Absence of renal hypoxia in the subacute phase of severe renal ischemia-reperfusion injury.

Tissue hypoxia has been proposed as an important event in renal ischemia-reperfusion injury (IRI), particularly during the period of ischemia and in the immediate hours following reperfusion. However, little is known about renal oxygenation during the subacute phase of IRI. We employed four different methods to assess the temporal and spatial changes in tissue oxygenation during the subacute phase (24 h and 5 days after reperfusion) of a severe form of renal IRI in rats. We hypothesized that the kidney is hypoxic 24 h and 5 days after an hour of bilateral renal ischemia, driven by a disturbed balance between renal oxygen delivery (Do2) and oxygen consumption (V̇o2). Renal Do2 was not significantly reduced in the subacute phase of IRI. In contrast, renal V̇o2 was 55% less 24 h after reperfusion and 49% less 5 days after reperfusion than after sham ischemia. Inner medullary tissue Po2, measured by radiotelemetry, was 25 ± 12% (mean ± SE) greater 24 h after ischemia than after sham ischemia. By 5 days after reperfusion, tissue Po2 was similar to that in rats subjected to sham ischemia. Tissue Po2 measured by Clark electrode was consistently greater 24 h, but not 5 days, after ischemia than after sham ischemia. Cellular hypoxia, assessed by pimonidazole adduct immunohistochemistry, was largely absent at both time points, and tissue levels of hypoxia-inducible factors were downregulated following renal ischemia. Thus, in this model of severe IRI, tissue hypoxia does not appear to be an obligatory event during the subacute phase, likely because of the markedly reduced oxygen consumption.

Epochs in the depressor/pressor balance of the renin-angiotensin system.

The renin-angiotensin system (RAS) plays a commanding role in the regulation of extracellular fluid homoeostasis. Tigerstadt and Bergman first identified the RAS more than two centuries ago. By the 1980s a voyage of research and discovery into the mechanisms and actions of this system led to the development of drugs that block the RAS, which have become the mainstay for the treatment of cardiovascular and renal disease. In the last 25 years new components of the RAS have come to light, including the angiotensin type 2 receptor (AT2R) and the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang(1-7)]/Mas receptor (MasR) axis. These have been shown to counter the classical actions of angiotensin II (AngII) at the predominant angiotensin type 1 receptor (AT1R). Our studies, and those of others, have demonstrated that targeting these depressor RAS pathways may be therapeutically beneficial. It is apparent that the evolution of both the pressor and depressor RAS pathways is distinct throughout life and that the depressor/pressor balance of the RAS vary between the sexes. These temporal patterns of expression suggest that therapies targeting the RAS could be optimized for discrete epochs in life.

Renal cellular hypoxia in adenine-induced chronic kidney disease.

We determined whether adenine-induced chronic kidney disease (CKD) in rats is associated with renal tissue hypoxia. Adenine (100 mg) or its vehicle was administered to male Sprague-Dawley rats, daily by oral gavage, over a 15-day period. Renal function was assessed before, and 7 and 14 days after, adenine treatment commenced, by collection of a 24-hour urine sample and a blood sample from the tail vein. On day 15, arterial pressure was measured in conscious rats via the tail artery. Renal tissue hypoxia was then assessed by pimonidazole adduct immunohistochemistry and fibrosis was assessed by staining tissue with picrosirius red and Masson's trichrome. CKD was evident within 7 days of commencing adenine treatment, as demonstrated by increased urinary albumin to creatinine ratio (30 ± 12-fold). By day 14 of adenine treatment plasma creatinine concentration was more than 7-fold greater, and plasma urea more than 5-fold greater, than their baseline levels. On day 15, adenine-treated rats had slightly elevated mean arterial pressure (8 mmHg), anaemia and renomegaly. Kidneys of adenine-treated rats were characterised by the presence of tubular casts, dilated tubules, expansion of the interstitial space, accumulation of collagen, and tubulointerstitial hypoxia. Pimonidazole staining (hypoxia) co-localised with fibrosis and was present in both patent and occluded tubules. We conclude that renal tissue hypoxia develops rapidly in adenine-induced CKD. This model, therefore, should prove useful for examination of the temporal and spatial relationships between tubulointerstitial hypoxia and the development of CKD, and thus the testing of the 'chronic hypoxia hypothesis'.

