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(Hypertension. 1997;30:358.)
© 1997 American Heart Association, Inc.

Articles

Growth-Dependent Induction of Angiotensin II Type 2 Receptor in Rat Mesangial Cells

Masahisa Goto; Masashi Mukoyama; Shin-ichi Suga; Tsunekazu Matsumoto; Masayo Nakagawa; Rieko Ishibashi; Masato Kasahara; Akira Sugawara; Issei Tanaka; Kazuwa Nakao

From the Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence to Masashi Mukoyama, MD, PhD, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. E-mail muko{at}kuhp.kyoto-u.ac.jp

	Abstract

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Abstract Angiotensin II acts on at least two receptor subtypes, AT₁ and AT₂. Although the physiological role of the AT₂ receptor is still poorly defined, it may be implicated in inhibition of cell growth, vasorelaxation, and apoptosis. In the present study, to investigate the role of the AT₂ receptor in the kidney and its implication in hypertensive states, we examined its expression using cultured mesangial cells (MC) from normotensive Wistar-Kyoto rats (WKY) and from stroke-prone spontaneously hypertensive rats (SHRSP). Receptor binding assays were performed using a nonselective ligand, [Sar¹,Ile⁸]angiotensin II, or AT₂-selective CGP42112A. Binding assays revealed that MC from WKY exhibited both AT₁ and AT₂ receptors, the ratio of which was confluence-dependent. In contrast, MC from SHRSP, whose proliferation activity was much higher than those from WKY, showed only the AT₁ subtype. In receptor binding and Northern blot analyses, expression of the AT₂ receptor of WKY-MC was low in the growing state but significantly induced upon confluence to become abundant in the postconfluent state, whereas that of SHRSP-MC was undetectable in either state. Gene expressions of AT_1A and AT_1B receptors were not significantly altered in either strain during the time in culture. These results indicate that the mesangial AT₂-receptor expression is growth-dependent and suggest a role in the inhibition of MC growth in WKY. Much lower expression of the AT₂ receptor in MC from SHRSP may suggest involvement in their higher proliferation activity and possibly in consequent renal disorders.

Key Words: receptors, angiotensin • angiotensin II • mesangium, glomerular • rats, inbred SHR

	Introduction

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Angiotensin II, a key regulator of cardiovascular homeostasis, exerts various actions in its diverse target tissues that control vascular tone, hormone secretion, tissue growth, and neuronal activities. These multiple actions are mediated by at least two pharmacologically distinct receptor subtypes, designated as AT₁ and AT₂ receptors.^{1 2 3 4} The AT₁ receptor further comprises two isoforms, AT_1A and AT_1B, with a striking similarity to each other in structure and pharmacological properties.^{5 6} Most known physiological effects of Ang II are thought to be mediated by the AT₁ receptor through the activation of the phosphoinositide/calcium or tyrosine phosphorylation pathways or through the inhibition of adenylate cyclase activity.^{3 4 5 6}

In contrast, the physiological role of the AT₂ receptor has been poorly understood. Recently, we and others have succeeded in cloning cDNA for the rat, mouse, and human AT₂ receptor and have revealed that it has limited identity to the AT₁ receptor.^{7 8 9 10} Moreover, the AT₂ receptor is abundantly and widely expressed in fetal tissues,^{7 8 9 10 11} but in adults its expression is limited to only several tissues, such as the adrenal gland, brain, lung, atretic ovarian follicles, and uterine myometrium.^{1 2 7 8 9 10 11 12 13} Its expression is also found in the adult heart and kidney but usually at much lower levels.^{14 15}

Although intracellular signaling mechanisms exerted by the AT₂ receptor stimulation are still unclear, the targeted disruption of the AT₂ receptor gene in mice has revealed that it may counteract classic pressor effects of Ang II mediated by the AT₁ receptor.^{16 17} Furthermore, it has been shown that the AT₂ receptor exerts antigrowth effects on VSMC¹⁸ and endothelial cells.¹⁹ More recently, the AT₂ receptor has been shown to mediate programmed cell death.²⁰ These observations, together with the findings that its expression can be enhanced in tissues undergoing remodeling or apoptosis,^{12 14 18 20} have led to the suggestion that the AT₂ receptor may participate in cardiovascular homeostasis and blood pressure control in multiple ways.

