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International audience; Proliferative glomerulonephritis is characterized by local inflammation and mesangial cell deterioration, followed by mesangial proliferation and glomerular healing. Parathyroid hormone-related peptide (PTHrP) is a mesangial cytokine-like growth factor implicated in mesangial proliferation and survival. No data are available about its role in glomerulonephritis. Herein, we analyzed the expression and role of PTHrP in glomerular inflammation and healing in an experimental model of glomerulonephritis induced by i.v. injection of Habu snake venom in mice. The temporal analysis showed marked renal damage in the first days after venom injection and the beginning of recovery within 7 days. Glomerular expression of PTHrP (transcript and protein) was observed in the early phase after venom injection (from day 1 to day 3), along with an inflammatory environment. The inactivation of secreted PTHrP with PTHrP-neutralizing antibody (PTH2E11; 120 μg i.p. daily) reduced the markers of local inflammation (expression of macrophage chemotactic protein-1; regulated upon activation, normal T cell expressed and secreted; cyclooxygenase 2; IL-6; and macrophage infiltration) and abolished the expression of PTHrP itself. Moreover, the glomerular cell proliferation was hampered, and the healing process was prevented on day 7 after venom injection. These results show that PTHrP has antinomic actions in glomerulonephritis, participating in both the proinflammatory condition and the healing process. Our work reveals the essential role of PTHrP in early glomerular repair in an experimental model of glomerulonephritis.

Injury of mesangial cells (MC) is a prominent feature of glomerulonephritis. Activated MC secrete inflammatory mediators that induce cell apoptosis. Parathyroid hormone-related peptide (PTHrP) is a locally active cytokine that enhances cell survival and is upregulated by proinflammatory factors in many cell types. The aim of this study was to analyze the regulation of PTHrP expression by inflammatory cytokines and to evaluate whether PTHrP itself acts as a proinflammatory and/or survival factor on male murine MC in primary culture. Our results showed that IL-1β (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1β were both transcriptional and posttranscriptional, with stabilization of the PTHrP mRNA by human antigen R (HuR). Proteome profiler arrays showed that PTHrP itself enhanced cytokines within 2 h in cell lysates, mainly IL-17, IL-16, IL-1α, and IL-6. PTHrP also stimulated sustained expression (2–4 h) of chemokines, mainly regulated upon activation normal T cell expressed and secreted (RANTES)/C-C motif chemokine 5 (CCL5) and macrophage inflammatory protein-2 (MIP-2)/C-X-C motif chemokine 2 (CXCL2), thymus and activation-regulated chemokine (TARC)/CCL17, and interferon-inducible T cell α-chemoattractant (I-TAC)/CXCL11. Moreover, PTHrP markedly enhanced cyclooxygenase-2 (COX-2) expression and elicited its autoinduction through the activation of the NF-κB pathway. PTHrP induced MC survival via the COX-2 products, and PTHrP overexpression in MC blunted the apoptotic effects of IL-1β and TNF-α. Altogether, these findings suggest that PTHrP functions as a booster of glomerular inflammatory processes and may be a negative feedback loop preserving MC survival.

Glomerulonephritis is characterized by the proliferation and apoptosis of mesangial cells (MC). The parathyroid-hormone related protein (PTHrP) is a locally active cytokine that affects these phenomena in many cell types, through either paracrine or intracrine pathways. The aim of this study was to evaluate the effect of both PTHrP pathways on MC proliferation and apoptosis. In vitro studies were based on MC from male transgenic mice allowing PTHrP-gene excision by a CreLoxP system. MC were also transfected with different PTHrP constructs: wild type PTHrP, PTHrP devoid of its signal peptide, or of its nuclear localization sequence. The results showed that PTHrP deletion in MC reduced their proliferation even in the presence of serum and increased their apoptosis when serum-deprived. PTH1R activation by PTHrP(1–36) or PTH(1–34) had no effect on proliferation but improved MC survival. Transfection of MC with PTHrP devoid of its signal peptide significantly increased their proliferation and minimally reduced their apoptosis. Overexpression of PTHrP devoid of its nuclear localization sequence protected cells from apoptosis without changing their proliferation. Wild type PTHrP transfection conferred both mitogenic and survival effects, which seem independent of midregion and C-terminal PTHrP fragments. PTHrP-induced MC proliferation was associated with p27Kip1 down-regulation and c-Myc/E2F1 up-regulation. PTHrP increased MC survival through the activation of cAMP/protein kinase A and PI3-K/Akt pathways. These results reveal that PTHrP is a cytokine of multiple roles in MC, acting as a mitogenic factor only through an intracrine pathway, and reducing apoptosis mainly through the paracrine pathway. Thus, PTHrP appears as a probable actor in MC injuries.

We investigated the involvement of the nitric oxide pathway in the inhibitory mechanisms of 5-hydroxytryptamine (5-HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in diabetic pithed rats. Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan. Four weeks later, the animals were anaesthetized, pretreated with atropine, and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. The inhibition of electrically induced pressor responses by 5-HT (10 microg/kg/min) in diabetic pithed rats could not be elicited after i.v. treatment with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microg/kg), a guanylyl cyclase inhibitor, or N-omega-L-Arginine methyl ester hydrochloride (L-NAME) (10 mg/kg), a nitric oxide synthase (NOS) inhibitor. The inhibitory effect produced by infusion of the selective 5-HT(1A) receptor agonist 8-hydroxydipropylaminotretalin hydrobromide (8-OH-DPAT) (20 microg/kg/min) was abolished in the presence of ODQ (10 microg/kg), or L-NAME (10 mg/kg) in diabetic pithed rats. The administration of L-Arginine (100 mg/kg) 30 min after L-NAME reproduced the inhibitory effect caused by 5-HT (10 microg/kg/min) and 8-OH-DPAT (20 microg/kg/min) on the electrically induced pressor responses, whereas in the presence of D-Arginine (100 mg/kg)+L-NAME the 5-HT or 8-OH-DPAT inhibitory effect on the pressor responses was abolished. In conclusion, in diabetic pithed rats, the inhibition produced by prejunctional 5-HT(1A) activation on electrically induced sympathetic pressor responses is mediated by the NO synthesis/pathway.

1. We investigated the effect of alloxan-induced diabetes on the inhibitory mechanisms of 5-hydroxytryptamine (5-HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in pithed rats, and analysed the type and/or subtype of 5-HT receptors involved. 2. Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan, then 4 weeks later, they were anaesthetized, pretreated with atropine and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. 3. Intravenous infusions of 5-HT (1-80 microg kg(-1) min(-1)) reduced the pressor effects obtained by electrical stimulation. The 5-HT(1) receptor agonist 5-carboxamidotryptamine, 5-CT (5 microg kg(-1) min(-1)), caused an inhibition of the pressor response, whereas the selective 5-HT(2) receptor agonist, alpha-methyl-5-HT (5 microg kg(-1) min(-1)) and the selective 5-HT(3) receptor agonist, 1-phenylbiguanide (40 microg kg(-1) min(-1)), did not modify the sympathetic pressor responses. 5-HT had no effect on exogenous noradrenaline (NA)-induced pressor responses. 4. The inhibition of electrically induced pressor responses by 5-HT (10 microg kg(-1) min(-1)) was unable to be elicited after i.v. treatment with methiothepin (100 microg kg(-1)) because of the marked inhibition produced by methiothepin alone. The 5-HT-induced inhibition was blocked after i.v. administration of WAY-100,635 (100 microg kg(-1)) and not affected by ritanserin (1 mg kg(-1)), MDL 72222 (2 mg kg(-1)). 5. The selective 5-HT(1A) receptor agonist, 8-hydroxydipropylaminotretalin hydrobromide (8-OH-DPAT) (5-20 microg kg(-1) min(-1)) but neither the rodent 5-HT(1B) receptor agonist, CGS-12066B (5 microg kg(-1) min(-1)), nor the selective nonrodent 5-HT(1B) and 5-HT(1D) receptor agonist, L-694,247 (5 and 40 microg kg(-1) min(-1)), inhibited the electrically induced pressor response. The selective 5-HT(1A) receptor antagonist, WAY-100,635 (100 microg kg(-1)), blocked the inhibition induced by 8-OH-DPAT (10 microg kg(-1) min(-1)). 8-OH-DPAT had no effect on exogenous NA-induced pressor responses. 6. Experimental diabetes produces changes in the inhibitory effect induced by 5-HT on electrically induced sympathetic pressor responses, such that the inhibitory action induced by 5-HT in diabetic pithed rats is mediated by prejunctional 5-HT(1A) receptors.

