Abnormal Renovascular Parathyroid Hormone-1 Receptor in ...

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Endocrinology 147(9):4384 – 4391 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2005-1517

Abnormal Renovascular Parathyroid Hormone-1 Receptor in Hypertension: Primary Defect or Secondary to Angiotensin II Type 1 Receptor Activation? Sandra Welsch, Eric Schordan, Catherine Coquard, Thierry Massfelder, Nathalie Fiaschi-Taesch, Jean-Jacques Helwig, and Mariette Barthelmebs Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 727, Strasbourg F-67085 France; and University Louis Pasteur, School of Medecine, Strasbourg F-67070 France We previously reported that PTHrP-induced renal vasodilation is impaired in mature spontaneously hypertensive rats (SHR) through down-regulation of the type 1 PTH/PTHrP receptor (PTH1R), a feature that contributes to the high renal vascular resistance in SHR. Here we asked whether this defect represents a prime determinant in genetic hypertension or whether it is secondary to angiotensin II (Ang II) and/or the mechanical forces exerted on the vascular wall. We found that the treatment of SHR with established hypertension by the Ang II type 1 receptor antagonist, losartan, reversed the down-regulation of PTH1R expression in intrarenal small arteries and restored PTHrP-induced vasodilation in ex vivo perfused kidneys. In contrast, the PTH1R deregulation was not found in intrarenal arteries isolated from prehypertensive SHR. Moreover, this defect, which is not seen in extrarenal vessels (aorta, mesenteric arteries) from mature SHR

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THrP WAS SHOWN, IMMEDIATELY after its identification in 1987, to reduce blood pressure (1), specifically bind to common PTH/PTHrP receptors in vascular smooth muscle (2), and relax renal arteries (3) or the intrarenal vascular bed (4). As a rule, exogenously added PTHrP was later shown to relax every vascular bed that had been tested (5) by activating the type 1 PTH/PTHrP receptor (PTH1R). Insight into the overall role of locally produced PTHrP in the vascular system initially came from studies in transgenic mice that constitutively overexpress PTHrP or the PTH1R in smooth muscle and exhibited lowered blood pressure (6, 7). Later a decrease in blood pressure has also been observed in adult rats that start to overexpress the PTH1R in vessels after iv delivery of PTH1R cDNA (8). In other respects, PTHrP has been reported to increase locally at sites of ischemic injury in stunned myocardium in which the release of endogenous PTHrP seemed protective for heart function (9). The PTHrP/ PTH1R system therefore appears as achieving an important First Published Online May 25, 2006 Abbreviations: Ang II, Angiotensin II; AT1R, Ang II type 1 receptor; DOCA, deoxycorticosterone-acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PTH1R, type 1 PTH/PTHrP receptor; RAS, reninangiotensin system; RvSMC, renovascular smooth muscle cell; SHR, spontaneously hypertensive rat; VSMC, vascular smooth muscle cell; WKY, Wistar-Kyoto. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

appeared kidney specific in accordance with the acknowledged enrichment of interstitial Ang II in this organ and its enhancement in SHR. In deoxycorticosterone-acetate-salt rats, an Ang II-independent model of hypertension, renovascular PTH1R expression and related vasodilation were not altered. In SHR-derived renovascular smooth muscle cells (RvSMCs), the PTH1R was spontaneously down-regulated and its transcript destabilized, compared with Wistar RvSMCs, both effects being antagonized by losartan. Exogenous Ang II elicited down-regulation of PTH1R mRNA in RvSMCs from Wistar rats. Together, these data demonstrate that Ang II acts via the Ang II type 1 receptor to destabilize PTH1R mRNA in the renal vessel in the SHR model of genetic hypertension. This process is kidney specific and independent from blood pressure increase. (Endocrinology 147: 4384 – 4391, 2006)

physiopathological task in the regulation of local and systemic hemodynamics. Whether the local PTHrP system is a candidate for the vascular or renovascular factors involved in the pathogenesis of hypertension is one of the legitimate questions raised by these studies. The first observation in this field was reported by Dipette et al. (10) in the spontaneously hypertensive rat (SHR) model of genetic hypertension. Although PTHrP caused similar blood pressure reductions in SHR and normotensive Wistar-Kyoto (WKY) rats, the specific binding of iodinated PTHrP was markedly reduced in renal tissue from SHR, pointing at a possible dysfunction of the PTH1R in the kidney. Robust and synergistic induction of PTHrP was later reported in aortic vascular smooth muscle cells (VSMCs) exposed to angiotensin II (Ang II) and mechanical stretch (11), two events that arise when hypertension develops. Subsequently the expression of PTHrP was shown to be increased in the aorta of SHR (12), suggesting that PTHrP up-regulation might mediate a negative-feedback regulation to oppose the myogenic contractile response linked to stretch and/or hypertension. Renal transplantation studies in both humans and animals with genetic hypertension have shown how much the kidney is important in the genesis of hypertension (13). In this context, we reported earlier not only a strong increase in PTHrP expression in the intrarenal arteries isolated from SHR but also a marked decrease in the expression of PTH1R (14, 15). Moreover, deregulation of the PTHrP system was accompa-

