Type 1 Parathyroid Hormone Receptor Expression Level Modulates Renal Tone and Plasma Renin Activity in Spontaneously Hypertensive Rat
Thierry Massfelder1,*,
Nathalie Taesch1,*,
Samuel Fritsch*,
Anne Eichinger*,
Mariette Barthelmebs*,
Andrew F. Stewart and
Jean-Jacques Helwig*
*Section of Renovascular Pharmacology and Physiology (INSERM-ULP), University Louis Pasteur School of Medicine, Strasbourg, France; and Division of Endocrinology and Metabolism, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
Correspondence to: Dr. Jean-Jacques Helwig, Pharmacologie & Physiologie Rénovasculaires, (Equipe Mixte INSERM-ULP 0015), 11, rue Humann, Bâtiment 4, 1er étage, F67085 Strasbourg Cedex, France. Phone: 333-90-24-34-54; Fax: 333-90-24-34-59; E-mail: jean-jacques.helwig{at}pharmaco-ulp.u-strasbg.fr
ABSTRACT. These studies examine whether PTHrP(1-36), a vasodilator,modulates BP and renal vascular resistance (RVR) in spontaneouslyhypertensive rat (SHR). Within the kidney of normotensive rats,PTHrP(1-36) was enriched in vessels. In vessels of SHR, PTHrPwas upregulated by 40% and type 1 PTH receptor (PTH1R) was downregulatedby 65% compared with normotensive rats. To investigate the roleof endogenous PTHrP in the regulation of BP and RVR, SHR weresubjected to somatic human (h)PTH1R gene delivery. Three weeksafter a single intravenous injection of pcDNA1.1 plasmid containingthe hPTH1R gene under the control of the cytomegalovirus promoter,hPTH1R mRNA was detected in all of the main organs. Within thekidney, the transgene was enriched in vessels. In the isolatedperfused kidney, RVR was reduced by 23% and PTHrP(1-36)–inducedvasodilation, which is depressed in SHR, was restored and avasoconstrictory response to PTH(3-34), a PTH1R antagonist,was revealed. These effects were not observed in control SHRtreated with empty plasmid. BP remained unchanged, and plasmarenin activity increased by 60%. Thus, in SHR renal vessels,a reduced number of PTH1R contributes to the high RVR, despitethe higher expression of vasodilatory PTHrP. Moreover, thesestudies provide evidence for a direct link between the densityof PTH1R and plasma renin activity, which might be responsiblefor the absence of effect of PTH1R gene delivery on BP in SHR.Overall, PTHrP significantly contributes to the homeostasisof renal and systemic hemodynamics in SHR.
Initially discovered as a tumor-derived humoral factor thatcauses humoral hypercalcemia of malignancy, parathyroid hormone-relatedprotein (PTHrP) is now primarily considered to be a locallyactive polyhormone, ubiquitously produced throughout the normalbody and endowed with cytokine- and growth hormone-like properties.Indeed, nascent PTHrP is posttranslationally processed to afamily a peptides, including PTHrP(1–36) (the PTH-likepart of the molecule), PTHrP(38–94) and PTHrP(107–139),each having its own biologic activities and receptors (1). Thetype 1 PTH receptor (PTH1R) (2) binds both PTHrP(1–36)and PTH(1–34), owing to structural homologies existingbetween both peptides. The list of the possible physiologicroles for PTHrP is continuously growing (1,3–5). PTHrPis a regulator of transepithelial calcium transport, vascularand extravascular smooth muscle tone, embryonic development,and cell proliferation, apoptosis, and differentiation.
A number of recent findings prompted us to explore the roleof endogenous PTHrP in the regulation of renal hemodynamicsin an animal model of genetic hypertension. First, transgenicmice that selectively overexpress PTHrP or the PTH1R in smoothmuscle exhibit a cardiovascular phenotype, including a decreaseof BP (6,7). However, whether endogenous PTHrP modulates regionalhemodynamics, including renal hemodynamics has not been clarifiedby these studies. Second, in both human and animal studies,the renovascular system appeared to be a privileged target ofthe vasodilatory properties of exogenous PTHrP(1–36),both in vitro an in vivo (8,9–13). In these studies, PTHrPnot only decreased renal vascular resistance (RVR) and increasedGFR, but also appeared to be a potent stimulator of renin releaseby direct interaction with juxtaglomerular cells (11). Howeveragain, a role for renovascular PTHrP in the regulation of renalhemodynamics has not been sought in these studies. Finally,in terms of genetic hypertension, the spontaneously hypertensiverat (SHR) remains the most widely used animal model of primaryhypertension (14). Genetically determined renal mechanisms playa major role in the development of primary hypertension in bothhumans and SHR (14). The expression of PTHrP gene in blood vesselshas been documented to be increased by vasoconstrictor agentsand mechanical forces (15,16). In the same vein, PTHrP transcripthas also been shown to be upregulated in the aorta in experimental(17) as well as genetic hypertension (18). Whether the vasoactivityof PTHrP(1–36) is altered in genetic hypertension hasnot been explored in these studies. In an earlier study, weprovided initial evidence for a marked decrease of PTHrP(1-36)–inducedvasodilation in the isolated perfused kidney of SHR (19). Overall,the central question as to whether vascular PTH1R and PTHrPplay a role in the modulation of renal as well as systemic hemodynamicsin genetically hypertensive rats has not yet been tackled.
Therefore, this study had two goals: first, to examine whetherthe expression of PTHrP and PTH1R are altered in the renal vasculatureof SHR, and second, to determine whether endogenous vascularPTHrP plays a significant role in the modulation of systemicand renal hemodynamics in these animals. PTH1R indeed appearedto be downregulated, and PTHrP upregulated in renal vesselsof SHR, raising the question of whether a low expression ofPTH1R could be responsible for the high BP and RVR in SHR. Recentstudies have demonstrated the potential of peripheral deliveryof naked DNA coding for vasodilators to reduce BP in SHR (20–23).To investigate the role of endogenous PTHrP in the regulationof systemic and renal hemodynamics in SHR, these studies promptedus to ask whether a replenishment of the PTH1R pool in peripheraland renal vessels by direct delivery of the human PTH1R (hPTH1R)gene would decrease RVR and systemic BP in SHR.