While parathyroid hormone type 1 receptor (PTH1R)-mediated vasodilatory, cardiac stimulatory, and renin-activating effects of exogenous PTH/PTH-related protein (PTHrP) are acknowledged, interactions of endogenous PTHrP with these systems remain unclear, mainly because the unavailability of viable PTHrP/PTH1R knockout mice. Transgenic mice overexpressing PTH1R in smooth muscle strongly have supported the PTHrP/PTH1R system as a cardiovascular system (CVS) regulator, but the consequences on renovascular (RVS) and renin-angiotensin systems (RAS) have not been explored in these studies. The aim was to develop a model in which one could study the consequences on CVS, RVS, and RAS of generalized PTH1R overexpression. Systemic PTH1R cDNA plasmid delivery was used in adult rats, a system that is amenable to studies in isolated perfused kidneys and that minimizes development-induced compensatory mechanisms. Intravenous administration of hPTH1R or green fluorescence protein-tagged hPTH1R in pcDNA3 resulted 3 wk later, in generalized expression of hPTH1R (mRNA and protein), especially in vessels, liver, heart, kidney, and central nervous system, where it is expressed physiologically. As expected, PTH1R overexpression decreased BP and renal tone. Unexpected, however, PTH1R overexpression decreased heart rate. These studies also revealed that endogenous PTHrP actually inhibits renin release and that hPTH1R overexpression tends to increase that effect. Striking, liver production and circulatory level of angiotensinogen and hence plasma renin activity were markedly reduced. Thus, abrupt PTH1R overexpression in adult rats profoundly alters the CVS, RVS, and RAS, strongly supporting the PTH/PTHrP/PTH1R system as crucial for heart and vascular tone regulation. In addition, these results revealed that PTH1R-mediated mechanisms might have protective effects against cardiovascular stress-induced responses, including stimulations in heart rate and RAS.

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.

These studies examine whether PTHrP(1-36), a vasodilator, modulates BP and renal vascular resistance (RVR) in spontaneously hypertensive rat (SHR). Within the kidney of normotensive rats, PTHrP(1-36) was enriched in vessels. In vessels of SHR, PTHrP was upregulated by 40% and type 1 PTH receptor (PTH1R) was downregulated by 65% compared with normotensive rats. To investigate the role of endogenous PTHrP in the regulation of BP and RVR, SHR were subjected to somatic human (h)PTH1R gene delivery. Three weeks after a single intravenous injection of pcDNA1.1 plasmid containing the hPTH1R gene under the control of the cytomegalovirus promoter, hPTH1R mRNA was detected in all of the main organs. Within the kidney, the transgene was enriched in vessels. In the isolated perfused kidney, RVR was reduced by 23% and PTHrP(1-36)-induced vasodilation, which is depressed in SHR, was restored and a vasoconstrictory response to PTH(3-34), a PTH1R antagonist, was revealed. These effects were not observed in control SHR treated with empty plasmid. BP remained unchanged, and plasma renin activity increased by 60%. Thus, in SHR renal vessels, a reduced number of PTH1R contributes to the high RVR, despite the higher expression of vasodilatory PTHrP. Moreover, these studies provide evidence for a direct link between the density of PTH1R and plasma renin activity, which might be responsible for the absence of effect of PTH1R gene delivery on BP in SHR. Overall, PTHrP significantly contributes to the homeostasis of renal and systemic hemodynamics in SHR.

We evaluated how renal vascular reactivity to vasopressin changes when nitric oxide (NO) synthesis varies, as has been reported to occur in the course of insulin-dependent diabetes mellitus. Renal vasoconstrictor responses to vasopressin were obtained in young and older Sprague-Dawley control rats (3 and 10 months old) and in age-matched diabetic rats that had been treated with streptozotocin (60 mg/kg i.v.) at the age of 2 months. In young rats, vasopressin (3-1000 ng/kg/min i.v.) induced in vivo a dose-dependent decrease in renal blood flow, which was diminished in streptozotocin diabetic rats (P0.05). Similarly, in in vitro perfused kidneys, the concentration-response curve for vasopressin (0.03-10 nM) was shifted 3-fold to the right in kidneys isolated from young diabetic rats (P0.05). This shift was abolished in the presence of an inhibitor of nitric oxide synthesis, N(G)-nitro-L-arginine (100 microM), in the perfusate. In 10-month-old rats, the in vivo renal vasoconstrictor dose-response curve to vasopressin was shifted 10-fold to the left as compared to that for young rats (P0.001). This shift was similar in both control and diabetic rats. In conclusion, the present study documented the existence of hyporesponsiveness to vasopressin in the early stage of diabetes, possibly related to nitric oxide overproduction. In contrast, renal vascular hyperreactivity to vasopressin occurs with aging, whether the rats are diabetic or not.

We recently showed that brain mineralocorticoid receptors (MRs) are involved in blood pressure and kidney function control in normotensive Wistar rats. We now assessed the involvement of brain MRs in spontaneously hypertensive rats (SHR), in which the presence of adrenocorticoids has been shown to be required for the development of hypertension. The effect of a single intracerebroventricular (ICV) injection of an MR antagonist (RU28318) on systolic blood pressure (SBP) and renal function was examined in conscious adult SHR and Wistar-Kyoto rats (WKY) maintained on a standard-sodium diet (0.4% Na + ). In WKY, a long-lasting decrease in SBP was caused by the ICV injection of 10 ng RU28318 as previously reported in Wistar rats, associated with increased urinary excretion of water and electrolytes. In SHR maintained on the standard diet, the ICV injection of RU28318 (10 or 100 ng) had no effect on cardiovascular and renal functions. However, the ICV injection of 10 ng RU28318 in SHR after 3 weeks of high sodium intake (8% Na + ) caused a long-lasting decrease in SBP. The effect was present at 8 hours (ΔSBP 34±2 mm Hg), persisted at 24 hours (ΔSBP 29±1 mm Hg), and disappeared at 48 hours after the injection. The hypotension was not associated with changes in heart rate, urinary excretion of water and electrolytes, and plasma renin activity, whereas renal denervation did not affect the decrease in SBP. A more pronounced decrease in SBP (49±3 mm Hg at 8 hours) was observed with 100 ng RU28318. This dose of the antagonist was without effect after subcutaneous administration. Thus, brain MRs appear to participate in the maintenance of hypertension in conscious adult SHR sensitized by sodium loading.

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 30 – 40% after inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine (L-NOARG) or of cyclo-oxygenase by indomethacin; a greater decrease (about 40 – 50%) 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. Keywords: Tyr(Me)8bradykinin, kinin B2 receptor, acetylcholine, endothelium, rat isolated kidney, nitric oxide, prostanoids, K channels, cytochrome P450, cannabinoid CB1 receptor Introduction After the description by Furchgott & Zawadzki (1980) of an absolute requirement of endothelium for the vasodilation elicited by acetylcholine (ACh) in rabbit aorta, endothelial cells were shown to release a variety of mediators which can act to alter the mechanical and electrical properties of the vascular smooth muscle cells (Furchgott & Vanhoutte, 1989). These factors include prostacyclin, endothelin, endothelium-derived relaxing factor and endothelium-derived hyperpolarizing factor (EDHF). Although endothelium-derived relaxing factor was rapidly recognized as nitric oxide (NO) or a compound releasing NO (Palmer et al., 1987; Ignarro et al., 1987), the identity of EDHF still remains controversial (Quilley et al., 1997; Mombouli & Vanhoutte, 1997). Endothelium-dependent vasodilation also occurs with bradykinin (BK), mainly by the activation of bradykinin B2 receptors. BK has been shown to increase the release of prostacyclin and NO from bovine aortic endothelial cells in culture (Busse et al., 1994; Blatter et al., 1995) and to produce hyperpolarization in coronary arteries with intact endothelium (Vanhoutte, 1993; Fisslthaler et al., 1999). The involvement of three major pathways, i.e. prostacyclin, NO and EDHF, in BK-induced relaxation was further documented in various blood vessels by the use of specific inhibitors of cyclo-oxygenase (COX), NO synthase (NOS) and K+ channels. These studies also revealed that, according to the vessel, each pathway did not contribute to the same degree to the vasorelaxation. In particular, NO-independent mechanisms appear to play a more important role in small resistance vessels (Garland et al., 1995). In the rat renal vasculature, we previously reported that the main response to BK existed of a relaxation via bradykinin B2 receptors (Bagate et al., 1999). BK-induced vasodilation was mimicked by Tyr(Me)8BK, a selective B2 receptor agonist (Regoli & Barabe, 1980), and was inhibited by icatibant, a specific B2 receptor antagonist. The question of kinin signalling pathways in renal vessels was previously addressed using BK (Fulton et al., 1992; Rapacon et al., 1996; Mieyal et al., 1998). The aim of the present study was to investigate these pathways in the isolated perfused rat kidney, with Tyr(Me)8BK. The kinin B2 receptor-mediated vasodilation was analysed before and after exclusion of transduction pathways by selective inhibitors. After having controlled requirement of intact endothelium, we evaluated the effects of NOS and/or COX inhibition. The NO/prostanoids-independent vasodilation was considered to be linked to so called ‘EDHF' although measurement of vascular smooth muscle cell membrane potential was not possible in our experimental preparation. However, suppression of the response with high extracellular K+ and by inhibitors of K+ channels supported the contribution of a hyperpolarizing factor. We finally investigated the effects of cytochrome P450 inhibition and cannabinoid CB1 receptor antagonism because epoxyeicosatrienoic acids (EETs) and endogenous cannabinoids are possible candidates for EDHF (Mombouli & Vanhoutte, 1997).

