Background: Women using oral contraceptives have higher ambulatory blood pressures (BPs) than other women. We sought to learn whether this was associated with an alteration in the balance of angiotensin II (Ang)/Ang (1-7) and whether this ratio and BP remained constant throughout the menstrual cycle.Method: In total, 30 (15 ovulatory, 15 taking oral contraceptives) healthy, normotensive women aged 18-30 years were studied. The ovulatory group was assessed within days 1-5 (follicular phase) and 19-23 (luteal phase) and the oral contraceptive group within days 19-23. Peripheral, central and 24-h BP, vascular wall stiffness, and body composition were measured along with plasma estradiol, progesterone, renin, aldosterone, Ang II, and Ang (1-7) concentrations.Results: In ovulatory women plasma renin activity (P<0.001), renin concentration (P<0.01) and aldosterone (P<0.05) were higher in the luteal than follicular phase, whereas BP, Ang II and the Ang II/Ang (1-7) ratio remained constant. In women taking oral contraceptives, plasma renin activity (P<0.001) and concentration (P<0.01) were higher than in follicular phase ovulatory women whereas 24-h BP, Ang II, Ang (1-7), and the Ang II/Ang (1-7) ratio (P<0.01) were higher than in both phases of the ovulatory group. However, there was no significant correlation between BP and the Ang II/Ang (1-7) ratio.Conclusion: This study confirms that BP is constant throughout the normal menstrual cycle along with a constant balance between the vasoconstrictor (Ang II) and vasodilator [Ang (1-7)] arms of the renin-Ang-aldosterone system. Women taking oral contraceptives have a higher Ang II/Ang (1-7) ratio associated with their BP elevation although no causal relationship has been found.

Angiotensin converting enzyme (ACE) 2 activity and angiotensin-(1-7) [Ang-(1-7)] levels are increased in experimental cirrhosis; however, the pathways of hepatic Ang-(1-7) production have not been studied. This study investigated the role of ACE2, ACE, and neutral endopeptidase (NEP) in the hepatic formation of Ang-(1-7) from angiotensin I (Ang I) and Ang II and their effects on portal resistance. Ang I or Ang II were administered to rat bile duct ligated (BDL) and control livers alone and in combination with the ACE inhibitor lisinopril, the ACE and NEP inhibitor omapatrilat, or the ACE2 inhibitor MLN4760 (n = 5 per group). BDL markedly upregulated ACE, ACE2, and NEP. Ang-(1-7) was produced from Ang II in healthy and in BDL livers and was increased following ACE inhibition and decreased by ACE2 inhibition. In contrast, Ang-(1-7) production from Ang I was minimal and not affected by ACE or NEP inhibition. Surprisingly, ACE2 inhibition in BDLs dramatically increased Ang-(1-7) production from Ang I, an effect abolished by ACE2/NEP inhibition. Ang II and Ang I induced greater portal pressure increases in BDL livers than controls. The effects of Ang I were closely correlated with Ang II production and were strongly attenuated by both ACE and ACE/NEP inhibition. These findings show that the major substrate for hepatic production of Ang-(1-7) is Ang II and this is catalyzed by ACE2. Ang I is largely converted to Ang II by ACE, and net conversion of Ang I to Ang-(1-7) is small. NEP has the ability to generate large amounts of Ang-(1-7) in the BDL liver from Ang I only when ACE2 activity is greatly decreased or inhibited.

