Effect of continuous infusion of vasopressin on glomerular growth response in spontaneously hypertensive rats

Regulatory Peptides 74 (1998) 11–18

Effect of continuous infusion of vasopressin on glomerular growth response in spontaneously hypertensive rats a, b a a a Kazushi Harada *, Toshio Ogura , Takayoshi Yamauchi , Fumio Otsuka , Yukari Mimura , Masami Hashimoto a , Tetsuya Oishi a , Hirofumi Makino a a

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

Received 15 October 1997; received in revised form 21 January 1998; accepted 21 January 1998

Abstract Vasopressin (VP) is thought to play an important role in the pressor and proliferative responses of renal glomeruli. We have utilized the spontaneously hypertensive rat (SHR) model to determine if glomerular proliferation is induced by chronic infusion of exogenous VP. SHR were continuously infused with 0.1 ng / kg / min VP (H-VP group), 1.0 ng / kg / min (H-VP group), or vehicle alone (control group) for fifteen days using osmotic minipumps, and the histological alterations and level of expression of platelet-derived growth factor B-chain (PDGF-B) and transforming growth factor (TGF)-b1 mRNA were determined. We observed no significant differences in systolic blood pressure, heart rate, serum electrolytes, protein and creatinine among the three groups of rats, but urine volume was found to be significantly decreased, and urine osmolality significantly increased, in the H-VP group. Kidney weight was significantly higher in the H-VP and L-VP groups than in the control group, and glomerular diameter was higher in the H-VP group. When we measured mesangial injury score and cellularity in the glomeruli of these animals, we observed VP dose-dependent proliferative changes. In the immunofluorescence study, although we did not find an obvious difference in depositions of collagen types III, IV and VI, a-smooth muscle actin and PDGF-B among the groups, the collagen type I and TGF-b1 increased in several glomeruli in the H-VP group. Reverse transcription polymerase chain reaction (RT-PCR) revealed no significant differences in the glomerular levels of PDGF-B mRNA among the three groups of rats, but the level of expression of TGF-b1 mRNA was significantly higher in the L-VP and H-VP groups than in the control group. These findings suggest that VP may contribute to glomerular proliferation, and that VP may exert its effects in part through the induction of TGF-b1 expression. These results also raise the possibility that blockade of VP receptors may be useful in the treatment of some forms of glomerular disease.  1998 Published by Elsevier Science B.V. Keywords: Osmotic minipump; TGF-b1; PDGF-B; Mesangial injury; RT-PCR

1. Introduction The glomerular mesangium is a branching-tree-shaped structure, composed of mesangial cells and the surrounding mesangial matrix, which supports the glomerular capillary network. The mesangium has been shown to be important in maintaining the microcirculation within the glomerulus. During the course of glomerular injury, mesangial cells are acted upon by a variety of mediators, which are either *Corresponding author: Tel.: 1 81 86 235 7235; fax: 1 81 86 222 5214.

released locally or are present in the systemic circulation [1]. Among these mediators are platelet-derived growth factor B-chain (PDGF-B), endothelin-1, angiotensin II (Ang II), interleukin-1, interleukin-6, epidermal growth factors [1–4] and vasopressin (VP), all of which have been shown to be potent mitogens for rat mesangial cells in culture [5,6]. In addition, transforming growth factor (TGF)-b1 has been reported to have both stimulatory [7] and inhibitory [7,8] effects on the proliferation of the renal glomerulus in vitro. We previously reported that the kidneys of spontaneously hypertensive rats (SHR) possess a higher number of

0167-0115 / 98 / $19.00  1998 Published by Elsevier Science B.V. All rights reserved. PII S0167-0115( 98 )00009-3