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 endogenous 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) decreased WKY and increased SHR cell proliferation. Additional studies indicated that the preferential coupling 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 translocation 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 effects of PTHrP on renovascular SMC proliferation appear to be reversed in the SHR model of genetic hypertension. A new concept emerges from these results, according to which a single molecule may have opposite effects on VSMC proliferation under physiological and pathophysiological conditions.

The interaction between an inhibitor of angiotensin I converting enzyme (ramipril) and renal lithium handling was analysed in conscious, normotensive Wistar rats in the absence or the presence of a specific bradykinin B2 receptor antagonist, icatibant. The rats were treated for 5 days with ramipril (1 mg/kg/day p.o.) or its vehicle, alone or together with icatibant (0.1 mg/kg/day, s.c. infusion). Lithium chloride (8.3 mg/kg i.p.) was given as a single dose on day 5. Systolic blood pressure and heart rate were measured by tail plethysmography on day 3 (3, 9 and 15 h after ramipril administration) and renal function on day 4 (0-6 and 6-24 h urine sampling) and day 5 (0-6 h urine sampling). In another group of rats, 24 h sodium excretion was assessed during the first 4 days of ramipril treatment. Ramipril decreased renal lithium clearance (90+/-8 vs. 142+/-10 microl/min/100 g, P0.001, n=24) and increased the fractional lithium reabsorption (74.3+/-1.9 vs. 66.7+/-1.7%, P0.05) and plasma lithium concentration (0.108+/-0.006 vs. 0.085+/-0.004 mM, P0.01). Alteration of renal lithium handling by ramipril was associated with a decrease in systolic blood pressure (-15% 3 h after ramipril administration) and sodium excretion (0-6 h after ramipril). The 24-h sodium excretion, however, tended to increase. Icatibant had no effect per se on renal function but attenuated the ramipril-induced decrease in renal lithium clearance (118+/-16 vs. 90+/-8 microl/min/100 g, n=12 and 24 respectively, P0.05 one-tailed test) and systolic blood pressure. These results suggest that endogenous bradykinin contributes to the ramipril-associated alteration in renal lithium handling. Bradykinin B2 receptor-mediated vasodilation seems to be involved.

Abstract —Nitric oxide attenuates both vasopressin-induced vasoconstriction and vasopressin release. We tested whether hypertension and renal dysfunction elicited by chronic inhibition of nitric oxide (NO) synthesis using N G -nitro- l -arginine (L-NNA) could be mediated in part by vasopressin V 1A receptors. Male rats were treated orally for 6 weeks with L-NNA (15 mg/kg per day), a nonpeptide V 1A receptor antagonist (2 S )-1-[(2 R ,3 S )-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1 H -indole-2-carbonyl]-pyrrolidine-2-carboxamide (SR 49059, 30 mg/kg per day), or a combination of SR 49059 and L-NNA (same doses), or they received no treatment. Both drugs were added to the food. Measurements were performed in conscious rats (urine collection in metabolic cages, tail-cuff arterial pressure) and at the end of the study in anesthetized rats (clearance measurements). L-NNA produced sustained hypertension, decreased glomerular filtration rate, and increased renal vascular resistance, plasma renin activity, and urinary albumin excretion. SR 49059 had no effect per se on these parameters and also did not attenuate the hypertension and renal dysfunction induced by L-NNA. Surprisingly, SR 49059 potentiated L-NNA–induced hypertension at the end of the 6-week treatment. However, the blood pressure response and the renal and mesenteric vasoconstriction elicited by exogenous vasopressin were attenuated in rats treated with SR 49059. L-NNA did not change plasma vasopressin concentration or 24-hour urinary vasopressin excretion. Our findings suggest that activation of vasopressin V 1A receptors does not contribute to the hypertension and renal dysfunction induced by chronic NO synthesis inhibition. They also document unchanged plasma vasopressin concentration in NO-deficient hypertension.

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 Tyrode's 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

1. 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 (100 microM). 2. Kidneys were perfused in the presence of cyclo-oxygenase inhibitor (10 microM indomethacin) with Tyrode's solution of relative viscosity zeta=1 (low viscosity perfusate, LVP) or, in order to augment shear stress, with Tyrode's solution containing 7% Ficoll 70 of relative viscosity zeta=2 (high viscosity perfusate, HVP). 3. 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). 4. ANGII (10 - 100 pM) 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. 5. 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 500 nM) gradually abolished the potentiation of ANGII vasoconstriction during NO inhibition, but did not affect vasoconstriction in response to ANGII in LVP kidneys. 6. 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.

1. Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II-induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT-R) antagonists on Ang II-induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single-pass circuit. 2. In the absence of NO blockade, the AT1-R antagonist L-158809 (500 nM) antagonized the Ang II-induced vasoconstrictions, while the AT2-R antagonist PD-123319 (500 nM) had no effect. 3. Perfusing kidneys in the presence of either NO synthase inhibitors, L-NAME (100 microM) or L-NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY-83583 (10 microM), significantly increased both molar pD2 (from 9.40+/-0.25 to 10.36+/-0.11) and Emax values (from 24.9+/-3.1 to 79.9+/-4.9 mmHg) of the concentration-response curve for Ang II-induced vasoconstriction. 4. In the presence of L-NAME, 500 nM L158809 abolished the Ang II-induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD-123319 reversed the left shift of the concentration-response curve for Ang II (molar pD2 value 9.72+/-0.13) leaving Emax value unaffected (91.3+/-7.6 mmHg). 5. In the presence of L-NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration-dependent manner by L-158809 (0.05-500 nM). By contrast, PD-123319 (0.5-500 nM) did not affect the 10 nM Ang II-induced vasoconstriction and concentration-dependently decreased the 0.1 nM Ang II-induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6. Similar to PD-123319, during NO blockade the AT2-R antagonist CGP-42112A at 5 nM decreased by 50% the 0.1 nM Ang II-induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II-induced vasoconstriction. 7. In conclusion, the renal Ang II-induced vasoconstriction, which is antagonized only by AT1-R antagonist in the presence of endogenous NO, becomes sensitive to both AT1- and AT2-R antagonists during NO synthesis inhibition. While AT1-R antagonist inhibited both L-NAME-potentiated and -augmented components of Ang II-induced vasoconstriction, AT2-R antagonists inhibited only the L-NAME-potentiated component.

Parathyroid hormone-related protein (PTHrP) belongs to vasoactive factors that regulate blood pressure and renal hemodynamics both by reducing vascular tone and raising renin release. PTHrP is expressed in systemic and renal vasculature. Here, we wanted to assess the contribution of vascular smooth muscle cell endogenous PTHrP to the regulation of cardiovascular and renal functions. We generated a mouse strain ( SMA-CreERT2/ PTHrPL2/L2or premutant PTHrPSM−/−), which allows temporally controlled, smooth muscle-targeted PTHrP knockdown in adult mice. Tamoxifen treatment induced efficient recombination of PTHrP-floxed alleles and decreased PTHrP expression in vascular and visceral smooth muscle cells of PTHrPSM−/−mice. Blood pressure remained unchanged in PTHrPSM−/−mice, but plasma renin concentration and creatinine clearance were reduced. Renal hemodynamics were further analyzed during clearance measurements in anesthetized mice. Conditional knockdown of PTHrP decreased renal plasma flow and glomerular filtration rate with concomitant reduction in filtration fraction. Similar measurements were repeated during acute saline volume expansion. Saline volume expansion induced a rise in renal plasma flow and reduced filtration fraction; both were blunted in PTHrPSM−/−mice leading to impaired diuresis. These findings show that endogenous vascular smooth muscle PTHrP controls renal hemodynamics under basal conditions, and it is an essential factor in renal vasodilation elicited by saline volume expansion.

