoassay kit (DuPont, Boston, MA). Losartan and PD-123,319 were obtained as gifts from E.I. DuPont De Nemours Pharmaceuticals (Wilmington, DE) and Parke-Davis (Ann Arbor, MI), respectively.

For studies on the effects of receptor-specific non-peptide ligands, washed cells were incubated in the control medium or in control medium containing angiotensin II (10⁷ M) alone, angiotensin II + losartan (1 µM), angiotensin II + PD-123,319 (1 µM), or with angiotensin II + losartan + PD-123,319 for 2 h at 37°C. Extraction with methanol and radioimmunoassay of cAMP were carried out as described. Protein concentration was measured by Bradford's method (3), using Coomassie brilliant blue reagent (Bio-Rad).

Statistical analysis. All values are expressed as means ± SE. cAMP concentrations were determined in triplicate for every experiment, and values were averaged, with N representing the total number of experiments. Values among various groups were compared using ANOVA. Significance among groups was defined as P < 0.05.

	RESULTS

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Validation of AT₂ receptor antibody. As shown in Fig. 1, the serum from rabbits immunized against a high-antigenicity AT₂ receptor peptide reacted at 1:10,000 dilution to the antigen (300 µg/blot) on a nitrocellulose membrane (Fig. 1A). The specificity of AT₂ antiserum is demonstrated by the ability of peptide conjugate (500 µg) to compete with the native AT₂ receptor on a Western blot (Fig. 1B). Quantitation of AT₂ receptor protein obtained from the Western blot is shown in Fig. 1C. The specificity of AT₁ and AT₂ receptor antisera is demonstrated by a Western blot using total adrenal protein as a source of AT₁ and AT₂ receptor proteins. As shown in Fig. 1D, each antiserum reacted at 1:5,000 dilution with its own antigen, whereas preimmune serum had no reactivity to adrenal proteins.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   AT2 receptor antibody validation. A: dot blot showing antigenicity against a range of peptide conjugate concentrations (600, 300, 150, 75 µg/dot, top to bottom). B: ability of peptide conjugate to compete for native AT2 receptor protein on Western blot. C: linear behavior of quantitation of AT2 receptor protein obtained from Western blot. D: multi-antisera screen of uniform distribution of adrenal protein on Western blot, demonstrating the specificity of the AT1 and AT2 receptor antisera: 1, AT1 receptor antiserum; 2, AT2 receptor antiserum; P, preimmune serum. Antiserum dilutions are indicated.

Documentation of AT₁ and AT₂ receptor proteins in glomerular epithelial cells membranes by immunoblot analysis. Glomerular epithelial cell membrane proteins were separated by SDS-PAGE, transferred to a membrane, and immunoblotted using antibodies (Ab) to AT₁ and AT₂ receptor proteins. As shown in Fig. 2, distinct binding was observed with each antibody separately (AT₁ Ab and AT₂ Ab) or together (AT₁ Ab + AT₂ Ab). The protein-antibody complex of the AT₁ receptor was visible just under the 44-kDa molecular mass marker, and that of the AT₂ receptor was visible between the 44- and 87-kDa markers.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Multiscreen immunoblot of glomerular epithelial cells (GEC). Membrane vesicles were obtained from GEC monolayer cultures as described in the text. Proteins were separated by SDS-PAGE and transferred to a nylon membrane immunoblotted with AT1 and AT2 antibodies (Ab) separately (left lane and second lane from left, respectively) and together (second lane from right). AT1 corresponded to a size of ~40 kDa, and AT2 was found to be between 44 and 87 kDa marker proteins. Goat anti-rabbit IgG (2' Ab) was used as nonspecific antibody in right lane.

Measurement of cAMP in glomerular epithelial cells. cAMP levels following a 2 h incubation of glomerular epithelial cells with angiotensin II at various concentrations (10¹¹-10⁵ M) were measured by radioimmunoassay. We found that 10⁷ M angiotensin II induced the maximum accumulation of cAMP (Fig. 3). This concentration of angiotensin II was selected for further experiments in the present studies. As shown in Table 1, angiotensin II caused a significant increase in intracellular levels of cAMP in glomerular epithelial cells compared with control. This effect of angiotensin II on cAMP was only partially inhibited when either AT₁ or AT₂ receptor ligand was used alone. The simultaneous use of losartan and PD-123,319 prevented the angiotensin II-induced increase in cAMP levels.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of various concentrations of angiotensin II (ANG II) on cAMP accumulation in GEC monolayers was determined by incubating 1011-105 M ANG II in medium. Concentration of cAMP was determined by radioimmunoassay as described in text. cAMP was found to be highest in cells treated with 107 M ANG II. N, no. of experiments.

