Jean-Jacques Helwig

Effect of intrarenally infused parathyroid hormone‐related protein on renal blood flow and glomerular filtration rate in the anaesthetized rat

1 Parathyroid hormone‐related protein (PTHrP) is expressed in the kidney and acts on vascular PTH/PTHrP receptors to vasodilate the isolated kidney and to stimulate renin release. However, effects of PTHrP on renal blood flow (RBF) and glomerular filtration rate (GFR) in vivo have not been assessed in the absence of its cardiac, peripheral and central effects. We investigated the renal effects of PTH and PTHrP infused into the left renal artery of anaesthetized rats. 2 Intrarenal infusions, adjusted to generate increasing concentrations of human PTHrP(1–34) and rat PTH(1–34) in renal plasma (2 × 10−11 to 6 × 10−9 m) produced a comparable dose‐dependent increase in RBF. The rise was 4% at the lowest and 34% at the highest concentrations of peptides. Up to a concentration of 2 × 10−9 m, mean arterial pressure (MAP) and heart rate were not affected, but at 6 × 10−9 m, intrarenally infused peptides reached the peripheral circulation, and caused a fall in MAP within a few minutes. While MAP returned to basal value after the last peptide infusion, RBF remained more than 10% above control for at least 30 min. 3 Two competitive PTH/PTHrP receptor antagonists, [Nle8,18, Tyr34]‐bPTH(3–34)amide and [Leu11, D‐Trp12]‐hPTHrP(7–34)amide (2 × 10−8 m) were devoid of agonist activity, but markedly antagonized the dose‐dependent increase in RBF elicited by PTHrP. 4 GFR and urine flow were measured in left PTHrP‐infused experimental kidney and right control kidney. Renal PTHrP concentration of 10−10 m elevated left RBF by 10%, and GFR by 20% without significantly increasing filtration fraction, and increased urine flow by 57%. In the right control kidney GFR and diuresis did not change. 5 The results indicate that PTHrP has similar renal haemodynamic effects as PTH and increases RBF, GFR and diuresis in anaesthetized rats.

Editorial: Parathyroid Hormone-Related Protein in Cardiovascular Development and Blood Pressure Regulation