Animals
All animal studies were approved by and in compliance with guidelinesof the European Community and the French Government (Ministryof Agriculture) concerning the animal experimentation. Nineor 12-wk-old SHR and Wistar Kyoto (WKY) rats (Iffa-Credo, l‘Arbresle,France) with free access to standard food and water were used.SHR are in a prehypertensive state up to 4 wk. Mean BP in 12-wk-oldSHR was 184 ± 3 mmHg (n = 8) as compared with 123 ±4 mmHg (n = 5) in normotensive WKY rats.
Isolation of Rat Intrarenal Arteries
Twelve-week-old WKY and SHR were anesthetized with ether anddecapitated. Kidneys were removed, rinsed, and placed in ice-coldphosphate-buffered saline (PBS). Renal arterial trees containingmainly arcuate and interlobular arteries were isolated by asieving method exactly as described previously (24). Isolatedvessels were frozen in liquid nitrogen and ground before processingfor mRNA, PTHrP(1–36), or membrane protein extractionas described below.
RIA of PTHrP(1-36)
Total kidneys or renal vessels were homogenized in 50 mM Tris-HClbuffer, pH 7.4, containing 2 mg·ml-1 aprotinin, 1 mg·ml-1leupeptin, 1 mg·ml-1 pepstatin, 10 mM ethylenediaminetetraaceticacid (EDTA), and 0.2 mM phenylmethylsulfonyl fluoride (PMSF).The tissues were then subjected to 3 consecutive freeze (liquidnitrogen)–thaw (37°C) cycles and centrifuged at 400,000g for 15 min, and PTHrP was measured in the supernatant witha commercially available RIA kit using an affinity-purifiedantibody directed against the 1-36 region of the peptide (PeninsulaLaboratories Ltd, St. Helens Merseyside, England). Results wereexpressed as pg PTHrP(1–36)·mg-1 protein.
Semiquantitative Reverse Transcriptase-PCR Analysis for PTH1R Transcript
Total RNA of isolated renal vessels was prepared by the Tri-Reagentmethod according to the protocol of the manufacturer (Sigma-Aldrich,St. Quentin Fallavier, France). Reverse transcription (RT) wasperformed on 4 µg (PTH1R) or 1.5 µg glyceraldehydephosphodehydrogenase (GAPDH) denaturated RNA using moloney murineleukemia virus reverse transcriptase (Perkin Elmer, Roissy,France) and nonspecific P(dT)15 primer (2 µM) at 37°Cfor 1 hr. PCR of the cDNA solutions was performed in the presenceof specific sense and antisense primers of PTH1R (13) or GAPDH(25). Concentrations of PTH1R primers (50 pM) and GAPDH primers(4.4 pM) were adjusted in preliminary experiments to obtainsimilar product amplification despite the different abundanceof transcripts. The PCR was run for 24 cycles by repeating denaturationat 94°C for 1 min, annealing at 60°C for 1 min, andpolymerization at 72°C for 1 min in the presence of Taqpolymerase (Sigma-Aldrich). The last cycle was followed by anadditional incubation at 72°C for 8 min. The PCR productswere size fractionated by a 2% agarose gel electrophoresis.PCR products were identified by their expected size of 817 bp(PTH1R) and 415 bp (GAPDH).
Western Blot Analysis for the Rat PTH1R
The expression of PTH1R protein in 12-wk-old WKY and SHR renalvessels was evaluated by Western blot analysis as compared withCOS7 cells taken as a negative control. Freshly isolated renalarteries were homogenized in homogenizing buffer consistingof 20 mM Tris-HCl, 10% glycerol, 100 mM NaCl, 2 mM PMSF, 2 mMEDTA, 2 mM ethyleneglycotetraacetic acid, 10 mM sodium orthovanadate,10 µg·ml-1, leupeptin and 10 µg·ml-1aprotinin. After 30-min centrifugation at 30,000 g, the pelletswere incubated at 4°C during 30 min in homogenizing buffercomplemented with 1% NP-40, 0.1% sodium dodecyl sulfate (SDS)and 1% deoxycholate and centrifuged for 30 min at 30,000 g,and the supernatant was used for Western blot analysis. COS-7cells were grown and lysed exactly as described before (26).Protein concentrations were determined according to the methodof Lowry et al. (27) with bovine serum albumin as standard.Samples were subjected to SDS-polyacrylamide gel electrophoresisexactly as described before (26).
pcDNA1.1-hPTH1R Plasmid DNA Preparation and Delivery
The isolation of the hPTH1R cDNA (HKrk) from phage librariesand subcloning of the 1.9-kb insert, containing the entire codingsequence, into the 4-kb mammalian expression vector pcDNA1.1has been described previously in detail (28). The plasmid DNA(pcDNA1.1-hPTH1R and empty pcDNA1.1) were amplified in TOP10Escherichea coli and purified in a giga-preparation with a plasmidpreparation kit (Qiagen, Chatsworth, CA) according to the manufacturer’sinstructions. Nine-week-old SHR were randomly divided into twogroups of 16 animals. The treated group received 500 µgper rat of hPTH1R-pcDNA1.1 in 1 ml of PBS-glucose into the tailvein (20–23). Simultaneously, the control group received500 µg per rat of empty pcDNA1.1.
The expression of hPTH1R transcript was evaluated by RT-PCRin renal vessels, total kidney, heart, aorta, lungs, liver,and brain 3 wk after hPTH1R DNA injection, using the same protocolas described above for endogenous rat PTH1R transcript, exceptfor the sense (5'322AGG AAC AGA TCT TCC TGC TGC A3433') andantisense primer sequences (5'813CAC AGC TAC GGT GAG GGA CGCCAG8363'). The PCR product was identified by its expected sizeof 515 bp.
