Effects of OPC-21268, a vasopressin V1-receptor antagonist, on expression of growth factors from glomeruli in spontaneously hypertensive rats

Regulatory Peptides 72 (1997) 87–95

Effects of OPC-21268, a vasopressin V1-receptor antagonist, on expression of growth factors from glomeruli in spontaneously hypertensive rats a, b a a a Fumio Otsuka *, Toshio Ogura , Takayoshi Yamauchi , Tetsuya Oishi , Masami Hashimoto , Yukari Mimura a , Hirofumi Makino a a

Department of Medicine III, Okayama University Medical School, Okayama 700, Japan b Health and Medical Center, Okayama University, Okayama 700, Japan

Received 20 May 1997; received in revised form 31 July 1997; accepted 7 August 1997

Abstract To assess the chronic in vivo effects of OPC-21268, a vasopressin-V1 receptor antagonist, on renal injury, we investigated the mRNA expressions of platelet-derived growth factor (PDGF) B-chain, transforming growth factor (TGF)-b 1 and proliferating cell nuclear antigen (PCNA) in the glomeruli of spontaneously hypertensive rats (SHR) treated with OPC-21268 for 3 weeks. SHR aged 10 weeks were given 2% NaCl in drinking water for 3 weeks. The OPC group was fed a 0.5% OPC-21268-containing diet for 3 weeks and the control group was given a normal diet. There were no significant changes in the time course of systolic blood pressure, heart rate, urine volume, or urinary sodium, protein and N-acetyl-b -glucosaminidase (NAG) excretion between the two groups. Serum electrolytes, protein and creatinine levels also did not differ between the groups. The mRNA expressions of PDGF B-chain, TGF-b 1 and PCNA in the glomerulus were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) methods. The mRNA expressions of PDGF B-chain and PCNA among these were significantly suppressed in the OPC group. No significant differences in renal histology including the organ weights were found between the two groups; however, the glomerular size tended to be enlarged in the OPC group. These findings suggest that chronic V1-receptor blockade directly inhibits the glomerular proliferative injury of salt-loaded SHR at the established hypertension stage.  1997 Elsevier Science Ireland Ltd. Keywords: Vasopressin; V1-receptor antagonist; Glomerulus; Spontaneously hypertensive rat (SHR); Platelet-derived growth factor (PDGF) B-chain; Proliferating cell nuclear antigen (PCNA)

1. Introduction Systemic hypertension is known to be a cause of progressive renal injury in humans and experimental animals. Although various factors appear to induce hypertensive nephropathy, such as hyperfiltration [1], intraglomerular hypertension [1,2], certain humoral factors [3], hydroxyl radicals [4] and so on, the precise mechanisms of their influence on renal histopathological changes are not clearly understood. We previously reported that angiotensin II causes mRNA expression of the proto-oncogene, c-fos, *Corresponding author. Tel.: 1 81 86 235 7235; fax: 1 81 86 222 5214.

in the cortex and medulla in vivo, as demonstrated by northern hybridization [5]. We also reported that the expression of platelet-derived growth factor (PDGF) Bchain in the glomerulus preceded the appearance of histological changes in spontaneously hypertensive rats (SHR) and that mRNA expression of PDGF B-chain was suppressed by administration of cilazapril, an angiotensin I-converting enzyme inhibitor (ACEI) [6]. Besides, treatment with cilazapril and L-158,809 (angiotensin II type I receptor antagonist) attenuated the mRNA expression of PDGF B-chain and TGF-b 1 from glomeruli in deoxycorticosterone acetate (DOCA)-salt treated hypertensive rats without any reduction in systemic blood pressure [7]. These findings indicate that the renin-angiotensin system

0167-0115 / 97 / $17.00  1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0167-0115( 97 )01041-0

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(RAS) plays an important role in renal injury via the expression of growth factors in the glomerulus. Vasopressin (VP) is a potent mitogenic factor in smooth muscle cells [8] and mesangial cells [9] in vitro. However, there is little information available about the in vivo mitogenic activity of VP. In preliminary experiments, we studied the expression of growth factors from the glomeruli of Wistar-Kyoto (WKY) rats during acute or continuous administration of VP, and observed marked expression of TGF-b 1 and a lesser degree of expression of PDGF B-chain in treated rats compared with vehicleinjected rats during continuous administration (unpublished observation). The receptors of VP are classified into V1 and V2 receptors, and recently, a non-peptide V1 receptor antagonist, OPC-21268, has been developed [10] that is available for oral administration. In the present study, to clarify whether the in vivo expression of growth factors in the glomerulus is suppressed by OPC-21268, we examined the histological changes and growth factor expression in the glomeruli of salt-loaded SHR. The aim of this study was to elucidate the chronic effects of V1 receptor blockade on glomerular injury in SHR.