	DISCUSSION

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We demonstrated the presence of angiotensin II receptor proteins in glomerular epithelial cells. Immunoblotting showed the presence of two distinct proteins that represent AT₁ and AT₂ receptors. These receptors are known to differ in their apparent molecular mass because of different levels of glycosylation (10). The presence of AT₁ receptor type has been extensively documented in various cells, including glomerular mesangial cells (8) and proximal tubular epithelial cells (4). AT₁ receptors in mesangial cells are believed to mediate angiotensin II-induced modulation of glomerular filtration rate and plasma flow (1, 7), and this receptor type is generally considered to be the mediator of all known biological effects of angiotensin II in adult tissues (5, 10, 25). Angiotensin II-induced inhibition of adenylate cyclase in most cell types is also believed to be mediated by AT₁ receptors (10). Cell-specific response to angiotensin II is perhaps due to the relative abundance of one of the two AT₁ subtypes. For instance, mesangial cells, which have a preponderance of the AT_1A subtype, respond to angiotensin II by activation of phospholipase C and subsequent changes in intracellular Ca²⁺. On the other hand, proximal tubular cells have mainly the AT_1B subtype, and respond to angiotensin II by activation of phospholipase A₂ (5, 8, 10).

In previous studies, the AT₂ receptor was not detectable in kidney tissue by autoradiography, nor was mRNA for this receptor protein detectable by Northern blot analysis. However, a very low message for the receptor protein could be found using reverse transcriptase-polymerase chain reaction (11). It is estimated that AT₂ receptors constitute only 5-10% of the total angiotensin II receptors in the kidney (27). The role of the AT₂ receptor type is not clear, but recent studies using specific antagonists have shown that these receptors blunt the pressure natriuresis in the kidney (15), mediate the increase in cGMP in renal interstitial fluid, and attenuate AT₁-mediated production of PGE₂ in sodium-depleted conscious rats (24).

Our results show that angiotensin II caused an accumulation of cAMP in glomerular epithelial cell monolayers. This increase was most marked at 10⁷ M, although it did not differ significantly from lower concentrations (Fig. 3), including the concentration of angiotensin II shown by Braam et al. (2) to be present in proximal tubule fluid. We have shown that angiotensin II at 10⁷ M caused increased intracellular levels of cAMP and cytoskeletal rearrangement in cultured glomerular epithelial cells (23). Other investigators have used this concentration to demonstrate changes in cAMP levels in isolated rat glomeruli (7). Angiotensin II generally causes inhibition of adenylate cyclase, which, in turn, attenuates protein kinase A-mediated phosphorylation of cellular proteins (10). In the kidney, it is reported to inhibit adenylate cyclase in proximal tubular cells (5). A few reports do suggest a stimulation of adenylate cyclase by angiotensin II (12, 13). We have demonstrated that angiotensin II causes increased levels of cAMP in cultured glomerular epithelial cells, indicating a possible stimulation of adenylate cyclase. This intracellular signaling mechanism is distinct from that found in mesangial cells or proximal tubule cells (5, 8, 10).

We demonstrated the presence of both AT₁ and AT₂ receptor types in glomerular epithelial cells and their synergistic effect on cAMP accumulation by using non-peptide angiotensin II receptor ligands. We found that the AT₁ ligand (losartan) or AT₂ ligand (PD-123,319) alone caused only partial inhibition of the angiotensin II-induced accumulation of cAMP. When both receptor ligands were included in the medium, complete inhibition of the increase in cAMP resulted. This finding suggests that angiotensin II binding to either receptor increases cAMP in glomerular epithelial cells (Table 1). Other cells are known to employ more than one receptor type to mediate the effects of angiotensin II. For example, the increase in nuclear and cytoplasmic levels of calcium in aortic smooth muscle cells caused by angiotensin II has been shown to be mediated by two receptor types (18). Similarly, angiotensin II increased cGMP and nitric oxide production in N1E-115 neuroblastoma cells by a mechanism that involved both AT₁ and AT₂ receptors (26).

Angiotensin II modulates glomerular blood flow, glomerular filtration rate, and glomerular capillary hydraulic conductivity. We have shown that hydraulic conductivity is diminished during volume depletion (21), after systemic or intrarenal infusion of angiotensin II (23), and after incubation of isolated rat glomeruli with angiotensin II or a cAMP analog. The decrease in hydraulic conductivity was prevented by the nonspecific angiotensin II receptor blocker, saralasin (14). In another set of experiments, neither losartan nor PD-123,319 completely blocked the decrease in hydraulic conductivity caused by angiotensin II. Concurrent use of losartan and PD-123,319 prevented the effect of angiotensin II (16). These observations confirmed that angiotensin II alters hydraulic conductivity and suggested the presence of both AT₁ and AT₂ receptors in glomeruli. Morphological and computational studies emphasize the importance of glomerular epithelial cells in the regulation of glomerular function (6, 17). The effect of angiotensin II on hydraulic conductivity and the apparent lack of AT₂ receptors on mesangial cells indicate a direct interaction of angiotensin II with glomerular epithelial cells.

Our results show the presence of both AT₁ and AT₂ receptors in glomerular epithelial cells. These receptors appear to act synergistically to increase intracellular cAMP and may have distinct biochemical and pharmacological properties. We propose that angiotensin II and its interaction with AT₁ and AT₂ receptors on glomerular epithelial cells play an important part in the regulation of the glomerular function.