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Welsch et al. • AT1R-Mediated Regulation of Renovascular PTH1R

nied by a marked decrease in PTHrP-induced renal vasodilation in the ex vivo perfused kidneys from SHR, strongly indicating that the density of PTH1R is the limiting factor for PTHrP-induced renal vasoactivity (14). In support of this, down-regulation of the PTH1R in intrarenal arteries has been proven to be responsible for the high renal vascular resistance in SHR. Indeed, replenishment of vascular PTH1R by the in vivo delivery of PTH1R cDNA reduced the renal vascular resistance of SHR back to the value measured in WKY rats (15). However, evidence that the abnormal expression of PTH1R in renal and perhaps other peripheral vasculatures is a significant prime determinant in genetic hypertension is still lacking. Because of the alterations in SHR of other regulators of vascular tone, in particular the renin-angiotensin system (RAS), it is probable that the down-regulation of the PTH1R is only a secondary event. In this context, Ang II acting through Ang II type 1 receptors (AT1R) appears as a valid candidate for PTH1R deregulation in renal vessels. Indeed, a decrease in PTH1R expression has been reported when VSMCs are exposed to Ang II (16). Moreover, Ang II levels are particularly high in the kidney in which abundant AT1R are widely distributed (17) and the local RAS is inappropriately activated in SHR with established hypertension (18). Another possible scenario that does not necessarily rule out the RAS is that the expression of PTH1R decreases in response to the mechanical forces exerted on the vascular wall on increase of blood pressure. The present experiments were therefore designed to test the hypothesis that the defect in PTH1R expression in SHR vessels may not represent a prime determinant in genetic hypertension but is secondary to AT1R-mediated actions and/or mechanical forces generated by hypertension on the vascular wall. Two specific questions were asked. First, does AT1R blockade reverse the down-regulation of PTH1R expression and related vasodilation in SHR vessels? Second, if Ang II is involved, does it act independently from the blood pressure increase? Our data demonstrate that endogenous Ang II, acting through the AT1R, down-regulates the PTH1R expression in arteries from SHR. This effect is independent from changes in blood pressure, kidney specific, and reproduced in vitro by Ang II in renovascular smooth muscle cells (RvSMCs) from the normotensive Wistar rat. Moreover, destabilization of the PTH1R transcript by Ang II seems responsible for the spontaneous down-regulation of PTH1R expression in RvSMCs from SHR.

Endocrinology, September 2006, 147(9):4384 – 4391

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ing solution for 3 wk. Concentrations were adjusted every 2 d to maintain constant dosing (20 –22 mg/kg䡠d). Control SHR and Wistar rats were run in parallel with free access to tap water. Systolic blood pressure was measured twice weekly in awake rats with a tail-cuff sphygmomanometric method (Letica, Barcelona, Spain). At the end of the 3-wk followup, a blood sample was obtained from abdominal aorta under ketamine/xylazine anesthesia (63 and 8 mg/kg, respectively) for plasma renin activity measurement as reported before (19). The 12-wk-old rats were thereafter used for tissue sampling or assessment of renal vascular reactivity (ex vivo perfused kidney).

Deoxycorticosterone-acetate (DOCA)-salt hypertensive rats To induce DOCA-salt low-renin hypertension, two pellets of DOCA (25 mg each) were implanted sc in 7-wk-old Wistar rats under ether anesthesia and rats were given 0.9% saline to drink as described previously (20). Sham-operated rats were subjected to the same procedure but did not receive DOCA pellets and had tap water to drink. Systolic blood pressure was measured once weekly in awake rats. At the end of the 5-wk follow-up, blood was obtained for plasma renin activity measurement under ketamine/xylazine anesthesia before tissue sampling or assessment of renal vascular reactivity (ex vivo perfused kidney).