The increase in renin secretion and the induction of the converting enzyme (ACE) observed during treatment by ACE inhibitors (CEIs) could result in increased angiotensin II (ang II) synthesis when the treatment is stopped. The object of this study was to compare changes in the components of the renin-angiotensin system with changes in arterial pressure in hypertensives, following the cessation of long-term ramipril treatment. Twenty hypertensives, treated for at least three months with ramipril, in monotherapy for the last three weeks, were randomly allocated to two parallel groups and received for fifteen days, on a double-bind basis, either a placebo (withdrawal group W, n = 12) or ramipril at the previous doses (treated group T, n = 8). Blood pressure was measured using four different techniques. The active renin (AR), angiotensinogen, angiotensin I (ang I), angiotensin II (ang II) and aldosterone plasma concentrations were measured, as was plasma angiotensin I converting enzyme (ACE) activity in vitro (colorimetric and fluorimetric method) and in vivo (the ang II/ang I ratio). The biological effects of cessation of long-term ramipril treatment in hypertensives were a decline in AR and angiotensin I concentrations, an increase in ACE activity and no significant changes in angiotensinogen, angiotensin II and aldosterone levels. Fifteen days after withdrawal, the different parameters of the renin-angiotensin system appear to have returned to basal value. A slow rise in blood pressure was also observed but no rebound increase was noted during the 15 days neither in angiotensin II levels nor in blood pressure. Following the cessation of prolonged ramipril treatment, in vivo converting enzyme inhibition disappears slowly, probably on account of the slow tight binding inhibitor properties of ramiprilat, the active metabolite of this CEI. The gradual decline in AF, plasma levels, together with the prolonged ACE inhibition as measured in vivo by the ang II/ang I ratio, explains the absence of a rise in ang II synthesis.

International audience; Human clear cell renal cell carcinoma (CCC) remains resistant to treatments despite the progress in targeted therapies. Several signaling pathways acting during renal development are reactivated during kidney tumorigenesis; this is the case of the sonic hedgehog (SHH)-Gli. Interestingly, the precursor of active vitamin D3 (VD3), cholecalciferol, has been demonstrated to be a strong inhibitor of SHH-Gli signaling. Here, we show the preclinical efficacy of cholecalciferol in CCC both in vitro and in vivo . A panel of CCC cell lines, tumors and normal corresponding tissues from CCC patients were used to evaluate the expression of the VD3 receptor and metabolizing enzymes and the effects of cholecalciferol treatment. Subsequently, xenografted mice were treated with cholecalciferol in a prophylactic or therapeutic manner; their response and the adverse effects were evaluated on the basis of weekly monitoring, followed by blood collection procedures and X-ray micro-computed tomography. VD3 receptor and metabolizing enzymes are dramatically decreased in human cell lines and tumors. Cholecalciferol decreases cell proliferation and increases cell death by inhibition of the SHH-Gli pathway. Xenografted mice treated with cholecalciferol exhibit absence of tumor development or substantial growth inhibition. The treatment was shown to be safe; it did not induce calcification or calcium reabsorption. These findings establish that, although VD3 receptors and metabolizing enzymes are absent in CCC, cholecalciferol supplementation is a strong tool to block the reactivation of SHH-Gli pathway in this pathology, leading ultimately to tumor regression. Cholecalciferol may have highly therapeutic potential in CCC.

The effects on renal function of quinapril, an angiotensin I converting enzyme (ACE) inhibitor, and of nifedipine, a dihydropyridine calcium antagonist, were studied in the early stages of diabetes in rats. Wistar rats received one injection of streptozotocin (STZ) to induce diabetes; the hyperglycaemia was then controlled with daily insulin therapy (2-3 units NPH insulin/rat). One week after STZ injection, rats were treated orally with quinapril (0.3 or 3 mg/kg/d) or nifedipine (30 mg/kg/day) for 1 week, after which renal functions were compared with those of untreated diabetic rats or non-diabetic control rats. At the end of these two weeks, diabetic rats had gained less weight and had developed renal hypertrophy and glomerular hyperfiltration (3.21 +/- 0.23 vs 2.36 +/- 0.09 ml/min for non-diabetic rats, mean +/- SEM, P < 0.01). Their urinary albumin excretion was higher, as was the urinary excretion of water, sodium, potassium, urea and glucose. One week treatment with quinapril or nifedipine had no significant effect on the increase in the glomerular filtration rate (respectively 2.97 +/- 0.18 and 2.99 +2- 0.15 ml/min). Quinapril and nifedipine differed with regard to their effects on urinary albumin excretion. Albuminuria was increased by nifedipine but not by quinapril (respectively 0.554 +/- 0.158 and 0.149 +/- 0.046 mg/day/100 g BW, P < 0.05). This difference between the effects of the dihydropyridine and the ACE inhibitor on albuminuria may be linked to different effects on the glomerular functions.

Effects of dietary protein and uninephrectomy on renal angiotensin converting enzyme in the rat. This study examines the effects of dietary protein and of uninephrectomy on angiotensin converting enzyme (ACE) in the normotensive rat, with particular regard to the kidney. Male Wistar Kyoto rats were fed isocaloric diets containing 5, 16 or 50% protein for three weeks. Other groups of rats were subjected to either left unilateral nephrectomy or sham operations, and the rats were killed eight days after surgery. ACE activity was measured in the kidney medulla, cortex, proximal tubule brush border membrane and in the plasma, heart and lung. Renal cortex and brush border ACE activity increased in parallel with protein intake, whereas plasma and lung ACE activity decreased; heart and kidney medulla ACE activity did not vary significantly. Uninephrectomy also led to a high increase in brush border ACE activity in the contralateral kidney, with no effect in the renal medulla or in the other tissues. The increase in ACE activity in the brush border membrane corresponded to a similar increase in the maximum number of binding sites of 3 H-ramiprilat. This suggested that the increase in ACE activity corresponded to an increase in ACE concentration. The increase in renal tubular ACE activity could result in higher angiotensin II levels, and could consequently play a role in the modification of sodium reabsorption and cellular growth which occurs in the proximal tubule in these experimental models.

Recent data have revealed biological and genetic variability in normotensive Wistar-Kyoto rats, which are considered to be the most appropriate control strain for spontaneously hypertensive rats. To investigate the possibility that angiotensin converting enzyme activity could be affected by this variability, we measured plasma and tissue (lung, heart, renal cortex, renal medulla, and adrenal gland) angiotensin converting enzyme activity in spontaneously hypertensive rats and normotensive Wistar-Kyoto rats from three commercial suppliers in France: Iffa-Credo, Janvier, and Charles River Laboratories. Angiotensin converting enzyme activity was measured in vitro with a fluorometric assay using carbobenzoxy-Phe-His-Leu as substrate. Angiotensin converting enzyme activity in both rat strains varied considerably from one supplier to another, and therefore, comparisons of spontaneously hypertensive rats and Wistar-Kyoto rats from the different suppliers produced conflicting results. For Wistar-Kyoto rats, angiotensin converting enzyme activity in the plasma, heart, kidney, and adrenal glands was highest in rats from Iffa-Credo and lowest in rats from Charles River. For spontaneously hypertensive rats, angiotensin converting enzyme activity in the plasma and tissues was highest in rats from Janvier, whereas no difference could be observed between rats from Iffa-Credo and Charles River. These data confirm the problem of how to interpret and compare studies that use spontaneously hypertensive and Wistar-Kyoto rat strains.

