Articles

Endothelin Production in Cultured Mesangial Cells of Spontaneously Hypertensive Rats

Miwako Ikeda, Masakazu Kohno, Tadanao Takeda
https://doi.org/10.1161/01.HYP.25.4.1
Hypertension. 1995;25:1-6
Originally published April 1, 1995

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Abstract

Abstract Cultured glomerular mesangial cells have been shown to produce a potent vasoconstrictive peptide, endothelin-1 (ET-1). We examined whether basal or stimulated ET-1 production by angiotensin II (Ang II) or arginine vasopressin (AVP) is enhanced in cultured mesangial cells of spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY). Basal ET-1 production in SHR mesangial cells was not different from that in WKY cells although a trend toward increased ET-1 production was observed in SHR cells. Ang II and AVP stimulated ET-1 production in a concentration-dependent manner in cells of both rat strains, but the agonist-induced stimulation was clearly greater in SHR cells than WKY cells. The protein kinase C–activating phorbol ester phorbol myristate acetate stimulated ET-1 production in a concentration-dependent manner in cells of both rat strains, but this stimulation was significantly greater in SHR cells than in WKY cells. These results suggest that Ang II– and AVP-induced mesangial cell production of ET-1 is clearly enhanced in SHR compared with WKY. Increased response of ET-1 production to protein kinase C activation appears to contribute in part to the observed enhancement of ET-1 production in SHR mesangial cells.

Endothelin-1 (ET-1) is a vasoconstrictive peptide of 21 amino acids that was first isolated from porcine vascular endothelial cells.1 This peptide acts not only on vascular smooth muscle cells1 2 but also on other cell types, including glomerular mesangial cells.3 4 In cultured mesangial cells, ET-1 binds its specific receptors, enhances phosphoinositide turnover, and induces contraction and proliferation of these cells.3 4 5 Previous studies demonstrated constitutive expression of ET-1 transcripts and peptide secretion in cultured glomerular mesangial cells.6 7 This ET-1 production by mesangial cells is found to be stimulated by angiotensin II (Ang II)8 9 and arginine vasopressin (AVP).10 11 Recent evidence indicates that activation of protein kinase C (PKC) may be involved in the stimulation of ET-1 production by these vasoactive peptides.8 11 12 In human endothelial cells, phorbol esters, activators of PKC, cause a marked and immediate stimulation of ET-1 production, accompanied by induction of preproendothelin mRNA.13 14

On the other hand, ET-1 has been reported to play a role in a variety of diseases that affect the kidney, including severe hypertension,15 16 17 acute renal failure,18 19 20 and cyclosporine nephrotoxicity.21 22 In fact, we have previously shown that plasma ET-1 levels are increased in hypertensive patients with impaired renal function15 and in severely hypertensive rats with renal damage.16 Therefore, if mesangial cell production of ET-1 is enhanced in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY), increased endogenous ET-1 in these cells may contribute to the pathophysiology of the renal involvement associated with the progression of hypertension in SHR. Accordingly, we designed the present study to test two hypotheses: (1) whether basal, Ang II–, or AVP-induced mesangial cell production of ET-1 is enhanced in SHR compared with WKY and (2) whether phorbol ester–induced mesangial cell production of ET-1 is enhanced in SHR compared with WKY. In addition, we examined which receptor subtypes of Ang II and AVP mediated ET-1 production in these cells.

Methods

Mesangial Cell Culture

Glomeruli from SHR and WKY rats (weight, 50 to 100 g) were isolated by sieving with stainless steel and nylon meshes under sterile conditions as previously reported.8 11 The isolated glomeruli were then cultured in RPMI 1640 medium containing 20% fetal calf serum (FCS) and antibiotics. The identity of the mesangial cells was confirmed by (1) morphology, (2) typical microfilaments seen by transmission electron microscopy, (3) survival in a medium containing d-valine substituted for l-valine, indicating the existence of d-amino acid oxidase, (4) resistance to puromycin aminonucleoside but susceptibility to mitomycin C, (5) the presence of receptors specific to Ang II and contraction in response to Ang II, and (6) the absence of immunofluorescence with factor VIII antibody. The cultures were maintained at 37°C with atmospheric air and 5% CO2, and subculture was carried out after treatment with Versene followed by trypsin. Cells after passages 4 to 8 were used for this experiment.

