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Cardiac surgery-associated acute kidney injury (CSA-AKI) is a high-risk complication with well-recognized increased morbidity and mortality after cardiac surgery attributable in large part to cardiopulmonary bypass (CPB)-associated factors contributing to AKI including hemodilution, hypothermia, hypotension, and exposure to artificial surfaces. These conditions disrupt the renal microcirculation and activate local and systemic inflammatory responses to nonpulsatile flow and low perfusion pressure. The underlying mechanisms of CSA-AKI in CPB are not fully understood, and the incidence of CSA-AKI remains high at around 30%. Furthermore, women appear to be more vulnerable than men to the renal injury associated with CPB even though the overall incidence of cardiovascular and kidney diseases is lower in premenopausal women. Nevertheless, estrogen elicits renoprotective effects in several ways including mitigating inflammation, promoting natriuresis, and endothelial protection as shown in preclinical studies. However, women have higher rates of CSA-AKI and these are exacerbated in postmenopausal women. This leads to the conundrum of whether sex, age, and hormonal status differences influence CSA-AKI. In this review, we briefly discuss the pathophysiology of CSA-AKI in CPB and sex differences in kidney functions with a focus on the possible role of estrogen-specific effects in CPB and also possible differences in CPB in women including greater hemodilution. Furthermore, we review strategies to prevent CSA-AKI in CPB with a highlight for potential sex-specific strategies. Improving our understanding of the impact of sex and sex hormones on CSA-AKI initiation and development will allow us to better manage the CPB strategies delivered to all patients. [ABSTRACT FROM AUTHOR]

Introduction: Mechanical stresses and strains exerted on the glomerular cells have emerged as potentially influential factors in the progression of glomerular disease. Renal autoregulation, the feedback process by which the afferent arteriole changes in diameter in response to changes in blood pressure, is assumed to control glomerular mechanical stresses exerted on the glomerular capillaries. However, it is unclear how the two major mechanisms of renal autoregulation, the afferent arteriole myogenic mechanism and tubuloglomerular feedback (TGF), each contribute to the maintenance of glomerular mechanical homeostasis. Methods: In this study, we made a mathematical model of renal autoregulation and combined this model with an anatomically accurate model of glomerular blood flow and filtration, developed previously by us. We parameterized the renal autoregulation model based on data from previous literature, and we found evidence for an increased myogenic mechanism sensitivity when TGF is operant, as has been reported previously. We examined the mechanical effects of each autoregulatory mechanism (the myogenic, TGF and modified myogenic) by simulating blood flow through the glomerular capillary network with and without each mechanism operant. Results: Our model results indicate that the myogenic mechanism plays a central role in maintaining glomerular mechanical homeostasis, by providing the most protection to the glomerular capillaries. However, at higher perfusion pressures, the modulation of the myogenic mechanism sensitivity by TGF is crucial for the maintenance of glomerular mechanical homeostasis. Overall, a loss of renal autoregulation increases mechanical strain by up to twofold in the capillaries branching off the afferent arteriole. This further corroborates our previous simulation studies, that have identified glomerular capillaries nearest to the afferent arteriole as the most prone to mechanical injury in cases of disturbed glomerular hemodynamics. Discussion: Renal autoregulation is a complex process by whichmultiple feedback mechanisms interact to control blood flow and filtration in the glomerulus. Importantly, our study indicates that another function of renal autoregulation is control of themechanical stresses on the glomerular cells, which indicates that loss or inhibition of renal autoregulation may have a mechanical effect that may contribute to glomerular injury in diseases such as hypertension or diabetes. This study highlights the utility of mathematical models in integrating data from previous experimental studies, estimating variables that are difficult to measure experimentally (i.e. mechanical stresses in microvascular networks) and testing hypotheses that are historically difficult or impossible to measure. [ABSTRACT FROM AUTHOR]

Renin was discovered more than a century ago. Since then, the functions of the renin–angiotensin system in the kidney have been the focus of intensive research revealing its importance in regulation of renal physiology and in the pathogenesis of heart, vascular, and kidney diseases. Inhibitors of renin–angiotensin system components are now foundational therapies for a range of kidney and cardiovascular diseases from hypertension to heart failure to diabetic nephropathy. Despite years of voluminous research, emerging studies continue to reveal new complexities of the regulation of the renin–angiotensin system within the kidney and identification of nonclassical components of the system like the prorenin receptor (PRR) and ACE2 (angiotensin-converting enzyme 2), with powerful renal effects that ultimately impact the broader cardiovascular system. With the emergence of a range of novel therapies for cardiovascular and kidney diseases, the importance of a detailed understanding of the renin–angiotensin system in the kidney will allow for the development of informed complementary approaches for combinations of treatments that will optimally promote health and longevity over the century ahead. [ABSTRACT FROM AUTHOR]

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been [...]