ß-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats.

We have previously shown that individual ß-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that ß-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel ß-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > ß-Pro(7) Ang III=Ang II > ß-Tyr(4) Ang III ≥ PD123319 >> ß-Phe(8) Ang III >> ß Arg(2) Ang III=ß-Val(3) Ang III >> ß-Ile(5) Ang III. The novel analogue ß-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that ß-Pro(7) Ang III was an AT2R agonist, we compared ß-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), ß-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and ß-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, ß-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.

Sex- and age-related differences in the chronic pressure-natriuresis relationship: role of the angiotensin type 2 receptor.

Sex hormones regulate the renin-angiotensin system. For example, estrogen enhances expression of the angiotensin type 2 receptor. We hypothesized that activation of the angiotensin type 2 receptor shifts the chronic pressure-natriuresis relationship leftward in females compared with males and that this effect is lost with age. Mean arterial pressure was measured by radiotelemetry in adult (4 mo old) and aged (14 mo old) wild-type and angiotensin type 2 receptor knockout male and female mice. Chronic pressure-natriuresis curves were constructed while mice were maintained on a normal-salt (0.26%) diet and following 6 days of high salt (5.0%) diet. Mean arterial pressure was lower in adult wild-type females than males (88 ± 1 and 97 ± 1 mmHg, respectively), a difference that was maintained with age, but was absent in adult knockout mice. In wild-type females, the chronic pressure-natriuresis relationship was shifted leftward compared with knockout females, an effect that was lost with age. In males, the chronic pressure-natriuresis relationship was not influenced by angiotensin type 2 receptor deficiency. Compared with age-matched females, the chronic pressure-natriuresis relationships in male mice were shifted rightward. Renal expression of the angiotensin type 2 receptor was fourfold greater in adult wild-type females than males. With age, the angiotensin type 2 receptor-to-angiotensin type 1 receptor balance was reduced in females. Conversely, in males, angiotensin receptor expression did not vary significantly with age. In conclusion, the angiotensin type 2 receptor modulates the chronic pressure-natriuresis relationship in an age- and sex-dependent manner.

Determinants of renal tissue hypoxia in a rat model of polycystic kidney disease.

Renal tissue oxygen tension (PO2) and its determinants have not been quantified in polycystic kidney disease (PKD). Therefore, we measured kidney tissue PO2 in the Lewis rat model of PKD (LPK) and in Lewis control rats. We also determined the relative contributions of altered renal oxygen delivery and consumption to renal tissue hypoxia in LPK rats. PO2 of the superficial cortex of 11- to 13-wk-old LPK rats, measured by Clark electrode with the rat under anesthesia, was higher within the cysts (32.8 ± 4.0 mmHg) than the superficial cortical parenchyma (18.3 ± 3.5 mmHg). PO2 in the superficial cortical parenchyma of Lewis rats was 2.5-fold greater (46.0 ± 3.1 mmHg) than in LPK rats. At each depth below the cortical surface, tissue PO2 in LPK rats was approximately half that in Lewis rats. Renal blood flow was 60% less in LPK than in Lewis rats, and arterial hemoglobin concentration was 57% less, so renal oxygen delivery was 78% less. Renal venous PO2 was 38% less in LPK than Lewis rats. Sodium reabsorption was 98% less in LPK than Lewis rats, but renal oxygen consumption did not significantly differ between the two groups. Thus, in this model of PKD, kidney tissue is severely hypoxic, at least partly because of deficient renal oxygen delivery. Nevertheless, the observation of similar renal oxygen consumption, despite markedly less sodium reabsorption, in the kidneys of LPK compared with Lewis rats, indicates the presence of inappropriately high oxygen consumption in the polycystic kidney.

Reduced sensitivity of the renal vasculature to angiotensin II in young rats: the role of the angiotensin type 2 receptor.