2. Materials and methods

2.1. Materials SHR aged 10 weeks were purchased from Charles River Japan (Kanagawa, Japan). All rats were housed in climatecontrolled metabolic cages with a 12-h light / 12-h dark cycle and food (MF, Oriental Yeast, Tokyo, Japan) and water provided ad libitum. Oligonucleotides for primers were synthesized with a model 380B DNA synthesizer (Applied Biosystems, Foster City, CA, USA). The locations of the oligonucleotides of primer pairs were as follows: platelet-derived growth factor (PDGF) B-chain, 1079–1098 and 1601–1620 [11]; transforming growth factor (TGF)-b 1, 1679–1699 and 1994–2014 [12]; and proliferating cell nuclear antigen (PCNA), 197–216 and 715–734 [13]. Special food containing 0.5% OPC-21268 (1-(1-[4-(3-acetylaminopropoxy)benzoyl] - 4 - piperidyl) - 3,4 - dihydro - 2(1H) quinolinone), a selective V1 receptor antagonist, was donated by Otsuka Pharmaceutical, (Tokushima, Japan).

2.2. Procedure for OPC-21268 treatment SHR aged 10 weeks were divided randomly into two groups. The OPC group was given 2% NaCl in drinking water and food containing OPC-21268 for 3 weeks, while the control group was given 2% NaCl and normal food (MF, Oriental Yeast Co., Tokyo, Japan). Systolic blood pressure and heart rate were measured every week, at 9 a.m. in conscious, restrained and warmed rats using tailcuff plethysmography (UR-5,000, Ueda Seisakusyo, Tokyo, Japan). Urine specimens were collected over a 24-h

period once a week and used for the measurement of urinary protein, sodium excretion and N-acetyl-b glucosaminidase (NAG) activity. Protein and urinary NAG activity were determined according to the procedure of the Bio-Rad protein assay kit (Bio-Rad Labs., Richmond, CA, USA) and NAG test pack (Shionogi Phrmaceutical, Osaka, Japan), respectively. Rats were killed by decapitation after week 3, and the kidneys were quickly removed for histological assessment and extraction of RNA. Trunk blood samples were collected and stored at 2 308C until they were assayed for electrolytes, creatinine, uric acid and total protein by an autoanalyzer system. Plasma concentrations of vasopressin were measured by radioimmunoassay.

2.3. Histological examination For observation by light microscopy, a portion of each kidney was fixed in 10% buffered paraformaldehyde, embedded in paraffin, sectioned at 4 m m, and stained with periodic acid Schiff (PAS) reagent. The diameter ( m m) of the glomeruli (200–300 glomeruli in each group), as observed by light microscopy, was measured at random using an objective micrometer (OB-M, Olympus Optical, Tokyo, Japan). Histograms of glomerular diameters were compared between the OPC and control groups. The glomerular damage in the two groups was compared using the mesangial injury score, which was described by Raij et al. [14]. A minimum of 100 glomeruli in each specimen was examined and lesion severity was graded from 0 to 4 1 according to the percentage of glomerular involvement. Thus, a 1 1 lesion represented an involvement of 25% of the glomerulus, while a 4 1 lesion indicated that 100% of the glomerulus was involved. An injury score was then obtained by multiplying the degree of damage (0 to 4 1 ) by the percentage of the glomeruli with that degree of injury, i.e. increase in mesangial matrix material or glomerulosclerosis. The extent of the injury in each individual tissue specimen was then obtained by summing these scores. In addition, glomerular cellularity was determined by measurement of total nuclear cell counts in each glomerulus (at least 100 glomeruli in each specimen).