BP Measurement and Analysis of Physiologic Parameters
Systolic BP of 12 control and 12 hPTH1R-treated SHR was measuredblindly 7 and 14 d after intravenous injections, with an electrosphygmomanometer(Letica Scientific Instruments LE5002, Barcelona, Spain) usingthe tail-cuff method. The conscious rats were placed in a thermostaticallycontrolled (37°C) plastic holder during measurements. Anaverage of at least five readings were taken for each animalafter they became acclimated to the environment. Control-injectedand hPTH1R-DNA–injected SHR (n = 12 each) were placedin individual metabolic cages with free access to tap waterand food 7, 14, and 21 d after the injections. After a 24-hperiod to accustom the rats, urine was collected over another24-h period for the determination of electrolytes (Na+, K+,Ca2+, PO43-), and creatinine. Water and food intakes were alsoassessed. At the end of the urine collection period, blood waswithdrawn from the tail vein for plasma electrolytes, creatinine,and renin activity measurements. Electrolytes were measuredwith ion-selective electrodes (EL-Ise, Beckman, Gagny, France),and creatinine was measured (Beckman autoanalyser) by standardcolorimetric methods. One ml of blood in an ice-cold EDTA-coatedtube was used for plasma renin activity (PRA) measurement. Theblood was immediately centrifuged at 4°C for 10 min at 2000g. Plasma samples were frozen in liquid nitrogen and storedat -80°C until assayed. PRA was measured by determiningthe level of angiotensin I (AngI) generated during a 30-minincubation of plasma at 37°C in the presence of 5 mM 8-hydroxyquinoleine.AngI was measured by RIA using rabbit anti-AngI. Antibodiesto AngI were raised in rabbits immunized against the peptidecoupled to bovine serum albumin by carbodiimide condensation(29). PRA is expressed as ng AngI·ml-1·hr-1 ofincubation.
The Isolated Perfused Rat Kidney (IPK)
Three weeks after intravenous injection, control pcDNA1.1-treatedand pcDNA1.1-hPTH1R–treated SHR were anesthetized by intraperitonealinjection of sodium pentobarbital (65 mg·kg-1). The rightkidney was isolated and perfused in an open single-pass circuitexactly as described before (19). The perfusion flow was adjustedduring a 60-min equilibration period to achieve a common pressurebaseline of 80 mmHg; thereafter, the flow thus adjusted wasmaintained constant. The resulting RVR at the end of the equilibrationperiod was expressed for 1 g left, nonperfused kidney. The vasodilatorresponses to PTHrP(1–36) (Neosystem, Strasbourg, France)were measured as pressure changes under constant flow. IPK havelittle, if any, vasoconstrictor tone under basal resting conditions;therefore, vascular tone was raised after the equilibrationperiod with phenylephrine (PE). For this purpose, PE (10 µM)was infused over 15-s periods every 2 min to induce repetitiveand transient pressure peaks of about 50 mmHg. The responsesto the continuous infusion of 10 nM PTHrP(1–36) were assessedby their ability to attenuate the PE-induced pressure peaks.In additional experiments, IPK of control pcDNA1.1-treated andpcDNA1.1-hPTH1R–treated SHR received after the equilibrationperiod 40 nM of the PTH1R antagonist PTHrP(3–36) (30)over a period of 6 min. These kidney preparations were not preconstrictedwith PE. The effect of the PTH1R antagonist on basal perfusionpressure was followed over a period of 30 min.
Calculations and Statistical Analyses
In RT-PCR studies, band intensities were evaluated by meansof a gel analysis software (Sigma Gel; Jandel Scientific, Erkrath,Germany) and ratios of product intensities between PTH1R andGAPDH (RT-PCR) in SHR were calculated. Ratios were normalizedwith respect to values obtained in WKY, which have been setto 1 in each experiment. In Western blot studies, the band intensitiesof pcDNA1.1-hPTH1R–treated rats were normalized with respectto values obtained in control pcDNA1.1-treated animals, whichhave been set to 1 in each experiment. The values of relativechanges were calculated as means ± SE and were statisticallyevaluated using the paired t test. All other values have beencalculated as means ± SE of the absolute values and werecompared using monofactorial or multifactorial ANOVA followedby the Student-Newman-Keul’s test for multiple comparisons.P values <0.05 were considered significant.
Expression of PTH1R and PTHrP(1-36) in Renal Vessels Expression of PTH1R.
A typical example of RT-PCR products for PTH1R in three independentrenal vessel preparations from 12-wk-old WKY and SHR is shownin the upper panel of Figure 1. The identity of the bands asPTH1R cDNA was confirmed by HindIII restriction analysis ofthe PCR products from WKY as well as from SHR. According todensitometric analysis of three separate studies (Figure 1,lower panel), the PTH1R mRNA level was approximately 43% lowerin SHR vessels than those of WKY.
Figure 1. Reverse transcriptase–PCR (RT-PCR) analysis comparing product intensities of the type 1 parathyroid hormone (PTH) receptor (PTH1R) (817 bp) and glyceraldehyde phosphodehydrogenase (GAPDH) (415 bp) gene expression in renal vessels isolated from 12-wk-old Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). The upper panel shows a typical example of an ethidium bromide–stained gel showing the product intensities of three separate WKY and three SHR, numbered 1, 2, or 3 as indicated. In the lower panel, band intensities were evaluated with a gel analysis software (Sigma Gel; Jandel Scientific, Erkrath, Germany) and ratios of product intensities between PTH1R and GAPDH were calculated. Ratios were normalized with respect to values obtained in WKY, which were set at 1 in each experiment. The values of relative changes were calculated as mean ± SE and were statistically evaluated using paired t test. Values are presented as mean ± SE. *P < 0.001 in five independent vessel preparations.
In Western blot studies, the polyclonal rabbit anti-PTH1R antibodydirected against an extracellular part (aminoacid 248 to 262)of the 3rd transmembrane domain recognized one band with theexpected size of 85 to 90 kD (Figure 2, upper panel), whichmost likely corresponded to the glycosylated form of the PTH1R(31–33). A second major band of 180 kD could be detectedas well, which may represent a dimer of the receptor (34,35).The 90- and 180-kD forms of PTH1R were reduced by 76 and 58%,respectively, in SHR compared with WKY (Fig. 2, lower panel).That the Western blot bands were really the PTH1R was supportedby the absence of their expression in COS-7 cells taken as negativecontrols as well as the absence of signal when the primary antibodywas replaced with nonimmune rabbit IgG (not shown).