BACKGROUND: The angiotensin type-2 receptor (AT2R) opposes the vasoconstrictor actions of angiotensin II (AngII) mediated through the angiotensin type-1 receptor (AT1R). Renal AT2R levels are high during fetal life, but decrease significantly during postnatal maturation. To provide insight into the functional role of the AT2R in the kidney during postnatal development, we investigated the effects of AT2R antagonism on cardiovascular responses to AngII in young and adult male rats. METHODS: In anesthetized 3- and 6-wk-old male Sprague-Dawley rats, mean arterial pressure (MAP) and renal blood flow (RBF) were measured in response to AngII in the presence of vehicle treatment or AT2R blockade with PD123319. RESULTS: The pressor effect of AngII and associated reduction in RBF were significantly less in 3-wk- than 6-wk-old rats. AT2R blockade potentiated the reduction in RBF in response to AngII in 3-wk-old rats only. CONCLUSION: In young rats, the AT2R modulates the response to AngII, blunting renal vasoconstriction. This effect is attenuated with age in association with a developmental reduction in renal AT2R expression. These findings may have implications for the development of novel therapies that target the renin-angiotensin system for the improvement of renal function in term and, in particular, preterm infants.

The "his and hers" of the renin-angiotensin system.

Sex differences exist in the regulation of arterial pressure and renal function by the renin-angiotensin system (RAS). This may in part stem from a differential balance in the pressor and depressor arms of the RAS. In males, the ACE/AngII/AT(1)R pathways are enhanced, whereas, in females, the balance is shifted towards the ACE2/Ang(1-7)/MasR and AT(2)R pathways. Evidence clearly demonstrates that premenopausal women, as compared to aged-matched men, are protected from renal and cardiovascular disease, and this differential balance of the RAS between the sexes likely contributes. With aging, this cardiovascular protection in women is lost and this may be related to loss of estrogen postmenopause but the possible contribution of other sex hormones needs to be further examined. Restoration of these RAS depressor pathways in older women, or up-regulation of these in males, represents a therapeutic target that is worth pursuing.

Unmasking the potential of the angiotensin AT2 receptor as a therapeutic target in hypertension in men and women: what we know and what we still need to find out.

Major sex differences exist in the development and progression of hypertension and cardiovascular disease. Prior to menopause, women have lower arterial pressure and, furthermore, are protected from hypertension and cardiovascular disease relative to age-matched men. However, after menopause this cardiovascular protection in women is lost. These sex differences have been linked to sexual dimorphism in the physiological mechanisms that regulate arterial pressure, including the renin-angiotensin system (RAS), which can also impact on the male and female response to different therapeutic approaches. This suggests that antihypertensive regimens need to be tailored according to sex. Newly discovered components of the RAS have emerged in recent years, allowing us to look beyond the classical RAS for novel therapeutic targets for hypertension. In this context, it is now well established that the angiotensin AT2 receptor (AT2 R) elicits depressor and natriuretic effects and that these effects are greater in females due to enhanced AT2 R levels modulated by oestrogen. In light of knowledge that AT2 R expression is regulated by oestrogen and that the prevalence of hypertension and cardiovascular risk is greater in women after menopause, AT2 R agonist therapy may represent an innovative therapeutic approach to treat hypertension. Consequently, understanding how ageing and changes in the sex hormone balance influence the RAS is vital if we are to evaluate the potential of the AT2 R as a therapeutic target in women and also in men.

The arterial depressor response to chronic low-dose angiotensin II infusion in female rats is estrogen dependent.

The complex role of the renin-angiotensin-system (RAS) in arterial pressure regulation has been well documented. Recently, we demonstrated that chronic low-dose angiotensin II (ANG II) infusion decreases arterial pressure in female rats via an AT(2)R-mediated mechanism. Estrogen can differentially regulate components of the RAS and is known to influence arterial pressure regulation. We hypothesized that AT(2)R-mediated depressor effects evident in females were estrogen dependent and thus would be abolished by ovariectomy and restored by estrogen replacement. Female Sprague-Dawley rats underwent ovariectomy or sham surgery and were treated with 17ß-estradiol or placebo. Mean arterial pressure (MAP) was measured via telemetry in response to a 2-wk infusion of ANG II (50 ng·kg(-1)·min(-1) sc) or saline. MAP significantly decreased in females treated with ANG II (-10 ± 2 mmHg), a response that was abolished by ovariectomy (+4 ± 2 mmHg) and restored with estrogen replacement (-6 ± 2 mmHg). Cardiac and renal gene expression of components of the RAS was differentially regulated by estrogen, such that overall, estrogen shifted the balance of the RAS toward the vasodilatory axis. In conclusion, estrogen-dependent mechanisms offset the vasopressor actions of ANG II by enhancing RAS vasodilator pathways in females. This highlights the potential for these vasodilator pathways as therapeutic targets, particularly in women.