2.4. Separation of glomeruli and extraction of total RNA The renal cortex was dissected out and minced in icecold phosphate-buffered saline (PBS). Glomeruli were isolated by the graded sieving technique [15]. The isolated glomeruli were rinsed with ice-cold PBS and treated with 2 mg / ml of collagenase (Wako Pure Chem. Ind., Osaka, Japan) in RPMI-1640 medium for 30 min at 378C. Total RNA was extracted by the acid–guanidium–phenol–chloroform method. The final RNA pellets were washed with 70% ethanol and resuspended in 100 m l of diethylpyrocarbonate-treated water. RNA was quantified by absorbance at 260 nm, and stored at 2 208C until the assay.

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2.5. Reverse transcription ( RT) of RNA and polymerase chain reaction ( PCR) Reverse transcription of RNA and amplification of cDNA were performed with a RT-PCR kit (Perkin Elmer Cetus Inst., Norwalk, CT, USA) from samples containing 10 ng of total RNA. Samples were then amplified by means of 30–40 cycles in a thermal cycler (TP Cycler-100, Toyobo, Osaka, Japan). Each cycle consisted of denaturation at 948C for 1 min, annealing at 55–658C for 2 min, and extension at 728C for 3 min. An aliquot of the PCR product was electrophoresed on 1.5% agarose gel, visualized by ethidium bromide staining and measured by a quantitative scanning densitometer (Scanning Imager 300SX, Molecular Dynamics, Sunnyvale, CA, USA). The relative mRNA levels for PDGF B-chain, TGF-b 1 and PCNA were determined after standardization with gene expression of G3PDH.

2.6. Statistics The results were expressed as mean6SEM and statistically analyzed by means of the unpaired Student’s t-test. Statistical significance was set at P , 0.05 or P , 0.01.

3. Results

3.1. Time courses of changes in systolic blood pressure, heart rate, urine volume and urinary samples Fig. 1 shows the time courses of changes in systolic blood pressure and heart rate; these showed no significant differences between groups at any time. Fig. 2 shows the

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time courses of changes in urine volume and urinary sodium, protein and NAG excretions. At no time, did these parameters differ significantly between the two groups. Urine volume and urinary sodium excretion gradually increased in both groups until week 13. The weekly intake of OPC-21268-containing or control food and the volume of water consumed showed no significant differences between the two groups at any time (data not shown). The daily administrated dose of OPC-21268 in the OPC group distributed from 151623 to 372629 mg / kg-body weight (mean6SEM).

3.2. Laboratory data for both groups on day 21 Table 1 shows the laboratory data for the two groups on day 21. Serum sodium, potassium, chloride, creatinine, uric acid and total protein in the trunk blood samples showed no significant differences between the two groups. Plasma VP concentration in the trunk blood also showed no significant difference between groups.

3.3. Organ weights and histological findings Table 2 shows the total body weight, kidney and heart weights, and kidney weight / body weight ratio and heart weight / body weight ratio in the control and OPC groups on day 21. There were no significant differences between the two groups in any of these factors. Fig. 3 shows the representative light microscopic appearance (PAS staining, 3 400) of renal tissue from the control and OPC groups on day 21. We found no remarkable differences between the two groups in renal tissue, such as proliferation, sclerosis, or tubulointerstitial abnormalities. The histograms of the glomerular diameter revealed tendency toward enlargement

Fig. 1. Time courses of changes in systolic blood pressure and heart rate in control SHR (open circles) and OPC-21268-treated SHR (closed circles). Values are expressed as mean6SEM obtained from 6 rats.

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Fig. 2. Time courses of changes in urine volume and urinary parameters in control SHR (open circles) and OPC-21268-treated SHR (closed circles). Values are expressed as mean6SEM obtained from 6 rats.

Table 1 Laboratory data in the control and OPC groups on day 21

Serum sodium (mEq / l) Serum potassium (mEq / l) Serum chloride (mEq / l) Serum creatinine (mg / dl) Serum uric acid (mg / dl) serum total protein (g / dl) Plasma VP concentration (pg / ml)

Control group (n 5 6)

OPC group (n 5 6)

138.060.41 8.5760.37 100.260.50 0.4560.05 1.2560.08 6.1760.14 2.960.59

139.760.99 8.6260.61 102.861.4 0.5060.03 1.3060.17 6.2060.32 3.360.70

Spontaneously hypertensive rats aged 13 weeks were given 2% NaCl in drinking water and food containing OPC-21268 (OPC group) or normal food (control group). Trunk blood samples were taken on day 21 and stored at 2 308C until the assay. Values are expressed as mean6SEM.

