Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Jul 10.
Published in final edited form as: Kidney Int. 2013 Jul 3;84(5):931–939. doi: 10.1038/ki.2013.193

Chronic AT2 receptor activation attenuates renal AT1 receptor function and blood pressure in obese Zucker rats

Quaisar Ali *, Yonnie Wu $, Tahir Hussain *
PMCID: PMC4091804  NIHMSID: NIHMS472472  PMID: 23823602

Abstract

Abnormal regulation of the renin angiotensin system such as enhanced renal AT1R function and reduced ACE2 activity contributes to obesity-related hypertension. Here we tested whether long-term AT2R activation affects renal function in obesity using lean and obese Zucker rats treated with the AT2R agonist CGP42112A for 2-weeks. This caused blood pressure to decrease by 13 mmHg which was associated with increased urinary sodium excretion in the obese rats. Cortical ACE2 expression and activity, the Mas receptor (MasR), and its ligand angiotensin-(1-7) were all increased in CGP-treated obese compared with control rats. Candesartan-induced natriuresis, a measure of AT1R function, was reduced but cortical AT1R expression and angiotensin II levels were similar in CGP-treated obese compared to control rats. Renin and AT2R expression in obese rats was not affected by CGP-treatment. In HK-2 cells in-vitro, CGP-treatment caused increased ACE2 activity and MasR levels but decreased AT1R levels and renin activity. Thus, long-term AT2R activation shifts the opposing arms of renin angiotensin system and contributes to natriuresis and blood pressure reduction in obese animals. Our study highlights the importance of AT2R as a target for treating obesity related hypertension.

Keywords: CGP42112A, obesity, RAS, renin. AT2 receptor

Introduction

Renin angiotensin system (RAS) is comprised of peptide hormones, whose production and biological activities are regulated by multiple enzymes and receptors. Renin is a critical regulator of RAS, it catalyzes the conversion of angiotensinogen into angiotensin I (Ang I), which is further converted to Ang II by angiotensin converting enzyme (ACE). ACE2, an isoform of the ACE, converts Ang I and Ang II to Ang-(1-9) and Ang-(1-7), respectively. Ang-(1-9) is further converted to Ang-(1-7) by ACE.1-3

Ang II is a pivotal hormone of RAS and works via activating angiotensin type 1 (AT1R) and type 2 (AT2R) receptors.3 Activation of AT1R promotes cell growth, induces vasoconstriction, antinatriuresis and blood pressure (BP) increase.3 Relative to the AT1R, AT2R is less studied,3 but has been shown to produce cellular and physiological responses that are opposite to those of the AT1R.4, 5 Thus, AT2R activation inhibits cell growth,6 promotes cell apoptosis7 and differentiation,8 contributes to natriuresis, 9-12 vaso-relaxation13 and potentially lowers BP.14-16 Ang-(1-7) is an important component of RAS and studies suggest that Ang-(1-7) causes vasodilatation and natriuresis by inhibiting Na-transport17-19 mainly via Mas receptors (MasR).20 However, there is evidence suggesting that Ang-(1-7) has affinity for AT1R and mediates a biphasic response on fluid absorption21 and inhibits cell growth in proximal tubules but induces mesangial cell growth.22, 23 Other non ACE2 enzymes such as neprilysin and prolyl-endopeptidase also catalyze the production of Ang-(1-7).24 Overall, ACE/Ang-II/AT1R and ACE2/Ang-(1-7)/MasR are considered as two arms of the RAS with opposite functions.

Abnormal renal Na excretion is believed to be a major risk factor in obesity-related hypertension.25 Hyperactivity of the RAS, mainly enhanced AT1R function is suggested to contribute to the excessive renal Na reabsorption and BP increase in obesity.26-28 Recently, we have demonstrated that the renal AT2R expression is increased in obese rats 9, 16, which inhibits proximal tubules Na-pump via NO/cGMP pathway and promotes natriuresis in these animals.12,29 We also reported that AT2R in the long-term may have a protective role against BP increase in obese rats.16 Additionally, there is evidence, including from our laboratory, suggesting that AT2R exerts anti-oxidative stress and anti-inflammatory activity.30 Both oxidative stress and inflammation, positively regulate each31, 32 other and have been implicated in the renal dysfunction and pathogenesis of hypertension via modulating RAS components such as increasing AT1R function.31