Materials

Ang II, AVP, [1-(β-mercapto-β,β-cyclopentamethylene propion acid), 2-(O-methyl) tyrosine]arginine vasopressin (PMP), phorbol myristate acetate (PMA), and 4α-phorbol-12,13-didecanoate (4α-PDD) were purchased from Sigma Chemical Co. RPMI 1640, trypsin, Versene, and FCS were purchased from GIBCO Laboratories. Flasks were purchased from Becton Dickinson and Co. ET-1, ET-2, ET-3, and big ET-1 [porcine (1-39)] were purchased from Peptide Institute, Inc. ET-1 antiserum was purchased from Peninsula Laboratories Inc. 125I–ET-1 was purchased from Amersham Japan Inc. The selective angiotensin subtype 1 (AT1) receptor antagonist losartan was donated by Merck Sharp & Dohme. The selective AT2 receptor antagonist PD 123319 was donated by Parke-Davis.

Pharmacological Treatment

The culture medium was removed, and the cell monolayers were washed twice with serum-free RPMI 1640. SHR and WKY mesangial cells were exposed to different concentrations (10−7, 10−8, and 10−9 mol/L) of Ang II and AVP for 24 and 48 hours. In separate experiments, losartan and PD 123319 were added to the well 5 minutes before addition of Ang II, and the cells were incubated for 24 hours. The V1 receptor antagonist PMP was added to the well 5 minutes before addition of AVP, and the cells were incubated for 24 hours.

To examine the effect of the PKC-activating phorbol ester PMA on ET-1 production in cells of both rat strains, we exposed cells to different concentrations (10−7, 10−8, and 10−9 mol/L) of PMA for 24 hours. In addition, to confirm the importance of PKC-dependent mechanisms in the stimulation of ET-1 production, we examined the effect of PKC depletion. PKC was depleted by preincubation with a high dose of PMA (10−7 mol/L) for 24 hours in cells of both rat strains.

All experiments were performed with 2 mL RPMI 1640 under quiescent (0.5% FCS) conditions. After incubation, the medium was aspirated and centrifuged at 3000g for 10 minutes and the supernatant collected and stored at 80°C until radioimmunoassay.

Measurement of ET-1 Concentration

ET-1 was extracted as previously described.23 Briefly, 1.5 mL of each sample was diluted with 4 mL of 4% acetic acid. After centrifugation, the solution was pumped at the rate of 1 mL/min through a Sep-Pak C18 cartridge (Millipore Corp). After evaporation of the eluate with 86% ethanol in 4% acetic acid by a centrifugal evaporator (model RD-31, Yamato Scientific Co), the dry residue was dissolved in the assay buffer described below. The recovery rate was found by addition of three different quantities of cold ET-1 (4, 20, and 40 pmol/L) to serum-free RPMI 1640. Recovery was 69±2%.

ET-1 concentration was assayed using ET-1 antiserum and 125I–ET-1 as a tracer. This antibody reacts 100% with ET-1 and cross-reacts 7% with ET-2, 7% with ET-3, and 35% with big ET-1 [porcine (1-39)]. The antiserum did not cross-react with somatostatin, β-endorphin, human secretin, Ang II, or AVP.

Radioimmunoassay was performed in an assay buffer of 0.01 mol/L sodium phosphate, pH 7.4, containing 0.05 mol/L NaCl, 0.1% bovine serum albumin, 0.1% Nonidet P-40, and 0.01% NaN3, as previously described.23 In brief, rehydrated antiserum (100 μL) was added to 100 μL of the sample or 100 μL of standard ET-1 dissolved in the assay buffer, and the mixture was incubated for 24 hours at 4°C. Approximately 15 000 cpm of 125I–ET-1 was added to each reaction and incubated for an additional 24 hours. After this incubation, 100 μL of diluted normal rabbit serum and 100 μL of diluted goat anti-rabbit IgG were added, and the mixture was again incubated for 24 hours. After the third incubation, the precipitate was collected by centrifugation at 1700g for 30 minutes. The supernatant was removed by aspiration and the pellet counted for 125I with a gamma counter. The detection level of this assay was 0.08 pmol/L (range, 0.08 to 80 pmol/L). The interassay variation was 13%, and the intraassay variation was 7%.