The inhibition of angiotensin converting enzyme by ramipril, ramiprilat, enalapril, enalaprilat, and captopril was studied in the plasma and various tissues (lung, heart, renal cortex, renal medulla) of normotensive rats and spontaneously hypertensive rats. Displacement curves for [3H]ramiprilat were established on each tissue with the converting enzyme inhibitors, and their potencies were expressed as the concentration that inhibited 50% of the specific [3H]ramiprilat binding. In the plasma, lung, and heart, the order of activities was: ramiprilat greater than enalaprilat greater than captopril greater than ramipril greater than enalapril. This order was different in the kidney (cortex and medulla): ramiprilat greater than enalaprilat greater than ramipril greater than captopril greater than enalapril. For ramiprilat, enalaprilat, and captopril, there were no differences in their respective potencies between tissues or between rat strains. However, the two prodrugs ramipril and enalapril were 10-30 times more active in the kidney than in the other tissues in both groups of rats. This was due to the deesterification of the prodrugs: in the presence of an esterase inhibitor (diethyl nitrophenyl phosphate, 10 microM), the potencies of ramipril in the kidney were not different from that obtained in the lung, which was not affected by the presence of the esterase inhibitor. These results suggest that the variations in the tissue activities of an angiotensin converting enzyme inhibitor are probably not due to differences in tissue affinities of the angiotensin converting enzyme inhibitor but depend on the concentration of this angiotensin converting enzyme inhibitor in each tissue.

International audience; PTHrP is produced in vessels and acts as a local modulator of tone. We recently reported that PTHrP(1–34) is able to induce vasorelaxation in rat uterine arteries, but in pregnancy, this response is blunted and becomes strictly endothelium dependent. The present study aimed to get insights into the mechanisms involved in these changes because the adaptation of uterine blood flow is essential for fetal development. On d 20 of gestation, RT-PCR analysis of uterine arteries showed that PTH/PTHrP receptor (PTH1R) mRNA expression was decreased, whereas that of PTHrP mRNA was increased. This was associated with a redistribution of the PTHrP/PTH1R system, with both PTH1R protein and PTHrP peptide becoming concentrated in the intimal layer of arteries from pregnant rats. On the other hand, the blunted vasorelaxation induced by PTHrP(1–34) in uterine arteries from pregnant rats was specifically restored by indomethacin and a specific cyclooxygenase-2 inhibitor, NS 398. This was associated with an increase in cyclooxygenase-2 expression and in 8-iso-prostaglandin F2α release when uterine arteries from pregnant rats were exposed to high levels of PTHrP(1–34). Most interestingly, 8-iso-prostaglandin F2α itself was able to increase PTHrP expression and reduce PTH1R expression in cultured rat aortic smooth muscle cells. These results suggest a local regulation of uterine artery functions by PTHrP during pregnancy resulting from PTH1R redistribution. Moreover, they shed light on a potential role of 8-iso-prostaglandin F2α.

Angiotensin converting enzyme (ACE) activity was measured by fluorimetry in the plasma, lung, heart, aorta and kidney (cortex and medulla) of 3-, 5-, 8- and 11-week-old spontaneously hypertensive rats (SHR) and compared with that of age-matched Wistar-Kyoto rats (WKY). In the plasma, lung and kidney (cortex and medulla), ACE activity was lower in SHR than in WKY. This was evident as early as the age of 3 weeks. In contrast, there were no differences between SHR and WKY in the aorta and the heart. Age-related variations in ACE activities differed in each tissue and in both groups of rats, but no major modifications were correlated with the development of hypertension. A binding assay was performed with [3H]ramiprilat; affinity (KD) and the maximum number of binding sites (Bmax) were determined in plasma and tissues of 3-week-old SHR and WKY. The KD values were identical in the two groups but Bmax was lower in all SHR tissues except in the heart; these results might be related to the decrease in ACE activity. Our results probably reflect genetic differences in ACE activity between SHR and WKY, and suggest that ACE regulatory mechanisms act differently in each tissue.

In the Wistar rat in vivo L-dopa (10 mg/kg, subcutaneously) was shown to have the characteristics of a kidney-directed dopamine (DA) prodrug: two daily injections increased 24 h urinary DA excretion 450-fold but had no systemic effects on blood pressure and heart rate. In inactin-anesthesized rats, L-dopa increased natriuresis, diuresis and renal blood flow; these effects were linked to endorenal DA synthesis and to DA-1 receptor stimulation since they were suppressed by both carbidopa and SCH 23390. In the isolated perfused rat kidney, DA was synthesized from L-dopa with a greater yield than from gludopa. In nonfiltering kidneys, L-dopa metabolism was not limited when the access to dopa decarboxylase was restricted to the basolateral membrane. The same was not true for gludopa, for which the basolateral metabolism was low.

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 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.

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in 40-80% of human conventional renal cell carcinomas (RCCs). We showed recently that VHL-deficient RCCs expressed large amounts of parathyroid hormone-related protein (PTHrP), and that PTHrP, acting through the PTH1 receptor (PTH1R), plays an essential role in tumor growth. We also showed that PTHrP expression is negatively regulated by the VHL gene products (pVHL). Our goal was to determine whether blocking the PTHrP/PTH1R system might be of therapeutic value against RCC, independent of VHL status and PTHrP expression levels. The antitumor activity of PTHrP neutralizing antibody and of PTH1R antagonist were evaluated in vitro and in vivo in a panel of human RCC lines expressing or not pVHL. PTHrP is upregulated compared with normal tubular cells. In vitro, tumor cell growth and viability was decreased by up to 80% by the antibody in all cell lines. These effects resulted from apoptosis. Exogenously added PTHrP had no effect on cell growth and viability, but reversed the inhibitory effects of the antibody. The growth inhibition was reproduced by a specific PTH1R antagonist in all cell lines. In vivo, the treatment of nude mice bearing the Caki-1 RCC tumor with the PTHrP antibody inhibited tumor growth by 80%, by inducing apoptosis. Proliferation and neovascularization were not affected by the antiserum. Anti-PTHrP treatment induced no side effects as assessed by animal weight and blood chemistries. Current therapeutic strategies are only marginally effective against metastatic RCC, and adverse effects are common. This study provides a rationale for evaluating the blockade of PTHrP signaling as therapy for human RCC in a clinical setting.

In vivo, vascular smooth muscle cells (VSMC) are continuously exposed to mechanical cyclic stretch as a result of the pulsatile blood flow from the cardiac contractile cycle. Stretch is altered in pathologic conditions and contributes to vascular remodeling by modulating VSMC proliferation and death. Parathyroid hormone-related protein (PTHrP) is a locally produced poly-protein that regulates cell growth. It was shown previously that PTHrP inhibits VSMC proliferation through the auto/paracrine pathway by interacting with its receptor, the PTH1R, but stimulates VSMC proliferation through the intracrine pathway by translocating into the nucleus. In the current study, VSMC that were isolated from both resistance and compliance vessels were used to study the role of PTHrP in VSMC proliferation under experimental stretch. It is shown that PTHrP gene expression is upregulated by stretch and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, it is demonstrated that PTHrP expression is controlled at the post-transcriptional level by stretch. Taken together, these results strongly suggest that PTHrP plays a critical role in the modulation of VSMC proliferation in response to stretch. Thus, in conditions in which stretch is increased, such as in hypertension or in restenosis after angioplasty, PTHrP may contribute to vessel hyperplasia.

Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in the kidney (cortex and medulla) and plasma of Wistar-Kyoto rats on a low-sodium (LS; 0.025% NaCl; n= 8), normal-sodium (NS; 1% NaCl; n = 7), or high-sodium (HS; 8% NaCl; n = 7) diet for 21 days. RA, ANG I, and ANG II levels increased in a manner inversely related to sodium content of the diet in both plasma and renal tissues. The LS diet resulted in a 16-, 2.8-, and 1.8-fold increase in plasma RA, ANG I, and ANG II levels, respectively, compared with those in HS rats. In the renal cortex and medulla, RA, ANG I, and ANG II levels were also increased by diminution of dietary salt content but, in contrast to plasma, ANG II levels increased much more than RA or ANG I levels [5.4 (cortex)- and 4.7 (medulla)-fold compared with HS rats]. In summary, we demonstrated variations of ANG II levels in the kidney during dietary salt modifications. Our results confirm that RA and ACE activity are not the steps limiting intrarenal ANG II levels. Nevertheless, despite RA and ACE activity differences between renal cortex and medulla, ANG I and ANG II levels are equivalent in these two tissues; these results argue against a compartmentalization of RAS in these two intrarenal areas.

This study was designed to determine the involvement of AT1receptors in the uptake of ANG II in the kidney of rats exposed to differing salt intake. Male Wistar-Kyoto rats were treated with a normal-salt (NS; 1% NaCl, n = 7) or a low-salt (LS; 0.025% NaCl, n = 7) diet combined with (LS+Los, n = 7; NS+Los, n = 7) or without losartan (30 mg · kg−1· day−1), an AT1receptor antagonist. Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in plasma, renal cortex, and medulla. In LS rats, in both plasma and renal cortex, the increase in RA was associated with an increase in ANG I and ANG II levels compared with NS rats, but intrarenal ANG II levels increased more than ANG I levels. In NS+Los rats, the increase in RA in plasma was followed by a marked increase in plasma ANG I and ANG II levels compared with NS rats whereas in the kidney the increase of renal RA was followed by a decrease of the levels of these peptides. The same pattern was observed in LS+Los rats, but the decrease in renal ANG II levels was much more pronounced in LS+Los rats than in NS+Los rats. Our results suggest that the increase in renal ANG II levels after salt restriction results mainly from an uptake of ANG II, via AT1receptors. Such elevated intrarenal ANG II levels could contribute to maintain sodium and fluid balance and arterial blood pressure during salt-deficiency states.