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renal VP receptors than do kidneys of age-matched Wistar Kyoto (WKY) rats [9,10]. We also showed that the acute administration of VP induced an enhanced pressor response in SHR [11], but not in deoxycorticosterone acetate (DOCA)-salt hypertensive rats [12], and that this enhancement was due to V1 stimulation. In addition, we found that chronic treatment of SHR with OPC-21268, a V1 receptor antagonist, attenuated the expression of proliferating cell nuclear antigen (PCNA) and PDGF-B in the glomeruli [13]. These findings suggest that VP may play an important role in the pressor and proliferative responses that occur in the glomeruli of SHR. To determine if the proliferative responses in the glomeruli would be induced by a chronic infusion of exogenous VP, we assayed the histological alterations resulting from chronic administration of two concentrations of VP to SHR, as well as any changes in the levels of expression of PDGF-B and TGF-b1 mRNA induced by VP.

paraformaldehyde, embedded in paraffin, sectioned at 4 mm, and stained with periodic acid Schiff (PAS) reagents. The diameter (mm) of the glomeruli (200–300 in each group) was measured with an objective micrometer (OBM, Olympus Optical Co. Ltd., Tokyo, Japan). Glomerular cellularity was determined by counting the number of nucleated cells in each glomerulus, assaying at least 100 glomeruli in each specimen. The severity of glomerular sclerosis was assessed using the mesangial injury score (MIS) [14], examining a minimum of 100 glomeruli in each specimen. The severity was graded from 0 to 4 1 according to the percentage of glomerular involvement. The 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 [13].

2.3. Immunofluorescence microscopy 2. Materials and methods

2.1. Implantation of osmotic minipump infusion of VP Eighteen male SHR (seven weeks old; 100–120 g), obtained from Charles River Japan (Kanagawa, Japan), were housed in climate-controlled metabolic cages with a 12-h light / 12-h dark cycle and provided food (MF, Oriental Yeast Co., Tokyo, Japan) and water ad libitum. Osmotic minipumps (Alza, CA, USA) were implanted subcutaneously into the backs of rats anesthetized with sodium pentobarbital. Arginine VP (Peptide Institute, Osaka, Japan) in lactated Ringer’s solution, was administered by subcutaneous infusion at concentrations of 0.1 and 1.0 ng / kg / min to the L-VP and H-VP groups of rats, respectively, while the control rats were infused with vehicle (lactated Ringer’s solution) alone. The systolic blood pressure and heart rate of conscious, restrained animals were measured at room temperature by tail-cuff plethysmography (UR-5000, Ueda Seisakusyo, Tokyo, Japan) at 9 a.m. on days 2 2, 2, 5, 10 and 15. Urine samples were collected from rats individually on days 2 1, 3 and 10. On day 15, the rats were sacrificed by decapitation. The kidneys were quickly removed and weighed, and portions of each were processed for histological evaluation and for extraction of total RNA. Truncal blood samples were collected and stored at 2 308C, and the concentration of electrolytes, creatinine, uric acid, total albumin and alanine aminotransferase (ALT) were measured by an autoanalyzer system.

2.2. Histological examination A portion of each kidney was fixed in 10% buffered

A portion of each kidney was immersed in OCT compound (TISSUE TEK, Miles Scientific, London, UK) and snap-frozen at 2 208C in a hexane / dry ice–acetone bath. Sections (4 mm) were cut using a cryostat microtome and incubated for 60 min at room temperature with rabbit anti-rat type I collagen antibody (Chemicon International, Temecula, CA, USA), rabbit anti-rat type III collagen antibody (Chemicon International), rabbit anti-bovine type IV collagen antibody (LSL, Tokyo, Japan), rabbit antibovine type VI collagen antibody (LSL), mouse antihuman a-smooth muscle actin (DAKO, Kyoto, Japan), rabbit anti-human PDGF-B (Genzyme, Cambridge, MA, USA), and rabbit anti-human TGF-b1 (King Brewing, Kakogawa, Japan). The samples were washed and incubated with fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG or goat anti-mouse IgG antibody for 30 min at room temperature and examined using a fluorescence microscope (Olympus, Tokyo, Japan).

2.4. Separation of glomeruli and extraction of total RNA [15] The renal cortex of each kidney was dissected out and minced in ice-cold phosphate-buffered saline (PBS). Glomeruli were isolated by the grade sieving technique, rinsed with ice-cold PBS, and incubated for 30 min at 378C with 2 mg / ml collagenase (Wako, Osaka, Japan) in RPMI1640 medium. Total RNA was extracted from each sample by the acid–guanidium–phenol–chloroform method. The resulting RNA pellets were washed with 70% ethanol and resuspended in 100 ml of diethylpyrocarbonate-treated water. The RNA was quantified by measuring the absorbance at 260 nm and stored at 2 208C.