Figure 2. The upper panel shows a representative example of immunoblot comparing the expression of endogenous rat PTH1R (approximately 90 and 180 kD) in renal vessels isolated from 12-wk-old WKY and SHR. The bar graph shows the densitometric analysis of the immunoblots. Values of the normalized ratio between product intensity of WKY (open bars) and SH (black bars) rats are presented as mean ± SE. *P < 0.01 in paired t test in three independent measurements.
Expression of PTHrP(1-36).
We compared extracts of renal vessels with extracts of totalkidney, which represents mainly the tubular component of theorgan (Figure 3). Within the kidney, endogenous PTHrP(1–36)appeared to be preferentially localized to vessels, in whichit was threefold higher than in total kidney. However, PTHrPis predominantly expressed in proximal and distal tubule; therefore,the ratio of vessels to isolated tubules would have been lower.Importantly, PTHrP(1–36) was increased by 40% in the vesselsof 12-wk-old SHR but not in total kidney compared with age-matchedWKY animals.
Figure 3. Immunoreactive PTHrP(1-36) content in WKY (open bars) and SH (black bars) rat renal vessels and in total kidney. *P < 0.05 versus total kidney; #P < 0.05 versus WKY rats in five independent measurements.
Detection of hPTH1R after Intravenous Gene Delivery
The pcDNA1.1-hPTH1R plasmid DNA (500 µg) was injectedintravenously into 3 SHR. Three weeks later, the hPTH1R transcriptwas detected in all tissues tested (Figure 4). Relative to GAPDHmRNA, high levels of expression were found in heart, liver,and aorta, and low levels of expression, if any, were foundin brain, lung, and total kidney. By contrast, an intermediatesignal was seen in isolated intrarenal vessels when comparedwith total kidney and corrected for GAPDH. Similar results wereobserved in tissues obtained from 3 to 5 separate experiments.
Figure 4. Expression of hPTH1R gene, which has been delivered 3 wk before in the vector pcDNA1.1 in the tail vein of 9-wk-old SHR. The upper panel shows a typical example of an ethidium bromide–stained gel showing the product intensities of three to five separate experiments. Reverse transcription in the presence of poly-dT was performed with 4 µg and 1.5 µg of total mRNA for hPTH1R (515 bp) and GAPDH (415 bp), respectively. cDNAs were amplified with specific primers as indicated in the Materials and Methods section. In the lower panel, the bar graph shows the band intensities evaluated by means of a gel analysis software (Sigma Gel), and ratios of product intensities between PTH1R and GAPDH were calculated.
To further assess that hPTH1R gene delivery, indeed increasedthe expression PTH1R in renal vessels, we performed Westernblot analysis using the same antibody as above. This antibodyrecognizes a region fully identical in human and rat PTH1R.Again, two bands of 90 and 180 kD were seen (Figure 5). In densitometricstudies, both forms of PTH1R were increased by 74 and 114%,respectively, in pcDNA1.1-hPTH1R–receiving SHR as comparedwith control pcDNA1.1-receiving SHR 3 wk after gene delivery.
Figure 5. Upper panel shows the immunoblot comparing the expression of hPTH1R (approximately 90 and 180 kD) in renal vessels isolated from 12-wk-old SHR that intravenously received 3 wk earlier either empty pcDNA1.1 plasmid (control-treated, white bars) or pcDNA1.1 with the 1.9-kb insert of the hPTH1R gene (hPTH1R-treated, hatched bars). The bar graph shows the densitometric analysis of the immunoblot. Values of the normalized ratio between product intensity of control-treated and hPTH1R-treated SHR are presented as mean ± SE for three independent experiments. *P < 0.05 in paired t test.
Effect of hPTH1R Gene Delivery on BP, PRA, and Other Physiologic Parameters
Intrarenal blood vessels of SHR are shown to be associated witha marked decrement in PTH1R content; therefore, we next askedwhether replenishment of the PTH1R pool would enable upregulatedPTHrP(1–36) to exert its vasodilatory effect and therebydecrease BP in SHR. In vivo delivery of hPTH1R gene was unableto significantly decrease systolic BP in SHR (Figure 6A). Previouslyproven stimulatory effect of PTHrP(1–36) on renal reninrelease (36) prompted us to ask whether endogenous PTHrP wouldbe responsible for an increase in PRA upon PTH1R gene deliveryand thereby counterregulate a putative effect on BP. PeripheralhPTH1R gene delivery had no effect on PRA at day 1 after plasmidinjection (Figure 6B). In contrast, PRA was markedly increasedin pcDNA1.1-hPTH1R–treated SHR at days 7, 10, and 14,with a maximum 60% augmentation at day 10. These results stronglysuggest that PRA was indeed upregulated.
Figure 6. Effect of in vivo hPTH1R gene delivery on systolic BP and plasma renin activity (PRA) in SHR, up to 14 d after intravenous plasmid injection. (A) Systolic BP was monitored simultaneously in 12 pcDNA1.1-hPTH1R–treated SHR (hatched bars) and in 12 control pcDNA1.1-treated SHR (open bars). (B) PRA in tail vein blood sample expressed as ng angiotensin I (AngI) formed by ml of plasma per hr of incubation was measured simultaneously in five pcDNA1.1-hPTH1R–treated SHR (hatched bars) and in five control pcDNA1.1-treated SHR (open bars) just before (0) and 1, 7, 10, and 14 d after plasmid injections. Values are shown as mean ± SE. *P < 0.05 versus control-treated SHR.
In a separate series of SHR, food and water intake, as wellas diuresis, natriuresis, and creatinine clearance, were assessed(Table 1). None of these parameters were affected by in vivoPTH1R gene delivery as compared with control SHR treated withempty pcDNA1.1. This was true also for blood and urinary excretionof calcium, potassium, and phosphate (not shown).