Ang-(1-7) (angiotensin-1-7), a peptide product of the recently described ACE (angiotensin-converting enzyme) homologue ACE2, opposes the harmful actions of AngII (angiotensin II) in cardiovascular tissues, but its role in liver disease is unknown. The aim of the present study was to assess plasma levels of Ang-(1-7) in human liver disease and determine its effects in experimental liver fibrosis. Angiotensin peptide levels were measured in cirrhotic and non-cirrhotic patients with hepatitis C. The effects of Ang-(1-7) on experimental fibrosis were determined using the rat BDL (bile-duct ligation) model. Liver histology, hydroxyproline quantification and expression of fibrosis-related genes were assessed. Expression of RAS (renin-angiotensin system) components and the effects of Ang-(1-7) were examined in rat HSCs (hepatic stellate cells). In human patients with cirrhosis, both plasma Ang-(1-7) and AngII concentrations were markedly elevated (P<0.001). Non-cirrhotic patients with hepatitis C had elevated Ang-(1-7) levels compared with controls (P<0.05), but AngII concentrations were not increased. In BDL rats, Ang-(1-7) improved fibrosis stage and collagen Picrosirius Red staining, and reduced hydroxyproline content, together with decreased gene expression of collagen 1A1, alpha-SMA (smooth muscle actin), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), ACE and mas [the Ang-(1-7) receptor]. Cultured HSCs expressed AT1Rs (AngII type 1 receptors) and mas receptors and, when treated with Ang-(1-7) or the mas receptor agonist AVE 0991, produced less alpha-SMA and hydroxyproline, an effect reversed by the mas receptor antagonist A779. In conclusion, Ang-(1-7) is up-regulated in human liver disease and has antifibrotic actions in a rat model of cirrhosis. The ACE2/Ang-(1-7)/mas receptor axis represents a potential target for antifibrotic therapy in humans.

A transport protein is described with 12 transmembrane spans. Within the cytoplasmic amino-terminal domain, several novel hexad repeats are conserved in human, mouse, rat and pig, four to six of which had the canonical form PS_S_H+. In the carboxyl-terminal domain, a polyglutamate sequence (5–8) is conserved. Restricted expression of the transporter was identified in acidophil cells of the adult pituitary that secrete growth hormone and prolactin. In the fetus, expression was restricted to osteoclasts, chondrocytes, thyroid, pituitary, central nervous system, eye, liver and heart. In particular, expression was found in structures associated with rapid calcium exchange including the retina, cardiomyocytes and in the intraplacental yolk sac that expresses calcitropic molecules. Furthermore, expression found in osteoclasts and kidney, within the distal portions of nephrons and collecting ducts, was consistent with a role in calcium homeostasis. In human pituitary, four mRNA transcripts, and in mouse kidney, three mRNA transcripts were expressed. In developing mouse kidney, the amount of each transcript varied that suggested the multiple transcripts might be differentially expressed in different physiological states. We propose that the transporter is specific for a calcium-chelator complex and is important for growth and calcium metabolism.

Neurotrophin therapy in the cochlea can potentially slow or reverse the degeneration of the auditory nerve that occurs during progressive deafness. Studies were performed to trace the diffusion and uptake of neurotrophin-3 (NT-3) following infusion into the cochlea. NT-3 labeled with 125I or coated onto fluorescent microspheres was introduced into the basal turn of normal hearing and deafened guinea pig cochleae via a single slow-rate injection. Cochleae were examined between 2 h and 28 days post-infusion by autoradiography or fluorescent microscopy to determine the number of turns labeled by NT-3, identify individual cells and tissues receiving NT-3 and quantify the proportion of signal in each tissue. In general, long-term infusions were required for all cochlear turns to receive NT-3. 125I NT-3 signal was strongest in cells lining the perilymphatic space of the scala tympani, basilar membrane, osseous spiral lamina and spiral ligament. Signal in the peripheral nerve tract and Rosenthal’s canal was only 1.3–2.1 times background levels of radiation. NT-3 microspheres were detected within neural areas of the cochlea (nerve tract and Rosenthal’s canal) in all cases, but not within neuronal cell bodies. NT-3 microspheres remained in the cochlea for at least 28 days, suggesting a low clearance rate within cochlear tissues.