The ex vivo isolated perfused kidney or mesenteric vascular bed After sodium pentobarbital anesthesia (50 mg/kg⫺1 ip; Centravet, Nancy, France), the right kidney was isolated and ex vivo perfused as previously described (14, 21). Perfusion was performed in an open single-pass circuit with a prewarmed (37 C), oxygenated (95% O2-5% CO2), colloid-free Krebs-Henseleit solution. After a 30-min equilibration period, the perfusion flow was adjusted to achieve a baseline perfusion pressure of 80 mm Hg. The isolated kidney was thereafter perfused at constant flow and perfusion pressure was continuously monitored. The vasodilator responses to PTHrP (1–36) (Neosystem, Strasbourg, France) were assessed by their ability to attenuate the phenylephrine-induced pressure peaks (15-sec infusion every 2 min). The mesenteric vascular bed was isolated and prepared according to McGregor (22). Ex vivo perfusion was performed with a Krebs-Henseleit solution after cannulation of the superior mesenteric artery and ligation of all branches of the superior mesenteric artery other than those irrigating the terminal ileum. The vasodilator responses to PTHrP (1–36) were determined as described for the perfused kidney.

Isolation of intrarenal arteries and other tissues After pentobarbital anesthesia, thoracic aorta and mesenteric arterial tree (mainly second- and third-order arterioles) were collected, whereas the intrarenal arterial tree (mainly arcuate and interlobular arteries) was isolated from the renal cortex as described previously (23). Tissues were rapidly homogenized in TRIzol and processed for total RNA extraction (Invitrogen, Cergy-Pontoise, France) according to the manufacturer’s protocol. For Western blot analysis, the organs were in situ perfused with an ice-cold glycerol-Tris-NaCl buffer before processing for membrane protein extraction (15).

RT-PCR Materials and Methods Animals Male SHR and age-matched Wistar Han rats were purchased from Charles River (l’Arbresle, France). They were housed in a constant temperature room with a 12-h dark, 12-h light cycle (light on at 0600 h) and free access to standard food (AO4; Safe, Villemoisson/Orge, France). Experiments were performed in compliance with guidelines of the European Community and the French government concerning the use of animals.

Losartan treatment of SHR Losartan (kindly provided by Merck Sharp & Dohme, Paris, France) treatment was randomly attributed to 9-wk-old SHR and given in drink-

The relative abundance of specific mRNA was analyzed by quantitative real-time PCR analysis with the LightCycler-FastStart DNA master SYBR green kit (Roche Diagnostics, Meylan, France) as described previously (24). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize mRNA expression. Reverse transcription was performed on 2 ␮g (aorta) or 5 ␮g (other samples) denatured RNA. PCRs were performed with the specific sense and antisense primer sets for rat PTH1R (sense, 5⬘-GGG CAC AAG AAG TGG ATC AT-3⬘; antisense, 5⬘-GGC CAT GAA GAC GGT GTA GT-3⬘); rat GAPDH (sense, 5⬘-CAT GGA GAA GGC TGG GGC TC-3⬘; antisense, 5⬘-AAC GGA TAC ATT GGG GGT GGT AG-3⬘); rat renin (sense, 5⬘-TTC TCT CCC AGA GGG TGC TA-3⬘; antisense, 5⬘-CCC TCC TCA CAC AAC AAG GT-3⬘); rat AT1a receptor (sense, 5⬘-ACC AGG TCA AGT GGA TTT CG-3⬘; antisense, 5⬘-ATC ACC ACC AAG CTG TTT CC-3⬘). PCRs were run as follows: 95 C for 10 min followed by 45 cycles at 95

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C for 10 sec, 60 C for 5 sec, and 72 C for 20 sec. PCR products were identified on agarose gels to verify for expected size: 210 bp (PTH1R), 415 bp (GAPDH), 211 bp (renin), and 209 bp (AT1aR). Data were quantified with the LightCycler analysis software (Roche Diagnostics) and normalized for GAPDH.

PTH1R Western blot analysis PTH1R protein was evaluated by Western immunoblots as described previously (15) on 25– 40 ␮g proteins according to the tissue, using a polyclonal rabbit antirat PTH1R antibody (Eurogentec, Angers, France). This antibody recognizes a major band of 90 kDa, consistent with the predicted molecular mass of the glycosylated receptor. Specificity was checked by competition with excess antigen peptide (rat peptide IV; Eurogentec). A polyclonal mouse anti-␤-actin antibody (Sigma-Aldrich, St. Quentin Fallaviers, France) was used for visualization of protein gel loading. Band intensities were quantified by a gel analysis software (Sigma Gel; Jandel Scientific, Erkrath, Germany) and normalized for ␤-actin.