Table 1. Body weight, food and water intake, diuresis (UV), netruresis (UVNa), and creatinine clearance (C-creatinine) in control pcDNA1.1-treated and pcDNA1.1-hPTH1R–treated SHRa
Effect of hPTHR1 Gene Delivery on Renal Tone In Vitro
Three weeks after plasmid injection, peripheral delivery ofPTH1R gene had no effect on the mean arterial pressure measuredin the aorta of the anesthetized animal in the course of IPKpreparation (Figure 7A), an observation similar to that seenin conscious SHR (Figure 6A). The RVR measured in the IPK ofpcDNA1.1-hPTH1R–treated SHR exhibited a 23% decrease ascompared with control-treated SHR (Figure 7B). In other words,RVR was 38% higher in control SHR, compared with SHR that receivedthe PTH1R gene. This change was quantitatively very similarto that described previously between SHR and WKY (19).
Figure 7. Effect of peripheral delivery of hPTH1R gene on renal vascular resistance (RVR) in vitro in SHR. (A) mean arterial pressure (MAP) measured in anesthetized SHR in the course of IPK preparations. (B) RVR measured in IPK after 1 h of equilibration. *P < 0.05 in t test for eight and nine independent IPK preparations from control pcDNA1.1-treated and pcDNA1.1-hPTH1R–treated SHR, respectively.
We then asked whether the decrease of RVR was due to endogenousvasodilatory PTHrP interacting with an increased number of receptorsafter pcDNA1.1-hPTH1R injection. To test this hypothesis, weasked whether PTHrP(3–36), a PTH1R antagonist (30), wouldbe able to increase basal tone in IPK derived from SHR, whichreceived pcDNA1.1-hPTHR1 plasmid 3 wk earlier (Figure 8). PTHrP(3–36)had no significant effect on basal perfusion pressure in controlIPK. In marked contrast, however, the PTHrP antagonist clearlyinduced a prolonged increase in basal perfusion pressure ofIPK derived from transfected SHR by about 10 mmHg (14%) comparedwith control animals.
Figure 8. Effect of peripheral delivery of hPTH1R gene in SHR on the action of the PTH1R antagonist, PTHrP(3-36), on basal tone of IPK prepared from control pcDNA1.1-treated (open circles, n = 3) and pcDNA1.1-hPTHR1–treated (black circles, n = 4) SHR. PTHrP(3-36) was infused over a period of 6 min.*P < 0.05 in ANOVA test.
PTHrP(1-36)–induced vasodilation has been proven to bemarkedly reduced in SHR IPK (19). We, therefore, also checkedthat renal vascular replenishment in PTH1R increases the renalvasodilation induced by exogenous PTHrP(1–36) (Figure 9).In IPK derived from pcDNA1.1-hPTH1R–treated SHR, thevasodilation induced by 10 nM PTHrP(1–36) was twice ofthat seen in control-treated SHR and was close to that seenearlier in normotensive rats (19).
Figure 9. Effect of peripheral delivery of hPTH1R gene in SHR on the PTHrP(1-36)–induced vasodilation in IPK prepared from control pcDNA1.1-treated (open bars, n = 5) and pcDNA1.1-hPTH1R-treated (hatched bars, n = 5) SHR. At the end of a 60-min equilibration period, phenylephrine (PE) was infused over 15-s periods every 2 min to induce repetitive, transient, and reproducible pressure peaks of about 50 mmHg in both series of rats. Each bar represents the peak value of the vasoconstriction induced by PE (10 µM). PTHrP(1-36) was continuously infused over a period of 8 min (arbitrary from 0 min on the graph). ND, not determined; #P < 0.05 versus control pcDNA1.1-treated SHR; *P < 0.05 versus the PE-induced vasoconstriction in the absence of PTHrP(1-36) in ANOVA test.
Mechanical stretch or distention of hollow organs, includingthe heart and vasculature, induce the expression of PTHrP (37).PTHrP thus produced may help to adapt wall compliance to lumenalfilling or pressure increase. The vasodilator effects of PTHrPmight also balance vasoconstrictor effects and might exert anegative feedback control of vascular tone. For example, constrictoryAngII (15) or endothelin-1 (37) upregulate dilatory PTHrP invascular smooth muscle cells (VSMC), which in turn stimulatesrenin secretion (11). It has therefore been hypothesized thatPTHrP expression might be enhanced in response to BP increase.In support of this, PTHrP mRNA and protein have been shown tobe increased in deoyxcorticsterone-acetate–treated andsaline-treated rats (17) as well as in both the mesenteric arteriesand the aorta of SHR with established hypertension (18). Overall,these findings suggested a negative feedback control of renovasculartone by PTHrP in hypertension. However, we recently demonstratedthat renal PTHrP(1-36)–induced vasodilation is bluntedin SHR with established hypertension (19). Also, PTH1R antagonistswere unable to affect RVR, which is elevated in these animals(19). One must conclude therefore that PTHrP is not able tocounteract the high renovascular tone in the SHR model of genetichypertension.