BACKGROUND Development in the metanephric-kidney transition period involves the precise expression of paracrine and autocrine events in an ordered spatio-temporal manner. Expression of these molecular events is tightly controlled and includes positive and negative growth factors and cognate receptors within close proximity in developing structures in the expanding renal cortex and medulla. The expression of calcitonin receptor (CTR) isoforms C1a and C1b in this context has not previously been described. Our current study also explored the relationship between the expression of CTR isoforms and amylin binding sites. METHODS Techniques included immunohistochemistry with novel antibodies that detect CTR isoforms, real time PCR for the quantification of CTR isoforms, Western blot and in vitro autoradiography, on tissues from embryo day 18 to postnatal day 30. RESULTS The CTR C1a isoform is expressed in the ureteric ducts of the metanephros and both isoforms are expressed in the developing distal convoluted tubules, ascending limbs of the loop of Henle and collecting ducts in the postnatal rat kidney. There was a 60-fold excess of C1a versus C1b isoforms. An apparent molecular weight of 63 kD was found. In vitro autoradiography demonstrated that while amylin binding sites were predominantly in the cortex, CTR expression was largely localized in the medulla in an earlier event, followed by cortical expression. CONCLUSIONS CTR C1a protein expression has been identified in the ureteric ducts in the metanephros and both isoforms expressed in the distal portions of the developing nephrons and collecting ducts. Since amylin binding sites have been localized on the proximal tubules of the cortex, it is unlikely that amylin receptors can be represented by modification of CTR affinity with receptor activity modifying proteins in the kidney.

Objective The effects of combined inhibition of neutral endopeptidase (NEP) with either angiotensin-converting enzyme (ACE), or endothelin-converting enzyme (ECE) on blood pressure, urinary albumin excretion and heart weight were explored in experimental diabetes. Design Streptozotocin-induced diabetic Sprague–Dawley rats were treated with vehicle, the NEP/ACE inhibitor S 21402, the NEP/ECE inhibitor CGS 26303, the NEP inhibitor SCH 42495, the ACE inhibitor captopril or the endothelin receptor antagonist bosentan for 4 weeks. Methods Blood pressure was measured by tail-cuff method and radiotelemetry. Albuminuria, plasma renin activity and plasma atrial natriuretic peptide (ANP) were determined by radioimmunoassay. NEP binding was assessed by in vitro quantitative autoradiography. Metabolic and biochemistry parameters including food intake, 24-h urine volume, plasma glucose, glycated hemoglobin, glomerular filtration rate (GFR) and urinary sodium excretion were also determined. Results Mean blood pressure over the 4-week study period after commencement of treatment was reduced to a similar extent by a range of treatments including the ACE inhibitor, NEP/ACE inhibitor, endothelin receptor antagonist, NEP/ECE inhibitor, but not the NEP inhibitor, compared with vehicle-treated diabetic rats. Heart to body weight ratio in diabetic rats was only reduced by the NEP/ACE and the NEP/ECE inhibitor. Increased albuminuria in diabetic rats (1.1 ×/÷ 1.2 mg/day) was reduced by the NEP/ACE (0.6 ×/÷ 1.2 mg/day) and the NEP/ECE inhibitors (0.4 ×/÷ 1.2 mg/day). Renal NEP was reduced by the NEP/ACE inhibitor (35 ± 4%) or NEP/ECE inhibitor (38 ± 4%) as well as by the pure NEP inhibitor (27 ± 4%) compared with the untreated diabetic group. Other abnormal metabolic and biochemical parameters in diabetic rats were not influenced by any drug treatment. Conclusions Combined inhibition of NEP/ACE or NEP/ECE confers beneficial effects on blood pressure, albuminuria and heart to body weight ratio in experimental diabetes.

OBJECTIVE Diabetic subjects have a high prevalence of hypertension, increased total body exchangeable sodium levels, and an impaired ability to excrete a sodium load. This study assessed the effect of dietary sodium restriction on the efficacy of losartan in hypertensive subjects with type 2 diabetes and albumin excretion rates of 10-200 microg/min. RESEARCH DESIGN AND METHODS In this study, 20 subjects were randomized to losartan 50 mg/day (n = 10) or placebo (n = 10). Drug therapy was given in two 4-week phases separated by a washout period. In the last 2 weeks of each phase, patients were assigned to low- or regular-sodium diets, in random order. In each phase, 24-h ambulatory blood pressure, urinary albumin-to-creatinine ratio (ACR), and renal hemodynamics were measured. RESULTS Achieved urinary sodium on a low-sodium diet was 85 +/- 14 and 80 +/- 22 mmol/day in the losartan and placebo groups, respectively. In the losartan group, the additional blood pressure-lowering effects of a low-sodium diet compared with a regular-sodium diet for 24-h systolic, diastolic, and mean arterial blood pressures were 9.7 mmHg (95% confidence interval [CI], 2.2-17.2; P = 0.002), 5.5 mmHg (2.6-8.4; P = 0.002), and 7.3 mmHg (3.3- 11.3; P = 0.003), respectively. In the losartan group, the ACR decreased significantly on a low-sodium diet versus on a regular-sodium diet (-29% [CI -50.0 to -8.5%] vs. + 14% [-19.4 to 47.9%], respectively; P = 0.02). There was a strong correlation between fall in blood pressure and percent reduction in the ACR (r = 0.7, P = 0.02). In the placebo group, there were no significant changes in blood pressure or ACR between regular- and low-sodium diets. There were no significant changes in renal hemodynamics in either group. CONCLUSIONS These data demonstrated that a low-sodium diet potentiates the antihypertensive and antiproteinuric effects of losartan in type 2 diabetes. The blood pressure reduction resulting from the addition of a low-sodium diet to losartan was of similar magnitude to that predicted from the addition of a second antihypertensive agent.

