Rat proximal tubule cell line transformed with origin-defective SV40 DNA: autocrine ANG II feedback

Julie R. Ingelfinger1, Flavia Jung1, Daniel Diamant1, Liam Haveran1, Edwin Lee1, Andrew Brem2, and Shiow-Shih Tang1

1 Pediatric Nephrology Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; and 2 Rhode Island Hospital, Brown University, Providence, Rhode Island 02903


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The renal proximal tubule (PT) is a major site for a complete tissue renin-angiotensin system (RAS) and produces endogenous angiotensin II (ANG II). The present studies demonstrate autocrine RAS feedback in a line of origin-defective SV40 plasmid transformed immortalized rat PT cells (IRPTC) designated as line 93-p-2-1, which are highly differentiated and express all RAS components. Receptor competition assays and Southern blot following RT-PCR demonstrated that these IRPTC express AT1 and AT2 angiotensin receptor subtypes. Autocrine RAS feedback was examined following exposure to ANG II (10-8 M), and it was noted that angiotensinogen mRNA increases significantly by 1 h and remains elevated through 24 h. The AT1 blocker losartan prevents this increase. Moreover, ANG II upregulates expression of ANG II receptor mRNA (both AT1 and AT2). Thus the present studies demonstrate positive ANG II feedback with angiotensinogen and ANG II receptors in PTC, suggesting that the main site of such intrarenal feedback in vivo is within PT. ANG II secreted by line 93-p-2-1 is increased by isoproterenol, suggesting beta -adrenergic regulation in IRPTC.

angiotensin II receptors; renin angiotensin system


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE PRESENCE of an intrarenal renin-angiotensin system (RAS) has become well accepted. Previous studies using the techniques of Northern blotting, in situ hybridization, and RT-PCR confirm that within the kidney, the proximal tubule (PT) contains a complete RAS and appears to be the primary site for local angiotensin synthesis (27, 28, 35). That angiotensin II (ANG II) is produced locally is supported not only by the high levels of this peptide but by the presence of a complete RAS in the PT. Renal angiotensinogen mRNA has been localized primarily to the PT (27, 28). Renin mRNA has been detected in low amounts in PT (34). Furthermore, renin protein is both filtered and present in peritubular lymphatics (60) and thus has access to the PT. Both angiotensin converting enzyme (ACE) mRNA and its protein are found in abundance in PT (29, 56). PT cells (PTC) express ANG II receptors (17), with both AT1 and AT2 subtypes being present (18). Thus, with endogenous production of physiological amounts of ANG II directly demonstrated in the PT using micropuncture technique (51), a fully active system is clearly present.

We previously have shown that ANG II exerts positive feedback on renal angiotensinogen mRNA expression in vivo (49). As recent data demonstrate PT ANG II levels to be 100-1,000 times higher than ANG II levels in the bloodstream (51), it seems likely that the positive feedback of ANG II on its substrate occurs in the PT, which is the primary site for local intrarenal angiotensinogen synthesis.

Locally produced ANG II appears to be intimately involved in modulating salt and water reabsorption and glucose and amino acid reabsorption and may play a role in cell growth and repair (5, 25, 61). Nonetheless, the modes of function of this PT RAS are still incompletely understood. Such questions would be well addressed by studies with cells in culture. Recently, we developed a cell line of immortalized rat PTC by transfecting PTC with SV40 temperature-sensitive mutant (tsA209) virus (54). These cells are transformed at permissive temperature (34°C) but return to an untransformed state at nonpermissive temperature (41°C). However, neither the permissive nor the nonpermissive temperature is convenient for the routine handling of cultured cells in general.

To demonstrate positive feedback of ANG II on its substrate in the PT, we developed a rapidly growing, stable PTC line, designated as line 93-p-2-1, from rat (the species which has been the focus of many physiological studies and in which the PT RAS is best demonstrated) by using origin-defective SV40 plasmid (23). This cell line, transformed at all times, has features distinct from the temperature-sensitive PTC line that we have previously reported (54) which make it useful in situations in which a large amount of material is required for experiments. The present work demonstrates positive ANG II-angiotensinogen feedback in immortalized rat PTC (IRPTC) line 93-p-2-1. Thus the present data confirm our previous finding that ANG II upregulates angiotensinogen mRNA expression in renal cortex in vivo following ANG II infusion via osmotic minipump in rats (48, 49). Additionally, ANG II feedback on its PT receptor subtypes is also evident in IRPTC, suggesting autoregulation of multiple components of the PT RAS.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Culture of primary rat proximal tubular cells. For each isolation, kidneys from four to five male Wistar rats (150 g) were removed and placed in 20 ml ice cold Hanks' buffered saline solution that had previously been gassed with 95% O2-5% CO2 for 30 min at room temperature. After decapsulation, kidneys were bisected, and the superficial cortex was dissected, removed, and minced. PTC were isolated via Percoll gradient as previously described (54). Cells with a density ~1.063 g/ml were removed, washed, and cultured in collagen-coated (rat tail collagen 0.1%) T-75 tissue culture flasks. DMEM with low glucose (5 mM) supplemented with 3.8 mg/ml NaHCO3, 25 mM HEPES buffer, pH 7.5, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM sodium pyruvate, 0.01 mM nonessential amino acids, and 5% FCS was used as the standard growth medium. Cells were cultured at 37°C and 5% CO2 and were fed every 2-3 days.