Culture of RvSMCs Intrarenal arterial trees were prepared from 12-wk-old SHR and Wistar rats, and primary cultures of RvSMCs were obtained by the explant method (23). RvSMCs were typically cultured on plates coated with rat collagen I (Sigma-Aldrich) in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at 37 C in 10% CO2. Cells were used in this study at passages 9 –16. For the evaluation of PTH1R expression, cells were grown for 24 h in serumfree medium in the presence or absence of losartan (1 ␮m) after a 48-h period of quiescence. In a different set of experiments, cells were exposed to Ang II (100 nm; Sigma-Aldrich) for 4 or 8 h in the absence or presence of losartan (1 ␮m). For the actinomycin D experiments, cells were grown for 0, 2, 4, or 8 h in serum-free medium supplemented with 5 ␮g/ml actinomycin D (Sigma-Aldrich) in the presence or absence of losartan or Ang II. Total RNA was isolated at each time point for PTH1R expression analysis by RT-PCR using GAPDH for normalization.

Statistical analysis Data are means ⫾ sem. Differences were tested for statistical significance by Student t test, one- or two-way ANOVA (repeated measurements) as appropriate, with post hoc Tukey test for multiple comparisons. Statistics were run with SigmaStat (Jandel Scientific). P ⬍ 0.05 was considered statistically significant.

Results Losartan treatment of SHR reverses down-regulation of both renovascular PTH1R expression and PTH1R-mediated vasodilation

As shown on Fig. 1A, treatment of SHR by losartan markedly decreased systolic blood pressure, although complete normalization was not obtained. The effectiveness of the AT1R antagonist was further documented by an approximately 20-fold increase in plasma renin activity (186 ⫾ 24 vs. 10 ⫾ 1 ng Ang I per milliliter⫺1 per hour⫺1 in SHR with and without losartan treatment, respectively, n ⫽ 6, P ⬍ 0.001). Under these conditions, losartan significantly corrected the decrease in PTH1R protein expression in intrarenal arterioles from 12-wk-old SHR vs. age-matched Wistar rats (Fig. 1B). Moreover, losartan restored PTH1R function as shown by the increase in PTHrP (1–36)-induced renal vasorelaxation in ex vivo perfused kidneys (Fig. 1C). These observations strongly suggest that the down-regulation of PTH1R in renal vessels cannot be considered as a primary defect but is mediated by Ang II acting through the AT1R in this model of genetic

FIG. 1. Effects of a 3-wk treatment of SHR by losartan (los) on systolic blood pressure, PTH1R protein expression in intrarenal arterioles, renal vascular reactivity to PTHrP (1–36), and PTH1R protein expression in intrarenal arterioles from prehypertensive 4-wk-old SHR. A, Systolic blood pressure was measured twice a week by a tail-cuff sphygmomanometric method in awake SHR (䡺, n ⫽ 11), losartantreated SHR (f, n ⫽ 11), and age-matched Wistar rats (E, n ⫽ 9). B, At the end of the 3-wk follow-up, intrarenal arterioles were prepared from 10 rats per group for the evaluation of PTH1R protein expression by Western blot analysis. Representative immunoblots are shown with a single band found at 90 kDa size for PTH1R and 42 kDa for ␤-actin. Densitometric analysis was used to calculate normalized protein ratio (PTH1R to ␤-actin), which was set at 1 for Wistar rats. C, At the same time, renal vasorelaxation elicited by exogenous PTHrP (1–36) was evaluated in the ex vivo isolated kidneys from the same rats. Kidneys were perfused at constant flow and preconstricted by sequential injections of phenylephrine (PE) during 15 sec every 2 min. Pressure peaks of 45–55 mm Hg were obtained at similar PE concentrations in SHR, SHR-Los, and Wistar rats (0.68 ⫾ 0.08, 0.70 ⫾ 0.18, and 0.83 ⫾ 0.12 ␮M, respectively). PTHrP (1–36) was tested at increasing concentrations, each being perfused over an 8-min period. Renal vasorelaxation was expressed as percent decrease in PE-induced vascular tone. D, Western blot analysis for PTH1R protein expression was performed on intrarenal arterioles prepared from young SHR and Wistar rats (n ⫽ 10). Results are given by means ⫾ # SEM. *, P ⬍ 0.05 vs. Wistar; , P ⬍ 0.05 SHR-Los vs. SHR.

hypertension. The observation that the expression of PTH1R was not altered in intrarenal small arteries prepared from prehypertensive 4-wk-old SHR (Fig. 1D) further supports this assumption. In these young SHR, the intrarenal RAS is indeed not yet activated (18).