Objective The aims of this study were to assess the renal expression of angiotensin type 1 (AT1) and type 2 (AT2) receptors in diabetic spontaneously hypertensive rats (SHR) and the effect of AT1 receptor blockade on the expression of these receptors. Design Diabetes was induced by injection of streptozotocin in SHRs. Irbesartan, an AT1 receptor antagonist, was given to diabetic SHRs for 32 weeks (15 mg/kg per day, n = 10). Diabetic (n = 10) and non-diabetic SHRs (n = 10) were studied concurrently. A separate group of control and diabetic Wistar–Kyoto (WKY) rats were also evaluated. Methods Gene and protein expressions of the AT1 and AT2 receptor were assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry with specific antibodies and in vitro autoradiography with [125I]Sar(1), Ile(8) angiotensin II or [125I]CGP42112B. Results Both AT1 and AT2 receptor mRNA levels in the kidney were reduced in diabetic SHRs compared to non-diabetic SHRs. Immunohistochemistry staining with specific antibodies showed a similar reduction in glomerular and tubulo-interstitial staining for both AT1 and AT2 receptors. Reduced binding for the AT1 and AT2 receptor was found in the kidney of diabetic SHRs. Diabetic SHRs developed albuminuria and had glomerular and tubulo-interstitial injury, which were prevented by treatment with irbesartan. Reduced expression of the AT1 receptor, but not the AT2 receptor, in diabetic SHRs was prevented by treatment with irbesartan. In diabetic WKY rats no such reduction in AT1 expression was observed, although there was a trend for reduced AT2 receptor expression. Conclusions These findings demonstrated that renal expression of both AT1 and AT2 receptor was reduced in long-term diabetic SHRs and that blockade of the AT1 receptor had disparate effects on expression of angiotensin II receptor subtypes.

Objective The aim of this study was to investigate the relative role of the angiotensin type 1 (AT1) and type 2 (AT2) receptors in mediating angiotensin II-induced regulation of AT2 receptor in mesenteric artery. Design Sprague–Dawley rats were infused with either angiotensin II or vehicle for 14 days at a dose of 58.3 ng/min. Ang II-infused rats were allocated to receive either an AT1 antagonist, valsartan at a dose of 30 mg/kg per day or the AT2 receptor antagonist PD123319 at a dose of 830 ng/min. Methods Gene and protein expression of the AT2 receptor in the mesenteric vasculature was assessed by quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry and by in vitro autoradiography with a specific radioligand, 125I-CGP 42112B. Results The AT2 receptor mRNA and protein were detected in the mesenteric artery from adult rats. Both nuclear emulsion and immunohistochemical staining showed expression of the AT2 receptor in the adventitial and medial layers. Compared to control rats, angiotensin II infusion was associated with a significant increase in the AT2 receptor expression. Valsartan treatment significantly reduced AT2 receptor gene expression, with no significant effect of PD123319 on this parameter. Conclusions This study confirms that the presence of the AT2 receptor in mesenteric arteries in adult rats, shows an up-regulation of the AT2 receptor following angiotensin II infusion and suggests a role for the AT1 receptor in this regulation. In view of the recently demonstrated effects of the AT2 receptor, these findings may be relevant to the role of the AT2 receptor in the pathophysiology of vascular remodeling.