The kidney is one of the major target organs on which Ang II acts. The fetal kidney abundantly expresses the AT₂ receptor, mainly in the undifferentiated nephrogenic mesenchymal tissues.^{21 22} After birth, its expression decreases markedly, but some areas still can express the AT₂ receptor, such as larger vessels²² and the glomerular area.¹⁵ The MC are the major site of the action of Ang II in the glomeruli controlling ultrafiltration.²³ Moreover, MC hyperplasia is one of the features of the hypertensive renal disease.²⁴ It is therefore interesting to examine whether the AT₂ receptor is expressed in MC and whether it has implications in the hyperplastic characteristics of MC as seen in hypertensive states. In the present study, we investigated the expression of the AT₂ receptor in cultured MC from normotensive WKY and from SHRSP, comparing it with expression of the AT₁ receptor.

	Methods

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Preparation of Cultured MC
Male WKY and SHRSP were obtained from Shionogi Research Laboratories (Osaka, Japan) at 20 weeks of age. All procedures were in accordance with our institutional guidelines for animal research. Tail-cuff pressure measurements²⁵ revealed an established severe hypertension in SHRSP (WKY, 125.0±2.3 mm Hg; SHRSP, 225.3±4.2 mm Hg; n=3 for each). After decapitation, six kidneys were obtained from each strain. Cortex was minced into pieces and passed through a graded series of metal sieves to isolate glomeruli.^{26 27} After centrifugation, pelleted glomeruli were resuspended and cultured in RPMI-1640 medium containing 10% fetal calf serum. MC purified by successive subcultures were used between passages 5 and 10.

To study the proliferation activity of MC from WKY and SHRSP, the number of cells cultured with 10% serum was counted from 2 days before reaching confluence through 8 days after confluence. Cellular DNA synthesis was assessed by ³H-thymidine incorporation into cells and labeled with 10 µCi/mL tracer for 8 hours in a serum-free condition.²⁸

Binding Studies with Cultured MC
Cultured MC on postconfluent days -2, 0, 2, 4, and 8 were used to examine the time-dependent expression of the Ang II receptors. Binding assays using intact cells were performed as described.²⁹ In brief, for competitive binding assays, cell monolayers in 24-well Falcon plates were washed twice, and ¹²⁵I-[Sar¹,Ile⁸]Ang II (2200 Ci/mmol, New England Nuclear) was added at a final concentration of 200 pmol/L in phosphate-buffered saline/0.1% bovine serum albumin. Cells were incubated at 22°C for 90 minutes with or without various concentrations of unlabeled Ang II, AT₁-selective CV-11974 (Takeda Chemical Industries), or AT₂-selective CGP42112A (Ciba-Geigy). After incubation, cell monolayers were washed, lysed with NaOH, and measured for their bound count. For AT₂-specific binding assays, cells were incubated with 500 pmol/L of ¹²⁵I-CGP42112A (1400 Ci/mmol) which had been iodinated by the chloramine-T method.¹³ For Scatchard analysis, cells were incubated with increasing concentrations of ¹²⁵I-CGP42112A with or without unlabeled 1 µmol/L CGP42112A.^{7 13 29} In both binding studies, nonspecific bindings with 10 µmol/L cold ligands were less than 10% of the maximal bindings.

Northern Blot Analysis
Total RNA was extracted from cultured MC at 90% confluence and 1-week postconfluence, using the acid guanidinium-phenol-chloroform extraction method with TRIzol reagent (GIBCO BRL).¹³ Total RNA (100 µg per lane) was separated by electrophoresis and transferred onto nylon membranes (Biodyne, Pall BioSupport). Hybridization was carried out using a ³²P-labeled HindIII/Sac I fragment (2.4 kb) of a rat AT₂ receptor cDNA clone⁷ or a Pst I–Xba I fragment (0.78 kb) of GAPDH clone (Clontech Labs). The mRNA concentrations were estimated by densitometry.