Ang II acts independently from the blood pressure increase to down-regulate the PTH1R in renal arteries from SHR

Because losartan treatment not only blocked the AT1R but also decreased blood pressure, we examined whether an increase in blood pressure was able to down-regulate the PTH1R by its own in renal vessels. We therefore used DOCAsalt hypertensive rats because in this model, hypertension developed with low plasma renin and little dependence on the RAS (25). Figure 2A shows the progressive increase in blood pressure in the DOCA-salt rats. After at least a 2-wk exposure to high blood pressure (systolic blood pressure ⬎ 160 mm Hg), neither renal vascular PTH1R protein expression (Fig. 2B) nor PTHrP-induced vasodilation in the ex vivo perfused kidney (Fig. 2C) was modified in DOCA-salt rats when compared with normotensive sham animals. As expected, very low plasma renin activity was measured in DOCA-salt hypertensive rats (1 ⫾ 0.2 vs. 18 ⫾ 3 ng Ang I per milliliter⫺1 per hour⫺1 in sham rats, n ⫽ 8, P ⬍ 0.001).

FIG. 2. Systolic blood pressure, renal vascular PTH1R expression, and related vasodilation in the DOCA-salt hypertensive rats. A, Systolic blood pressure was measured once a week by a tail-cuff sphygmomanometric method in awake DOCA-salt rats (F, n ⫽ 13) and age-matched sham Wistar rats (E, n ⫽ 11). B, Intrarenal arterioles were obtained for Western blot analysis of PTH1R protein expression. Shown is a representative immunoblot from 10 independent determinations. PTH1R to ␤-actin ratio was set at 1 for sham Wistar rats. C, Renal vascular reactivity elicited by PTHrP (1–36) was evaluated in ex vivo isolated perfused kidneys. Pressure peaks of 52–54 mm Hg were induced by phenylephrine (PE) concentrations of 0.47 ⫾ 0.06 and 0.83 ⫾ 0.12 ␮M in DOCA-salt and sham rats, respectively. Renal vasorelaxation was expressed as percent decrease in PE-induced vascular tone. Results are given by means ⫾ SEM. *, P ⬍ 0.05 vs. sham.


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Because intrarenal Ang II levels have been reported to be enhanced in 14-wk-old SHR but not 7-wk-old rats (18), we performed a time-course evaluation of the changes in renovascular PTH1R expression in SHR (Fig. 3). The deregulation in PTH1R mRNA became evident only in 12-wk-old SHR. Of particular interest, PTH1R expression was not modified in 8-wk-old SHR, although these rats were obviously hypertensive (systolic blood pressure 165 ⫾ 8 vs. 121 ⫾ 6 mm Hg in age-matched Wistar rats, n ⫽ 6, P ⬍ 0.001). Present data thus demonstrate that an increase in blood pressure per se, in the absence of an activated intrarenal RAS, is unable to down-regulate the PTH1R expression. We then asked the reverse question: is Ang II acting through the AT1R still able to change the PTH1R expression in renal arteries under conditions in which blood pressure is no longer increased? For this purpose, we examined the regulation of PTH1R in vitro, in RvSMCs derived from 12wk-old SHR and Wistar rats. Previous studies have indicated that VSMCs from SHR generate high endogenous levels of Ang II, mainly because of overexpression of components of the Ang II-generating system (26). Figure 4A shows that this may be true also in VSMCs derived from small intrarenal arteries isolated from SHR kidney. Indeed, the expression of renin mRNA was 3-fold higher in RvSMCs from SHR than cells from Wistar rats, although the Ang II levels in conditioned medium were too low for detection by RIA in our hands. AT1aR mRNA expression remained unchanged (Fig. 4A). Basal PTH1R mRNA level was decreased by half in SHR-derived RvSMCs and, importantly, losartan abrogated this difference (Fig. 4B), pointing to a key contribution of endogenous Ang II. Moreover, exposure of Wistar-derived RvSMCs to Ang II for 4 or 8 h decreased PTH1R mRNA by half, and this effect was also reversed by losartan (Fig. 4C). Exogenous Ang II, however, induced no additive effect on the already deregulated expression of PTH1R in SHR-derived RvSMCs (Fig. 4D). Taken together, these data show that Ang II acts independently from changes in blood pressure by down-regulating the PTH1R in RvSMCs and that this occurs spontaneously in SHR.