PTH-related protein (PTHrP) was isolated in 1987 as the tumoral factor responsible for the humoral hypercalcemia of malignancy (reviewed in Ref. 1). PTHrP is produced not only by many tumors, but also by virtually all normal cells and tissue types throughout the body during development and adult life. PTHrP and PTH/PTHrP receptor (PTH-R) are present in smooth muscle-rich organs such as bladder, uterus, stomach, intestine, and the chicken oviduct, as well as throughout the cardiovascular system. In the latter system, PTHrP and the PTH-R are produced in the heart as well as in vascular smooth muscle (VSM) and in endothelial cells of all vessels examined so far. Lessons from PTHrP or PTH-R gene knockout mice, which die at birth or in utero, emphasize the critical role of PTHrP for normal development (2). The PTHrP and PTH genes arise from a common ancestral gene. The PTHrP pre-messenger RNA is alternatively spliced to give rise to three initial translation products of 139, 141, and 173 amino acids, depending on the cell type and the species. These forms are in turn posttranslationally processed to form a family of mature peptides, of which PTHrP (1–36) (structurally and functionally related to PTH), PTHrP (38–94) and PTHrP (107–139) are the major secretory forms. Receptors for mid-region and carboxy-terminal PTHrP species have only been defined by indirect pharmacological tools and are still under investigation. On the other hand, the PTH-R, which recognizes PTHrP (1–36) as well as PTH, has been cloned and well characterized in terms of structure, localization, pharmacological properties, and physiological regulation. The PTH-R is an heptahelical, G protein-coupled receptor that binds PTH as well as all of the amino-terminal containing PTHrP species. Over the last decade, a growing number of studies have provided clear evidence for PTHrP as a novel class of multifunctional peptides. First, PTHrP is a regulator of transepithelial calcium transport in renal tubules, placenta, and possibly the mammary gland. Second, PTHrP is a regulator of development, growth, and differentiation in all tissues that have been examined so far. Finally, PTHrP is a potent regulator of smooth muscle. This latter area has been extensively studied, and PTHrP is now appreciated to be involved in the control of vascular tone as a vasodilator, as well as of cardiac functions as a positive chronotropic and inotropic factor. The PTHrP gene has often been compared with an early gene. This has been proven to be true in smooth muscle where PTHrP expression is quickly and transiently up-regulated by mechanical forces, by vasoconstrictors such as angiotensin II, and growth factors (for complete reviews, see Refs. 3–7). In two papers appearing in the present issue of Endocrinology, Clemens and co-workers (8, 9) describe for the first time a cardiovascular phenotype of transgenic mice overexpressing PTHrP and/or PTH-R in smooth muscle. These studies not only strongly support PTHrP as a cardiovascular regulatory peptide but also raise a number of intriguing questions concerning the role of PTHrP during the development of the cardiovascular system and the mechanisms by which PTHrP modulates blood pressure in adult animals. While the expression of PTHrP and PTH-R are known to be spatially and temporally regulated during development of various organs such as bone, skin, mammary gland, or kidney (2–7), no clear evidence has been obtained to date for a role of PTHrP in the development of the cardiovascular system. In the PTHrP knockout mouse, which dies at birth from a severe skeletal disorder, the cardiovascular system appears to develop normally, suggesting that PTHrP is not an essential factor for the development of the cardiovascular system (2). However, accurate vascular morphometric investigations, such as vessel lumen-to-wall ratio measurements, have not been specifically performed in these mice, and no cardiovascular functional data are available. Massfelder et al. (10) have recently demonstrated that introduction of PTHrP into A10 VSM cells by gene transfer stimulates proliferation of these cells by transport of PTHrP into the nucleus. Paradoxically, the opposite occurred with the addition of exogenous PTHrP: in this setting, PTHrP was antimitogenic. Moreover, the number of dividing cells in the aortic media of PTHrP knockout fetuses was reduced compared with their normal littermates. These studies demonstrate that, in VSM, PTHrP can operate in both a paracrine or autocrine pathway but may also operate via an “intracrine” (nuclear targeting) pathway. PTHrP could thus be involved in both paracrine as well as intracrine signaling pathways of VSM proliferation during angiogenesis and vasculogenesis. The coordinated distribution of PTHrP and PTH-R between adjacent cell types in a wide variety of fetal sites, including bone, kidney, skin, and mammary gland (3–7), strongly suggests that PTHrP and the PTH-R are involved in the modulation of cell differentiation. Similarly, because PTHrP and PTH-R are present in both VSM cells and endothelial cells, it appears also likely that PTHrP could be involved in the modulation of angiogenesis and vasculogenesis through paracrine signaling pathways. It is now accepted that continuous exposure of the PTH-R to ligand (PTH or PTHrP) leads to receptor desensitization (11). As discussed below, the PTH-R is rapidly desensitized in vascular tissue in response to sustained exposure to PTHrP Received January 20, 1999. Address all correspondence and requests for reprints to: Jean-Jacques Helwig, Ph.D., Laboratoire de Physiologie et de Pharmacologie (CJF INSERM 9409, EA MENRT 2307), 11 rue Humann, 67085 Strasbourg Cedex, France. E-mail: jean-jacques.helwig@pharmaco-ulp.u-strasbg.fr. 0013-7227/99/

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

03.00/0 Vol. 140, No. 4 Endocrinology Printed in U.S.A. Copyright

Signal transduction pathways involved in kinin B2 receptor‐mediated vasodilation in the rat isolated perfused kidney

In previous studies, added parathyroid hormone‐related protein (PTHrP) inhibits whereas transfected PTHrP stimulates the proliferation of A10 aortic smooth muscle cells by nuclear translocation of the peptide. In the present studies, we asked whether these paradoxical trophic actions of PTHrP occur in smooth muscle cells (SMC) cultured from small intrarenal arteries of, and whether they are altered in, 12‐wk‐old spontaneously hypertensive rats (SHR) as compared to normotensive Wistar‐Kyoto (WKY) rats. SHR cells grew faster than WKY cells. PTHrP transcript was increased in SHR‐derived cells whereas PTH1 receptor (PTH1R) transcripts were similar in both cell lines. In both strains of cells, stable transfection with human PTHrP(1–139) cDNA did not further induce proliferation, suggesting maximal effect of endoge‐nous PTHrP in wild cells. In contrast, transfection with antisense hPTHrP(1–139) cDNA, which abolished PTHrP mRNA, decreased WKY but increased SHR cell proliferation. Added PTHrP(1–36) (1–100 pM) de¬creased WKY and increased SHR cell proliferation. Additional studies indicated that the preferential cou¬pling of PTH1‐R to G‐protein Gi was responsible for the proliferative effect of exogenous PTHrP in SHR cells. Moreover, PTHrP was detected in the nucleolus of a fraction of WKY and SHR renal SMC, in vitro as well as in situ, suggesting that the nucleolar transloca¬tion of PTHrP might be involved in the proliferative effects of endogenous PTHrP. In renovascular SMC, added PTHrP is antimitogenic, whereas endogenously produced PTHrP is mitogenic. These paradoxical ef¬fects of PTHrP on renovascular SMC proliferation appear to be reversed in the SHR model of genetic hypertension. A new concept emerges from these re¬sults, according to which a single molecule may have opposite effects on VSMC proliferation under physio¬logical and pathophysiological conditions.—Mass¬felder, T., Taesch, N., Endlich, N., Eichinger, A., Escande, B., Endlich, K., Barthelmebs, M., Helwig, J.‐J. 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)