The RAS components apart from having independent effects also influence the function and generation of other RAS components and these altogether contribute to the net renal function and BP change. It has been reported that AT2R is implicated in attenuation of AT1R-mediated myocyte growth and chronotrophic effect33 and provides negative feedback on renin activity.34 There is evidence showing that Ang II via AT1R activation decreases ACE2 activity 35 and Ang-(1-7) production. Since evidence clearly suggests an enhanced renal AT1R function, which contributes to excessive renal Na-absorption and obesity-related hypertension,9,23,30 it is not known whether chronic AT2R activation attenuates renal AT1R function and enhances ACE2-Ang-(1-7) activity/expression in obesity. We hypothesize that AT2R activation attenuates AT1R-mediated antinatriuresis and increases cortical ACE2 activity/expression and Ang-(1-7) production in obese rat. We tested this hypothesis by treating obese rats with AT2R agonist for 2-weeks, followed by measuring renal AT1R function and the expression of ACE2-Ang-(1-7)-MasR. The changes in these RAS components were correlated with renal Na excretion and BP changes.

Results

General Parameter

Compared with lean, obese rats had greater body weight (lean 295±8 vs obese 473±12 gm) (Table 1) and consistently consumed more food (Fig 2A) and water (Fig 2B) over the treatment period. The CGP-treatment did not affect the food and water intake in either of the rat strains.

Table 1.

Parameters/Rat groups LCT LCGP OCT OCGP
MAP, mm Hg ND ND 106 ± 3 93 ± 3#
Heart rate, beats per min ND ND 392 ± 11 409 ± 9ns
Renal blood flow, ml/hr 3±0.3 4±0.4 7.3 ± 0.8* 9 ± 2ns
Kidney wt, gm 2.2±0.1 2.3±0.1 2.8±0.1* 2.7±0.1ns
Body wt, g 295±8 291±8 473±12* 461±11ns
Urinary Nitrates at day 2,
nmol/min		 0.1±0.06 0.1±0.04 0.4±0.11* 1±0.26#
Urinary Nitrates at day 14,
nmol/min		 0.07±0.01 0.08±0.01 0.58±0.3* 0.47±.03ns
Open in a new tab

ND: Not Determined

*

compared to LCT

#

compared to OCT (LCT-lean control, LCGP-lean CGP42112A-treated, OCT-obese control, OCGP-obese CGP42112A-treated), ns-not significant-compared to OCT.

Fig.2.

Fig.2

Fig.2

Fig.2

Fig.2

Fig.2

Open in a new tab

(A) Food (B) water intake (C) urinary volume (D) urinary sodium excretion (UNaV) and (E) glomerular filtration rate (GFR) measured using FITC-inulin clearance method in conscious control and CGP-treated lean and obese Zucker rats. *significantly different compared with lean control rats, #significantly different compared with obese control rats. Values are represented as mean±SEM; One way ANOVA followed by Neuman-Keuls test, p<0.05; N=5-7 in each group). (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

Hemodynamic and Renal Functions

As shown in table 1, treatment with CGP for 2-weeks decreased the BP in conscious obese animals by 13 mm Hg without affecting the heart rate. Since we have reported earlier that CGP42112A treatment did not change the BP of lean Zucker rats,30 we did not measure BP in lean rats in this study. However, we measured BP in obese rats to ensure whether the BP changes reported earlier under anesthesia30 are reproducible in conscious animals. Renal blood flow was significantly higher in obese compared to lean rats and was not affected by CGP-treatment. The kidney weight of obese rats was higher compared to that of lean rats and was also not affected by CGP-treatment. Obese rats excreted higher amount of total urinary nitrates/nitrites compared to lean rats. Consistent with previous studies,36 excretion of urinary nitrates/nitrites, as measured on days 2 and 14 of the treatment period was greater in obese rats compared to lean rats. CGP-treatment had no effect on nitrates/nitrites excretion in lean rats but caused an increase in obese rats only during the initial (measured at 2 day) treatment period. The daily urinary volume (UV) in obese rats was greater than that of lean rats (Fig 2C). However, the daily urinary Na excretion was significantly increased in obese CGP-treated rats during early days of the treatment and mostly stayed high through remaining treatment period (Fig 2D). The GFR as measured by FITC-inulin clearance on days 6, 9 and 14 in obese rats was elevated when compared to that of lean rats. Treatment with CGP for 2-weeks did not cause any change in GFR in either lean or obese rat (Fig 2E).