FIG. 3. Time course of the evolution of PTH1R mRNA expression in intrarenal arterioles from SHR and Wistar rats. Real-time quantitative RT-PCR analysis was performed for the evaluation of PTH1R mRNA in renal arterioles isolated from 4-, 8-, and 12-wk-old rats. Expression of target transcripts was compared with the expression of GAPDH used as a housekeeping gene. Normalized ratios were calculated (PTH1R to GAPDH mRNA) and set at 1 for Wistar rats (n ⫽ 6/group). Results are given by means ⫾ SEM. *, P ⬍ 0.05 vs. Wistar.

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FIG. 4. Contribution of endogenous Ang II in the regulation of PTH1R mRNA in primary culture of RvSMCs from SHR and effects of exogenous Ang II. RvSMCs from 12-wk-old SHR and Wistar rats were grown for 24 h in serum-free medium after a 48-h period of quiescence. Cells were also grown in serum-free medium supplemented with losartan (los; 1 ␮M), Ang II (100 nM), or both. Real-time quantitative RT-PCR analysis was performed for the evaluation of renin (A), AT1aR (A), and PTH1R mRNA (B). The effects of exogenous Ang II on PTH1R mRNA were tested on RvSMCs from Wistar rats (C) and SHR (D). Expression of target transcripts was compared with the expression of GAPDH used as a housekeeping gene. Normalized ratios were calculated (specific to GAPDH mRNA) and set at 1 for Wistar rats (n ⫽ 3/group). Results are given by means ⫾ SEM. *, P ⬍ 0.05 vs. Wistar; #, P ⬍ 0.05 SHR-Los vs. SHR.

PTH1R down-regulation is specific for the renal vasculature in SHR

Because Ang II concentrations are particularly high in the kidney when compared with other tissues and plasma (27), we further hypothesized that the PTH1R deregulation might preferentially occur in renal arteries, compared with other vessels or tissues from SHR, even if they are, in most cases, exposed to higher hypertensive stretch than intrarenal resistance arterioles. The expressions of PTH1R in mesenteric arterioles and aorta were comparable in 12-wk-old hypertensive and normotensive rats, at both the mRNA (Fig. 5A) and protein levels (Fig. 5B). In support of this, the PTHrP (1–36)-induced vasodilation of the ex vivo perfused mesenteric vasculature isolated from mature SHR and age-matched Wistar rats were comparable, too (Fig. 5C). The decrease in vascular PTH1R expression and function therefore appears as a kidney-specific event, which further supports the key role of Ang II in this process.


FIG. 5. Aortic and mesenteric vascular PTH1R expression and function in SHR. Real-time quantitative RT-PCR analysis for PTH1R mRNA expression (n ⫽ 6 per group) (A) and Western blot analysis for PTH1R protein expression (n ⫽ 10 per group) (B) were performed in the mesenteric vasculature and aorta from 12-wk-old SHR and Wistar rats. GAPDH was used as the housekeeping gene to normalize PTH1R gene expression, and ␤-actin was used to normalize for protein load of the membranes; both ratios were set at 1 for Wistar rats. C, Mesenteric vasorelaxation elicited by exogenous PTHrP (1–36) was evaluated in the ex vivo isolated mesenteric vascular bed. Perfusion was performed at constant flow and sequential injections of phenylephrine (PE) during 15 sec every 2 min were used to restore vascular tone. Pressure peaks of 50 –55 mm Hg were obtained at PE concentrations of 2.8 ⫾ 0.7 and 2.1 ⫾ 0.4 ␮M in SHR and Wistar rats, respectively. PTHrP (1–36) was tested at increasing concentrations, each being perfused over an 8-min period. Mesenteric vasorelaxation was expressed as percent decrease in PE-induced vascular tone. Results are given by means ⫾ SEM. No difference was statistically significant.

Ang II decreases PTH1R expression by destabilizing PTH1R mRNA

To gain a first insight into the mechanism underlying the down-regulation of PTH1R expression by Ang II, we examined whether Ang II acts on PTH1R mRNA levels through a posttranscriptional process. Under transcription blockade with actinomycin-D, the PTH1R mRNA decay was followed up over 8 h in both Wistar- and SHR-derived RvSMCs (Fig. 6A). The decay was faster in SHR-derived cells, with a corresponding lower mRNA half-life (5.7 ⫾ 0.3 vs. 12.4 ⫾ 2.6 h in Wistar-derived cells, n ⫽ 5– 6, P ⬍ 0.001). Losartan restored PTH1R mRNA stability in SHR-derived RvSMCs, as shown by the increase in half-life (9.4 ⫾ 0.2 h, P ⬍ 0.001 vs. SHR) up