Transformation with origin-defective SV40 plasmid. Primary rat PTC at 30% confluence were used for transformation. The calcium phosphate transfection method (47) was used to transfect the cells with origin-defective SV40 DNA mutant 6-1 in pMK 16 (20 µg per T-75 flask) for 20 h. After transfection, cells grew in fresh medium that was changed weekly. As anticipated, most cells died after reaching confluence (1-2 wk) and formed "ghost" cells. After 5-8 wk, distinct foci of outgrowing cells became apparent and were picked up enzymatically with trypsin-EDTA using a cloning cylinder for subculture in T-75 tissue culture flasks without collagen coating. These subcultures were then expanded into cell lines. One cell line, designed 93-p-2-1, used for the present studies, has been characterized and appears stable up to 50 passages.

Immunohistochemistry. Cells were grown on coverslips, and each protein of interest was then detected by indirect immunofluorescence as described below using passages 16-20. Multiple antibodies were utilized. We used polyclonal rabbit anti-rat angiotensinogen antisera provided by Dr. Jacob Bouhnik (INSERM, Paris, France) at a 1:1,000 dilution (6). An anti-rabbit ACE antibody produced in goat, which reacts with rat cells and tissues (14) at a 1:1,000 dilution, was provided by Dr. Richard Soffer (Cornell University). A polyclonal anti-rat renin antibody from Dr. Tadashi Inagami (Vanderbilt University), which has a titer of 1:42,000 and stains at a dilution of 1:10,000, was used to identify renin (39). Polyclonal and monoclonal anti-rat antibodies to gp330 were provided by Dr. John Niles and by Drs. David Bachinsky and Mansum Sy, respectively (Massachusetts General Hospital, Boston, MA), and used at a dilution of 1:200 (concentration 10 µg/ml) (2). Na-K-ATPase antibody was a kind gift from Dr. Dennis Brown. Dr. William Sly (St. Louis University, St. Louis, MO) provided antibody to carbonic anhydrase (used at 1:200) (8), and Dr. S. H. Lin (University of Texas, Houston, TX) provided antibody to ecto-ATPase (used at 1:100) (45). Anti-rat antibody to the aquaporin CHIP-28 (AQP-1) was obtained from Drs. Ivan Sabolic' and Dennis Brown (Massachusetts General Hospital and used at dilution 1:400) (46). Dr. Edward Harlow, Massachusetts General Hospital, provided antibody to large T antigen. Anti-factor VIII and alkaline phosphatase antibodies were obtained commercially from DAKO (Carpinteria, CA). GLUT-2 and SGLT-2 antibodies were obtained from East Acres Biological Laboratories.

Immunofluorescence studies were carried out as follows (46). Cells were on 12-mm coverslips, grown in 35-mm diameter petri dishes in DMEM to 95% confluence and then fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Prior to staining for renin, cells were switched to defined serum-free medium for 24 h to be sure that adsorbed renin from the serum was not the source of positive immunostaining. Nonspecific staining was blocked by washing cells with PBS containing 0.5% BSA. Cells on coverslips were incubated with the appropriate primary antibody (or control, preimmune serum) for 2 h followed by washing and incubating for 1 h with FITC-labeled second antibody. Coverslips were mounted on slides in 50% glycerol in 0.2 M Tris · HCl, pH 8.0, containing 2% n-propylgallate to retard quenching of the fluorescence signal. The slides were examined and photographed with a Nikon FXA photomicroscope equipped for epifluorescence (Nikon, Melville, NY) or a confocal microscope (Bio-Rad 600, Cambridge, MA) and then photographed using Kodak Tmax 400 film "push-processed" to 1,600 ASA.