With this background, we now report that within the kidney ofnormotensive rats, PTHrP(1-36) is enriched in vessels. Withinthe kidney of SHR with established hypertension, PTHrP(1-36)is selectively upregulated in vessels. Conversely, the presentfindings also reveal that the expression of both PTH1R transcriptand protein are markedly reduced in renal vessels of SHR, suggestingtranscriptional regulation of PTH1R gene expression. WhetherPTH1R downregulation takes place before or after PTHrP upregulationremains an unanswered question. Vascular PTH receptors rapidlydesensitize to PTHrP (38,39); therefore, it is possible thatPTHrP upregulation may be responsible for PTH1R downregulation.In support of this, PTH1R downregulation has been associatedwith PTHrP upregulation induced by AngII in VSMC (40) or duringneointimal formation after angioplasty in rat carotids and humancoronary arteries (41). An increased level of circulating PTHin SHR, together with a reduced density of PTH1R in renal corticalmembranes, further supports this hypothesis (42). Accordingly,downregulation of PTH1R may also explain why hyperparathyroidismis associated with an increased incidence of hypertension andcardiovascular disease. Alternatively, downregulation of PTH1Rwith resultant compensatory increases in endogenous PTHrP maybe characteristic of the SHR. Whether the upregulation of PTHrPand downregulation of PTH1R in renal vessels takes place beforeor in response to BP increase is another question that remainsunanswered. That BP may be responsible for PTHrP upregulationhas been documented by Noda et al. (18). In these studies, performedon the aorta, PTHrP upregulation was not observed in young prehypertensiveSHR. Moreover, antihypertensive treatment of mature SHR withAngII type I receptor antagonist or hydralazine resulted ina concomitant decrease in the PTHrP transcript level with loweringof BP. However, AngII is known to upregulate PTHrP; therefore,it is not clear whether PTHrP downregulation was due to BP decreaseor to the inhibition of AngII action. In this context, PTH1Rand PTHrP expressions have been proven to be similar and increased,respectively, in VSMC cultured from intrarenal vessels isolatedfrom SHR as compared with WKY (26). Thus, upregulation of PTHrPin renal VSMC of SHR is conserved in vitro, and downregulationof PTH1R is not. These observations strongly suggest that BPwould play a major role in PTH1R downregulation and that PTHrPupregulation would reflect a genetic defect in SHR. Overall,the question of whether the alteration of the PTHrP/PTH1R systemreflects a genetic defect in SHR requires much further investigation.Also of note is the fact that the results of Noda et al. (18)are in conflict with another study performed on the aorta (43).These latter authors found a decrease in PTHrP mRNA expressionin the aorta of hypertensive SHR. This discrepancy is not clear.Interestingly enough, Noda et al. (18) saw a PTHrP mRNA increasein another resistance vessel, the mesenteric vascular bed, whichis consistent with our own results.
These results also strongly suggest that, although upregulated,PTHrP is unable to fully exert its vasodilatory effect for lackof a sufficient number of renovascular PTH1R in SHR renal vessels.They further suggest that increasing or restoring the vasculardensity of PTH1R would in turn oppose the high renal and systemictone in these animals. The present study shows that somaticgene delivery of human PTH1R plasmid DNA under the control ofa constitutive promoter significantly decreased in vitro RVRof SHR back to the value seen in normotensive animals. The reductionof the in vitro renal tone appeared to be long-lasting, as itwas maintained for at least 3 wk after the intravenous injectionof pcDNA1.1-hPTH1R. Expression of hPTH1R was detected in allof the main organs. In the kidney, the expression of hPTH1Rappeared preferentially localized in the vessels, suggestingthat, after intravenous injection, hPTH1R gene is preferentiallydelivered to the vascular structures, at least in the kidney.However, the precise cellular localization of transgene expressionwithin the vascular wall has not been explored and will requirespecific studies. Altogether, these results clearly demonstratethat systemic gene delivery of hPTH1R in genetically hypertensiverats results in expression of the foreign gene and that thepool of PTH1R thus replenished induces a long-lasting reductionin the intrinsic value of RVR measured in the IPK. That thereduction in vascular tone was due to endogenous vasodilatoryPTHrP was further documented by the appearance of a vasoconstrictoryresponse to a PTH1R antagonist; it was not observed in the controlvector-treated SHR or in normotensive rats (19).
PTHrP is upregulated not only in renal vessels but also in theaorta and the mesenteric arteries (18); therefore, it appearsreasonable to anticipate that the expression of PTHrP is increasedin all vessels of the SHR. Hence it appears conceivable thatreplenishment of the PTH1R pool would improve not only renaltone but also BP in SHR. We, however, found that the intravenousdelivery of the PTH1R gene to SHR had no significant effecton BP. We have previously observed that exposure of either IPK,renal cortical cells enriched in juxtaglomerular cells, or isolatedglomeruli, to subnanomolar or nanomolar concentrations of PTHrP(1-36)caused a strong and immediate increase in renin activity release(11,36). Consistent with these earlier studies, we now reportthat peripheral delivery of PTH1R gene increased PRA, an effectthat lasted at least 2 wk after gene delivery. Importantly,this finding would imply that endogenous PTHrP stimulates reninrelease through interaction with juxtaglomerular PTH1R, as thekidney is the major source of renin. Thus, the lack of an effecton BP might reasonably be attributed to the simultaneous effecton PRA. Tissue angiotensinogen level in the kidney is commonlyconsidered to be very low compared with plasma angiotensinogen.Therefore, an increase in renin production by the IPK does notresult in the formation of AngII, which could counteract thebeneficial effect of hPTH1R gene delivery on renal tone in vitro.On the other hand, it is conceivable that the effect of hPTH1Rgene delivery on renin release would attenuate its beneficialeffect on RVR under in vivo conditions, as well. Further studiesare required to answer these questions. In any event, this studyprovides initial evidence for a cause-and-effect relation betweenPTH1R density and PRA, strongly supporting the PTH/PTHrP systemas a regulator of the renin-angiotensin system and, in turn,as a regulator of BP.
In conclusion, we report that in renal vessels of the SHR PTHrPis upregulated and that the PTH1R is downregulated. Which ofthese is an upstream central abnormality causing BP increasein SHR remains to be defined. Although it is generally acceptedthat the efficiency and uptake of somatic naked PTH1R plasmidDNA by direct intravenous injection is rather low, it appearedeffective enough to restore vasodilation in response to exogenousPTHrP in the IPK, and to decrease basal RVR owing to endogenousPTHrP in this in vitro model. Thus, in SHR renal vessels, areduced number of PTH1R contributes to the high RVR, despitethe high expression of vasodilatory PTHrP. Significant findingsof these studies also suggest a direct link between the levelof PTH1R expression and the activity of circulating renin, whichmight be responsible for the absence of beneficial effect ofPTH1R gene delivery on BP. Most importantly, the findings reportedherein strongly support the view that the PTHrP system contributesin a significant way to the homeostasis of renal and systemichemodynamics in the SHR model of genetic hypertension. Althoughsomatic PTH1R gene therapy appears to be compromised by itseffect on PRA, these findings could open other perspectivesin the management of hypertension.