RAS components

AT1R and AT2R

Western blot showed presence of AT2R as two bands (44 and 39 kDa), which is likely due to various degrees of glycosylation.9 Earlier we reported that deglycosylation of AT2R protein shifted all bands to one band, which was displaced by the AT2R-blocking peptide.9 Also we have validated the antibody using AT2R knock-down (using siRNA) kidney samples (unpublished data). Analysis of AT2R bands revealed that the AT2R expression was significantly elevated in obese compared to lean rats, as reported earlier.16 The CGP-treatment caused a modest but insignificant increase in the AT2R expression in obese rat (Fig 3A). Analysis of AT1R bands at 43 kDa suggests that the AT1R expression was similar in lean and obese rats and CGP-treatment did not cause any change in its expression (Fig 3B).

Fig. 3.

Fig. 3

Fig. 3

Open in a new tab

Expression of (A) AT1R and (B) AT2R in the kidney cortex of control and CGP-treated lean and obese Zucker rats. Upper panels: Representative Western blots for respective proteins with loading control β-actin. Bar graphs: represent the ratios of densities of the respective protein bands and β-Actin i.e. AT1/β-Actin, AT2/β-Actin *significantly different compared with lean control rats. Values are represented as mean±SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05; N=5-7 in each group). (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

Renin and ACE

Analysis of the renin band at 41 kDa revealed that the renin expression in obese rats was significantly lower by approximately 45% compared to lean rats and was not affected by CGP-treatment (Fig 4A). Analysis of the ACE bands at 170 kDa revealed that the ACE expression in obese rats was significantly higher compared to lean rats. The CGP-treatment of lean and obese rats did not affect the ACE expression (Fig 4B).

Fig. 4.

Fig. 4

Fig. 4

Open in a new tab

Expression of (A) renin and (B) ACE in the kidney cortex of control and CGP-treated lean and obese Zucker rats. Upper panels: Representative Western blots for respective proteins with loading control β-actin. Bar graphs: represent the ratios of densities of respective protein bands and β-Actin i.e. renin/β-Actin, ACE/β-Actin *significantly different compared with lean control rats. Values are represented as mean±SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05; N=5-7 in each group). (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

ACE2 and Mas receptor

Western blot demonstrates the presence of ACE2 as approximately 90 kDa which is significantly less expressed in obese rats compared with lean rats. The CGP-treatment of obese rats caused approximately 3-fold increase in the ACE2 expression as compared to obese control rats (Fig 5A), but it had no effect in lean rats. ACE2 activity in the kidney cortex of obese rats was lower than in lean rats, and CGP-treatment significantly increased this activity in obese rats (Fig 5B). Changes in ACE2 protein expression and activity were not parallel which could be due to the unavailability of newly synthesized ACE2 protein post-translates into active protein. Analysis of the Mas receptor bands at 47 kDa revealed that the CGP-treatment increased Mas receptor expression in both lean and obese rats (Fig 5C).

Fig. 5.

Fig. 5

Fig. 5

Fig. 5

Open in a new tab

(A) ACE2 expression (B) ACE2 activity and (C) Mas receptor expression in the kidney cortex of control and CGP-treated lean and obese Zucker rats. Upper panels: Representative Western blots for respective proteins with loading control β-actin. For Western blot only, bar graphs represent the ratios of densities of respective protein bands and β-Actin i.e. ACE2/β-Actin, MasR/β-Actin $significantly different compared with lean control rats, #significantly different compared with obese control rats. Values are represented as mean±SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05; N=5-7 in each group). (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

Ang II and Ang-(1-7) peptides

The LC/MS quantification of angiotensin peptides revealed that the levels of Ang II and Ang-(1-7) were similar in control lean and obese rats. CGP-treatment caused no changes in Ang II levels but a 2-fold increase in Ang-(1-7) peptide levels in the kidney cortex of obese rats (Fig 6 A-B).

Fig 6.