Angiotensinogen (AGT) expression in renal proximal tubular cells (RPTCs) and intrarenal tumor necrosis factor-[alpha] (TNF-[alpha]) levels are increased in hypertension and renal diseases However, the contribution of TNF-[alpha] to AGT expression in RPTCs has not been established. Therefore, the objective of the present study was to determine influence of TNF-[alpha] on AGT expression in RPTCs. Human kidney-2 (HK-2) cells, immortalized human RPTCs, were treated with several concentrations of TNF-[alpha] up to 24 h. AGT mRNA and protein expression were evaluated by RT-PCR and ELISA, respectively. Activation of nuclear factor-[kappa]B (NF-[kappa]B) by TNF-[alpha] was evaluated by Western blot analysis, immunocytochemistry, and electrophoretic mobility shift assay (EMSA). TNF-[alpha] suppressed AGT mRNA expression in a dose- and time-dependent manner. Maximum AGT mRNA reduction was caused by 40 ng/ml of TNF-[alpha] (0.52 [+ or -] 0.09, ratio to control, at 24 h) and at 24 h (0.66 [+ or -] 0.05, ratio to control, by 10 ng/ml TNF-[alpha]). TNF-[alpha] reduced AGT protein accumulation in the medium between 8 and 24 h (0.62 [+ or -] 0.13 by 40 ng/ml TNF-[alpha], ratio to control). TNF-[alpha] activated and induced translocalization of p50 and p65, which are NF-[kappa]B subunits. Elevated formation of p50/p65 and p50/p50 dimers by TNF-[alpha] were observed by EMSA and supershift assay. Gene silencing of p50, but not p65, attenuated the effect of TNF-[alpha] on reduction of AGT expression in RPTCs. These results indicate that TNF-[alpha] suppresses AGT expression through p50/p50 homodimer formation in human RPTCs, suggesting a possible counteracting mechanism that limits excessive intrarenal AGT production. nuclear factor-[kappa]B; human kidney-2 doi: 10.1152/ajpcell.00078.2010.

A deficiency in nitric oxide (NO) generation leads to salt-sensitive hypertension, but the role of increased superoxide ([O.sup.-.sub.2]) in such salt sensitivity has not been delineated. We examined the hypothesis that an enhancement in [O.sup.-.sub.2] activity induced by high-salt (HS) intake under deficient NO production contributes to the development of salt-sensitive hypertension. Endothelial NO synthase knockout (eNOS KO; total n = 64) and wild-type (WT; total n = 58) mice were given diets containing either normal (NS; 0.4%) or high-salt (HS; 4%) for 2 wk. During this period, mice were chronically treated with a [O.sup.-.sub.2] scavenger, tempol (400 rag/l), or an inhibitor of NADPH oxidase, apocynin (1 g/l), in drinking water or left untreated (n = 6-8 per group). Blood pressure was measured by radiotelemetry and 24-h urine samples were collected in metabolic cages. Basal mean arterial pressure (MAP) in eNOS KO was higher (125 [+ or -] 4 vs. 106 [+ or -] 3 mmHg) compared with WT. Feeding HS diet did not alter MAP in WT but increased it in eNOS KO to 166 [+ or -] 9 mmHg. Both tempol and apocynin treatment significantly attenuated the MAP response to HS in eNOS KO (134 [+ or -] 3 and 139 [+ or -] 4 mmHg, respectively). Basal urinary 8-isoprostane excretion rates ([U.Iso] V), a marker for endogenous [O.sup.-.sub.2] activity, were similar (2.8 [+ or -] 0.2 and 2.4 [+ or -] 0.3 ng/day) in both eNOS KO and WT mice. However, HS increased [U.sub.Iso] V more in eNOS KO than in WT (4.6 [+ or -] 0.3 vs. 3.8 [+ or -] 0.2 ng/day); these were significantly attenuated by both tempol and apocynin treatment. These data indicate that an enhancement in [O.sup.-.sub.2] activity contributes substantially to the development of salt-sensitive hypertension under NO-deficient conditions. superoxide anion; nitric oxide; kidney function; hypertension doi: 10.1152/ajprenal.00047.2010.