Induction of hyperglycemia in adult intrauterine growth-restricted rats: effects on renal function.

Intrauterine growth restriction (IUGR) leads to a reduction in nephron endowment at birth and is linked to renal dysfunction in adulthood. The aim of the present study was to determine whether kidneys of IUGR rat offspring are more vulnerable to a secondary insult of hyperglycemia. IUGR was induced in Wistar-Kyoto rats by maternal protein restriction. At 24 wk of age, diabetes was induced in male IUGR and non-IUGR offspring by streptozotocin injection; insulin was injected daily to maintain blood glucose levels at either a mild (7-10 mmol/l; n=8/group) or a moderate (10-15 mmol/l; n=8/group) level. At 32 wk of age, renal function was assessed using ultrasound and [(3)H]inulin and [(14)C]para-aminohippurate clearance techniques. Conscious mean arterial blood pressure and heart rate were unchanged in IUGR offspring. Relative kidney length was increased significantly in IUGR offspring, and renal function was altered significantly; of importance, there was a significant increase in filtration fraction, indicative of glomerular hyperfiltration. Induction of hyperglycemia led to marked impairment of renal function. However, the response to hyperglycemia was not different between IUGR and non-IUGR offspring. Maintaining blood glucose levels at a mild hyperglycemic level led to marked improvement in all measures of renal function in IUGR and non-IUGR offspring. In conclusion, while the IUGR offspring showed evidence of hyperfiltration, the response to hyperglycemia was similar in IUGR and non-IUGR kidneys in adulthood. Importantly, maintaining blood glucose levels at a mild hyperglycemic level markedly attenuated the renal dysfunction associated with diabetes, even in IUGR offspring.

Hypertension-mediated albuminuria is associated with reduced lysosomal activity in the kidney and the heart.

BACKGROUND: Recent studies suggest that expression of the transforming growth factor-beta (TGF-beta)-inducible gene-h3 (betaig-h3) and its anti-lysosomal activity may be responsible for the development of albuminuria and cardiovascular disease associated with hypertension. METHODS: We evaluated the proposed linkage using the spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat models. The kidney and left ventricular weight/body weight ratios were measured and cardiac collagen deposition was analyzed by Masson's trichrome stain. Renal and cardiac TGF-beta(1) and betaig-h3 expression were determined by real-time reverse transcription-polymerase chain reaction, and renal and cardiac cathepsin B and L activities were measured as an indicator of lysosomal proteolytic activity. RESULTS: SHR exhibited increased levels of intact urinary albumin without significant change in total albumin (intact plus albumin-derived material) excretion. This was accompanied by renal hypertrophy, increased renal betaig-h3 expression, and reduced renal cathepsin B and L activities. At the same time, increased cardiac TGF-beta(1) and betaig-h3 expression and reduced cardiac cathepsin B activity was identified in SHR in addition to cardiac hypertrophy and increased collagen deposition. All these changes could be ameliorated with ramipril treatment. CONCLUSIONS: These findings implicate for the first time betaig-h3 expression and lysosomal activity as a key factor in the induction of albuminuria and cardiovascular disease associated with hypertension.

Chronic recurrent dehydration associated with periodic water intake exacerbates hypertension and promotes renal damage in male spontaneously hypertensive rats.

Epidemiological evidence links recurrent dehydration associated with periodic water intake with chronic kidney disease (CKD). However, minimal attention has been paid to the long-term impact of periodic water intake on the progression of CKD and underlying mechanisms involved. Therefore we investigated the chronic effects of recurrent dehydration associated with periodic water restriction on arterial pressure and kidney function and morphology in male spontaneously hypertensive rats (SHR). Arterial pressure increased and glomerular filtration rate decreased in water-restricted SHR. This was observed in association with cyclic changes in urine osmolarity, indicative of recurrent dehydration. Additionally, water-restricted SHR demonstrated greater renal fibrosis and an imbalance in favour of pro-inflammatory cytokine-producing renal T cells compared to their control counterparts. Furthermore, urinary NGAL levels were greater in water-restricted than control SHR. Taken together, our results provide significant evidence that recurrent dehydration associated with chronic periodic drinking hastens the progression of CKD and hypertension, and suggest a potential role for repetitive bouts of acute renal injury driving renal inflammatory processes in this setting. Further studies are required to elucidate the specific pathways that drive the progression of recurrent dehydration-induced kidney disease.