Fig 6

Fig 6

Open in a new tab

LC/MS quantification of (A) Angiotensin II and (B) Angiotensin-(1-7) in the kidney cortex of control and CGP-treated lean and obese rats. #significantly different compared with obese control rats, Values are represented as mean±SEM; One-way ANOVA with Newman-Keuls test, p<0.05; N =5-7 in each group. (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

Effect of AT1R antagonist candesartan-induced diuresis and natriuresis

To study the AT1R function on diuresis and natriuresis in obese rats we infused candesartan (100 μg/kg, i.v. bolus) under anesthesia. As shown in figure 7(A-B), infusion of candesartan in control obese rats increased diuresis and natriuresis, which were significantly attenuated in obese CGP-treated rats. Administration of AT1R antagonist candesartan did not affect the BP or heart rate in either of the groups (Fig 7C-D). Previously this dose of candesartan had been reported ineffective for BP in obese rats.37, 9

Fig 7.

Fig 7

Fig 7

Fig 7

Fig 7

Open in a new tab

Effect of candesartan (100 μg/kg bolus) on (A) diuresis (B) natriuresis (C) mean arterial pressure and (D) heart rate in control and CGP-treated obese Zucker rats. *significantly different compared with control basal, #significantly different compared with control candesartan. Values are represented as mean ± SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05, N=5 rats in each group). (OBCT-obese control, OCGP-obese treated with CGP42112A).

Effect of AT2R agonist and antagonist on RAS components

To study the effect of AT2R agonist and antagonist on RAS components under controlled non-pathological conditions, we treated HK-2 cells with CGP42112A (10 nM), PD123319 (1 μM) alone or in combination. Treatment with CGP caused an increase in the ACE2 activity and MasR expression, while it caused a decrease in the renin activity and AT1R expression (Fig 8 A-D). These changes were blocked by simultaneous treatment with PD. Treatment with PD alone did not affect the activity/expression levels of these RAS components (Fig 8 A-D).

Fig 8.

Fig 8

Fig 8

Fig 8

Fig 8

Open in a new tab

Effect of AT2R agonist and antagonist on (A) ACE2 activity (B) MasR expression (C) Renin activity and (D) AT1R expression. Upper panels: Representative Western blots for respective proteins with loading control β-actin. Bar graphs: represent the ratios of densities of respective protein bands and β-Actin i.e AT1/β-Actin, Mas/β-Actin. *significantly different compared with control. Values are represented as mean ± SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05, N=6-7 in each group). (CT-control, CGP-CGP42112A, PD-PD123319).

Discussion

The novel findings of this study are that AT2R agonist treatment caused changes in two arms of the RAS, namely decrease in AT1R function and increase in the levels of ACE2-Ang-(1-7)-MasR axis, and that these changes likely are directly related to AT2R activation as suggested by the in-vitro studies. These two arms of RAS have been implicated in exerting opposing effects on natriuresis and blood pressure.17, 38-40 The enhanced AT1R function is one of the mechanisms responsible for enhanced Na reabsorption and BP increase in obese rats. 9, 26, 28 In this study, decreased candesartan-induced natriuresis in CGP-treated obese rats suggests a reduced AT1R function. The AT1R activation has been shown to down-regulate ACE2 expression and activity.35 Therefore it is likely that the reduced ACE2 activity in obese rats was due to the higher AT1R function, which is blunted by AT2R treatment leading ACE2 activity bounce back to the normal level. These findings were also supported by cell culture studies, wherein we found that CGP-treatment of HK-2 cells significantly increased ACE-2 activity while lowering the AT1 receptor expression. The reduced expression of renal ACE2 in obesity is consistent with previous studies.41