Adenosine is an important paracrine agent regulating renal vascular tone via adenosine [A.sup.1] and [A.sup.2] receptors. While A2B receptor message and protein have been localized to preglomerular vessels, functional evidence on the role of [A.sup.2]B receptors in mediating the vasodilator action of adenosine on afferent arterioles is not available. The present study determined the role of [A.sup.2]B receptors in mediating the afferent arteriolar dilation and compared the effects of [A.sup.2]B and [A.sup.2]A receptor blockade on afferent arterioles. We used the rat in vitro blood-perfused juxtamedullary nephron technique combined with videomicroscopy. Single afferent arterioles of Sprague-Dawley rats were visualized and superfused with solutions containing adenosine or adenosine [A.sup.2] receptor agonist (CV-1808) along with adenosine [A.sup.2]B and [A.sup.2]A receptor blockers. Adenosine (10 [micro]mol/1) caused modest constriction and subsequent superfusion with SCH-58261 (SCH), an [A.sup.2A] receptor blocker, at concentrations up 10 [micro]mol/1 elicited only slight additional decreases in afferent arteriolar diameter with maximum effect at a concentration of 1 [micro]mol/l (- 11.0 [+ or -] 2.5%, n = 6, P < 0.05). However, superfusion of adenosine-treated vessels with MRS-1754 (MRS), an [A.sup.2]B receptor blocker, elicited greater decreases in afferent arteriolar diameter (-26.0 [+ or -] 4.7%, n = 5, P < 0.01). SCH did not significantly augment the adenosine-mediated afferent constriction elicited by MRS; however, adding MRS after SCH caused further significant vasoconstriction. Superfusion with CV-1808 dilated afferent arterioles (17.2 [+ or -] 2.4%, n = 6, P < 0.01). This effect was markedly attenuated by MRS (-22.6 [+ or -] 2.0%, n = 5, P < 0.01) but only slightly reduced by SCH (-9.0 [+ or -] 1.1%, n = 5, P < 0.05) and completely prevented by adding MRS after SCH (-24.7 [+ or -] 1.8%, n = 5, P < 0.01). These results indicate that, while both [A.sup.2A] and [A.sup.2B] receptors are functionally expressed in juxtamedullary afferent arterioles, the powerful vasodilating action of adenosine predominantly involves [A.sup.2B] receptor activation, which counteracts [A.sup.1] receptor-mediated vasoconstriction. adenosine receptor antagonists; renal microcirculation; vascular biology; afferent arterioles; kidney doi: 10.1152/ajprenal.00149.2010.

Am J Physiol Renal Physiol 298: F150-F157, 2010. First published October 21, 2009; doi:10.1152/ajprenal.00477.2009.--Angiotensin-converting enzyme (ACE) inhibition (ACEi) ameliorates the development of hypertension and the intrarenal ANG II augmentation in ANG II-infused mice. To determine if these effects are associated with changes in the mouse intrarenal renin-angiotensin system, the expression of angiotensinogen (AGT), renin, ACE, angiotensin type 1 receptor ([AT.sub.1]R) mRNA (by quanitative RT-PCR) and protein [by Western blot (WB) and/or immunohistochemistry (IHC)] were analyzed. C57BL/6J male mice (9-12 wk old) were distributed as controls (n = 10), ANG II infused (ANG II = 8,400 ng x [kg.sup.-1] x [min.sup.-1] for 12 days), ACEi only (ACEi = 10, lisinopril, 100 mg/1), and ANG II infused + ACEi (ANG II + ACEi = 11). When compared with controls (1.00), AGT protein (by WB) was increased by ANG II (1.29 [+ or -] 0.13, P < 0.05), and this was not prevented by ACEi (ACEi + ANG II, 1.31 [+ or -] 0.14, P < 0.05). ACE protein (by WB) was increased by ANG II (1.21 [+ or -] 0.08, P < 0.05), and it was reduced by ACEi alone (0.88 [+ or -] 0.07, P < 0.05) or in combination with ANG II (0.80 [+ or -] 0.07, P < 0.05). [AT.sub.1]R protein (by WB) was increased by ANG II (1.27 [+ or -] 0.06, P < 0.05) and ACEi (1.17 [+ or -] 0.06, P < 0.05) but not ANG II + ACEi [1.15 [+ or -] 0.06, not significant (NS)]. Tubular renin protein (semiquantified by IHC) was increased by ANG II (1.49 [+ or -] 0.23, P < 0.05) and ACEi (1.57 [+ or -] 0.15, P < 0.05), but not ANG II + ACEi (1.10 [+ or -] 0.15, NS). No significant changes were observed in AGT, ACE, or [AT.sub.1]R mRNA. In summary, reduced responses of intrarenal tubular renin, ACE, and the [AT.sub.1]R protein to the stimulatory effects of chronic ANG II infusions, in the presence of ACEi, are associated with the effects of this treatment to ameliorate augmentations in blood pressure and intrarenal ANG II content during ANG II-induced hypertension. angiotensin-converting enzyme; angiotensinogen; renin; lisinopril