Vascular kinin B1 and B2 receptor-mediated effects in the rat isolated perfused kidney–differential regulations

The signal transduction pathways involved in kinin B2 receptor‐related vasodilation were investigated in rat isolated perfused kidneys. During prostaglandin F2α or KCl‐induced constriction, the vasodilator response to a selective B2 receptor agonist, Tyr(Me)8bradykinin (Tyr(Me)8BK), was assessed. Tyr(Me)8BK produced a concentration‐ and endothelium‐dependent relaxation that was decreased by about 30u2003–u200340% after inhibition of nitric oxide (NO) synthase by NG‐nitro‐L‐arginine (L‐NOARG) or of cyclo‐oxygenase by indomethacin; a greater decrease (about 40u2003–u200350%) was observed after concomitant inhibition of the two pathways. High extracellular K+ diminished Tyr(Me)8BK‐induced relaxation by about 75% suggesting a major contribution of endothelium‐derived hyperpolarization. The residual response was almost completely suppressed by NO synthase and cyclo‐oxygenase inhibition. The K+ channel inhibitors, tetrabutylammonium (non‐specific) and charybdotoxin (specific for Ca2+‐activated K+ channel), suppressed Tyr(Me)8BK‐induced relaxation resistant to L‐NOARG and indomethacin. Inhibition of cytochrome P450 (clotrimazole or 7‐ethoxyresorufin) decreased the NO/prostanoids‐independent relaxation to Tyr(Me)8BK by more than 60%, while inhibition of the cannabinoid CB1 receptor (SR 141716A) had only a moderate effect. Acetylcholine induced a concentration‐dependent relaxation with characteristics nearly similar to the response to Tyr(Me)8BK. In contrast, the relaxation elicited by sodium nitroprusside was potentiated in the absence of NO (L‐NOARG or removal of endothelium) but remained unchanged otherwise. These results indicate that the activation of kinin B2 receptors in the rat isolated kidney elicits an endothelium‐dependent vasorelaxation, mainly dependent on the activation of charybdotoxin‐sensitive Ca2+‐activated K+ channels. In addition, cytochrome P450 derivatives appear to be involved.

Role of shear stress in nitric oxide-dependent modulation of renal angiotensin II vasoconstriction.

Bradykinin (BK) and analogs acting preferentially at kinin B1 or B2 receptors were tested on the rat isolated perfused kidney. Kidneys were perfused in an open circuit with Tyrodes solution. Kidneys preconstricted with prostaglandin F2α were used for the analysis of vasodilator responses. BK induced a concentration‐dependent renal relaxation (pD2=8.9±0.4); this vasodilator response was reproduced by a selective B2 receptor agonist, Tyr(Me)8‐BK (pD2=9.0±0.1) with a higher maximum effect (Emax=78.9±6.6 and 55.8±4.3% of ACh‐induced relaxation respectively, n=6 and 19, P<0.02). Icatibant (10u2003nM), a selective B2 receptor antagonist, abolished BK‐elicited relaxation. Tachyphylaxis of kinin B2 receptors appeared when repeatedly stimulated at 10u2003min intervals. Des‐Arg9‐BK, a selective B1 receptor agonist, induced concentration‐dependent vasoconstriction at micromolar concentration. Maximum response was enhanced in the presence of lisinopril (1u2003μM) and inhibited by R 715 (8u2003μM), a selective B1 receptor antagonist. Des‐Arg9‐[Leu8]‐BK behaved as an agonist. A contractile response to des‐Arg9‐BK occurred after 1u2003h of perfusion and increased with time by a factor of about three over a 3u2003h perfusion. This post‐isolation sensitization to des‐Arg9‐BK was abolished by dexamethasone (DEX, 30u2003mgu2003kg−1 i.p., 3u2003h before the start of the experiment and 10u2003μM in perfusate) and actinomycin D (2u2003μM). Acute exposure to DEX (10u2003μM) had no effect on sensitized des‐Arg9‐BK response, in contrast to indomethacin (30u2003μM) that abolished it. DEX pretreatment however had no effect on BK‐induced renal vasodilation. Present results indicate that the main renal vascular response to BK consists of relaxation linked to the activation of kinin B2 receptors which rapidly desensitize. Renal B1 receptors are also present and are time‐dependently sensitized during the in vitro perfusion of the rat kidneys.