Objective We have previously shown that brain mineralocorticoid receptors (MR) participate in the control of arterial pressure and renal excretory function in normotensive rats. In the present study, we evaluate the influence of sodium intake on the control of cardiovascular and renal function by brain MR in normotensive conscious rats. We hypothesize that modulation of sodium intake affects the cardiovascular and renal effects of brain MR blockade. Design and methods We examined the effect of intracerebroventricular (ICV) administration of MR antagonist (RU28318) on systolic blood pressure (SBP), heart rate, and urinary excretion of water and electrolytes in normotensive Wistar rats subjected to different dietary sodium content. Rats were fed high (8%), normal (0.4%), or depleted (0%) sodium for 3 weeks. SBP and heart rate measurements were performed using an indirect tail cuff method. Metabolic cages were used to assess renal function. Results ICV injection of 100 ng RU28318 induced a long-lasting decrease ( 0.01) in SBP in rats submitted to different sodium intake. At 8 h, the decrease in SBP did not differ between rats on high (30 +/- 5 mmHg), normal (28 +/- 6 mmHg), and low (21 +/- 3 mmHg) sodium diets. At 24 h, the decrease in SBP was also comparable between rats on different sodium diets. Increased diuresis was observed during the period 0-8 h after ICV injection of RU28318; this was less pronounced in rats on the low sodium diet. Urinary excretions of potassium and chloride were also increased during this period, particularly in rats on the high and normal sodium diets compared with rats with low sodium intake. Urinary excretion of sodium was increased only in the rats fed high and normal sodium diets. Measurement of plasma renin activity, which was suppressed and stimulated, respectively, by high and low sodium intake, indicated that the effects on SBP and renal function induced by ICV RU28318 were independent from the level of activation of the renin-angiotensin system. Conclusion In contrast to our hypothesis, in normotensive Wistar rats, sodium intake does not affect the hypotension induced by brain MR blockade. However, sodium depletion attenuated the renal effects of brain MR blockade.

Although lower than in brain, the type 2 PTH receptor (PTH2-R) has been shown to be expressed throughout the cardiovascular system. Tuberoinfundibular peptide (TIP) purified from brain is thought to be the endogenous selective ligand of the PTH2-R. In the present studies, TIP and PTH2-R mRNA expressions were evidenced by RT-PCR in rat intrarenal arteries as well as in renovascular smooth muscle cells cultured from these arteries. In the isolated perfused rat kidney (IPK), peptides known to bind to both PTH1- and PTH2-Rs, such as rat PTH (1-34) and the hybrid PTH/PTHrP peptide, [Ile(5), Trp(23)]PTHrP (1-36), failed to exhibit improved vasodilatory effect, compared with human PTHrP (1-36), which binds only to the PTH1-R. Thus, a non-PTH1-R seemed not to be involved in the vasodilatory effects of these peptides. On the other hand, TIP exhibited complex vasoactivity, constricting the IPK at 10 nM and dilating the IPK at 1, 100, and 1000 nM. Moreover, [p-benzoyl-L-Phe(4),Ile(5),Trp(23)]PTHrP (1-36), initially described as a selective PTH2-R antagonist, also displayed a strong vasodilatory effect and therefore could not be used to check that TIP-induced vasoactivity was mediated by the PTH2-R. However, both [p-benzoyl-L-Phe(4),Ile(5),Trp(23)]PTHrP (1-36) and TIP displayed similar or even enhanced vasodilation in IPK in which PTH1-R-induced vasodilation was fully desensitized by sustained exposure to human PTHrP (1-36). Importantly, in IPK desensitized to the vasodilatory action of PTHrP (1-36), the hybrid PTH/PTHrP peptide and rat PTH (1-34), whose vasodilatory responses appeared exclusively PTH1-R dependent in naive IPK, produced a new and strong vasodilation. In conclusion, TIP and PTH2-R mRNAs are expressed in renal vessels and TIP appears as a new vasoactive peptide. Whether TIP interacts with PTH2-R could not be shown. However, these studies reveal the ability of TIP, as well as of other peptides known to bind to the PTH2-R, to dilate renal vessels in a PTH1-R-independent manner. Moreover, results obtained in IPK desensitized to the vasodilatory action of PTHrP (1-36) strongly suggest that TIP, along with PTHrP, might be coordinately involved in the regulation of renal hemodynamics.

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 8 ml/min with Tyrode's solution (relative viscosity eta=1) or, in order to increase shear stress, with Tyrode's solution supplemented with 4.7% Ficoll 400 (Ficoll 400; eta=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 N(omega)-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 P(450) 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.

In normotensive rats, we have previously demonstrated a role of brain mineralocorticoid receptors in blood pressure and renal function control. In the present study, the coordinate cardiovascular and renal effects of brain mineralocorticoid receptor blockade were examined by intracerebroventricular (i.c.v.) administration of a selective mineralocorticoid receptor antagonist (RU28318; 3,3-oxo-7 propyl-17-hydroxy-androstan-4-en-17yl-propionic acid lactone) in rats with hypertension induced by deoxycorticosterone acetate (DOCA) and salt. DOCA pellets were implanted s.c. in male Wistar rats given 0.9% NaCl as drinking solution 3 or 5 weeks before assessment of the effects of i.c.v. injection of RU28318 on cardiovascular and renal functions. Changes in expression of brain angiotensinogen, atrial natriuretic peptide (ANP) and mineralocorticoid receptor mRNA in specific brain areas in 3-week DOCA–salt rats were evaluated by in situ hybridization. The rise in systolic blood pressure induced by DOCA–salt treatment was most marked during the first 3 weeks. At 3 and 5 weeks after implantation of the DOCA-pellets a single i.c.v. injection of 10 ng of RU28318 significantly decreased systolic blood pressure during 24 h as assessed at 2, 8 and 24 h, while heart rate was not altered. Increased urinary excretion of water and electrolytes was observed in 3- and 5-week DOCA–salt rats during the period 0–8 h after i.c.v. injection of RU28318 while the suppressed plasma renin activity was not affected. The expression of brain angiotensinogen, ANP and mineralocorticoid receptor mRNA was not altered by 3-week DOCA–salt treatment, but 3 h after i.c.v. injection of RU28318, mineralocorticoid receptor mRNA expression in hippocampal cell fields responded with an increase of about 40%. In conclusion, these results demonstrate that in rats with hypertension induced by DOCA–salt, brain mineralocorticoid receptor blockade affects renal function and blood pressure regulation.

The aim of this study was to evaluate the renal vascular effects of oxytocin in Sprague-Dawley rats and in Brattleboro heterozygous or homozygous rats, the latter being genetically deficient in vasopressin synthesis. Studies were performed in vitro, in the isolated kidney perfused in an open circuit with a Tyrode's solution. Oxytocin induced a concentration-dependent renal vasoconstriction in Sprague-Dawley rats, at rather high concentrations (EC50=170±39 nM, mean ± SEM, n=6) with a maximum response amounting to 44% of that elicited by vasopressin (increase in renal vascular resistance: 11.5±0.9 mmHg min ml–1 vs. 26.2±2.2 mmHg min ml–1). Oxytocin-evoked renal vasoconstriction was abolished by SR 49059, a selective vasopressin V1A receptor antagonist (10 nM), but not by d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr-NH2 9]vasotocin, an oxytocin receptor antagonist (10 nM). In the presence of SR 49059, oxytocin did not induce renal vasorelaxation. Oxytocin induced renal vasoconstriction in Brattleboro homozygotes and heterozygotes (EC50=59±12 nM and 262±110 nM; E max=7.8±1.1 mmHg min ml–1 and 6.9±0.4 mmHg min ml–1, n=5 respectively) with characteristics similar as observed in Sprague-Dawley rats concerning partial agonist activity, low potency and antagonism by SR 49059. Responsiveness to vasopressin did not differ in Brattleboro homozygotes and heterozygotes (EC50 ~0.25 nM) and was similar as we reported in Sprague-Dawley rats. These findings indicate that high concentrations of oxytocin induce renal vasoconstriction in the rat by activating vasopressin V1A receptors. The low agonist activity makes it unlikely that oxytocin can substitute functionally for vasopressin at the renal vascular V1A receptor in Brattleboro homozygous rats which are deficient in endogenous vasopressin.