Am J Physiol Renal Physiol 298: F103-F108, 2010. First published November 11, 2009; doi:10.1152/ajprenal.00479.2009.--Atrial natriuretic peptide (ANP) elicits natriuresis; however, the relative contributions of proximal and distal nephron segments to the overall ANP-induced natriuresis have remained uncertain. This study was performed to characterize the effects of ANP on distal nephron sodium reabsorption determined after blockade of the two major distal nephron sodium transporters with amiloride (5 [micro]g/g body wt) plus bendroflumethiazide (12 [micro]g/g body wt) in male anesthetized C57/BL6 and natriuretic peptide receptor-A gene (Npr1) targeted four-copy mice. The lower dose of ANP (0.1 ng x g body [wt.sup.-1] x [min.sup.-1], n = 6) increased distal sodium delivery (DSD, 2.4 [+ or -] 0.4 vs. 1.6 [+ or -] 0.2 [micro]eq/min, P < 0.05) but did not change fractional reabsorption of DSD compared with control (86.3 [+ or -] 2.0 vs. 83.9 [+ or -] 3.6%, P > 0.05), thus limiting the magnitude of the natriuresis. In contrast, the higher dose (0.2 ng x g body [wt.sup.-1] x min-1, n = 6) increased DSD (2.8 [+ or -] 0.3 [micro]eq/min, P < 0.01) and also decreased fractional reabsorption of DSD (67.4 [+ or -] 4.5%, P < 0.01), which markedly augmented the natriuresis. In Npr1 gene-duplicated four-copy mice (n = 6), the lower dose of ANP increased urinary sodium excretion (0.6 [+ or -] 0.1 vs. 0.3 [+ or -] 0.1 [micro]eq/min, P < 0.05) and decreased fractional reabsorption of DSD compared with control (72.2 [+ or -] 3.4%, P < 0.05) at similar mean arterial pressures (91 [+ or -] 6 vs. 92 [+ or -] 3 mmHg, P > 0.05). These results provide in vivo evidence that ANP-mediated increases in DSD alone exert modest effects on sodium excretion and that inhibition of fractional reabsorption of distal sodium delivery is requisite for the augmented natriuresis in response to the higher dose of ANP or in Npr1 gene-duplicated mice. atrial natriuretic peptide; renal sodium reabsorption; distal nephron segments; amiloride; bendroflumethiazide

In angiotensin II (ANG II)-induced hypertension, intrarenal ANG II levels are increased by [AT.sub.1] receptor-mediated ANG II internalization and endogenous ANG II generation. The objective of the present study was to determine the relative contribution of de novo formation of endogenous ANG II. Male Sprague-Dawley rats were divided into three groups: sham operated (n = 6), [Val.sup.5]-ANG II infused (n = 16), and [Ile.sup.5]-ANG II infused (n = 6). [Val.sup.5]-ANG II and [Ile.sup.5]-ANG II were infused at 80 ng/min via subcutaneous osmotic minipump for 13 days, followed by harvesting of blood and kidney samples. In six [Val.sup.5]-ANG II-infused rats, urine was collected on the day before infusion and on day 12 of infusion. Extracted samples were subjected to HPLC to separate [Val.sup.5]-ANG II from [Ile.sup.5]-ANG II followed by RIA. Systolic blood pressure increased significantly from 121 [+ or -] 2 to 206 [+ or -] 4 mmHg in the [Val.sup.5]-ANG II-infused rats and from 124 [+ or -] 3 to 215 [+ or -] 5 mmHg in the [Ile.sup.5]-ANG II-infused rats. In the [Val.sup.5]-ANG II-infused rats, the plasma [Ile.sup.5]-ANG II levels increased 196.2 [+ or -] 70.1% compared with sham plasma [Ile.sup.5]-ANG II concentration. [Val.sup.5]-ANG II levels were 150.0 [+ or -] 28.2 fmol/ml which accounted for 53.5 [+ or -] 10.1% of the total ANG II in plasma. The kidney [Ile.sup.5]-ANG II levels in the [Val.sup.5]-ANG II-infused rats increased 69.9 [+ or -] 30.7% compared with sham kidney [Ile.sup.5]-ANG II concentrations. Intrarenal accumulation of [Val.sup.5]-ANG II accounted for 52.5 [+ or -] 5.3% of the total kidney ANG II during [Val.sup.5]-ANG II infusion while endogenous [Ile.sup.5]-ANG II accounted for 47.5 [+ or -] 8.6%. The urinary [Ile.sup.5]-ANG II excretion rate on day 12 increased 93.2 [+ or -] 32.1% compared with preinfusion level indicating increased formation of endogenous ANG II. Thus, the increases in intrarenal ANG II levels during chronic ANG II infusions involve substantial stimulation of endogenous ANG II formation which contributes to overall augmentation of intrarenal ANG II. angiotensin II-induced hypertension; renin-angiotensin system; high-performance liquid chromatography