Pressor responsiveness to angiotensin II in female mice is enhanced with age: role of the angiotensin type 2 receptor.

BACKGROUND: The pressor response to angiotensin II (AngII) is attenuated in adult females as compared to males via an angiotensin type 2 receptor (AT2R)-dependent pathway. We hypothesized that adult female mice are protected against AngII-induced hypertension via an enhanced AT2R-mediated pathway and that in reproductively senescent females this pathway is no longer operative. METHODS: Mean arterial pressure was measured via telemetry in 4-month-old (adult) and 16-month-old (aged) and aged ovariectomized (aged-OVX) wild-type and AT2R knockout (AT2R-KO) female mice during baseline and 14-day infusion of vehicle (saline) or AngII (600 ng/kg/min s.c.). Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to determine renal gene expression of angiotensin receptors and angiotensin-converting enzyme 2 in response to 14-day treatment with vehicle or AngII. RESULTS: Basal mean arterial pressure was similar between the groups. The pressor response to AngII was augmented in adult AT2R-KO compared to adult wild-type mice (29 ± 3 mmHg versus 10 ± 4 mmHg, respectively, on day 14 as compared to basal mean arterial pressure, P = 0.002). In wild-type mice, pressor responsiveness to AngII was augmented with age, such that the pressor response to AngII was similar between aged AT2R-KO and wild-type female mice (31 ± 4 mmHg versus 34 ± 3 mmHg, respectively, on day 14, P = 0.9). There were no significant differences in pressor responsiveness to AngII between aged and aged-OVX mice. Vehicle-treated aged wild-type mice had a lower renal AT2R/AT1R balance as compared to adult counterparts. In response to AngII, the renal AT2R/AT1R balance in aged wild-type females was greater than that observed in vehicle-treated aged wild-type females and adult wild-type females, yet the protective effects of AT2R activation were not restored. CONCLUSIONS: The protective role of the AT2R depressor pathway is lost with age in female mice. Therefore, targeting deficits in AT2R expression and/or signaling may represent a novel anti-hypertensive approach in aged females.

Impaired ability to modulate glomerular filtration rate in aged female sheep following fetal uninephrectomy.

Fetal uninephrectomy (uni-x) results in hypertension at a later age in female than male sheep. We hypothesized that dysregulation of tubular sodium handling contributes to the reduced ability to regulate extracellular fluid (ECF) homeostasis in older females born with a congenital nephron deficit. Following renal excretory balance studies, the response to inhibition of the Na(+)K(+)2Cl(-) cotransporter with furosemide (0.5 mg/kg bolus + 1 mg/kg per hour, i.v) or vehicle treatment was examined in conscious 5-year-old female uni-x (n = 7) and sham (n = 7) sheep. Balance studies in meal-fed sheep demonstrated that while average 24 h sodium excretion over 6 days was not different between the groups, the daily variation in sodium excretion was significantly greater in uni-x compared to sham sheep (31 ± 4% vs. 12 ± 2%; P < 0.001). Basal plasma renin activity (PRA) and renal cortical cyclooxygenase-2 (COX-2) gene expression were lower in uni-x sheep (both, P < 0.01). The increases in glomerular filtration rate (GFR) and renal blood flow observed in sham sheep in response to furosemide were significantly attenuated in uni-x sheep (both P GROUP×TREAT < 0.05). However, fractional sodium excretion increased by a greater extent in the uni-x (4.4 ± 1.0%) as compared to the sham sheep (2.0 ± 0.4%; P GROUP×TIME < 0.05) in response to furosemide. In conclusion, fetal uni-x was associated with altered renal sodium handling and hypertension in aged females. The impaired ability to modulate PRA and GFR in the adults with a congenital nephron deficit may reduce the capacity of the kidney to respond to gains or losses in ECF to maintain a stable internal environment.

Role of inflammation and the angiotensin type 2 receptor in the regulation of arterial pressure during pregnancy in mice.