Short-circuit current. IRPTC were seeded heavily on Transwell clear membranes measuring 24 mm in diameter with an 0.4-µm pore size (Costar, Cambridge, MA) and grown to confluence in an intact monolayer. The transepithelial electrolyte transport was estimated by short-circuit current (Isc) under resting conditions using Ussing chambers (MRA International, Naples, FL) modified to accommodate Transwell 24 support membranes (Costar) (4, 7, 43). Voltage clamps (model AVC 300; Buck, Franklin, MA) were used for Isc studies. The background was subtracted for calculating the resistance. Studies were conducted with the cells grown to confluence. Both sides of the membranes were bathed in mammalian Ringer solution (122 mM NaCl, 25 mM NaHCO2, 5 mM KCl, 1.3 mM MgSO4, 2 mM CaCl2, 1 mM KH2PO4, and 25 mM glucose) and bubbled with 95% O2-5% CO2 (pH 7.4) at 37°C.

Tissue mRNA expression studies. Homogenization of cells (or whole kidney control tissue) was carried out in 4 M guanidine thiocyanate, 0.5% sodium-n-lauryl sarcosine, 25 mM sodium citrate, 0.1 M beta -mercaptoethanol, and 2 M CsCl; and RNA was pelleted by ultracentrifugation (12, 47). The RNA was resuspended in 0.2 M sodium acetate, pH 5.5, rocked at 4°C for 30 min, and precipitated in two volumes of ethanol. The precipitated RNA was dissolved in water, and the amount was quantitated by absorbance at 260 nm in duplicate. Relative levels of mRNA were compared using identical amounts of total RNA applied per sample. Standard Northern blot analysis, using the formaldehyde-agarose method (21), was carried out. In addition, to examine the effect of ANG II on angiotensinogen message more quantitatively, slot blots were carried out. Samples were denatured in formaldehyde and then serially diluted in 15× SSC. Three amounts of each sample (2, 4, and 8 µg) were blotted onto nylon filters using a slot blot manifold. Northern and slot blots were hybridized to probes of interest using the Rapid-Hyb buffer system (Amersham Life Sciences). After prehybridization for 1 h, the blots were hybridized for 2 h in a buffer to which [alpha -32P]cDNA probes were added. After hybridization, blots were washed in 2× SSC with 0.1% SDS at room temperature for 10 min, then three times in 0.2× SSC with 0.1% SDS for 15 min each at 65°C. Blots were exposed for 2 days to X-ray film (Kodak XAR; Eastman Kodak, Rochester, NY).

The relative amounts of mRNA were determined by scanning the autoradiograms of both Northern and slot blots with a computer scanner and measuring the mean density of signals (Desk Scan II, version 1.5.1, Hewlett Packard; followed by Image, version 1.37, by Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). Blots were normalized with results from hybridizing the same blots with beta -actin cDNA. Units are expressed as relative densitometric signals ± SE.

cDNA probes. We used full-length rat cDNA probes for rat angiotensinogen and rat renin, both provided by Dr. Kevin Lynch (9, 32). We used cDNA for rat ACE (provided by Dr. J. Krieger) (29). AT1 cDNA was provided by Dr. T. J. Murphy, Emory University (37). AT2 cDNA was provided by Dr. Victor Dzau (36). Probes were labeled by random oligonucleotide priming using [32P]dCTP to an activity of 1 × 108 dpm/µg DNA (21).

RT-PCR studies. To detect renin mRNA, we utilized the RT-PCR technique (22, 35). Total RNA (1 ng) was used for cDNA synthesis in the presence of 20 U MMLV reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD), 5 µM oligo(dT)15, 1 mM dNTPs, and 3 mM MgCl2. In some samples, reverse transcriptase was omitted to control for contamination or amplification from renin genomic DNA. Samples were denatured at 65°C for 5 min. Then enzyme was added, followed by extension at 42°C for 1 h and termination at 93°C for 5 min. Samples were then placed at 5°C for 5 min. PCR was subsequently performed with the following segment-specific primers
5′-TGCCACCTTGTTGTGTGAGG-3′ (sense primer)
5′-ACCCGATGCGATTGTTATGCCG-3′ (reverse primer)
In each reverse transcription reaction set, a sample was run without reverse transcription as a control. The PCR reactions had 4 µM primers, 4 mM MgCl2, 200 µM dNTPs, reaction buffer, and cDNA in a 98-µl reaction volume. Samples were placed in a thermocycler prewarmed to 94°C (Ericomp, San Diego, CA) to minimize nonspecific amplification. After 2 min, 2.5 U of Taq polymerase (Promega) was added to the reaction tubes. The PCR cycles (n = 35) were as follows: 94°C for 30 s (denaturation), 58°C for 1 min (annealing), and 72°C for 1.25 min (extension). These cycles were followed by an additional final extension at 72°C for 5 min. Subsequently, the PCR reaction products were subjected to size fractionation on a 1.5% agarose gel, transferred to nylon filters, and probed with alpha -32P-labeled renin cDNA. The amplified fragment was 374 bp. Blots were prehybridized with Rapid-Hyb buffer (Amersham) for 1.5 h at room temperature. Blots were then hybridized with the probe for 1 h at 65°C. Washing conditions included a prewash at room temperature (2× SSC, 0.1% SDS) and two washes at 65°C for 15 min in 0.1× SSC with 0.1% SDS. Washed filters were exposed to Kodak XAR film over an intensifier screen.