RT-PCR Analysis
RT-PCR was performed as described.^{14 30} PCR primers used for the AT_1A receptor were as follows: 5'-GCACACTGGCAATGTAATGC-3' and 5'-GTTGAACAGAACAAGTGACC-3'. For identifying the AT_1B receptor, PCR primers were 5'-GCCTGCAAGTGAAGTGATTT-3' and 5'-TTTAACAGTGGCTTTGCTCC-3'. PCR primers for the AT₂ receptor were 5'-TTGCTGCCACCAGCAGAAAC-3' and 5'-GTGTGGGCCTCCAAACCATTGCTA-3'. PCR was carried out with cycles of 30 seconds of denaturation at 94°C, 45 seconds of annealing at 58°C, and 1 minute of extension at 72°C. Since PCR amplifications for the AT_1A, AT_1B, and AT₂ receptor cDNAs occurred linearly between 24 and 32 cycles, we analyzed their message expression in this range. Similarly, RT-PCR analysis for GAPDH mRNA as a control was performed at a range of 20 to 24 cycles.

Statistical Analysis
Results are presented as mean±SE. The ANOVA followed by Scheffé’s test was used for statistical analysis. A value of P<.05 was considered significant.

	Results

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Growth of Cultured MC
SHRSP-MC grew more rapidly than WKY-MC after plating in low cell density. After reaching confluence, the growth of WKY-MC was retarded, whereas SHRSP-MC continued to grow, resulting in 10 times more cells than WKY-MC on postconfluent day 8 (Fig 1A). ³H-Thymidine incorporation was also significantly higher in SHRSP-MC than in WKY-MC even in an 8-day postconfluent state (Fig 1B).

View larger version (13K): [in this window] [in a new window]   Figure 1. A, Growth curves of cultured WKY-MC () and SHRSP-MC (•). Cells were cultured in RPMI-1640/10% fetal calf serum, fed every other day. Means of three determinations for each culture are shown. B, 3H-Thymidine incorporation into 8-day postconfluent MC from WKY and SHRSP. Twenty-four hours after medium was changed to RPMI-1640 without serum, cells were labeled with 3H-thymidine (10 µCi/mL) for 8 hours. Values are mean±SE; n=6 for each. *P<.01 vs WKY-MC.

Ang II Receptor Subtypes in Cultured MC
In WKY-MC, the displacement of ¹²⁵I-[Sar¹,Ile⁸]Ang II binding with CGP42112A yielded a biphasic pattern (Fig 2A and 2B), indicating the presence of two receptor subtypes in either state. In 2-day postconfluent WKY-MC, CV-11974 displaced 80% of ¹²⁵I-[Sar¹,Ile⁸]Ang II binding at 10^-7 mol/L, while CGP42112A inhibited 20% of its binding, indicating the ratio of the AT₁ to the AT₂ receptor in WKY-MC as 8:2 (Fig 2A). In 8-day postconfluent WKY-MC, however, the ratio was changed to 2:8 for the AT₁ to the AT₂ receptor (Fig 2B). In contrast, almost all of the radioligand binding to 2-day or 8-day postconfluent SHRSP-MC was displaced by CV-11974 but not by CGP42112A at 10^-7 mol/L, indicating that most of the Ang II receptor in SHRSP-MC in either state was that of the AT₁ subtype (Fig 2C and 2D).

View larger version (28K): [in this window] [in a new window]   Figure 2. Displacement of 125I-[Sar1,Ile8]Ang II binding with Ang II (•), CGP42112A (), or CV-11974 () in 2-day postconfluent WKY-MC (A), 8-day postconfluent WKY-MC (B), 2-day postconfluent SHRSP-MC (C), and 8-day postconfluent SHRSP-MC (D). Cells were incubated with 200 pmol/L 125I-[Sar1,Ile8]Ang II for 90 minutes at 22°C in the presence of indicated concentrations of competitors. Means of duplicate data for each point from a representative experiment out of the two are shown.