Table 2 Organ weights and the body weight ratio in the control and OPC groups on day 21

Body weight [BW] (g) Kidney weight [KW] (g) Heart weight [HW] (g) KW/ BW (%) HW/ BW (%)

Control group (n 5 6)

OPC group (n 5 6)

311.063.0 1.2060.009 1.2860.01 0.3960.004 0.4160.007

314.0614.5 1.1560.03 1.2960.03 0.4060.02 0.4260.02

Spontaneously hypertensive rats aged 13 weeks were given 2% NaCl in drinking water and food containing OPC-21268 (OPC group). Values are expressed as mean6SEM.

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Fig. 3. Photomicrographs show representative histological findings in glomeruli from 13-week-old control SHR (A) and age-matched SHR treated with OPC-21268 (B) (PAS 3 400). No remarkable differences, such as proliferation, sclerosis or tubulointerstitial abnormalities, are visible between the two groups.

in the OPC group compared with the control group (Fig. 4). Mesangial injury score and glomeular cellularity in each group are shown in Table 3, and these parameters

showed no significant differences between the two groups, but the injury score in the OPC group was lower than in the control group.

Fig. 4. Bar graphs show the frequency distribution of glomerular diameters in 13-week-old control SHR (open columns), and age-matched SHR treated with OPC-21268 (closed columns). The histograms of these bar graphs were added in each graph. The glomerular diameters in SHR treated with OPC-21268 tended to be larger than those in control SHR.

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Table 3 Mesangial injury score and cellularity in the control and OPC groups on day 21

Mesangial injury score Cellularity (/ glomerulus)

Control group (n 5 6)

OPC group (n 5 6)

27.663.60 48.561.40

21.662.48 48.360.99

Spontaneously hypertensive rats aged 13 weeks were given 2% NaCl in drinking water and food containing OPC-21268 (OPC group) or normal food (control group). Values are expressed as mean6SEM. No statistically significant differences were found between groups.

3.4. mRNA expressions of growth factors and PCNA in both groups on day 21 Representative mRNA expressions of PDGF B-chain, TGF-b 1 and PCNA in the glomeruli from the control and OPC groups on day 21 are shown in Fig. 5. The values for mRNA expressions of the growth factors and PCNA were all standardized by G3PDH mRNA expression and quantified with a scanning densitometer (Fig. 6). Among these growth factors, PDGF B-chain and PCNA expressions were significantly lower in the OPC group than in the control group. The mRNA expression of TGF-b 1 showed a slight decrease in the OPC group but not significant.

4. Discussion

Fig. 5. Blots show representative mRNA expressions of PDGF B-chain, TGF-b 1, PCNA and G3PDH from glomeruli of 13-week-old control SHR (control group) and age-matched SHR treated with OPC-21268 (OPC group) according to RT-PCR, which was performed using total RNA extracted by the acid–guanidium–phenolchloroform method as described in Section 2. The left lanes in each show molecular weight markers. PDGF B-chain and PCNA mRNA expressions in the OPC group were markedly suppressed in comparison with those in the control group.

We previously reported enhanced pressor response to V1 stimulation in SHR after acute administration of combined VP, V1 and V2 antagonists, suggesting that SHR possess high-density V1 receptors [16], unlike DOCA-salt hypertensive rats [17]. Our data also showed that renal V1 and V2 receptors were increased in SHR compared with age-matched WKY rats, according to radiolabelled receptor assay [18] and quantified autoradiography findings [19,20]. Our data suggested that responsiveness to exogenous VP infusion may be exaggerated in SHR. We detected significant expression of TGF-b 1 mRNA and a lesser degree of PDGF B-chain mRNA in glomeruli from AVPinfused (0.1 and 1 ng / min / kg) SHR, using an osmotic mini-pump for 15 days (unpublished data). We hence designed this study to clarify whether V1 blockade affects renal damage and physiological values associated with hypertension and renal impairment. Although VP is known to be a potent mitogenic agent in vitro [8,9], its effects in vivo have not been clarified. OPC-21268 is a nonpeptide, orally active, selective V1 receptor antagonist [10] that lowers blood pressure acutely in DOCA-salt hypertensive rats [21], in Dahl salt-sensitive hypertensive rats [22], and in the malignant phase of hypertension in stroke-prone SHR [23]. In this study, we observed chronic effects of V1 blockade on glomerular PDGF B-chain and PCNA expressions, but no significant accompanying effect on biochemical or histological changes. Expecting that various factors other than VP affect the glomeruli around the time when proliferative and sclerotic changes visible by light microscopy appear, we used 10week-old SHR, after the establishment of hypertension but before the development of nephropathy. In addition, since a high salt intake is believed to stimulate intrinsic VP secretion [24], suggesting that circulating VP plays a role in hypertension and renal damage in this model, we administered 2% NaCl to 10-week-old SHR for 3 weeks in drinking water. PDGF has been found in mesangial cells in culture [25–27]. In this study, we examined gene expression of