Peroxynitrite ([ONOO.sup.-]) is formed endogenously by the reaction of nitric oxide (NO) and superoxide ([O.sub.2.sup.-]). To examine the hypothesis that [OONO.sup.-] cause renal vasodilation at low concentrations but cause vasoconstriction at higher concentrations, we examined renal responses to intraarterial infusion of incremental doses of [OONO.sup.-] (10, 20, and 40 [micro]g x [kg.sup.-1] x [min.sup.-1]; 45 min each) in anesthetized rats. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearance. In control rats (n = 6), low dose (10 [micro]g x [kg.sup.-1] x [min.sup.-1]) of [OONO.sup.-] increased RBF by 10 [+ or -] 3% and GFR by 15 [+ or -] 5%. The higher doses (20 and 40 [micro]g x [kg.sup.-1] x [min.sup.-1]) mostly reversed these responses which were -7 [+ or -] 4 and -27 [+ or -] 7% (P < 0.05) in RBF and -0.1 [+ or -] 4.8 and -14 [+ or -] 12% in GFR, respectively. There were no appreciable changes in urine flow (V) and sodium excretion ([U.sub.Na]V) during [OONO.sup.-] infusion. However, in rats pretreated with NO synthase (NOS) inhibitor, L-NAME (50 [micro]g x [kg.sup.-1] x [min.sup.-1]; n = 5), these doses of [OONO.sup.-] significantly reduced RBF (-26 [+ or -] 7, -27 [+ or -] 6, and -44 [+ or -] 3%) and GFR (-21 [+ or -] 6, -25 [+ or -] 8, and -32 [+ or -] 12%) with variable increases in V or [U.sub.Na]V. Long-term infusion of [OONO.sup.-] (10 [micro]g x [kg.sup.-1] x [min.sup.-1] for 75 min) in another set of control rats (n = 5) also showed similar vasodilator and hyperfiltration responses. These data indicate that [OONO.sup.-] acts as an oxidant at high concentration but provides renoprotective function at low concentration that depends on intact NOS activity. nitric oxide; superoxide; oxidative stress; renal blood flow; glomerular filtration rate

Heme oxygenases (HO-1, HO-2) catalyze conversion of heme to iron, carbon monoxide (CO), and biliverdin/bilirubin. We studied the effects of renal HO-1 induction on afferent arteriole (Aft-Art) autoregulatory responses to increases in renal perfusion pressure (RPP). Rats were treated with hemin and Sn[Cl.sub.2] to induce HO-1, and Aft-Art autoregulatory responses were evaluated using the rat blood-perfused juxtamedullary nephron preparation. Renal HO-1 expression was significantly increased in hemin- and Sn[Cl.sub.2]-treated rats, while HO-2 was not altered. Aft-Art autoregulatory constrictor responses to increases in RPP from 100 to 150 mmHg were attenuated in hemin- and Sn[Cl.sub.2]-treated rats compared with control rats (+ 1.1 [+ or -] 3.3, n = 9 and +4.4 [+ or -] 5.3, n = 9 vs. -14.2 [+ or -] 1.5%, n = 10, respectively) (P < 0.05). Acute HO inhibition with chromium mesoporphyrin (CrMP; 15 [micro]mol/1) restored Aft-Art autoregulatory responses in hemin- and Sn[Cl.sub.2]-treated rats. Superfusing Aff-Arts from control rats with 100 [micro]mol/1 biliverdin did not alter autoregulatory responses; however, superfusion with 1 mmol/l CO significantly attenuated autoregulatory responses to increases in RPP from 100 to 150 mmHg (+3.3 [+ or -] 5.4 vs. -16.6 [+ or -] 3.8%, n = 6) (P < 0.05). Acute soluble guanylate cyclase inhibition with 10 [micro]mol/l ODQ restored Aft-Art autoregulatory responses in hemin-treated rats. Immunohistochemistry shows HO-2 to be expressed mainly in epithelial cells with weak staining in proximal tubules, interlobular arteries, and Aft-Arts. In hemin- and Sn[Cl.sub.2]-treated rats, HO-I was induced in tubular epithelial cells but not interlobular arteries and Aft-Arts. We conclude that induction of renal HO- I attenuates Aft-Art constrictor responses to increases in RPP via increasing CO production from tubular epithelial cells, suggesting that an augmented HO system in pathophysiological conditions modulates renal autoregulation. hemin; carbon monoxide; biliverdin; bilirubin; autoregulation; kidney; tin chloride; soluble guanylate cyclase