Ang II and AVP did not interfere with the radioimmunoassay.

Calculations and Statistical Analysis

The statistical significance of differences in the results was evaluated by ANOVA, and probability values were calculated by Scheffé’s method.24 Values are expressed as mean±SD.

Results

Effects of Ang II and AVP on ET-1 Production

Confluent cultured mesangial cells of both rat strains produced ET-1 in a time-dependent manner. Under quiescent conditions (0.5% FCS), basal ET-1 production of SHR cells was not different from that of WKY cells, although a trend toward increased ET-1 production was observed in SHR cells (Figs 1⇓ and 2⇓).

Figure 1.
Figure 1.

Bar graphs show effects of angiotensin II (Ang II) on endothelin-1 production in cultured mesangial cells of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Cells were exposed to different Ang II concentrations for 24 and 48 hours at 37°C. Each value is the mean±SD of six cell cultures. *P<.05 vs basal production in WKY; **P<.05 vs basal production in SHR; †significant difference between SHR and WKY (P<.05).

Figure 2.
Figure 2.

Bar graphs show effects of arginine vasopressin (AVP) on endothelin-1 production in cultured mesangial cells of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Cells were exposed to different AVP concentrations for 24 and 48 hours at 37°C. Each value is the mean±SD of six cell cultures. *P<.05 vs basal production in WKY; **P<.05 vs basal production in SHR; †significant difference between SHR and WKY (P<.05).

Ang II stimulated ET-1 production in a concentration-dependent manner between 10−7 and 10−9 mol/L in cells of both rat strains. However, the stimulation of ET-1 production was clearly greater in SHR cells than in WKY cells (Fig 1⇑).

AVP stimulated ET-1 production in a concentration-dependent manner between 10−7 and 10−9 mol/L in cells of both strains. The stimulation by AVP of ET-1 production appeared to be greater than that by Ang II in cells of both rat strains. However, the stimulation of ET-1 production was clearly greater in SHR cells than in WKY cells (Fig 2⇑).

Effects of Ang II Receptor Antagonists on ET-1 Production

Effects of losartan and PD 123319 on Ang II–stimulated production of ET-1 in cultured mesangial cells of both rat strains are shown in Fig 3⇓. Preincubation of the cells with 10−6 mol/L losartan 5 minutes before the addition of 10−7 mol/L Ang II abolished the Ang II–mediated increase of ET-1 production in both strains. However, preincubation of the cells with 10−6 mol/L PD 123319 5 minutes before the addition of 10−7 mol/L Ang II had no effect on ET-1 production. This suggests that the AT1 receptor is coupled to Ang II–mediated ET-1 production in cultured mesangial cells of both rat strains.

Figure 3.
Figure 3.

Bar graph shows effects of losartan and PD 123319 (PD) on angiotensin II (Ang II)–stimulated production of endothelin-1 in cultured mesangial cells of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Cells were preincubated with 10−6 mol/L losartan or PD 123319 for 5 minutes before the addition of 10−7 mol/L Ang II and then were incubated for 24 hours. Each value is the mean±SD of four cell cultures. *P<.05 vs basal production; †P<.05 vs Ang II alone.

Effects of V1 Receptor Antagonist on ET-1 Production

Effects of the selective V1 receptor antagonist PMP on AVP-stimulated production of ET-1 in cultured mesangial cells of both rat strains are shown in Fig 4⇓. Preincubation of the cells with 10−6 mol/L PMP 5 minutes before the addition of 10−7 mol/L AVP abolished the AVP-mediated increase of ET-1 production. This effect of PMP was observed in cells of both strains, suggesting that the V1 receptor is coupled to AVP-mediated ET-1 production in cultured mesangial cells of both rat strains.

Figure 4.
Figure 4.