Nitric oxide, but not vasopressin V2 receptor-mediated vasodilation, modulates vasopressin-induced renal vasoconstriction in rats

Renal vasoconstriction in response to angiotensin II (ANGII) is known to be modulated by nitric oxide (NO). Since shear stress stimulates the release of a variety of vasoactive compounds from endothelial cells, we studied the impact of shear stress on the haemodynamic effect of ANGII in isolated perfused kidneys of rats under control conditions and during NO synthase inhibition with L‐NAME (100u2003μM). Kidneys were perfused in the presence of cyclo‐oxygenase inhibitor (10u2003μM indomethacin) with Tyrodes solution of relative viscosity ζ=1 (low viscosity perfusate, LVP) or, in order to augment shear stress, with Tyrodes solution containing 7% Ficoll 70 of relative viscosity ζ=2 (high viscosity perfusate, HVP). Vascular conductance was 3.5±0.4 fold larger in HVP as compared with LVP kidneys, associated with an augmentation of overall wall shear stress by 37±5%. During NO inhibition, vascular conductance was only 2.5±0.2 fold elevated in HVP vs LVP kidneys, demonstrating shear stress‐induced vasodilatation by NO and non‐NO/non‐prostanoid compound(s). ANGII (10–100u2003pM) constricted the vasculature in LVP kidneys, but was without effect in HVP kidneys. During NO inhibition, in contrast, ANGII vasoconstriction was potentiated in HVP as compared with LVP kidneys. The potentiation of ANGII vasoconstriction during NO inhibition has been shown to be mediated by endothelium‐derived P450 metabolites and to be sensitive to AT2 receptor blockade in our earlier studies. Accordingly, in HVP kidneys, increasing concentrations of the AT2 receptor antagonist PD123319 (5 and 500u2003nM) gradually abolished the potentiation of ANGII vasoconstriction during NO inhibition, but did not affect vasoconstriction in response to ANGII in LVP kidneys. Our results demonstrate, that augmentation of shear stress by increasing perfusate viscosity induces vasodilatation in the rat kidney, which is partially mediated by NO. Elevated levels of shear stress attenuate renal ANGII vasoconstriction through enhanced NO production and are required for AT2 sensitive potentiation during NO inhibition.