Augmented intrarenal ANG II stimulates 1L-6, which contributes to renal injury. The expression of intrarenal angiotensinogen (AGT) is enhanced by increased intrarenal ANG II in human renin/human AGT double transgenic mice. ANG II also augments AGT expression in hepatocytes and cardiac myocytes. However, the mechanisms underlying AGT augmentation by ANG II and the contribution of IL-6 to this system are poorly understood. This study was performed in human renal proximal tubular epithelial cells (HRPTECs) to test the hypothesis that IL-6 contributes to the upregulation of AGT expression by ANG II. Human kidney-2 (HK-2) cells, immortalized HRPTECs, were incubated with [10.sup.-7] M ANG II and/or 10 ng/ml IL-6 for up to 24 h. AGT mRNA and protein expressions were measured by real-time RT-PCR and ELISA, respectively. The activities of NF-[kappa]B and STAT3 were evaluated by Western blotting and EMSA. Stimulation with either ANG II or IL-6 did not significantly alter AGT mRNA or protein expression. In contrast, costimulation with ANG II and IL-6 significantly increased AGT mRNA and protein expressions (1.26 [+ or -] 0.10 and 1.16 [+ or -] 0.13 over control, respectively). Olmesartan, an ANG II type 1 receptor blocker, and an IL-6 receptor antibody individually inhibited this synergistic effect. NF-[kappa]B was also activated by costimulation with ANG II and 1L-6. Phosphorylation and activity of STAT3 were increased by stimulation with IL-6 alone and by costimulation. The present study indicates that IL-6 plays an important role in ANG II-mediated augmentation of AGT expression in human renal proximal tubular cells. angiotensinogen; kidney; renin-angiotensin system; cytokines

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 [+ or -] 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 [+ or -] 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 [+ or -] 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 [+ or -] 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 [+ or -] 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 [+ or -] 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 [+ or -] 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 [+ or -] 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases. enzyme-linked immunosorbent assay; renin-angiotensin system; plasma; urine; mouse; rat

Chronic ANG II infusions lead to increases in intrarenal ANG II levels, hypertension, and tissue injury. Increased blood pressure also elicits increases in renal interstitial fluid (RIF) ATP concentrations that stimulate cell proliferation. We evaluated the contribution of purinergic receptor activation to ANG II-induced renal injury in rats by treating with clopidogrel, a P2Y12 receptor blocker, or with PPADS, a nonselective P2 receptor blocker, [alpha]-Actin expression in mesangial cells, afferent arteriolar wall thickness (AAWT), cortical cell proliferation, and macrophage infiltration were used as early markers of renal injury. Clopidogrel and PPADS did not alter blood pressure, renin or kidney ANG II content. [alpha]-Actin expression increased from control of 0.6 [+ or -] 0.4% of mesangial area to 6.3 [+ or -] 1.9% in ANG II-infused rats and this response was prevented by clopidogrel (0.4 [+ or -] 0.2%) and PPADS. The increase in AAWT from 4.7 [+ or -] 0.1 to 6.0 [+ or -] 0.1 mm in ANG II rats was also prevented by clopidogrel (4.8 [+ or -] 0.1 mm) and PPADS. ANG II infusion led to interstitial macrophage infiltration (105 [+ or -] 16 vs. 62 [+ or -] 4 cell/[mm.sup.2]) and tubular proliferation (71 [+ or -] 15 vs. 20 [+ or -] 4 cell/[mm.sup.2]) and these effects were prevented by clopidogrel (52 [+ or -] 4 and 36 [+ or -] 3 cell/[mm.sup.2]) and PPADS. RIF ATP levels were higher in ANG II-infused rats than in control rats (11.8 [+ or -] 1.9 vs. 5.6 [+ or -] 0.6 nmol/l, P < 0.05). The results suggest that activation of vascular and glomerular purinergic P2 receptors may contribute to the mesangial cell transformarion, renal inflammation, and vascular hypertrophy observed in ANG II-dependent hypertension. extracellular ATP; renal inflammation; vascular hypertrophy