Kinins in the circulation are rapidly metabolized by several different peptidases. The purpose of this study was to evaluate the contribution of membrane-bound peptidases to kinin metabolism in the renal circulation. Experiments were performed in vitro, in isolated rat kidneys perfused at a constant flow rate (8 ml/min) with Tyrode's solution. The effects of peptidase inhibitors were evaluated on the functional vasodilator response caused by bradykinin (30 nM) or [Tyr(Me)(8)]bradykinin (10 nM) via activation of bradykinin B2 receptors in kidneys precontracted with prostaglandin F2alpha. Angiotensin converting enzyme inhibitors, enalaprilat (3 microM), ramiprilat (1 microM) or lisinopril (1 microM), increased the bradykinin-induced renal vasodilation by 40% or more. Inhibitors of neutral endopeptidase (thiorphan or phosphoramidon, 10 microM), basic carboxypeptidase (DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid or MGTPA, 10 microM) and aminopeptidase P (apstatin, 20 microM) however did not enhance the renal vasodilator response elicited by kinins, whatever tested alone or in the presence of lisinopril. These findings indicate that angiotensin converting enzyme is the major peptidase whose inhibition potentiates the renal bradykinin B2 receptor mediated vasodilator response of kinins. The relative contribution in this potentiation of inhibition of kinin inactivation and of cross-talk of angiotensin converting enzyme with bradykinin B2 receptor remains however to be clarified.

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 3 ng/kg per min and reached a maximum at 100 ng/kg per min. The dose required for a half-maximal response (ED50) was 24±4 ng/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±0.6 nM. 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 (1–1000 ng/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 (1 mg/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 N ω-nitro-l-arginine (L-NNA, 100 µg/kg for 10 min and 7.5 µg/kg per min, i.r.a.) enhanced vasopressin-induced renal vasoconstriction (EC50 0.6±0.1 nM, P

Abstract —Brain mineralocorticoid receptors appear to contribute to mineralocorticoid hypertension and may be involved in blood pressure control in normotensive rats. We examined the effect of blockade of central mineralocorticoid receptors with the use of a selective antagonist (RU28318) on cardiovascular and renal function in conscious normotensive rats. The contribution of renal innervation was evaluated in rats with bilaterally denervated kidneys. Young adult, male Wistar rats were trained for systolic blood pressure measurement by a tail sphygmographic method and accustomed to metabolic cages for collection of urine. One week before experimentation, an intracerebroventricular cannula was implanted. Systolic blood pressure was diminished 30 minutes after an intracerebroventricular dose of 10 ng of RU28318. The effect was maximal at 8 hours and was still present after 24 hours. Blood pressure returned to the basal level by 48 hours. During the period 0 to 8 hours after intracerebroventricular injection, rats treated with the antagonist showed an increase in diuresis and urinary electrolyte excretion. No significant effect on plasma renin activity, measured 8 and 30 hours after administration of RU28318, was observed. In denervated rats, the decrease in systolic blood pressure after administration of RU28318 was reduced. The difference was statistically significant compared with controls at 2 hours but not at 8 hours, and blood pressure returned to the basal value by 24 hours. The increases in diuresis and urinary electrolyte excretion induced by RU28318 were abolished in denervated rats. These results show that brain mineralocorticoid receptors are involved in blood pressure regulation and kidney function homeostasis in conscious normotensive rats. The renal nerves appear to participate in the brain mineralocorticoid receptor control of blood pressure.

The purpose of the present study was to determine whether the physiological dopamine prodrug, L-dopa, could suppress streptozotocin-induced diabetic glomerular hyperfiltration, and thus prevent further evolution of the diabetic nephropathy. Male Wistar rats treated with streptozotocin (60 mg/kg, intravenously) rapidly developed hyperglycemia which was stabilized (congruent to 4 g/L) by a daily insulin injection. Within two weeks, a significant increase in glomerular filtration rate (GFR) and a rise in the filtration fraction were observed as described in the early stage of diabetic nephropathy. Increase in both GFR and in the filtration fraction were normalized by treating the rats with L-dopa (10 mg/kg, subcutaneously, twice a day for one week). The effects of L-dopa were linked to endorenal DA synthesis and to DA-1 receptor stimulation since both carbidopa and SCH 23390 suppressed them.

To compare the effects of an angiotensin-converting enzyme inhibitor on circulating and tissue renin-angiotensin system (RAS), we measured different RAS parameters during the first day of treatment (Day1) as well as after two weeks of treatment (Day14). Ramipril was given orally once daily to adult male spontaneously hypertensive rats (SHR). Renin activity (RA), angiotensin converting enzyme (ACE) activity and levels of angiotensin I (ang I) and angiotensin II (ang II) in the plasma, renal cortex and renal medulla were assessed at Day1 and Day14 of the treatment. In the plasma, both RA and ang I increased 10 to 15 fold one to four hours after acute as well as at Day14 of ramipril treatment and then returned to basal values within 24 hours. Plasma ang II levels were not significantly decreased at Day1 or Day14. The decrease in the ang II/ang I ratio suggested a sustained inhibition of plasma ACE at Day14. In the renal cortex and medulla, a clearly different pattern was observed: in ramipril treated rats, RA in the renal cortex and medulla did not change at Day1 but at Day14 we observed a slight and sustained increase in RA. Despite very high basal levels of RA, ang I levels in the renal cortex were comparable to those in the plasma. The ang I level increased only one-fold one hour after ramipril intake at Day1 and Day14. This suggests that angiotensinogen may have a limiting role in the synthesis of ang I in the kidney. Ang II levels were slightly higher in the renal cortex and medulla than in the plasma suggesting local synthesis of the peptide. In the kidney, ang II levels decreased one and four hours after the acute or prolonged ramipril treatment and the ang II/ang I ratio was reduced at the same time. Our results show that the responses of the plasma and kidney components of the RAS to ACE inhibition are different in the plasma and the kidney suggesting that the circulating and tissue RAS are at least in part independent.

The renal vascular effects of [Arg8]vasopressin (vasopressin) were investigated in the isolated perfused rat kidney. Vasopressin (0.01-3 nM) elicited a dose-dependent vasoconstriction in kidneys from Sprague Dawley rats, with a EC50 value of 0.206 +/- 0.044 nM. Inhibition of nitric oxide synthase by N omega-nitro-L-arginine (100 microM) shifted the vasopressin-induced vasoconstrictor response curve to the left. Inhibition of cyclooxygenase by indomethacin (10 or 30 microM) blunted the constriction induced by low concentrations of the peptide. Vasopressin, like angiotensin II but not noradrenaline, induced tachyphylaxis, SR 49059 ((2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene- sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2- carboxamide) (1-30 nM), a new potent and selective non-peptide vasopressin V1A receptor antagonist, shifted the concentration-response curve for vasopressin to the right without decreasing the maximum contraction. Antagonism became competitive with a pA2 value (+/- S.D.) of 9.72 +/- 0.20 during inhibition of nitric oxide release. [Mpa1,D-Arg8]Vasopressin (desmopressin; 0.1-100 nM), or vasopressin (0.01-1 nM) after blockade of the vasopressin V1A receptor by SR 49059, induced no vasopressin V2 receptor-related renal relaxation in kidneys with vascular tone previously restored by noradrenaline or prostaglandin F2 alpha. These findings indicate that in the isolated perfused rat kidney vasopressin is a potent renal vasoconstrictor. The constriction depends on activation of smooth muscle vasopressin V1A receptors and is modulated by endothelial nitric oxide but not by prostacyclin or vasopressin V2 receptor-related vasodilation.