AT₂ Receptor Binding in Cultured MC
We next examined time-dependent changes of the AT₂-receptor binding in cultured MC using the AT₂-selective binding assay. The number of AT₂ binding sites expressed in WKY-MC was very low in growing state; after cells became confluent, however, there was a marked increase in the number of AT₂ binding sites, reaching a plateau within 1 week after confluence (Fig 3). Scatchard analyses revealed a significant increase in B_max (11.0±0.18 and 34.5±0.55 fmol/10⁶ cells for 2-day and 8-day postconfluent cells, respectively; n=4 for each) without a significant change in the affinity for CGP42112A (0.46±0.18 and 0.26±0.06 nmol/L for 2-day and 8-day postconfluent cells, respectively; n=4 for each). On the other hand, no AT₂ binding sites were detected in SHRSP-MC even in an 8-day postconfluent state (Fig 3).

View larger version (15K): [in this window] [in a new window]   Figure 3. Time-dependent expression of the AT2 receptor (AT2R) after confluence in cultured rat MC. WKY-MC () and SHRSP-MC (•) were incubated with 500 pmol/L 125I-CGP42112A for 90 minutes at 22°C, and bound ligand was expressed as moles. Values are mean±SE; n=6 for each.

Gene Expression of Ang II Receptor Subtypes in Cultured MC
To examine the AT₂ receptor gene expression, we performed Northern blot analysis in preconfluent and 1-week postconfluent MC. As shown in Fig 4A, in WKY-MC, AT₂ receptor mRNA expression at 3.5 kb was very low in the preconfluent state but was markedly increased 1 week after confluence. In SHRSP-MC, no message was detected in either state.

View larger version (47K): [in this window] [in a new window]   Figure 4. A, Northern blot analysis for the AT2 receptor mRNA in cultured rat MC. One hundred micrograms of total RNA in each lane was separated by electrophoresis, transferred onto the membrane, and hybridized to a probe for the AT2 receptor (AT2R), and GAPDH was used as a control. W indicates WKY-MC; S, SHRSP-MC. B, Gene expression of Ang II receptor subtypes in cultured rat MC using the RT-PCR method. From the upper panel, bands for the AT2 receptor (AT2R), the AT1A receptor (AT1AR), the AT1B receptor (AT1BR) (30 cycles each), and GAPDH (24 cycles) are displayed. Results of a representative experiment out of four are shown. Lane 1, WKY-MC in 90% confluence; lane 2, WKY-MC 1 week after confluence; lane 3, SHRSP-MC in 90% confluence; lane 4, SHRSP-MC 1 week after confluence. C, The quantification of the mRNA levels shown in panel B, analyzed with linear regression at 24 to 32 PCR cycles and normalized relative to the GAPDH mRNA level in each sample. The mean of relative mRNA levels for the AT1A receptor in preconfluent WKY-MC is 1.0 arbitrary unit. Values are mean±SE; n=4 for each. *P<.01. ND indicates not detectable.

We then performed RT-PCR analysis to compare the expression of AT_1A and AT_1B receptors with that of the AT₂ receptor. Messages for both AT_1A and AT_1B receptors were detected at similar levels between the two strains (Fig 4B and 4C). Again, the AT₂ receptor message of WKY-MC was more abundant in the confluent state than in the growing state (4.58±0.10 versus 0.24±0.10 units, respectively; P<.01), whereas no AT₂ receptor message was detected in SHRSP-MC (Fig 4B and 4C). Unlike the AT₂ receptor, expressions of the AT_1A and AT_1B receptors did not change significantly after confluence in either strain (Fig 4B and 4C).

	Discussion

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The mesangium, directly controlling the glomerular function through its contractile and proliferative nature, is one of the primary sites of Ang II action in the kidney.²³ Since the AT₂ receptor may exhibit vasorelaxant and antigrowth properties,^{16 17 18 19 31} it is important to know whether the AT₂ receptor is expressed in MC and whether it has some role in mesangial function in hypertensive conditions.