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2.5. Reverse transcription ( RT) of RNA and polymerase chain reaction ( PCR) Reverse transcription of each RNA sample was performed with MMLV reverse transcriptase (United States Biochemical, Cleveland, OH, USA) following the manufacturer’s protocol. Amplification of sequences specific for the PDGF-B and TGF-b1 genes was performed in a thermal cycler (TP Cycler-100, Toyobo, Osaka, Japan) using Takara Taq DNA polymerase (Takara Shuzo, Otsu, Shiga, Japan) and oligonucleotide primers, synthesized on a Model 380-B DNA synthesizer (Applied Biosystems, Foster City, CA, USA), corresponding to 1079–1098 and 1601–1620 of the PDGF-B gene [16] or 1679–1699 and 1994–2014 of the TGF-b1 gene [17]. Amplification was done for 30–35 cycles of denaturation at 948C for 1 min, annealing at 55–608C for 2 min, and extension at 728C for 3 min. Aliquots of the PCR products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. The intensity of the bands was measured with a quantitative scanning densitometer (Scanning Imager 300SX, Molecular Dynamics, Sunnyvale, CA, USA). Relative PDGF-B and TGF-b1 mRNA levels were determined after normalization against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message.

2.6. Statistics Results are expressed as means6SEM. Statistical analysis utilized the paired Student’s t-test and the ANOVA test. A level of p , 0.05 was accepted as being statistically significant.

Fig. 1. Time course of (A) systolic blood pressure and (B) heart rate in spontaneously hypertensive rats administered vasopressin. s, control; d, L-VP; h, H-VP. n 5 6 in each group.

3. Results

3.1. Effect of VP infusion on blood pressure and heart rate Although we found that the systolic blood pressure in all three groups of rats increased during the course of the infusion, we observed no significant differences in systolic blood pressure among the three groups (Fig. 1A). We also observed no significant differences in heart rate among the three groups of animals (Fig. 1B).

3.2. Effect of VP infusion on urine and blood chemistry In the H-VP group of rats, the volume of urine decreased with time; in contrast, we observed no significant change in urine volume in the L-VP and control groups (Fig. 2). The urine osmolality of the H-VP group increased significantly ( p , 0.01), from 865696 mOsm / kg on day 2 1 to 2095687 mOsm / kg on day 10. In contrast, we detected no significant differences in urinary protein and sodium excretion among the three groups of rats (Table 1).

Fig. 2. Changes in urine volume in the control, L-VP and H-VP groups. Values in each group are expressed relative to that on day 2 1 in that group. ** p , 0.01 compared with the value on day 2 1. n 5 4 in each group.

We observed no significant differences among the three groups of animals when we measured the serum concentrations of sodium, potassium, chloride, calcium, albumin, creatinine, uric acid and ALT in trunk blood on day 15 (Table 2).

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Table 1 Urinary protein and sodium excretion in VP-infused SHR Control group Urinary protein excretion (mg /day) Day 2 1 3.361.3 Day 10 27.465.0 Urinary sodium excretion (mEq /day) Day 2 1 1.1160.05 Day 10 0.8660.12

L-VP group

H-VP group

4.361.0 25.265.1

8.666.7 21.460.6

1.3160.05 0.6960.18

1.4560.04 0.8060.10

SHR were infused with lactated Ringer’s solution (control group), 0.1 ng / kg / min VP (L-VP group) or 1.0 ng / kg / min VP (H-VP) group. n 5 4 in each group.