Bar graph shows effects of the selective V1 receptor antagonist PMP on arginine vasopressin (AVP)–stimulated production of endothelin-1 in cultured mesangial cells of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Cells were preincubated with 10−6 mol/L PMP for 5 minutes before the addition of 10−7 mol/L AVP and then were incubated for 24 hours. Each value is the mean±SD of four cell cultures. *P<.05 vs basal production; †P<.05 vs AVP alone.

Effects of PMA on ET-1 Production

Effects of the PKC-activating phorbol ester PMA on ET-1 production in cultured mesangial cells of both rat strains are shown in Fig 5⇓. PMA stimulated ET-1 production in a concentration-dependent manner between 10−9 and 10−7 mol/L in cells of both rat strains, although the increase by 10−9 mol/L PMA in WKY cells was not statistically significant. However, this stimulation of ET-1 production was clearly greater in SHR cells than in WKY cells. An inactive enantiomer of phorbol ester, 4α-PDD, had no effect on the ET-1 production in these cells of both rat strains (Fig 5⇓).

Figure 5.
Figure 5.

Bar graph shows effects of the protein kinase C (PKC)–activating phorbol ester phorbol myristate acetate (PMA) and an inactive enantiomer of phorbor estel, 4α-PDD, on endothelin-1 production in cultured mesangial cells of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Cells were incubated with different concentrations of PMA or 4α-PDD (10−7 mol/L) for 24 hours. Each value is the mean±SD of six cell cultures. *P<.05 vs basal production in WKY; †significant difference between SHR and WKY (P<.05).

To confirm the importance of PKC-dependent mechanisms in the stimulation of ET-1 production, we examined the effect of PKC depletion. The ET-1 production by PKC-depleted cells was not increased by addition of 10−7 mol/L Ang II or 10−7 mol/L AVP (Tables 1⇓ and 2⇓).

View this table:
Table 1.

Effect of Ang II on ET-1 Production With or Without PKC Depletion in Cultured SHR and WKY Mesangial Cells

View this table:
Table 2.

Effect of AVP on ET-1 Production With or Without PKC Depletion in Cultured SHR and WKY Mesangial Cells

Discussion

First, we have shown that cultured mesangial cells of both SHR and WKY produce ET-1 in a time-dependent manner. Under quiescent conditions (0.5% FCS), basal ET-1 production of SHR cells was not different from that of WKY cells, although a trend toward increased ET-1 production was observed in SHR cells.

Second, we have confirmed previous observations reported by Bakris et al9 10 and us11 16 25 that Ang II and AVP stimulate mesangial cell production of ET-1. Under the current experimental conditions, the stimulatory effect of AVP appeared to be greater than that of Ang II. This stimulation by Ang II and AVP of ET-1 production was observed in mesangial cells of both SHR and WKY. However, Ang II– and AVP-induced stimulation of ET-1 production was clearly greater in SHR cells than in WKY cells. Bakris and Re9 demonstrated that Ang II increases ET-1 production in human mesangial cells, but this increase is only significant in cells incubated with 10% FCS, not in quiescent or serum-free conditions. The precise reasons for the difference between our present data and the data of Bakris and Re are not clear at present.

One possible explanation is that age or species of the tissue from which cells are isolated may be related to this difference. Bakris and Re9 performed their experiment using adult human mesangial cells, whereas we used cells isolated from young SHR and WKY. For example, the proliferative action of Ang II has been shown to be much more apparent in fetal human mesangial cells under serum-free conditions compared with cells isolated from adults.26

Alterations in Ang II receptors may contribute to the variable response of mesangial cells to Ang II. It has been reported that the number of Ang II binding sites in cultured mesangial cells might be decreased with the increase of passage number.27

The amount of Ang II– and AVP-stimulated ET-1 production was much lower even in mesangial cells of SHR compared with that in cultured vascular endothelial cells. Nevertheless, the ET-1 concentration in the culture media of Ang II– and AVP-stimulated SHR mesangial cells appears to attain levels that are within the biologically effective range for this peptide.1 Furthermore, it is important to note that a low concentration of exogenous ET-1 potentiates the vasoconstrictive or mitogenic action of other vasoconstrictors or growth factors, such as norepinephrine,28 29 serotonin,30 and platelet-derived growth factor.31 Therefore, ET-1 produced by Ang II and AVP may act, together with Ang II, AVP, or other endogenous substances, to stimulate the contraction or proliferation even at a low concentration.