This study was performed in transgenic mice to test the hypothesis that the selective intrarenal overproduction of ANG II increases intrarenal mouse (m) angiotensinogen (AGT) expression. We used the following three groups: 1) single transgenic mice (group A, n = 14) expressing human (h) AGT only in the kidney, 2) double-transgenic mice (group D, n = 13) expressing human renin systemically in addition to hAGT only in the kidney, and 3) wild-type (group W, n = 12) mice. Exogenous hAGT protein is inactive in group A because endogenous mouse renin cannot cleave hAGT to ANG I because of a high species specificity. All mice were monitored from 12 to 18 wk of age. Systolic blood pressure progressively increased from 116 [+ or -] 5 mmHg (12 wk) to 140 [+ or -] 7 (18 wk) in group D. This increase was not observed in groups A or W. Intrarenal hAGT levels were similar in groups A and D; however, hAGT was not detectable in kidneys of group W. Kidney ANG II levels were increased in group D (216 [+ or -] 43 fmol/g) compared with groups A (117 [+ or -] 16) and W (118 [+ or -] 17). However, plasma ANG II concentrations were similar among the three groups. Endogenous renal mAGT mRNA was increased significantly in group D (1.46 [+ or -] 0.19, ratio) compared with groupsA (0.97 [+ or -]0.12) and W (1.00 [+ or -] 0.08). Endogenous renal mAGT protein was also significantly increased in group D compared with groups A and W. Interstitial collagen-positive area, interstitial macrophage/monocyte infiltration, and afferent arteriolar wall thickness were increased significantly in group D compared with groups A and W. These data indicate for the first time that the selective stimulation of intrarenal production of ANG II from hAGT augments endogenous intrarenal mAGT mRNA and protein expression. hypertension; transgenic mouse; angiotensin II; renal injury

We and others have previously shown that high levels of ANG II are accumulated in the rat kidney via a type 1 (A[T.sub.1]) receptor-mediated mechanism, but it is not known which A[T.sub.1] receptor is involved in this process in rodents. We tested the hypothesis that A[T.sub.1a] receptor-deficient mice (Agtr1a-/-) are unable to accumulate ANG II intracellularly in the kidney because of the absence of A[T.sub.1a] receptor-mediated endocytosis. Adult male wildtype (Agtr1a+/+), heterozygous (Agtr1a+/-), and Agtr1a-/- were treated with vehicle, ANG II (40 ng/min ip via osmotic minipump), or ANG II plus the A[T.sub.1] antagonist losartan (10 mg*[kg.sup.-1]* [day.sup.-1] po) for 2 wk. In wild-type mice, ANG II induced hypertension (168 [+ or -] 4 vs. 113 [+ or -] 3 mmHg, P < 0.001), increased kidney-to-body weight ratio (P < 0.01), caused pressure natriuresis (P < 0.05), and elevated plasma and whole kidney ANG II levels (P < 0.001). Concurrent administration of ANG II with losartan attenuated these responses to ANG II. In contrast, Agtr1a-/- mice had lower basal systolic pressures (P < 0.001), smaller kidneys with much fewer A[T.sub.1b] receptors (P < 0.001), higher basal 24-h urinary sodium excretion (P < 0.01), as well as basal plasma and whole kidney ANG II levels (P < 0.01). However, intracellular ANG II levels in the kidney were lower in Agtr1a-/- mice. In Agtr1a-/- mice, ANG II slightly increased systolic pressure (P < 0.05) but had no effect on the kidney weight, urinary sodium excretion, and whole kidney ANG II levels. Losartan restored systolic pressure to basal levels and decreased whole kidney ANG II levels by -20% (P < 0.05). These results demonstrate a predominant role of A[T.sub.1a] receptors in blood pressure regulation and in the renal responses to long-term ANG II administration, that A[T.sub.1b] receptors may play a limited role in blood pressure control and mediating intrarenal ANG II accumulation in the absence of A[T.sub.1a] receptors. kidney; losartan; receptor endocytosis; urinary sodium excretion

Effects of aldosterone receptor (AR) blockade with eplerenone (epl) on renal [Na.sup.+] excretion, arterial blood pressure, intra-adrenal and renal ANG II, and plasma aldosterone levels during ANG II-dependent hypertension were evaluated. Rats from one cohort (n = 10/group) 1) control, 2) control + epl (25 mg/day), 3) ANG II (60 ng/min), and 4) ANG II + epl were maintained in metabolic cages for 28 days for daily urine collections. Systolic blood pressure (SBP) was measured weekly by tail-cuff. In a second cohort (n = 12/group), daily SBP was measured by telemetry (n = 6 rats/group) 1) control, 2) ANG II, and 3) ANG II + epl. A diet containing epl (0.1%) was provided after 1 wk of ANG II infusion. Direct monitoring of BP by telemetry showed that epl delayed the onset of the increase in SBP by 2 days and slightly reduced SBP (186 [+ or -] 6 vs. 177 [+ or -] 8 mmHg). Epl transiently increased [Na.sup.+] excretion within 24 h of treatment in both normo- and hypertensive rats; however, balance was reestablished within 5 days suggesting that alternative mechanisms for conserving [Na.sup.+] are activated. Cortical [alpha]-epithelial [Na.sup.+] channel content ([alpha]-ENaC) was not altered after 21 days of epl treatment. Epl exacerbated the ANG II-mediated increases in intrarenal ANG II (226 [+ or -] 16 vs. 365 [+ or -] 38 fmol/g) and further increased intra-adrenal ANG II (3.9 [+ or -] 0.3 vs. 8.2 [+ or -] 0.9 fmol/mg) and aldosterone (255 [+ or -] 55 vs. 710 [+ or -] 87 pmol/mg) content. Exacerbation of intrarenal ANG II levels likely contributes to the maintenance of [alpha]-ENaC protein content and thus [Na.sup.+] reabsorption, which helps explain the ineffectiveness of AR blockade in reducing SBP in ANG II-infused models of hypertension. eplerenone; mineralocorticoids; sodium; spironolactone doi:10.1152/ajprenal.00504.2006