Table 2 Blood chemistry of VP-infused SHR

Sodium (mEq / l) Potassium (mEq / l) Chloride (mEq / l) Calcium (mg / dl) Albumin (g / dl) Creatinine (mg / dl) Uric acid (mg / dl) ALT (IU / l)

Control group

L-VP group

H-VP group

13860.6 8.860.5 10360.4 10.160.2 3.360.2 0.4160.02 1.760.2 3061.1

13860.3 9.260.4 10360.2 10.160.2 3.360.1 0.4360.02 1.860.2 3260.6

13761.5 9.260.5 10260.9 10.360.3 3.460.2 0.4060.02 1.760.2 3061.2

The groups of rats are described in the legend to Table 1. All measurements were performed on day 15. ALT: alanine aminotransferase; n 5 6 in each group.

Fig. 3. Photomicrographs of PAS-stained glomeruli on day 15 in (A) control, (B) L-VP and (C) H-VP rats. 3 400.

glomeruli in the control group, whereas we observed moderate proliferative changes in several glomeruli in the H-VP group (Fig. 3). Typical tubular and interstitial changes, however, were not detected in any of these specimens. These findings were quantitatively confirmed when we assayed MIS and glomerular cellularity in the kidneys of these rats. Compared with the control group, both of these parameters were elevated in both VP-infused groups (Table 4). In measuring glomerular size in the three groups of rats, we observed larger glomeruli in the H-VP group than in the L-VP and control groups (Fig. 4). We also assayed the expression of collagens, a-smooth muscle actin, PDGF-B and TGF-b1 in the glomeruli by immunohistochemistry. Although we did not find an obvious difference in depositions of collagen types III, IV and VI, a-smooth muscle actin and PDGF-B among the

3.3. Effect of VP infusion on organ weight Although body weight was similar in the three groups of rats, the kidney weight and the ratio of kidney weight to body weight were significantly higher in both groups of rats infused with VP than in the control group. In addition, the heart weight and the ratio of heart weight to body weight in the H-VP group were significantly increased compared with the control group (Table 3).

3.4. Effect of VP infusion on renal histology

Table 4 Mesangial injury score (MIS) and cellularity in VP-infused SHR Control group MIS Cellularity

19.064.9 48.961.7

L-VP group 23.366.9 50.561.7

a

H-VP group 37.5611.7 a c 55.061.5 a

The groups of rats are described in the legend to Table 1. All measurements were performed on day 15. MIS, mesangial injury score. Cellularity, nuclear cells / glomerulus; a p , 0.05 compared with control group; c p , 0.05 compared with L-VP group. n 5 6 in each group.

When we examined the renal histology of these rats after fifteen days of infusion, we found virtually intact

Table 3 Body and organ weights in VP-infused SHR

BW (g) KW (g) HW (g) KW/ BW HW/ BW

Control group

L-VP group

H-VP group

244.063.2 0.9860.02 0.9960.01 0.4060.01 0.4160.01

247.563.3 1.0760.04 b 1.0660.02 0.4460.01 b 0.4360.02

242.563.1 1.0860.03 1.2160.06 0.4460.01 0.5060.02

b b b ac

The groups of rats are described in the legend to Table 1. All measurements were performed on day 15. BW, body weight; KW, kidney weight; HW, heart weight; a p , 0.05, b p , 0.01 compared with control group; c p , 0.05 compared with L-VP group. n 5 6 in each group.

Fig. 4. Frequency distribution of glomerular diameters on day 15 in VP-infused SHR. h, control; , L-VP; j, H-VP.

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rats (Fig. 6). When we normalized the expression of these two transcripts against that of GAPDH, we detected a significant increase in the expression of TGF-b1 mRNA in both the L-VP and H-VP groups compared with the control group (Fig. 7). In contrast, the expression of PDGF-B message was only slightly higher in the L-VP and H-VP rats than in the control rats, but the differences were not statistically significant (Fig. 7).

4. Discussion

Fig. 5. Photomicrographs of representative immunofluorescent reactions of the control, L-VP and H-VP glomeruli with antibodies directed against (A) collagen type I, (B) TGF-b1. 3 200.

Fig. 6. RT-PCR determination of the expression of PDGF-B, TGF-b1 and GAPDH mRNA in glomeruli of a representative control (Lane 1), L-VP (Lane 2) and H-VP (Lane 3) rats on day 15. Molecular size markers are shown in Lane lt.

groups, collagen type I and TGF-b1 increased within several glomeruli in the H-VP group (Fig. 5).