Angiotensin II (Ang II) AT(1A) receptors are localized to renomedullary interstitial cells (RMIC) in the inner stripe of the outer medulla but not in the inner medulla. Thus, there seems to be a correlation between decreases in AT(1A) receptor binding to RMIC and increases in interstitial osmolality, suggesting that osmolality is important in determining Ang II binding to RMIC. Cultured RMIC were incubated in media of differing osmolalities (330, 630, 930, and 1230 mOsm/kg H(2)O). (125)I-[Sar(1), Ile(8)] Ang II binding to AT(1A) receptors on RMIC grown in hyperosmolal media (930 mOsm/kg H(2)O) was reduced compared with isoosmolal (330 mOsm/kg H(2)O) media and was progressively reduced with further increases of osmolality. Similar studies were performed using bradykinin (BK) as a control peptide. Binding of the BK receptor ligand (125)I-[HPP-Hoe 140] to B(2) receptors was not affected by varying osmolality of the media. Reverse transcriptase-PCR demonstrated the presence of the mRNA expression for both AT(1A) and B(2) receptors at each osmolality. The conclusion is that osmolality modulates Ang II binding to RMIC; in these cells, this phenomenon is restricted to Ang II as BK binding is not affected. Osmolality-induced changes in Ang II binding may modulate the actions of this peptide on RMIC and provide an important mechanism by which these cells modulate renal medullary function.

BACKGROUND Angiotensin II (Ang II) is associated with cell proliferation and apoptosis. The role of the angiotensin type 2 receptor (AT2R) in these processes remains controversial. Conventional radioligand binding of 125I-Sar1, Ile8 Ang II in adult kidney has failed to demonstrate the binding for the AT2R. METHODS The presence of the AT2R was explored in adult rat kidney by in vitro and in vivo autoradiography using the selective AT2R radioligand 125I-CGP 42112B. The roles of the angiotensin type 1 receptor (AT1R) and the AT2R in mediating cellular proliferation and apoptosis were assessed using selective AT1R or AT2R antagonists in Ang II-infused Sprague-Dawley (SD) rats. RESULTS 125I-CGP 42112B binding was demonstrated by in vitro and in vivo autoradiography techniques in the glomeruli and proximal tubules of SD rats. This binding could be displaced by Ang II and the AT2R antagonist PD123319 but not by the AT1R antagonist valsartan. Subcutaneous infusion of Ang II for 14 days in eight-week-old SD rats induced proliferation of proximal tubular epithelial cells, as assessed by a twofold increase in proliferating cell nuclear antigen (PCNA)-positive cells and apoptosis, as assessed by a threefold increase in terminal dUTP nick end labeling (TUNEL)-positive cells. The administration of the AT2R antagonist PD123319 or the AT1R antagonist valsartan was associated with attenuation of the increases in both PCNA- and TUNEL-positive cells following Ang II infusion. Ang II infusion was associated with increased osteopontin gene and protein expression, which could be reduced by treatment with either valsartan or PD123319. CONCLUSION These findings indicate that there is significant expression of the AT2R in the adult kidney, and that the AT2R has a role in mediating Ang II-induced proliferation and apoptosis in proximal tubular epithelial cells and expression of osteopontin.