FIG. 6. Contribution of Ang II to the regulation of PTH1R mRNA stability in SHR- and Wistar-derived RvSMCs. PTHrP mRNA was evaluated by real-time quantitative RT-PCR analysis in cells grown in the presence of actinomycin D (5 ␮g/ml) in serum-free medium. The relative intensity of PTH1R transcript signal (PTH1R to GAPDH mRNA ratio) was expressed as a percentage of the signal obtained at the time of actinomycin D addition (0 h) set at 100%. In SHR-derived RvSMCs (A), the spontaneous down-regulation of PTH1R mRNA level was concomitant with lower stability of PTH1R transcript; both were reversed by losartan (Los; 1 ␮M). In Wistar-derived RvSMCs (B), a similar decrease in PTH1R mRNA level and stability was elicited by exogenously added Ang II (100 nM). Results are given by means ⫾ SEM (n ⫽ 3– 6). *, P ⬍ 0.05 vs. Wistar; #, P ⬍ 0.05 SHR-Los vs. SHR.

to a value close to that seen in Wistar cells. Conversely, exposure of Wistar-derived RvSMCs to Ang II increased the PTH1R mRNA decay, reducing its half-life from 12.4 ⫾ 2.6 to 5.8 ⫾ 0.2 h (n ⫽ 5– 6, P ⬍ 0.001). Thus, these results show that endogenous Ang II acts via the AT1R to destabilize PTH1R transcript in SHR-derived RvSMCs. In addition, the same process occurs in response to exogenous Ang II in Wistar cells. Discussion

We previously reported that the down-regulation of PTH1R in renal arterioles from mature SHR, compared with WKY rats, was associated with a loss of function as shown by the marked impairment of PTHrP (1–36)-induced vasodilation in the ex vivo perfused kidney from hypertensive rats (14, 15). The present study adds further support to this deregulation by comparing SHR with Wistar rats. Importantly, impaired PTH1R-mediated renal vasodilation also occurs under in vivo conditions as the response to direct perfusion of PTHrP (1–36) into the renal artery is decreased in SHR (Welsch, S., personal results). Because the density of PTH1R in the renal vasculature is the limiting factor for PTHrP-


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induced vasorelaxation in SHR and because this defect significantly contributes to the high renal vascular resistance in these rats (15), it became relevant to further analyze PTH1R deregulation in SHR. We particularly wonder whether the abnormal expression of PTH1R in renal and perhaps other peripheral vessels is a prime defect in SHR or whether it is dependent from other defects, namely the activation of the RAS and/or the increase in blood pressure. The present results demonstrate the key contribution of endogenous Ang II acting through the AT1R in the vascular PTH1R down-regulation. In the last years, the focus on the systemic RAS shifted to an emphasis on local systems, particularly in the kidney. Renal interstitial fluid Ang II concentrations are about 30fold higher than plasma levels or concentrations in aorta and heart, suggesting local synthesis (17, 27). Indeed, all the components needed for Ang II generation are present in the renal interstitium and the positive feedback of Ang II on local angiotensinogen production leads to sustained intrarenal Ang II production (17). Moreover, Kobori et al. (18) recently reported that the enhanced intrarenal angiotensinogen production in SHR contributes to increased local Ang II levels, leading to hypertension and renal injury in this strain. Of interest, this inappropriate activation of the intrarenal RAS was observed in 14-wk-old SHR but not 7-wk-old SHR and occurred without any change in the plasma Ang II levels. In light of these particularities of the intrarenal RAS, our present data strongly support that high endogenous Ang II levels acting through the AT1R may be the key factor in vascular PTH1R down-regulation. Indeed, treatment of SHR by losartan blunted the deregulation of PTH1R expression in intrarenal arteries and enhanced PTH1R-mediated function, as shown by the increase in renal vasodilation in the ex vivo perfused kidney. On other vessels, in which the local RAS does not seem to be activated in SHR, PTH1R expression remained unchanged and was not affected by losartan. Moreover, losartan reversed the PTH1R down-regulation found under basal conditions in RvSMCs derived from SHR, and these cells no longer respond to exogenous PTHrP, suggesting that they produce high endogenous levels of Ang II. This observation is in line with the 50-fold increase in Ang II-like immunoreactivity in SHR-derived VSMCs as reported by Fukuda et al. (26), mainly through overexpression of angiotensinogen, cathepsin D, and angiotensin-converting enzyme. In RvSMCs derived from SHR, we found a 3-fold increase in renin mRNA. Our experiments in the presence of actinomycin D lead to the obvious conclusion that endogenous Ang II, acting via the AT1R, modulates PTH1R expression in SHR RvSMCs by decreasing the stability of its mRNA. Our results demonstrating that endogenous Ang II is responsible for the vascular PTH1R down-regulation in SHR are in line with the own effects of exogenous Ang II on Wistar-derived VSMCs. Indeed, Ang II was able to decrease PTH1R mRNA in these cells, as was previously reported by Okano et al. (16) on aortic VSMCs from normotensive rats. In contrast, however, a transient increase in immunoreactive PTH1R was observed in renal arteries after a 3-d sc infusion of Ang II (28). The raisons for this discrepancy are not clear at the present time. Changes in blood pressure may appear as a confusing