3.5. Effect of VP infusion on the expression of glomerular PDGF-B and TGF-b1 mRNA We also assayed the expression of PDGF-B and TGF-b1 mRNA by RT-PCR in the glomeruli of the three groups of

Fig. 7. Densitometric analysis of the relative expression of (A) PDGF-B and (B) TFG-b1 mRNA in h, control; , L-VP; j, H-VP rats. All values were normalized against GAPDH expression in the same sample. * p , 0.01 vs. control group. n 5 6 in each group.

VP plays important physiological roles in vasoconstriction and antidiuresis, suggesting that it may be involved in the regulation of systemic blood pressure in hypertension. Using the SHR animal model of hypertension, several groups of researchers have attempted to elucidate the contribution of VP to the hypertensive process [18–20]. In a previous study, we demonstrated that the acute administration of VP induced a pressor response in both SHR and WKY rats, and that acute V1 stimulation induced a greater pressor response in SHR than in WKY rats [11]. In addition, a larger number of renal VP receptors was detected in hypertensive SHR than in age-matched WKY rats [9,10], suggesting a mechanism for the enhanced response to exogenous VP infusion observed in SHR. We therefore sought to determine if the chronic infusion of VP induces a pressor response and glomerular proliferation in SHR through the expression of growth factors. We demonstrated here that the chronic infusion of VP does not significantly affect the blood pressure or heart rate in SHR. It has been shown in dogs that direct intra-arterial infusion of high doses ( $ 10 ng / kg / min) of VP causes a pressor response in a dose-dependent manner [21]. In addition, it was demonstrated that administration of the V1 receptor antagonist, OPC-21268, for four weeks attenuated the development of hypertension in young SHR, suggesting that VP contributes to hypertension in SHR [22]. In contrast, the administration of physiological doses of VP does not elevate the blood pressure [23]. Chronic and selective VP blockade, using the V1 and V2 receptor antagonists, OPC-21268 and OPC-31260, respectively, resulted in a lower than expected effect on blood pressure by circulating VP [24]. These results suggest that VP affects the systemic blood pressure in SHR only when administered in high doses or in the presence of VP receptor antagonists. When administered chronically, however, the effects of VP on systemic blood pressure may be offset by homeostatic regulation or by the compensatory effect of other vasoactive substances. We have also shown that the chronic infusion of VP causes a significant and dose-dependent increase in glomerular proliferation and mesangial expansion in nineweek-old SHR. Notably, these changes were accompanied by a significant quantitative increase in TGF-b1, but not in

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PDGF-B message in the glomeruli. Besides, we detected an increase in protein level expression on TGF-b1 in an immunofluorescence study, but the depositions were observed in several (possibly injured) glomeruli, but not in all glomeruli in the H-VP group. In vascular smooth muscle cells (VSMCs), VP stimulation has been shown to induce rapid contraction [25], and prolonged VP stimulation, and, in the absence of other growth-promoting agents, has been shown to cause hypertrophy [26]. TGF-b1 has been believed to have biphasic effects, i.e. it acts primarily as an inhibitor, but may also occasionally act as a stimulator, of cell proliferation [27]. TGF-b1 has been reported to be secreted from the cortex and glomeruli in an inactive (latent) form and then to become activated in response to tissue injury or local inflammation [28]. This continuous and slow induction of the active form of TGF-b1 may be characteristic of TGF-b1-induced tissue injuries in chronic models of renal disease. A second functional activity of TGF-b1 is its ability to increase the extracellular matrix by stimulating the production of collagen and non-collagenous protein components and by inhibiting their degradation [29,30]. Since collagen types I and III were found to be increased in expanded mesangial cells and in hyalinized or crescentic lesions in biopsy specimens from patients with various types of glomerulonephritis, it suggested that the level of expression of these types of collagen may be markers of glomerular proliferation [31]. It has also been reported that collagen types I, III, IV and V have been shown to be produced in mesangial cells in certain pathological conditions [32] and in cultured rat kidney epithelial (NRK52E) cells [33]. Recently, we found that the increase in collagen type III in the glomerulus of Dahl-salt-sensitive rats was attenuated by treatment with an Ang II type 1 (AT 1 ) receptor antagonist, TCV 116 [34]. In the present study, when we assayed the levels of various collagens in the glomeruli of VP-infused and control SHR, we detected an increase in only collagen type I in the glomeruli of the H-VP group. Concerning the a-smooth muscle actin, which is used as a marker of glomerular damage, we observed no differences in its expression between VP-infused and control SHR. Since a-smooth muscle actin has been detected within sclerotic glomeruli in salt-loaded SHR, resulting from the phenotypic modulation of mesangial cells [35], it may not be detected within the mildly proliferative glomeruli induced by chronic infusion with VP. The B chain of PDGF is believed to be responsible for the action of PDGF on the kidney [36]. PDGF, a potent mitogen for mesangial cells in culture, is also thought to induce the proliferation of glomerular cells in vivo [36,37]. We have previously shown that PDGF-B mRNA expression in SHR [15] and DOCA [38] glomeruli was attenuated by treatment with an angiotensin-converting enzyme (ACE) inhibitor or with an AT 1 receptor antagonist, suggesting that Ang II may induce glomerular prolif-