During normal pregnancy the renin-angiotensin system is activated, yet pregnant women are resistant to the pressor effects of angiotensin II. Our aim was to determine the role of the angiotensin type 2 receptor (AT2R) in the regulation of arterial pressure, natriuresis, and immune cell infiltration during pregnancy. Mean arterial pressure was measured via telemetry, and flow cytometry was used to enumerate immune cell infiltration in 14-week-old wild-type and AT2R knockout mice during gestation. In wild-type mice, mean arterial pressure decreased during gestation, reaching a nadir at gestational day 9 (-6±2 mm Hg) and returned to near preconception levels during late gestation. In AT2R-deficient mice, the midgestational decrease in mean arterial pressure was absent. Furthermore, mean arterial pressure was significantly increased during late gestation compared with wild-type mice (≈10 mm Hg). As expected, circulating immune cell activation was suppressed during pregnancy. However, this response was absent in AT2R-deficient mice. While renal immune cell infiltration was similar between the genotypes, there was a significant T cell phenotypic switch toward a proinflammatory T-helper 1 phenotype in AT2R-deficient mice. These data indicate that the AT2R plays an important role in arterial pressure regulation and may modulate T cell activation and renal cytokine production during pregnancy. Therefore, deficits in AT2R expression may contribute to pregnancy-induced hypertension and thus represents a potential therapeutic target.

Angiotensin type 2 receptor stimulation increases renal function in female, but not male, spontaneously hypertensive rats.

Accumulating evidence suggests that the protective pathways of the renin-angiotensin system are enhanced in women, including the angiotensin type 2 receptor (AT2R), which mediates vasodilatory and natriuretic effects. To provide insight into the sex-specific ability of pharmacological AT2R stimulation to modulate renal function in hypertension, we examined the influence of the AT2R agonist, compound 21 (100-300 ng/kg per minute), on renal function in 18- to 19-week-old anesthetized male and female spontaneously hypertensive rats. AT2R stimulation significantly increased renal blood flow in female hypertensive rats (PTreatment<0.001), without influencing arterial pressure. For example, at 300 ng/kg per minute of compound 21, renal blood flow increased by 14.3±1.8% from baseline. Furthermore, at 300 ng/kg per minute of compound 21, a significant increase in urinary sodium excretion was observed in female hypertensive rats (+180±59% from baseline; P<0.05 versus vehicle-treated rats). This was seen in the absence of any major change in glomerular filtration rate, indicating that the natriuretic effects of AT2R stimulation were likely the result of altered renal tubular function. Conversely, we did not observe any significant effect of AT2R stimulation on renal hemodynamic or excretory function in male hypertensive rats. Finally, gene expression studies confirmed greater renal AT2R expression in female than in male hypertensive rats. Taken together, acute AT2R stimulation enhanced renal vasodilatation and sodium excretion without concomitant alterations in glomerular filtration rate in female hypertensive rats. Chronic studies of AT2R agonist therapy on renal function and arterial pressure in hypertensive states are now required to establish the suitability of AT2R as a therapeutic target for cardiovascular disease, particularly in women.

Blunted sodium excretion in response to a saline load in 5 year old female sheep following fetal uninephrectomy.

Previously, we have shown that fetal uninephrectomy (uni-x) causes hypertension in female sheep by 2 years of age. Whilst the hypertension was not exacerbated by 5 years of age, these uni-x sheep had greater reductions in renal blood flow (RBF). To further explore these early indications of a decline in renal function, we investigated the renal response to a saline load (25 ml/kg/40 min) in 5-year old female uni-x and sham sheep. Basal mean arterial pressure was ∼15 mmHg greater (P(Group)<0.001), and sodium excretion (∼50%), glomerular filtration rate (∼30%, GFR) and RBF (∼40%) were all significantly lower (P(Group)<0.01) in uni-x compared to sham animals. In response to saline loading, sodium excretion increased significantly in both groups (P(Time)<0.001), however this response was blunted in uni-x sheep (P(GroupxTime)<0.01). This was accompanied with an attenuated increase in GFR and fractional sodium excretion (both P(GroupxTime)<0.05), and reduced activation of the renin-angiotensin system (both P<0.05), as compared to the sham group. The reduction in sodium excretion was associated with up-regulations in the renal gene expression of NHE3 and Na(+)/K(+) ATPase α and ß subunits in the kidney cortex of the uni-x compared to the sham animals (P<0.05). Notably, neither group completely excreted the saline load within the recovery period, but the uni-x retained a higher percentage of the total volume (uni-x: 48±7%; sham: 22±9%, P<0.05). In conclusion, a reduced ability to efficiently regulate extracellular fluid homeostasis is evident in female sheep at 5 years of age, which was exacerbated in animals born with a congenital nephron deficit. Whilst there was no overt exacerbation of hypertension and renal insufficiency with age in the uni-x sheep, these animals may be more vulnerable to secondary renal insults.