Chronic angiotensin (ANG) II infusions into rats lead to augmented intrarenal levels of ANG II and inflammatory factors, impaired renal function, and progressive hypertension. Residual effects persist after cessation of ANG II infusions, as manifested by a hypertensive response to high-salt intake. This study was performed to determine the residual cytokines and chemokines following the cessation of ANG II infusion. Male Sprague-Dawley rats, maintained on a normal diet, received either a sham operation or continuous ANG II infusion (120 ng/min) subcutaneously via minipumps. The ANG H-infused rats were further subdivided into three subgroups. Minipumps were removed on day 12 with subsequent harvesting of kidneys at 0, 3, and 6 days after cessation of ANG II infusion. After 12 days of ANG II infusion, systolic blood pressure, interstitial fibrosis, preglomerular hypertrophy, and interstitial macrophage infiltration were significantly enhanced compared with the shams. By 3 days following the cessation of ANG II infusion, systolic blood pressure was normalized; however, interstitial fibrosis and preglomerular hypertrophy were still present. Furthermore, increased interstitial macrophage infiltration was still present 6 days after cessation of ANG II infusion. Importantly, augmented mRNA levels of monocyte chemotactic protein (MCP)-1 (1.55 [+ or -] 0.15 vs. 1.00 [+ or -] 0.13, relative ratio) and transforming growth factor (TGF)-[[beta].sub.1] (1.52 [+ or -] 0.16 vs. 1.00 [+ or -] 0.08) persisted 6 days after the withdrawal of ANG II infusion (1.60 [+ or -] 0.20 for MCP-1 and 1.43 [+ or -] 0.17 for TGF-[[beta].sub.1]). Thus, the ANG II-induced activation of MCP-1 and TGF-[[beta].sub.1] is sustained and may account for the persistent effect of chronic ANG II infusions on interstitial macrophage infiltration, suggesting a possible mechanism for the development of salt sensitivity in ANG II-dependent hypertension. kidney; tissue injury; cytokines; chemokines; residual effects

Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 [micro]mol/1 CO solution, or 15 [mmicro]mol/1 chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/1 [N.[omega]]-nitro-L-arginine (L-NNA) to inhibit NO synthase (NOS) or 10 [micro]mol/1 1H-[1,2,4]oxadia-zolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 [micro]mol/1 CORM-2 or with CO (10 [micro]mol/1) significantly dilated AAs (22.0 [+ or -] 0.9 and 22.8 [+ or -] 0.9 vs. 18.3 [+ or -] 0.9 [micro]m, n = 5, P < 0.05; and 26.0 [+ or -] 1.4 vs. 18.8 [+ or -] 0.7 [micro]m, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 [+ or -] 0.7 vs. 17.2 [+ or -] 0.6 [micro]m, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 [+ or -] 1.2 [micro]m, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 [+ or -] 1.6 [micro]m, n = 6, P < 0.05). CORM-2 attenuated the L-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 [+ or -] 0.4 vs. 18.8 [+ or -] 0.4 [micro]m, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 [+ or -] 0.7 [micro]m, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction. heme oxygenase; renal circulation; soluble guanylate cyclase

Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG H is taken up by proximal tubule cells (PTCs) through A[T.sub.1] receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase[dependent mechanisms, and 3) A[T.sub.1] receptor-mediated endocytosis of ANG H has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [[sup.125]I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and 'colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the A[T.sub.1] receptor iblocker losartan (16.5 [+ or -] 4.6%, P < 0.01 vs. ANG II, 78.3 [+ or -] 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 [+ or -] 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by ~58% (basal, 229.8 [+ or -] 11.4 vs. ANG II, 361.3 [+ or -] 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < '0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 [+ or -] 25.5 pg ANG II/mg protein, not 'significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 [+ or -] 2.8 vs. ANG II, 9.1 [+ or -] 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 [+ or -] 8.6 vs. forskolin + ANG II, 42.8 [+ or -] 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through A[T.sub.1] receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling. kidney; receptor-mediated endocytosis doi:10.1152/ajprenal.00405.2005