Plasma membrane preparation of cultured cells. Membrane preparation was as previously described (54, 55). Briefly, cells (from 20 T-75 flasks) were rinsed twice with 5 ml of 1× PBS, then harvested using a cell scraper in 5 ml of buffer A [1 mM NaHCO3, 0.25 M sucrose, 2 mM MgCl2, 1 mM CaCl2, 5 mM KCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] (3). Cell suspensions were pooled and centrifuged at 800 g at 4°C for 10 min, then resuspended in 12 ml buffer B (1 mM NaHCO3, 2 mM MgCl2, 1 mM CaCl2, 5 mM KCl, and 1 mM PMSF) and incubated at 4°C for 30 min. Swollen cells in the low ionic strength buffer were homogenized in a glass homogenizer 10 times on ice, followed by addition of 2 M sucrose added to a final concentration of 0.25 M. The homogenate was spun at 800 g at 4°C for 10 min. The membrane preparation was prepared by resuspending the pellet in 0.4 ml of buffer A after ultracentrifugation of the supernatant at 23,000 rpm (49,000 g) at 4°C for 30 min. The membrane preparation was frozen in liquid N2 and then stored at -70°C.

ANG II receptor assays. Receptor binding studies were performed by saturation assays in which plasma membranes (30 µg) were incubated in binding buffer [50 mM Tris, 10 mM MgCl2, 100 mM NaCl, 1 mg/ml bovine serum albumin (heat inactivated), 1 mM PMSF, and 1 mM 8-hydroxyquinoline] with various concentrations of 125I-labeled ANG II (New England Nuclear-Dupont, Boston, MA) for 45 min at 25°C in the presence of various concentrations of unlabeled ANG II (3, 53). After incubation, bound and free radioactivity were separated by filtration through glass-fiber filters (Whatman GF/C) presoaked with assay buffer. Each filter was rinsed three times with 5 ml ice-cold 1× PBS, and radioactivity was counted in a gamma counter (Rockville, MD) with a counting efficiency of 73%. Nonspecific binding was determined by the addition of 10 µM unlabeled ANG II. On the basis of competition curves, dissociation constant (Kd) and maximum binding capacity (Bmax) were determined by use of the EBDA/LIGAND curve-fitting computer program (Biosoft).

Receptor competition studies were performed by incubating plasma membranes (30 µg) with 125I-ANG II for 30 min in the presence of 10-5 M ANG II, losartan, or PD-123319. Studies were repeated on four separate occasions. Data were plotted as the negative logarithm of the concentration of the competitor (in molar concentration) vs. the level of 125I-ANG II bound radioactivity (as percentage of control).

Reciprocal inhibition studies were performed following preincubation with 1 µM of losartan, followed by competition with ANG II or PD-123319; or with 1 µM of PD-123319, followed by competition with ANG II or losartan. Studies were done on three separate occasions, in duplicate on each occasion.

ANG II RIA. After confluence, IRPTC were grown in serum-free medium for 24 h. In certain experiments, as noted in RESULTS, isoproterenol (10-5 M) was added to the serum-free medium. Cells were harvested in 1.5 ml of trifluoracetic acid (TFA), and then cell lysate was adjusted with water to 0.5% TFA, followed by boiling for 3 min. The protein precipitate was removed by centrifugation, and the supernatant was applied to an RP18 Sep-Pak column (Waters Associates, Milford, MA). The column was washed with 20% methanol, and ANG II was eluted with 80% methanol. ANG II was concentrated in a Sep-Pak column. ANG II extracted from medium or cells was determined by RIA (49). The primary antibody was an anti-ANG II antibody purchased from Arnel (no. 1711), and secondary antibody was a donkey anti-rabbit IgG. This assay recognizes radioimmune ANG II and does not cross-react with ANG I.