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variable in the regulation of PTH1R expression as well as the previously observation that mechanical cyclic stretch decreases PTH1R expression in RvSMCs derived from Wistar rats (24). This effect was, however, inhibited by losartan and was therefore Ang II dependent (Welsch, S., personal results). In support of this, no change in vascular PTH1R expression was found in the present study in the DOCA-salt hypertensive rats with a low renin status. These rats might also have low intrarenal levels of Ang II as was measured previously after a high-sodium diet (19). Finally, our results on Wistar-derived RvSMCs clearly show that Ang II acts independently from blood pressure changes or stretch to elicit vascular down-regulation of PTH1R. In recent years, a number of studies have reported that the expression of PTHrP and PTH1R are regulated in a reciprocal fashion. Induction of PTHrP was indeed concomitant with a decrease in PTH1R expression in various models of renal or vascular damage, including renal failure (29, 30), renal or cerebral ischemia (31, 32), and balloon angioplasty (33). In these settings in which the RAS is often activated, Ang II may contribute to the down-regulation of vascular PTH1R rather than PTHrP itself. Indeed, PTHrP can transiently desensitize the PTH1R but is not able, at least by the autocrine/paracrine pathway, to change PTH1R expression as reported previously on VSMCs exposed to exogenous PTHrP (1–36) (16). In the evaluation of the contribution(s) of PTHrP in these pathologies, it is important to keep in mind that the PTHrPinduced effects related to the autocrine/paracrine pathway might be damped, whereas its intracrine effects will be preserved. The intracrine pathway seems to be involved in the modulation of cell proliferation and/or apoptosis (24, 34 – 36), and antimitogenic effects have been previously reported in SHR-derived RvSMCs (37). The question as to whether PTHrP exerts a negative feedback on intrarenal arteries hyperplasia in SHR needs further specific studies. In conclusion, the present findings establish that the abnormal renovascular PTH1R expression and the marked decrease in PTHrP-induced renal vasodilation are not a primary defect but is secondary to the activation of the RAS in the SHR model of genetic hypertension. Significant findings obtained with losartan demonstrate that locally produced Ang II acts independently from blood pressure changes through the AT1R to destabilize PTH1R mRNA, which in turn down-regulates the PTH1R in renal vessels. The vascular deregulation of PTH1R seems kidney specific in SHR, in accordance with the previously described enhancement of Ang II in the renal interstitium of this strain. Overall, the defect in PTH1R expression in renal vessels therefore appears as a factor involved in the maintenance rather than the development of altered renal hemodynamics in genetic hypertension. Acknowledgments The authors warmly thank A. F. Stewart (Department of Medecine, University Pittsburgh, Pittsburgh, PA) for helpful discussions. They gratefully acknowledge the skillful technical assistance of Virginie Roques, Sylvie Rothhut, and Jacques Steger as well as the secretarial assistance of Daniel Kuhlwein. They also thank Merck Sharp & Dohme (Paris, France) for the gift of losartan. Received November 30, 2005. Accepted May 18, 2006.


Address all correspondence and requests for reprints to: Dr. Mariette Barthelmebs, Unite´ 727, Institut National de la Sante´ et de la Recherche Me´dicale, Pharmacologie et Physiopathologie Re´nales, Faculte´ de Me´decine, 11 rue Humann, 67085 Strasbourg cedex, France. E-mail: [email protected]. This work was supported by grants from the French National Institute of Health (Institut National de la Sante´ et de la Recherche Me´dicale) (to J.-J.H.), the Louis Pasteur University of Strasbourg (to J.-J.H.), and the French Ministry of Higher Education (to J.-J.H.). This work is part of the Ph.D. thesis of S.W., who was supported by a fellowship from Institut National de la Sante´ et de la Recherche Me´dicale and the Alsace Region. M.B. is a Research Director in the French National Center for Scientific Research. Disclosure: S.W., E.S., C.C., T.M., N.F.-T., J.-J.H., and M.B. have nothing to declare.

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