The therapeutical use of cyclosporine A (CsA) is hampered by the development of nephrotoxicity characterized by a marked increase in renal vascular resistance (RVR). We investigated vascular functions in kidneys of rats treated with CsA. The ex vivo vascular reactivity of kidneys from control rats and animals treated subacutely with CsA [50 mg/kg/day subcutaneously (s.c.) for 16-21 days] or an olive oil vehicle (1 ml/kg) was analyzed in male Wistar rats. The right kidney was isolated and perfused with Tyrode's or Krebs solution in an open circuit. The effects of acetylcholine (Ach), fenoldopam (FEN), and sodium nitroprusside (SNP) on norepinephrine (NE) preconstricted kidneys were studied. In control kidneys (untreated or vehicle-treated), Ach induced a relaxation (EC 50 = 0.56 ± 0.05 x 10 -9 M ; E max = 88.2 ± 2.1% decrease in the vascular tone restored by NE) which was endothelium-dependent [near-complete abolition after treatment with a detergent, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane-sulfonate (CHAPS) treatment] but only partially inhibited by indomethacin (EC 5O = 1.71 ± 0.39 x 10 -9 M, p < 0.05 ; E max = 87.1 ± 4.9%, NS) or indomethacin with N G -nitro-L-arginine methyl ester (L-NAME : EC 50 = 1.04 ± 0.38 x 10 -9 M, NS ; E max = 63.8 ± 2.5%, p < 0.01). CsA treatment induced a marked decrease in creatinine clearance and natriuresis measured in vivo but had no effect on systolic blood pressure (SBP). In CsA-treated rats, Ach-induced renal relaxation was partly blunted (EC 50 = 1.88 ± 0.34 x 10 -9 M, p < 0.01 ; E max = 82.8 ± 4.6%, NS), with both a defect in prostaglandin (PG) and nitric oxide (NO)-related responses. CsA treatment had no effect on endothelium-independent relaxations induced by FEN and SNP. These results show that subacute CsA treatment selectively impairs renal endothelium-dependent relaxation related to PGs and NO release.

1. The interaction of ramipril, an inhibitor of angiotensin I converting enzyme, with renal lithium handling was analysed in conscious normotensive Wistar rats and compared with the known increase in renal tubular lithium reabsorption induced by the non-steroidal anti-inflammatory drug, indomethacin. 2. The rats were treated for five days with ramipril (1 mg kg-1 day-1 orally), indomethacin (2.5 mg kg-1 day-1 intramuscularly) or their solvents. Lithium chloride (16.7 mg kg-1 intraperitonealy) was given as a single dose on the fifth day and renal functions were measured. 3. Ramipril induced a decrease in renal lithium clearance which was correlated with the decrease in the quantity of filtered lithium and the increase in the tubular fractional reabsorption of the metal. Ramipril also reduced the systolic blood pressure of the rats by about 15 mmHg. 4. In the absence of any effect on creatinine clearance or systolic blood pressure, indomethacin increased renal fractional lithium reabsorption and led to an increase in plasma lithium levels, as previously reported by our group. 5. In conclusions, our results indicate that ramipril decreases renal lithium excretion in Wistar rats, when given orally at a dose of 1 mg kg-1 day-1 over five days.

1. The interaction of losartan, a non-peptide specific AT1 receptor antagonist with the renal handling of lithium was analysed in conscious normotensive Wistar rats and compared with the known increase in renal tubular lithium reabsorption induced by the non-steroidal anti-inflammatory drug, indomethacin. 2. The rats were treated for five days with losartan (10 mg kg-1 day-1, orally), indomethacin (2.5 mg kg-1 day-1, intramuscularly) or their solvents. Lithium chloride (16.7 mg kg-1, i.p.) was given as a single dose on the fifth day; renal functions were then measured. 3. Indomethacin, in the absence of any effect on creatinine clearance, increased renal fractional lithium reabsorption and led to an increase in plasma lithium levels. 4. Losartan did not modify renal lithium handling and its plasma level. No change was observed in renal lithium clearance, the quantity of filtered lithium or the fractional reabsorption of the metal. As expected, losartan had no effect on systolic blood pressure in normotensive rats. 5. In conclusion, our results indicate that losartan, when given orally in the rat at a dose of 10 mg kg-1 day-1 over five days, does not modify renal lithium handling. They suggest that blockade of the angiotensin II receptors does not interfere with renal lithium reabsorption, which occurs mainly at a proximal tubular site.

The renal effects of i.v. injections of (+/-)-ozolinone, its enantiomers (-)-ozolinone and (+)-ozolinone and its prodrug (+/-)-etozoline, were compared with those of furosemide, in pentobarbital anesthetized dogs. Renal blood flow (electromagnetic flow-meter) and glomerular filtration rate (polyfructosan clearance) were assessed on the left denervated kidney together with renin secretion and urinary electrolyte excretion. (-)-Ozolinone (15.5 mg/kg i.v.) behaves as a stereoselective loop diuretic equipotent to 20 mg/kg of furosemide and 45 mg/kg of (+/-)-ozolinone; (+)-ozolinone induced only minor salidiuretic effects. Both ozolinone enantiomers markedly increased the renal blood flow and decreased the filtration fraction, suggesting that the vosodilating effect predominates on the efferent glomerular arterioles. (-)-Ozolinone also induced an acute rise in renin secretion. The inhibition of prostaglandin synthesis (indomethacin or meclofenamate) prevented renin hypersecretion in response to (-)-ozolinone and modified its salidiuretic effects but had no effect on the vascular response. The inhibition of the kallikrein-kinin system by aprotinin had no effect on the overall renal response to (-)-ozolinone. The inhibition of the renin-angiotensin system by captopril decreased blood pressure, prolonged the (-)-ozolinone-induced decrease in renal vascular resistance and increased renin secretion. Our results demonstrate that the loop diuretic, ozolinone, induces stereoselective and prostaglandin-dependent renin secretion, which is involved in the regulation of intra-renal hemodynamics.

Both insulin-dependent diabetes mellitus (IDDM) and unilateral nephrectomy (UNX) are associated with an increase in the glomerular filtration rate. Glomerular hyperfiltration has been linked to intraglomerular hypertension in both conditions, although it has only been linked to the development of nephropathy in diabetes. In this study, we examined the possibility of preventing diabetic nephropathy through early dopamine (DA) prodrugs treatment and we also investigated the participation of endogenous DA in the acute functional adaptation of the remaining kidney after UNX. In an animal model of IDDM (steptozotocin-treated Wistar rats), the early increase in the glomerular filtration rate was prevented by treatment with DA prodrugs (L-dopa or gludopa), an effect which was mimicked by fenoldopam (a D1 agonist) and suppressed by carbidopa and SCH 23390 (a D1 antagonist). An increase in endorenal DA synthesis and the subsequent stimulation of vascular D1 receptors appears to prevent early glomerular hyperfiltration in diabetic rats. However, in a long-term study lasting more than one year, streptozotocin-diabetic Wistar rats (unlike to diabetic Munich Wistar rats) failed to develop overt nephropathy characterized by albuminuria and systemic hypertension. During long-term treatment of diabetic rats with L-dopa, the renal availability of DA was diminished. The acute adaptation of the remaining kidney to UNX took the form of an early transient pressor effect with a moderate increase in the glomerular filtration rate and renal blood flow, and a marked decrease in tubular sodium reabsorption. SCH 23390 suppressed the hemodynamic and tubular responses to UNX, suggesting that endogenous DA plays a key role.

Objectives Recent evidence suggests that tissue generation of angiotensins I and II depends on the level of the plasma components of the renin-angiotensin system and on tissue-specific processes. The present study was undertaken to clarify the possible relationship between plasma renin activity (PRA) and tissue angiotensin converting enzyme (ACE) activity in the heart, lung, kidney cortex and kidney medulla of Wistar-Kyoto rats. In the kidney cortex particular attention was focused on renal brush-border ACE. Methods Different experimental models known to have opposite effects on PRA were used: changes in salt intake, deoxycorticosterone acetate (DOCA) with or without salt supplements, and the Goldblatt two-kidney, one clip (2-K,1C) model. Two weeks after the start of the experiments the rats were killed, and PRA, and plasma and tissue ACE activity, were measured. Results At the end of the study the blood pressure in the treated rats was not significantly different from control. As expected, the PRA were highest in the 2-K,1C and depleted-salt groups and lowest in the DOCA, DOCA-salt and high-salt groups. ACE responses were different in different types of tissue, with no relationship between PRA and plasma or tissue ACE activity. For example, DOCA treatment led to increased ACE activity in the heart and the kidney only if the rats were maintained on a high salt intake. DOCA or salt alone failed to have this effect. In the 2-K,1C model the unclipped kidneys did not show any significant variation in ACE activity, but the clipped kidneys exhibited increased ACE activity compared with sham-operated rats. This increase, coupled with increased renal renin secretion, could play a role in the acceleration of local angiotensin II formation, and could thus initiate and sustain the development of hypertension in this model. Conclusion The present results show that variations in ACE activity were organ-specific and were not linked either to hypertension or to changes in PRA.