Statistical analysis. Statistical analysis was performed using one-way analysis of variance (comparisons among 3 or more groups) followed by Bonferroni modification to analyze preplanned comparisons. Linear regression or multivariate analysis was used to test for significant correlations between variables for the receptor binding assays.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

PTC morphology and markers. Rat PTC in primary culture demonstrate centrally located dome formation (Fig. 1A), a feature that has been associated with cells derived from transporting epithelia. These cells displayed strong activity for alkaline phosphatase, a brush border marker enzyme for PTC. The primary cultured PTC were grown to 30% confluence and then transfected with origin-defective SV40 plasmid. The cells appeared viable 24 h after transfection (Fig. 1B). Subsequently, as can be seen, most cells appeared to have died (Fig. 1C), and only after several weeks did individual colonies begin to arise from "ghost" cells (Fig. 1D). Figure 1E shows a clonal cell colony, denoted as line 93-p-2-1, which has now been passaged over 50 times and retains consistent morphological characteristics and PT markers (Table 1). These cells express large T antigen, which appears on immunofluorescence immunohistochemistry in a nuclear staining pattern (data not shown), indicating incorporation of SV40 cDNA plasmid into the genome.


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Fig. 1.   Transformation in cell line 93-p-2-1: progression of transformation (phase-contrast micrographs, ×85). A. primary cells prior to transfection. B: cells 24 h after transformation. C: ghost cells 2 wk after transfection. D and E: new cells arising and forming colony, respectively.

                              
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Table 1.   Proximal tubule markers found in line 93-p-2-1

Line 93-p-2-1 expresses PT characteristics as defined by the presence of characteristic antigens for each segment (S1, S2, and S3) of the tubule. Carbonic anhydrase, ecto-ATPase, and GLUT-2 are all present. The cells also express alkaline phosphatase, aquaporin-1 (CHIP-28), and Heymann antigen (gp330) while not expressing factor VIII. These characteristics are summarized in Table 1. In general, 90-95% of cells were positive by immunostaining for the antigens tested (as well as for RAS components as noted below).

This line generates Isc, demonstrating that it polarizes (7). We observed the unstimulated potential difference to be 0.73 ± 0.13 mV, the steady-state Isc was 5.13 ± 0.79 µA/cm2, and the resistance was 128 ± 25.7 Omega  · cm2 (±SE, n = 26). These values are similar to those reported for other PTC-like cell lines (4, 43).

Expression of RAS components. Line 93-p-2-1 expresses components of the RAS: angiotensinogen, renin, ACE, and AT1 and AT2 mRNA and protein. As can be seen in Fig. 2, top, a representative Northern blot, angiotensinogen mRNA is present in low levels in these cells and increases with exposure to ANG II. In addition, the cells express angiotensinogen protein, as shown by immunostaining in Fig. 2, bottom. However, no staining is seen when preimmune sera are used or second antibody omitted (data not shown). ACE mRNA is present, as is shown in a representative Northern blot (Fig. 3A), and ACE immunostaining (Fig. 3B).


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Fig. 2.   Angiotensinogen in cell line 93-p-2-1: demonstration of angiotensinogen mRNA and protein. A: representative Northern blot; a typical band as expected is seen at 1,800 kb. Lanes 1 and 2, control; lanes 3 and 4, 10-9 M ANG II; lanes 5-7, 10-8 M ANG II; and lanes 8-10, 10-7 M ANG II. Cells were exposed to ANG II for 24 h. B: positive immunostaining for angiotensinogen, 1:100. Magnification, ×725.


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Fig. 3.   Angiotensin converting enzyme (ACE) in cell line 93-p-2-1: evidence of ACE mRNA and protein. A: a representative Northern blot. Lanes 1-3, cells; lane 4, rat kidney. B: positive immunostaining for ACE, 1:100. Magnification, ×660.

Renin mRNA, present in small amounts, is clearly demonstrated in line 93-p-2-1 using RT-PCR (Fig. 4, top). Renin immunostaining demonstrates renin protein present in the cytoplasm and cell surface of the cells (Fig. 4, bottom).


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Fig. 4.   Renin in cell line 93-p-2-1: renin mRNA as demonstrated by RT-PCR (top) and renin immunostaining (bottom).

Specific 125I-ANG II binding to plasma membrane is saturable, and Scatchard analysis from saturation binding assays reveals a single class of binding sites (Kd = 2.9 nM; Bmax = 92.7 fmol/mg protein) (Fig. 5).