Oxytocin-induced renin secretion by denervated kidney in anaesthetized rat

Abstract. The renal vascular response to vasopressin and its modulation were evaluated in vivo by infusing the peptide directly into the renal artery of anaesthetized rats. The intra-renal artery (i.r.a) infusion of vasopressin induced a dose-dependent decrease in renal blood flow. Vasoconstriction was obvious at a dose of 3xa0ng/kg per min and reached a maximum at 100xa0ng/kg per min. The dose required for a half-maximal response (ED50) was 24±4xa0ng/kg per min (mean±SEM, n=8), corresponding to an estimated concentration in renal arterial blood required for a half-maximal response (EC50) of 1.9±0.6xa0nM. Thiobutabarbitone anaesthesia markedly increased plasma vasopressin concentration. This increase was prevented partially by hypotonic hydration of the rats without any change in the renal vascular response to exogenous vasopressin. Vasopressin-induced vasoconstriction dose/response curves were similar in homozygous and heterozygous Brattleboro rats. Infusion of desmopressin (1–1000xa0ng/kg per min, i.r.a.), a vasopressin V2 receptor-selective agonist, failed to induce renal vasodilation or vasoconstriction. In the presence of SR 49059 (1xa0mg/kg i.v.), a vasopressin V1A receptor antagonist that completely abolished the vasopressin-induced renal vasoconstriction, desmopressin again failed to induce vasodilation. Inhibition of nitric oxide synthase by Nω-nitro-l-arginine (L-NNA, 100xa0µg/kg for 10xa0min and 7.5xa0µg/kg per min, i.r.a.) enhanced vasopressin-induced renal vasoconstriction (EC50 0.6±0.1xa0nM, P<0.05). In contrast, cyclooxygenase blockade by indomethacin (5xa0mg/kg, i.v.) neither modified the vasopressin-induced decrease in renal blood flow nor altered the potentiation of vasoconstriction by L-NNA.These results show that the constrictor response of the rat renal vascular bed in vivo is observed only with high local concentrations of vasopressin. This hyporeactivity in vivo was not explained by an anaesthesia-elicited increase in endogenous vasopressin, nor by a modulatory effect linked to V2 receptor activation or prostanoid release. In contrast, NO release contributed to the attenuation of vasopressin-induced renal vasoconstriction.

Shear stress modulates vasopressin-induced renal vasoconstriction in rats.

The effects of oxytocin on renin secretion by denervated kidney were investigated in vivo, by infusing the peptide directly into the renal artery of anaesthetized rats. Renin secretion was calculated by the renal veno-arterial difference in plasma renin activity multiplied by renal plasma flow. The intra-renal arterial (i.r.a.) infusion of oxytocin (1.5 or 15 ng/kg/min, 10 min) induced a sixfold increase in renin secretion as compared to vehicle-treated controls, without effects on renal blood flow, mean arterial blood pressure, glomerular filtration rate or natriuresis. The effect of oxytocin (1.5 ng/kg/min) was prevented by pretreatment with an oxytocin receptor antagonist, desGly-NH(2),d(CH(2))(5)[D-Tyr(2),Thr(4),Orn(8)]vasotocin] (5.6 microg/kg bolus i.v. 20 min before oxytocin infusion, followed by 2.8 microg/kg/min i.r.a.). Nadolol (2.5 mg/kg i.v.), a beta-adrenoceptor antagonist, also blocked the oxytocin-induced increase in renin secretion. These results show that oxytocin is able to stimulate renin release by activating oxytocin receptors but that beta-adrenoceptors also seem to be involved.

Binding of Parathyroid Hormone and Parathyroid Hormone-Related Protein to Vascular Smooth Muscle of Rabbit Renal Microvessels*

Abstract. Vasopressin is a potent renal vasoconstrictor in vitro which elicits relatively minor renal vascular effects in vivo. Efficient modulation might occur through shear stress-elicited release of vasodilator compounds from endothelial cells. The aim of this study was to evaluate in vitro, in the isolated perfused kidney, the influence of shear stress and related nitric oxide (NO) release on basal renal vascular tone and on vasopressin-induced renal vasoconstriction.Rat kidneys were perfused at a constant flow rate of 8xa0ml/min with Tyrodes solution (relative viscosity η=1) or, in order to increase shear stress, with Tyrodes solution supplemented with 4.7% Ficoll 400 (Ficoll 400; η=2.3), which is representative of the relative viscosity found in small vessels. Renal shear stress was further elevated during vasoconstriction elicited by vasopressin.Basal renal true vascular conductance, which reflects mean blood vessel radius, was 2.5-fold higher and overall wall shear stress doubled in Ficoll 400 – as compared to Tyrode-perfused kidneys. The decrease in vascular conductance elicited by NO synthase inhibition with Nω-nitro-L-arginine (L-NNA) increased with the viscosity of the perfusate. Shear stress was elevated during vasopressin-induced renal vasoconstriction, all the more kidneys were Ficoll 400-perfused. In these kidneys, the concentration-response curve to vasopressin was shifted to the right, giving evidence of hyporeactivity to the peptide. L-NNA potentiated vasoconstriction to vasopressin particularly in Ficoll 400-perfused kidneys, although additional inhibition of cyclooxygenase and/or cytochrome P450 was without effect. These results provide in vitro evidence that shear stress enhanced by perfusate viscosity increases basal renal vascular conductance by an NO-dependent mechanism. Together with shear stress enhanced during vasoconstriction, it blunts vasopressin-induced renal vasoconstriction.