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Fig. 6. Bar graphs show the relative mRNA levels, standardized by G3PDH mRNA expression, for PDGF B-chain, TGF-b 1 and PCNA from glomeruli of 13-week-old control SHR (open columns) and age-matched SHR treated with OPC-21268 (closed columns). Values are expressed as mean6SEM obtained from 6 rats. *P , 0.05, **P , 0.01 versus control SHR.

PDGF B-chain, which is considered to be responsible for the action of PDGF on the kidney [26,27], as an index of the proliferation of glomerular cells. TGF-b is believed to act primarily as an inhibitor but also occasionally as a stimulator of cell proliferation, but a consistent mode of action has not been confirmed [28]. TGF-b has also been reported to increase extracellular matrix by stimulating the production of collagen and non-collagenous protein components and inhibiting their degradation [29–32]. PCNA is an intranuclear polypeptide whose expression and synthesis are linked with cell proliferation [33–35]. The steady-state levels of PCNA mRNA are growth-regulated [36,37]. Since the onset of cellular DNA synthesis is certainly one of the most critical steps in the regulation of cell cycle progression, a study on the regulation of PCNA gene is considerable help in understanding how cellular proliferation is regulated in animal cells [38]. In this study, we examined PCNA mRNA expression in glomeruli, to clarify the extent of alteration of cell proliferation. Yamamura et al. reported that the increase in diastolic blood pressure induced by VP before and after oral administration of 30 mg / kg-body weight of OPC-21268 was about 90% for 4 h in rats [10]; however, in our experiment, there were no differences in systolic blood pressure or heart rate between the two groups during 3-week experimental period under feeding with the adequate dose of OPC-21268. Time courses of changes in urinary volume and sodium, protein, NAG also did not differ significantly between groups. Nevertheless, there were significant differences in PDGF B-chain and PCNA expressions in glomeruli between the OPC and control groups. These observations suggest that chronic V1 receptor blockade directly inhibits glomerular growth and proliferative mechanisms. Furthermore, in this experiment, no histopathological changes, including glomerular or interstitial proliferation, were seen with the administration of OPC-21268. This glomerular histopathological finding was confirmed by the lack of significant alteration of the mesangial injury score and cellularity in either group.

However, the diameter of the glomeruli tended to increase in the OPC group. The enlargement of glomeruli in the OPC group may be attributable to the volume retention induced by the enhanced V2 actions due to both V1 blockade and salt-loading, but the plasma VP concentration in the trunk blood samples did not differ significantly under the chronic blockade of the V1 receptor from the control. Besides, the glomerular relaxation induced by extinction of the mesangial contractile effects via the V1 receptor [39,40] may be involved in the glomerular enlargement in the OPC group. In hypertensive renal damage, it is well-known that the hypertensive state enhances renal proliferative injury, and that the amelioration of hypertension can itself inhibit the progression of such glomerular damage [41,42]. In our chronic study, a V1 receptor antagonist appeared to inhibit glomerular cell proliferative factors such as PDGF B-chain and PCNA directly, since the V1 antagonist exerted these effects without altering blood pressure, heart rate or biochemical values. Although suppression of the PDGF B-chain by OPC-21268 administration is probably followed by PCNA suppression, the differences between the suppressive effects on PCNA and on PDGF B-chain expression in glomeruli suggest that other growth factors may also involve in this cell proliferative cycle. This study first elucidated that a chronic V1 blockade directly inhibited the hypertensive glomerular injury in vivo. In summary, this in vivo study showed that chronic V1 antagonism using OPC-21268 suppressed the expressions of PDGF B-chain and PCNA mRNA in the renal glomeruli of SHR in the established hypertension stage. It is possible that this agent may have been beneficial in the prevention of hypertensive nephropathy.

Acknowledgements We thank Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan), for the donation of OPC-21268.

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