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Fig. 5.   A: saturation binding of 125I-ANG II to plasma membranes of immortalized rat proximal tubular cells (IRPTC). Plasma membranes (30 µg) were incubated at 25°C for 30 min with various concentrations of 125I-ANG II in presence or absence of 10-4 M ANG II to determine specific ANG II binding. B: Scatchard plot of the data. Data shown are representative of 2 separate experiments performed in duplicate. B/F, bound/free ratio.

To characterize ANG II receptor subtypes further, losartan (an AT1-selective antagonist) or PD-123319 (an AT2-selective antagonist) were tested. Figure 6 shows that these selective antagonists displaced 125I-ANG II binding in a biphasic manner, suggesting the presence of two receptor subtypes in IRPTC line 93-p-2-1. Figure 7, A and B, demonstrates reciprocal inhibition studies of 125I-ANG II binding to plasma membranes using varying concentrations of cold ANG II following preincubation with either 1 µM losartan (to block AT1 binding sites) or 1 µM PD-123319 (to block AT2 binding sites). These data demonstrate that AT1 and AT2 receptors account for the ANG II binding seen in the plasma membrane of line 93-p-2-1.


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Fig. 6.   Competition of 125I-ANG II-specific binding to plasma membranes of IRPTC by ANG II, losartan, and PD-123319. Plasma membranes (30 µg) were incubated with 125I-ANG II for 30 min at 25°C in presence of varying concentrations of ANG II (), losartan (bullet ), and PD-123319 (). 125I-ANG II binding in presence of 10-4 M unlabeled ANG II was subtracted from each value, and data shown are means ± SE of 3 separate experiments, performed in duplicate.


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Fig. 7.   Reciprocal blocking experiments. A: AT1 receptor subtypes were studied by first blocking AT2 receptor subtypes with 1 µM PD-123319, followed by competition with ANG II () or losartan (). B: AT2 receptor subtypes were studied by first blocking AT1 receptor subtypes with 1 µM losartan, followed by competition with ANG II () or PD-123319 (). Data shown are means ± SE of 3 separate experiments, performed in duplicate.

ANG II (10-8 M) exposure upregulates several RAS components. As may be seen in Fig. 2, top, ANG II exposure results in upregulation of angiotensinogen mRNA (shown at a 24-h time point in this Northern blot). The time course of this change as determined by slot blot analysis is shown quantitatively in Table 2; the angiotensinogen mRNA increases by 1 h and remains elevated up through the 24-h time point. This effect could be blocked by the addition of losartan (10-5 M) to the media during incubation (Fig. 8). Densitometry revealed relative densitometric units of 536 ± 44 in control cells, compared with 913.4 ± 79 in ANG II-treated cells. Losartan brought this stimulation back to control levels, even in the presence of ANG II (586 ± 191 relative densitometric units). Losartan in the absence of ANG II resulted in a slightly lower steady-state angiotensinogen mRNA level than controls (ANOVA, P < 0.025).

                              
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Table 2.   Time course of angiotensinogen mRNA increase after ANG II exposure



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Fig. 8.   Losartan blocking of ANG II-induced increase in angiotensinogen mRNA in a representative Northern blot. Cells were exposed to ANG II for 24 h. Lanes 1 and 2, control; lanes 3 and 4, ANG II; lanes 5 and 6, losartan (10-5 M) plus ANG II; and lane 7, losartan alone. Note that the increase in angiotensinogen after ANG II exposure is blocked by addition of losartan (10-5 M). Densitometry revealed relative densitometric units of 536 ± 44 in controls, compared with 913.4 ± 79 in ANG II-treated cells. Losartan brought this back to control levels, even in presence of ANG II. P < 0.025.

Additionally, ANG II (10-8 M) appears to upregulate both AT1 and AT2 receptor mRNA, as shown in Fig. 9.


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Fig. 9.   Representative Northern blots for ANG II receptor subtype mRNA. Top: representative Northern blot for AT1 mRNA. Bottom: representative Northern blot for AT2. ANG II exposure for 24 h led to upregulation of steady-state mRNA for both AT1 and AT2 receptor subtypes.

To examine line 93-p-2-1 for evidence of intracellular ANG II and secreted ANG II, cells were grown in serum-free media for 24 h after confluence was reached and were then harvested, as was media. ANG II extracted from media or cells was determined by RIA. At basal state, IRPTC secrete ANG II into the culture medium and contain ANG II in the cell layer [2.59 ± 0.32 pg/106 cells (n = 5) in media and 1.27 ± 0.29 pg/106 cells (±SE; n = 4) in cell lysates]. Thus approximately 1/3 of the ANG II would appear to be intracellular, where we speculate that it might act within the cell in intracrine fashion.