1. Renomedullary interstitial cells (RMIC), abundant throughout the medulla of the kidney, have been demonstrated to have binding sites for many vasoactive peptides, including atrial natriuretic peptide, endothelin, angiotensin II and bradykinin (BK). These observations would support the hypothesis that interactions between RMIC and vasoactive peptides are important in the regulation of renal function.2. We aimed to localize the BK B2 receptor binding site to RMIC in vivo and to also demonstrate that these receptors are biologically active in vitro.3. The present study demonstrates BK B2 binding sites on RMIC of the inner stripe of the outer medulla and the inner medulla of the rat kidney in vivo.4. We further demonstrate that the BK B2 radioligand [125I]‐HPP‐Hoe140 specifically bound to rat RMIC in vitro. In addition, reverse transcription–polymerase chain reaction detected the mRNA for the BK B2 receptor subtype in cell extracts.5. For RMIC in vitro, cAMP levels were increased at 1 min and cGMP levels were increased at 2 min after treatment with 10–10 and 10–7 mol/L BK, respectively. Inositol 1,4,5‐trisphosphate was increased at 10 s treatment with both 10–6 and 10–7 mol/L BK.6. For RMIC in vitro, BK induced an increase in cell proliferation ([3H]‐thymidine incorporation) and an increase in extracellular matrix synthesis (ECM; trans‐[35S] incorporation), both effects mediated by BK B2 receptors.7. We conclude that BK B2 receptors are present on RMIC both in vivo and in vitro. These receptors are coupled to intracellular second messenger systems and, in vitro, their stimulation results in cellular proliferation and synthesis of ECM.

In continuation of our tradition of joint congresses, the meeting in Feiburg will focus on new experimental and clinical data. The program, consisting of posters, free contributions, and invited reviews, will have three focuses: (1) basic research; (2) experimental clinical science, and (3) lectures on hot clinical topics. The congress also encompasses free symposia, plenary sessions, and a social program. The major topics will be defined by the Joint Program Committee of the Gesellschaft für Nephrologie and the Deutsche Arbeitsgemeinschaft für Klinische Nephrologie. The congress language will be German. Free communications or posters should be submitted by using the specific abstract form which, together with the preliminary program, will be available in February 1999. The success of the meeting will depend firstly on broad participation of nephrologists and those interested in nephrology and secondly on the contributions submitted. We will try to generate an interesting program and secure sufficient time also for social activities.

The aim of this study was to explore the regulation of angiotensin receptors after chronic infusion with angiotensin II (Ang II) and to clarify the relative roles of the angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors in the mediation of Ang II-induced mesenteric vascular hypertrophy. In male Sprague-Dawley rats, Ang II infusion at a dose of 58.3 ng/min by subcutaneous osmotic minipumps for 14 days led to increased mesenteric weight and wall:lumen ratio of the vessels and proliferation of smooth muscle cells. These vascular changes were attenuated by either valsartan, an AT(1) receptor antagonist, at a dose of 30 mg. kg(-1). d(-1) by gavage, or PD123319, an AT(2) receptor antagonist, at a dose of 830 ng/min by intraperitoneally implanted osmotic minipumps. Ang II infusion was associated with hypertension, which was prevented by valsartan, but not PD123319. (125)I-Sar(1), Ile(8) Ang II binding to mesenteric vasculature was increased after Ang II infusion. Valsartan treatment was associated with reduced Ang II binding to both receptor subtypes, whereas PD123319 was associated with reduced Ang II binding to only the AT(2) receptor subtype. These findings suggest that the trophic and proliferative effects of Ang II on the mesenteric vasculature are mediated by both AT(1) and AT(2) receptors.

We have previously reported that amylin has mitogenic actions on tubular epithelial cells isolated from mature rat kidney and cultured in vitro. In experiments using in situ hybridization, we have demonstrated that amylin mRNA can be detected transiently in rat metanephros from embryo day 17 (E17) to postnatal day 3 (PN3). These transcripts are localized in the sub-nephrogenic zone. RT-PCR was performed using oligonucleotide primers for rat amylin and mRNA extracted from fetal body (E19), PN1 and PN5 metanephroi, and adult rat kidney. These results corroborate the finding, using in situ hybridization, that there is a window of expression of rat amylin in the developing kidney in the perinatal period. During this period tubular elongation is evident and amylin peptide, detected by immunohistochemical staining, is found associated with developing tubules. Some of these tubules also express a brush border glycoprotein, detected by immunohistochemical staining. Amylin acts as a mitogen with primary cultures of proximal tubular epithelial cells from PN4 renal cortex. An amylin antagonist inhibited this mitogenic action suggesting that this was mediated by amylin receptors as previously described. We suggest that amylin peptide is biosynthesized in the developing proximal tubules, acts in an autocrine fashion to promote the proliferation and differentiation of brush border epithelial cells and hence plays an important role as a growth factor in the development of the kidney.