Sex differences in the pressor and tubuloglomerular feedback response to angiotensin II.

Awareness of sex differences in the pathology of cardiovascular disease is increasing. Previously, we have shown a role for the angiotensin type 2 receptor (AT(2)R) in the sex differences in the arterial pressure response to Ang II. Tubuloglomerular feedback (TGF) contributes in setting pressure-natriuresis properties, and its responsiveness is closely coupled to renal Ang II levels. We hypothesize that, in females, the attenuated pressor response to Ang II is mediated via an enhanced AT(2)R mechanism that, in part, offsets Ang II-induced sensitization of the TGF mechanism. Mean arterial pressure was measured via telemetry in male and female wild-type (WT) and AT(2)R knockout (AT(2)R-KO) mice receiving Ang II (600 ng/kg per minute SC). Basal 24-hour mean arterial pressure did not differ among the 4 groups. After 10 days of Ang II infusion, mean arterial pressure increased in the male WT (28±6 mm Hg), male AT(2)R-KO (26±2 mm Hg), and female AT(2)R-KO (26±4 mm Hg) mice, however, the response was attenuated in female WT mice (12±4 mm Hg; P between sex and genotype=0.016). TGF characteristics were determined before and during acute subpressor Ang II infusion (100 ng/kg per minute IV). Basal TGF responses did not differ between groups. The expected increase in maximal change in stop-flow pressure and enhancement of TGF sensitivity in response to Ang II was observed in the male WT, male AT(2)R-KO, and female AT(2)R-KO but not in the female WT mice (P between sex and genotype <0.05; both). In conclusion, these data indicate that an enhanced AT(2)R-mediated pathway counterbalances the hypertensive effects of Ang II and attenuates the Ang II-dependent resetting of TGF activity in females. Thus, the enhancement of the AT(2)R may, in part, underlie the protection that premenopausal women demonstrate against cardiovascular disease.

Sex-specific influence of angiotensin type 2 receptor stimulation on renal function: a novel therapeutic target for hypertension.

The renin-angiotensin system is a powerful regulator of arterial pressure and body fluid volume. Increasing evidence suggests that the angiotensin type 2 receptor (AT(2)R), which mediates the vasodilatory and natriuretic actions of angiotensin peptides, is enhanced in females and may, therefore, represent an innovative therapeutic target. We investigated the therapeutic potential of direct AT(2)R stimulation on renal function in 11- to 12-week-old anesthetized male and female Sprague-Dawley rats. Renal blood flow was examined in response to a graded infusion of the highly selective, nonpeptide AT(2)R agonist, compound 21 (100, 200, and 300 ng/kg per minute), in the presence and absence of AT(2)R blockade (PD123319; 1 mg/kg per hour). Direct AT(2)R stimulation significantly increased renal blood flow in both males and females, without influencing arterial pressure. This was dose dependent in females only and occurred to a greater extent in females at the highest dose of compound 21 administered (males: 13.1±2.4% versus females: 23.0±3.2% change in renal blood flow at 300 ng/kg per minute versus baseline; P<0.01). In addition, AT(2)R stimulation significantly increased sodium and water excretion to a similar extent in males and females (P(Group)=0.05 and 0.005). However, there was no significant change in glomerular filtration rate in either sex, suggesting that altered tubular function may be responsible for AT(2)R-induced natriuresis rather than hemodynamic effects. Taken together, this study provides evidence that direct AT(2)R stimulation produces vasodilatory and natriuretic effects in the male and female kidney. The AT(2)R may, therefore, represent a valuable therapeutic target for the treatment of renal and cardiovascular diseases in both men and women.