Since the beta -adrenergic agonist isoproterenol increases the expression of angiotensinogen in vitro (33), we examined its effect as an agent that should also influence ANG II secretion. As shown in Fig. 10, isoproterenol (10-5 M) increases secretion of ANG II from IRPTC into media. This finding suggests that the secretion of ANG II by IRPTC is regulatable by a beta -adrenergic mechanism.


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Fig. 10.   Effects of beta -adrenergic stimulation on accumulation of ANG II in cell line 93-p-2-1 culture media. Line 93-p-2-1 cells were incubated with isoproterenol (10-5 M) for 24 h at 37°C. Bars are means, and error lines are ±SE (n = 4).


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The immortalized rat proximal tubular cell line (93-p-2-1) described here polarizes and expresses enzymes and proteins that are normally present in normal PTC, thus displaying a highly differentiated phenotype. This cell line, cultured at 37°C, is transformed and grows rapidly, thus facilitating studies requiring large numbers of cells. Cell line 93-p-2-1 expresses all components of the RAS: angiotensinogen, renin, ACE and both AT1 and AT2 receptors. Furthermore, ANG II appears to upregulate angiotensinogen and both receptor subtypes.

That ANG II is directly involved in PT functions is supported by studies such as those of Seikaly et al. (51), which demonstrate endogenous production of ANG II within PT. ANG II has multiple and diverse well-described effects in this segment of the nephron, including sodium and bicarbonate resorption (5, 13, 25, 50), volume reabsorption (5, 25), and cellular hypertrophy (61). Cellular actions of ANG II within the PT appear to be mediated by several modes of signal transduction following direct occupancy of receptors on the cell membrane (17): via reduction of adenyl cyclase activity, via increase in phospholipase C, or via 5,6-epoxyeicosatrienoic acid (18). The present studies indicate that IRPTC secrete ANG II and that a beta -adrenergic mechanism, at least in part, may regulate secretion. Furthermore, ANG II appears to exhibit positive feedback on angiotensinogen mRNA, mediated via AT1 receptors.

Angiotensinogen mRNA levels in renal cortex are roughly 25-fold lower than reported in liver (27), with virtually all intrarenal angiotensinogen mRNA being synthesized in the PT. Thus the primary source of intrarenal angiotensinogen is the PT, a fact supported by the present results. In vivo renal angiotensinogen mRNA is differentially regulated, increasing with salt-depletion by approximately two- to threefold (26), whereas hepatic angiotensinogen is far less affected by changes in sodium diet. There is also strain variability in renal angiotensinogen mRNA expression in the rat, as shown by differences in angiotensinogen steady-state mRNA levels and regulation by sodium diet in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto rats (40). The SHR, a model of salt-sensitive essential hypertension, has a low steady-state angiotensinogen mRNA level and fails to modulate this level when exposed to a low-salt diet (40). Angiotensinogen mRNA in the kidney also exhibits androgen dependence (19). Cell line 93-p-2-1 would appear to be a potentially useful tool for additional studies concerning the regulation of angiotensinogen mRNA. Furthermore, studies examining RNA stability and rate of production have yet to be fully addressed in PTC, and a cell line such as 93-p-2-1 may prove helpful in this regard.

How angiotensinogen, the source of angiotensins, is processed within the kidney is incompletely studied. In other tissues, it appears that rat angiotensinogen is secreted constitutively (16). Chan et al. (10) have examined production of angiotensinogen by transiently transfecting opossum kidney (OK) cells with portions of the 5'-flanking region of the angiotensinogen gene linked to a growth hormone reporter gene, which then was translated into a detectable product (growth hormone). Chan et al. (10) found evidence of constitutive secretion of angiotensinogen using that system. However, since growth hormone itself is normally secreted constitutively, the modes of native intracellular processing of angiotensinogen within the PT may differ from the transiently transfected system that Chan and his colleagues (10) employed.

Angiotensinogen immunostaining has been detected in subapical granules within PTC in vivo (56). In rabbit PTC in culture, Yanagawa and colleagues (63) found a time-dependent rise in angiotensinogen levels using an indirect assay, suggesting de novo synthesis followed by secretion. Since there is evidence that intracellular ANG II receptors exist (53, 55), it is attractive to hypothesize that PTC with such intracellular receptors may process angiotensinogen within the cells. Thus intracellular angiotensinogen might provide a ready source of ANG II for intracrine responses.