eration via expression of PDGF-B. We also showed that the glomerular expression of PDGF-B and PCNA in SHR fed a high salt diet was reduced by chronic treatment with a V1 receptor antagonist [13]. In SHR, however, VP infusion had no effect on either the protein or message level of PDGF-B abundance. It is possible that the discrepant results on PDGF-B expression between previous reports and the present experiments depend upon the different conditions of salt loading or V2 action, which may be activated under V1 antagonism. In contrast, it has been shown, in cultured rat aortic smooth muscle cells, that increases in protein synthesis induced by Ang II and VP are not dependent on autocrine secretion of PDGF [39]. It is not yet clear, however, whether or not the effects of VP on PDGF-B expression in SHR glomeruli differ from those of Ang II. It is especially uncertain because of the unusual nature of the biological effect of VP in SHR, including enhanced intracellular sodium concentration, due to an increase in V1 receptor numbers [40], enhanced pressor activity due to an impairment in baroreflex activity [41] and attenuated chloride-induced contraction of mesangial cells [42]. Recently, the relationship between glomerular hypertension and proliferation has been actively investigated, and it has been hypothesized that glomerular hypertension-induced proliferation arising from mechanical stress occurs via an autocrine or paracrine mechanism, through the activation of protein kinase C [43] or the expression of various cytokines [44]. We have shown here that infusion of the higher VP concentration (1.0 ng / kg / min) induced increases in glomerular size and kidney weight. Although these rats also had the highest MIS and cellularity, we cannot confirm that the increases in glomerular size and kidney weight are due to increased glomerular capillary pressure, inasmuch as we did not measure the latter in these animals. It is also possible that VP, via its V2 receptors, may have an antidiuretic effect, resulting in increased kidney weight and glomerular size. In fact, in a previous study, we observed that the increased kidney weight and glomerular size in DOCA-salt hypertensive rats, which also have significant glomerular proliferation, were attenuated by treatment with an ACE inhibitor, suggesting a relationship between glomerular size and glomerular proliferation [38]. We have thus demonstrated that continuous infusion of SHR with VP causes a significant and dose-dependent increase in glomerular proliferation without altering systemic blood pressure, as well as a significant increase in TGF-b1 mRNA expression. These findings suggest that VP may be involved in glomerular proliferation and in the expansion of the mesangial matrix in vivo, and that the effects of VP may be mediated, at least in part, via induction of TGF-b1 expression. In addition, since we did not clarify whether these effects of VPs were unique to SHR, we should investigate the difference of glomerular growth response induced by VP between SHR and WKY

K. Harada et al. / Regulatory Peptides 74 (1998) 11 – 18

rats in a future study. Although the mechanism underlying the contribution of VP to hypertension and mesangial proliferation is not yet known, our results suggest that chronic blockade of the proliferative effects of VP may be useful in treating some forms of glomerular disease.

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We thank Professor emeritus Z. Ota of Okayama University for helpful advice and encouragement during this study.

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