Acknowledgments
This work was supported through grants from French NationalInstitute of Health (E INSERM 0015), the University Louis Pasteur(EA 2307), and the French Medical Research Foundation. A. F.S. and N. T. are supported by NIH grant Ro-I DK 54308. Thiswork is part of the PhD thesis of N. T., who was supported bya fellowship of the French Ministry of Higher Education. Wewarmly thank Dr. A. B. Abou-Samra (Endocrine Unit, MassachusettsGeneral Hospital, Boston, MA) for kindly providing us the pcDNA1.1plasmid construct. The authors also gratefully acknowledge Drs.J. P. Girolami and C. Pecher (U 388 INSERM, Toulouse, France)for their advice regarding the Western blot studies and TheLaboratoire d’Analyses de Biologie Médicale, (CHU,Strasbourg, Drs. M. Offner and M. Grima) for measuring bloodand urinary parameters. Mrs. D. Kuhlwein and S. Rothhut arethanked for secretarial and technical help, respectively.
Footnotes
1Thierry Massfelder and Nathalie Taesch participated equallyin this work.
Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH, Vasavada RC, Weir EC, Broadus AE, Stewart AF: Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 76: 127–173, 1996[Abstract/Free Full Text]
Juppner H, Abou-Samra AB, Freeman M, Kong XF, Schipani E, Richards J, Kolakowski LF Jr, Hock J, Potts JT Jr, Kronenberg HM, Segre GV: A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254: 1024–1026, 1991[Abstract/Free Full Text]
Amizuka N, Henderson JE, Hoshi K, Warshawsky H, Ozawa, H Goltzman D, Karaplis AC: Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion. Endocrinology 137: 5055–5067, 1996[Abstract]
Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra AB, Juppner H, Segre GV, Kronenberg HM: PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273: 663–666, 1996[Abstract]
Strewler GJ: The physiology of parathyroid hormone-related protein. New England J Med 342: 177–185, 2000[Free Full Text]
Maeda S, Sutliff RL, Qian J, Lorenz JN, Wang J, Tang H, Nakayama T, Weber C, Witte D, Strauch AR, Paul RJ, Fagin JA, Clemens TL: Targeted overexpression of parathyroid hormone-related protein (PTHrP) to vascular smooth muscle in transgenic mice lowers blood pressure and alters vascular contractility. Endocrinology 140: 1815–1825, 1999[Abstract/Free Full Text]
Qian J, Lorenz JN, Maeda S, Sutliff RL, Weber C, Nakayama T, Colbert MC, Paul RJ, Fagin JA, Clemens TL: Reduced blood pressure and increased sensitivity of the vasculature to parathyroid hormone-related protein (PTHrP) in transgenic mice overexpressing the PTH/PTHrP receptor in vascular smooth muscle. Endocrinology 140: 1826–1833 1999[Abstract/Free Full Text]
Massfelder T, Helwig JJ:, Parathyroid hormone-related protein in cardiovascular development and blood pressure regulation. Endocrinology 140: 1507–1510, 1999[Free Full Text]
Massfelder T, Fiaschi-Taesch N, Stewart AF, Helwig JJ: Parathyroid hormone-related peptide - a smooth muscle tone and proliferation regulatory protein. Curr Opin Nephrol Hypertension 7: 27–32, 1997
Trizna W, Edwards RM: Relaxation of renal arterioles by parathyroid hormone and parathyroid hormone-related protein. Pharmacology 42: 91–96, 1991[Medline]
Saussine C, Massfelder T, Parnin F, Judes C, Simeoni U, Helwig JJ: Renin stimulating properties of parathyroid hormone-related peptide in the isolated perfused rat kidney. Kidney Int 44: 764–773, 1993[Medline]
Lee K, Brown D, Urena P, Ardaillou N, Ardaillou R, Deeds J, Segre GV: Localization of parathyroid hormone/parathyroid hormone-related peptide receptor mRNA in kidney. Am J Physiol 270: F186–F191, 1996[Abstract/Free Full Text]
Yang T, Hassan S, Huang YG, Smart AM, Briggs JP, Schnermann JB:, Expression of PTHrP, PTH/PTHrP receptor, and Ca(2+)-sensing receptor mRNAs along the rat nephron. Am J Physiol 272: F751–F758, 1997[Abstract/Free Full Text]
Rettig R, Bandelow N, Patschan O, Kuttler B, Frey B, Uber A: The importance of the kidney in primary hypertension: Insights from cross-transplantation. J Hum Hypertens 10: 641–644, 1996[Medline]
Noda M, Katoh T, Takuwa N, Kumada M, Kurokawa K, Takuwa Y: Synergistic stimulation of parathyroid hormone-related peptide gene expression by mechanical stretch and angiotensin II in rat aortic smooth muscle cells. J Biol Chem 269: 17911–17917, 1994[Abstract/Free Full Text]
Pirola CJ, Wang HM, Strgacich MI, Kamyar A, Cercek B, Forrester JS, Clemens TL, Fagin JA: Mechanical stimuli induce vascular parathyroid hormone-related protein gene expression in vivo and in vitro. Endocrinology 134: 2230–2236, 1994[Abstract]
Takahashi K, Inoue D, Ando K, Matsumoto T, Ikeda K, Fujita T: Parathyroid hormone-related peptide as a locally produced vasorelaxant: Regulation of its mRNA by hypertension in rats. Biochem Biophys Res Commun 208: 447–455, 1995[CrossRef][Medline]
Noda M, Katoh T, Kurokawa K, Takuwa Y: Increased expression of parathyroid hormone-related peptide gene in blood vessels of spontaneously hypertensive rats. Hypertension 30: 1284–1288, 1997[Abstract/Free Full Text]
Fiaschi-Taesch N, Endlich N, Massfelder T, Endlich K, Stewart AF, Helwig JJ: Renovascular parathyroid hormone-related protein in spontaneously hypertensive rats: dilator or trophic factor? Kidney Int 67: S207–S210, 1998