The present work demonstrates that the RAS in cell line 93-p-2-1 exhibits autocrine feedback. Our previous in vivo studies, in which ANG II was infused via osmotic minipumps in rats, indicated that ANG II has a positive feedback upon its substrate, with the result that ANG II upregulated intrarenal angiotensinogen (48, 49). Those findings are supported by the present in vitro results, which demonstrate an increase in angiotensinogen mRNA in response to ANG II, and that this is blocked by the AT1 blocker losartan. Thus this positive feedback would appear to be mediated via the AT1 receptor, since losartan prevents this increase. Whether losartan modulates the increase in angiotensinogen induced by ANG II by also blocking the effects of endogenous ANG II has not been determined by the present studies. However, angiotensinogen mRNA steady-state levels appeared slightly lower than controls in the presence of losartan alone.

Ming et al. (33) have examined the role of isoproterenol in stimulating expression of the angiotensinogen gene in OK cells. In these studies, isoproterenol also increased angiotensinogen mRNA. With renal nerve stimulation, one would expect beta agonist release, and Nakamura and Johns (38) have shown that low levels of renal nerve stimulation increase angiotensinogen mRNA levels in vivo. The present studies support this type of stimulation, although, based on studies examining angiotensinogen expression in these cells in more detail, it would appear that dexamethasone plus isoproterenol may act synergistically, likely with intracellular cAMP to increase angiotensinogen production.

Our data suggest that ANG II may be present both within and secreted by cells. We have previously shown that ANG II-like receptors are present both on the plasma membrane and within the nucleus of endothelial cells (55). Preliminary data would suggest that both nuclear and plasma membrane ANG II receptors are present in PTC. Thus the finding of both intracellular and extracellular ANG II would be consistent with the localization of receptors that we find.

AT1 and AT2 receptors are both expressed in this cell line. Cheng et al. (11) have recently shown that ANG II increases proximal AT1 receptor expression in rabbit PT. Our data also indicate that ANG II may upregulate AT1 receptors. In contrast, Robillard et al. (42) have shown that ANG II has little effect on AT1 receptors but may downregulate AT2 receptors in the fetal sheep. The finding that AT2 receptors are upregulated by ANG II in this cell line is in contradistinction to this recent report of Robillard et al. (42). However, we would point out that the present cell line is derived from 4- to 6-wk-old male rats, and that Robillard's data were obtained from fetal sheep kidney.

Several studies using in situ hybridization and ligand binding autoradiography have reported that AT2 subtype is detected in rat fetal kidneys but disappears as the kidney develops (1, 52). In contrast, the AT2 subtype can be detected in the adult kidney of human and opossum (15). On the basis of results we have obtained with both receptor binding and Northern blot analysis, we conclude that our IRPTC line 93-p-2-1 expresses AT2 receptor subtype.


    ACKNOWLEDGEMENTS

We thank Dr. Kevin Lynch for providing the rat renin and angiotensinogen cDNA probes, Dr. T. J. Murphy for the AT1 cDNA probe, and Dr. Russell Medford for beta -actin cDNA. We thank Dr. Jose Krieger and Dr. Victor Dzau for providing rat cDNA for ACE. We thank Dr. V. J. Dzau for providing the cDNA for AT2. We also thank Dr. Laurie Glimcher for providing origin-defective SV40. We are grateful to Dr. Richard Soffer for antiserum to ACE and to Dr. Tadashi Inagami for the antiserum to rat renin. We thank Drs. John Niles, Robert W. McCluskey, and Giuseppe Andres for anti-gp330 antisera and advice concerning gp330. We thank Dr. William Sly (St. Louis University, St. Louis, MO) for antibody to carbonic anhydrase IV and Dr. S. H. Lin (University of Texas, Houston, TX) for antibody to ecto-ATPase. We thank Dr. Dennis Brown for providing antibody to Na-K-ATPase. We thank Dr. Edward Harlow (Massachusetts General Hospital) for antibody to large T antigen.


    FOOTNOTES

These studies were supported by National Institutes of Health Grants HL-40210 and HL-48455 (to J. R. Ingelfinger) and HL-43131 and DK-50836 (to S.-S. Tang) and by research grants from the Milton Fund (to S.-S. Tang), Merck (to J. R. Ingelfinger), and the American Heart Association of Rhode Island (to A. Brem).

Address for reprint requests: J. R. Ingelfinger, Bartlett 4X-411, Massachusetts General Hospital, Boston, MA 02114.

Received 14 October 1997; accepted in final form 1 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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