As mentioned above, Bakris and Re9 have shown that Ang II acts as a mitogen under certain culture conditions in cultured human mesangial cells, and this mitogenic effect of Ang II is mediated through mesangial cell ET-1 production. Bakris et al10 have also shown in another report that AVP acts as a mitogen in part by increasing mesangial cell production of ET-1. Furthermore, the local renin-angiotensin system within the kidney has been found to play an important role in the maintenance or development of the high blood pressure in SHR, although plasma Ang II levels in SHR are not elevated above levels in WKY.32 33 On the other hand, plasma AVP concentration has been found to be high in SHR34 and is markedly increased in the malignant or severe stage of hypertension.35 36 Taken together with our data, these observations raise the possibility that the excess ET-1 production caused by Ang II or AVP in glomerular mesangial cells of SHR induces the contraction and proliferation of these cells probably in concert with Ang II and AVP, thereby contributing to the alteration of glomerular function associated with the progression of hypertension in SHR. However, it remains to be clarified whether Ang II and AVP have physiological roles as modulators of ET-1 production in glomerular mesangial cells in vivo, because not only high concentrations of Ang II and AVP are required to stimulate ET-1 production, but also high concentrations of ET-1 are required to induce the contraction and proliferation of these cells.

Next, we showed in the current experiment that Ang II–induced mesangial cell ET-1 production was abolished by the selective AT1 receptor antagonist losartan. On the other hand, this stimulation by Ang II was not affected by the selective AT2 receptor antagonist PD 123319. These results indicate that Ang II stimulates ET-1 production via AT1 receptors in mesangial cells of both rat strains. Furthermore, we showed that AVP-induced mesangial cell ET-1 production was abolished by the selective V1 receptor antagonist PMP, and therefore, AVP stimulates ET-1 production via V1 receptors in these cells.

Finally, we showed that Ang II– and AVP-stimulated mesangial cell production of ET-1 is PKC dependent in both SHR and WKY. In fact, the PKC-activating phorbol ester PMA stimulated ET-1 production in mesangial cells of both rat strains, and neither Ang II nor AVP stimulated ET-1 production in PKC-depleted mesangial cells. Furthermore, an inactive enantiomer of phorbol ester, 4α-PDD, had no effect on mesangial cell production of ET-1, indicating that the effect of PMA was not a nonspecific action of phorbol ester. We also showed that the stimulation by PMA of ET-1 production was significantly greater in SHR mesangial cells than in WKY cells. This suggests that an increased response of ET-1 production to PKC activation contributes in part to the observed enhancement of ET-1 production in SHR mesangial cells. However, further studies will be necessary to elucidate the exact cellular mechanism of the above-mentioned difference in cells of both rat strains.

Overall, our results suggest that AT1 receptor– and V1 receptor–mediated mesangial cell production of ET-1 is clearly enhanced in SHR compared with WKY. This difference appeared to be partly due to the different response of ET-1 production to PKC activation in mesangial cells of both rat strains. It is well established that Ang II and AVP stimulate ET-1 production in vascular endothelial cells.13 23 37 Therefore, in the glomerulus, Ang II and AVP appear to stimulate both mesangial and endothelial cell production of ET-1. The excess ET-1 production in the glomerulus may contribute to the renal involvement associated with the progression of hypertension in SHR.

Acknowledgments

This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and by a grant of the Osaka Heart Club. We greatly acknowledge the technical assistance of Machiko Johchi, Atsumi Ohnishi, and Tomoko Okuno.

  • Received June 6, 1994.
  • Accepted November 7, 1994.

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  • Endothelin Production in Cultured Mesangial Cells of Spontaneously Hypertensive Rats
    Miwako Ikeda, Masakazu Kohno and Tadanao Takeda
    Hypertension. 1995;25:1-6, originally published April 1, 1995
    https://doi.org/10.1161/01.HYP.25.4.1
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