The present study demonstrates the confluence-dependent expression of the AT₂ receptor in WKY-MC but not in SHRSP-MC. The AT₁ receptor is predominant (80%) in newly confluent WKY-MC, consistent with the previous report by Ernsberger et al.³² The binding property of their remaining fraction was a little different from ours in that they reported it as PD123319-sensitive but CGP42112A-insensitive.³² The reason for this discrepancy is currently unclear. One important finding in the present study is that the AT₂ receptor is easily inducible in WKY-MC after confluence is reached. By contrast, the AT₂ receptor was undetectable, and only the AT₁ receptor was expressed in SHRSP-MC even after confluence. Although it is not known whether this situation also occurs in vivo, it is conceivable that the induction of the mesangial AT₂ receptor expression in response to proper stimuli would occur more easily in WKY than in SHRSP. However, this speculation should await further clarification.

The mechanism of how the AT₂ receptor is induced in WKY-MC is unclear. Dudley and Summerfelt³³ have already shown that R3T3 cells, a mouse fibroblast cell line, exhibit a similar confluence-dependent AT₂ receptor expression. Recently, Horiuchi et al³⁴ revealed that IRF binding motif is located in the promoter region of the mouse AT₂ receptor gene and that the expression of the AT₂ receptor in R3T3 cells is regulated by these transcriptional factors (IRF-1 and -2). Although it is unclear whether IRFs are active in the promoter region of the rat AT₂ receptor gene, similar IRF-binding motif-like sequences can be found,³⁵ suggesting the possibility that IRF may modulate the expression of the AT₂ receptor in the rat also.

This study revealed higher proliferation activity of SHRSP-MC than WKY-MC. Similar results have already been reported in SHR-MC,³⁶ and this would be at least partly due to increased intracellular calcium responses.³⁷ The AT₂ receptor is shown to exert an antiproliferative effect in VSMC and endothelial cells^{18 19} and to induce apoptosis in PC12W cells.²⁰ It is therefore probable that the AT₂-receptor stimulation would cause antiproliferation and/or apoptosis in postconfluent WKY-MC. This hypothesis is currently under extensive study in our laboratory. It might be unlikely that the growth of MC is regulated by the endogenously secreted Ang II in an autocrine manner, but it would be possible that the induced mesangial expression of the AT₂ receptor may modulate the cell growth in vivo where the local Ang II production is enhanced. Conversely, if SHRSP-MC were less able to induce the AT₂ receptor expression, it might result in their higher proliferation activity in the pathological condition. Indeed, it is postulated that the abnormal renin-angiotensin system with enhanced AT₁ receptor–mediated responses during early life could have a critical role in the development of hypertension and related renal and vascular complications in SHR.³⁸ Ichiki et al³⁹ revealed the expression of the AT₂ receptor in WKY-VSMC but not in SHR-VSMC by serum depletion and insulin stimulation. It is interesting that the AT₂ receptor is inducible in cells other than MC. The lack of induction of the AT₂-receptor expression in SHR or SHRSP may prompt the cell proliferation that would result in hypertensive vascular changes or renal diseases.

In conclusion, we have reported that the AT₂ receptor in WKY-MC was expressed after reaching confluence, whereas that in SHRSP-MC was not expressed in any state. These results indicate that the AT₂ receptor expressed in WKY-MC may modulate cell growth in the confluent state. Lower expression of the AT₂ receptor in SHRSP-MC may suggest involvement in the higher mesangial proliferation activity.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 or AT2 receptor = type 1 or type 2 Ang II receptor GAPDH = glyceraldehyde-3-phosphate dehydrogenase IRF = interferon regulatory factor MC = mesangial cells RT-PCR = reverse transcription–polymerase chain reaction SHRSP = stroke-prone spontaneously hypertensive rats VSMC = vascular smooth muscle cells WKY = Wistar-Kyoto rats

	Acknowledgments

 
This work was supported in part by research grants from the Japanese Ministry of Education, Science and Culture, Japanese Ministry of Health and Welfare, and the Yamanouchi Foundation for Research on Metabolic Disorders. We thank Dr Marc de Gasparo (Ciba-Geigy, Basel, Switzerland) for donating CGP42112A and Dr Kohei Nishikawa (Takeda Chemical Industries, Osaka, Japan) for CV-11974.

Received September 18, 1996; first decision October 15, 1996; accepted January 31, 1997.

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