Chao J, Jin L, Lin KF, Chao L:, Adrenomedullin gene delivery reduces blood pressure in spontaneously hypertensive rats. Hypertension Res 20: 269–277, 1997[Medline]
Lin KF, Chao J, Chao L: Human atrial natriuretic peptide gene delivery reduces blood pressure in hypertensive rats. Hypertension 26: 847–853, 1995[Abstract/Free Full Text]
Lin KF, Chao J, Chao L: Atrial natriuretic peptide gene delivery reduces stroke-prone mortality rate in Dahl salt-sensitive rats. Hypertension 33: 219–224, 1999[Abstract/Free Full Text]
Wang C, Chao L, Chao J: Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats. J Clin Invest 95: 1710–1716, 1995
Endlich K, N Endlich, N Fiaschi-Taesch, JJ Helwig: Culture of vascular smooth muscle cells from small arteries of the rat kidney. Kidney Int 57: 2468–2475, 2000[CrossRef][Medline]
Celic S, Chilco PJ, Zajac JD, Martin TJ, Findlay DM: A type I collagen substrate increases PTH/PTHrP receptor mRNA expression and suppresses PTHrP mRNA expression in UMR106–06 osteoblast- like cells. J Endocrinol 150: 299–308, 1996[Abstract/Free Full Text]
Massfelder T, Taesch N, Endlich N, Eichinger A, Escande B, Endlich K, Barthelmebs M, Helwig JJ: Paradoxical actions of exogenous and endogenous parathyroid hormone-related protein on renal vascular smooth muscle cell proliferation: reversion in the SHR model of genetic hypertension. FASEB J 15: 707–718, 2001[Abstract/Free Full Text]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951[Free Full Text]
Schipani E, Karga H, Karaplis AC, Potts JT Jr, Kronenberg HM, Segre GV, bou-Samra AB, Juppner H:. Identical complementary deoxyribonucleic acids encode a human renal and bone parathyroid hormone (PTH)/PTH-related peptide receptor. Endocrinology 132: 2157–2165, 1993[Abstract]
Menard J, Catt KJ: Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I. Endocrinology 90: 422–430, 1972[Medline]
Rabbani SA, Mitchell J, Roy DR, Hendy GN, Goltzman D: Influence of the amino-terminus on in vitro and in vivo biological activity of synthetic parathyroid hormone-like peptides of malignancy. Endocrinology 123: 2709–2716, 1988[Abstract]
Karpf DB, Bambino T, Alford G, Nissenson RA: Features of the renal parathyroid hormone-parathyroid hormone-related protein receptor derived from structural studies of receptor fragments. J Bone Miner Res 6173: 82n, 1991
Orloff JJ, Goumas D, Wu TL, Stewart AF: Interspecies comparison of renal cortical receptors for parathyroid hormone and parathyroid hormone-related protein. J Bone Miner Res 6: 279–287, 1991[Medline]
Bisello A, Greenberg Z. Behar, V Rosenblatt, M. Suva, LJ and Chorev: M. Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor. Biochemistry 35: 15890–15895, 1996[CrossRef][Medline]
Karpf, DB Arnaud CD Bambino, T Duffy, D. King KL Winer, J and Nissenson: R.A. Structural properties of the renal parathyroid hormone receptor: Hydrodynamic analysis and protease sensitivity. Endocrinology 123: 2611–2620, 1988[Abstract]
Overton MC, Blumer KJ. G-protein-coupled receptors function as oligomers in vivo. CurrBiol 10: 341–344, 2000
Saussine C, Judes C, Massfelder T, Musso MJ, Simeoni U, Hannedouche T, elwig JJ: Stimulatory action of parathyroid hormone on renin secretion in vitro: A study using isolated rat kidney, isolated rabbit glomeruli and superfused dispersed juxtaglomerular cells. Clinical Sci (Colch) 84: 11–19, 1993
Noda M, Takuwa Y, Katoh T, Kurokawa K. Mechanical force regulation of vascular parathyroid hormone-related peptide expression. Kidney Int Suppl 55: S154–S155, 1996[Medline]
Massfelder T, Stewart AF, Endlich K, Soifer N, Judes C, Helwig JJ: Parathyroid hormone-related protein detection and interaction with NO and cyclic AMP in the renovascular system. Kidney Int 50: 1591–1603, 1996[Medline]
Nyby MD, T Hino, ME Berger, BL Ormsby, MS Golub, AS Brickman: Desensitization of vascular tissue to parathyroid hormone and parathyroid hormone-related protein. Endocrinology 136: 2497–2504, 1995[Abstract]
Okano K, Wu S, Huang X, Pirola CJ, Juppner H, Abou-Samra AB, Segre GV, Iwasaki K, Fagin JA, Clemens TL: Parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor and its messenger ribonucleic acid in rat aortic vascular smooth muscle cells and UMR osteoblast-like cells: cell-specific regulation by angiotensin- II and PTHrP. Endocrinology 135: 1093–1099, 1994[Abstract]
Ozeki S, Ohtsuru A, Seto S, Takeshita S, Yano H, Nakayama T, Ito M, Yokota T, Nobuyoshi M, Segre GV, Yamashita S, Yano K: Evidence that implicates the parathyroid hormone-related peptide in vascular stenosis. Increased gene expression in the intima of injured carotid arteries and human restenotic coronary lesions. Arterioscler Thromb Vasc Biol 16: 565–575, 1996[Abstract/Free Full Text]
DiPette DJ, Christenson W, Nickols MA, Nickols GA: Cardiovascular responsiveness to parathyroid hormone (PTH) and PTH- related protein in genetic hypertension. Endocrinology 130: 2045–2051, 1992[Abstract]
Garcia SI, Clemens TL, Fagin JA, Finkielman S, Pirola CJ: Parathyroid hormone-related protein expression in vascular smooth muscle of spontaneously hypertensive rats: Evidence for lack of response to angiotensin II. J Hypertension 16: 1467–1474, 1998[CrossRef][Medline]
Received for publication June 12, 2001.
Accepted for publication August 21, 2001.
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Abstract
Introduction