The ACE2 is an important regulatory enzyme which converts Ang II to Ang-(1-7). Analysis of angiotensin peptides revealed that treating obese rats with CGP led to an increased formation of Ang-(1-7) in the kidney cortex. This increase in the levels of Ang-(1-7) could have been due to increased activity of ACE2 leading to a shift in the metabolism of Ang II by converting Ang II to Ang-(1-7), or less efficiently through conversion of Ang I to Ang-(1-9). Surprisingly, we found no significant changes in the Ang II levels in obese CGP-treated rats. Ang-(1-7) binds to MasR and exerts beneficial effects such as vasodilatation and opposes action of Ang II mediated via AT1R.20, 42-44 Ang-(1-7) in the kidney also has been implicated in natriuresis. 45 Ang-(1-7) upregulates central nitric oxide synthase in spontaneously hypertensive rats and this increase is suggested as a compensatory and protective mechanism against hypertension.46 Interestingly, although ACE2 activity was significantly lower in obese rats but Ang-(1-7) was similar in both lean and obese rats. Furthermore, the enhanced activity of ACE2 in CGP-treated obese rats was equal to the lean rats, but the Ang-(1-7) in CGP-treated obese rats was un-proportionally high. These un-parallel changes in ACE2 activity and Ang-(1-7) levels suggest that a non-ACE pathway may be involved in Ang-(1-7) production. Neprilysin and prolyl-endopeptidase are abundantly expressed in the proximal tubules and catalyze Ang I to Ang-(1-7).24 Recently, proangiotensin 12 [Ang-(1-12)] has been identified and is believed to be a precursor to the formation of Ang II and Ang-(1-7) via non-renin dependent pathway.47 Although renin is the critical regulator of RAS pathway, these observations also suggest the importance of formation of Ang II and Ang-(1-7) from non-renin/ACE2 dependent sources. In pathological conditions like those in obese rats and in the present study, Ang-(1-7) production could also have been contributed via non-renin/ACE2 enzymatic pathways. Role of AT2R in regulation of non-renin/ACE2 pathways needs to be investigated in obese kidney.

AT2R is believed to be a functional antagonist of the AT1R functions.4, 5, 48 As indicated above, our renal function study revealed that candesartan produced marked increase in Na and urine excretion in obese rats which was significantly blunted in CGP-treated obese rats and may have contributed to the enhanced natriuresis and BP reduction. The reduced AT1R function is not related to the reduction of AT1R expression or its ligand Ang II in obese rats treated with CGP, as both the cortical AT1R expression and Ang II levels were similar in control and CGP-treated obese rats. Our earlier studies using western blotting, RT-PCR47 and ligand binding9 also showed no difference in the AT1R expression in the lean and obese rats. On the other hand, Xu et al.,49 have reported an increased AT1R expression in obese rat kidney. Also, we have reported in one study using western blotting a modest increase in the cortical AT1R expression in obese rats compared to lean rats.16 The basis of discrepancy is not clear; however, the batch and source of animals and the antibody used could be reasons of such discrepancy. Overall, we believe that there is either no difference or a modest increase in AT1R expression in obese rats compared to lean rats. However, enhanced renal AT1R function has been reported from multiple laboratories, including ours.9,23,30 We speculate a post-receptor signaling that contributes to enhanced AT1R function in obese animals. The AT2R agonist treatment via a post-receptor cross-talk may have caused an attenuation in AT1R expression and function as supported by a recent study, where AT2R is shown to decrease AT1R function in renal proximal tubules cells from Wistar-Kyoto rats via cGMP pathway.50 Consistent to these findings, we also observed that selective stimulation of AT2R in HK-2 cell lines decreased AT1R expression. Considering all findings together, it appears that AT2R agonist treatment lowered AT1R function, which in turn allowed ACE2-Ang-(1-7) to increase. However, molecular mechanisms are yet to be determined as to how AT2R attenuates renal AT1R function in obese rats, and if AT2R directly regulates ACE2 expression/activity and other alternate pathways leading to enhanced Ang-(1-7) levels.

Most of the effects on RAS components, kidney function or BP associated with AT2R agonist treatment were observed only in obese rats; whereas MasR expression in response to AT2R agonist treatment was increased in both the lean as well as obese rat kidneys. These selective changes in RAS could plausibly be attributed to the two sets of AT2R signaling in proximal tubules of obese and lean rats.29 We have shown that the proximal tubule AT2Rs stimulate cGMP/NO only in obese rats, but are linked to cAMP inhibition to similar extent in both the lean and obese rats. 29 Therefore, it is possible that AT2R via cGMP decreases AT1R function, which in turn allows ACE2 expression to go up in obese rats. MasR regulation by AT2R agonist may be linked to the AT2R’s ability to affect cAMP in both of the rat strains.

The AT2R activation has been reported to exert a negative feedback on renin production. 34 We have shown that renal renin expression was lower in obese compared to lean rats.51 However, in the present study we found that despite renin expression being lower in obese rats, Ang II levels were similar in lean and obese rats. This could be due to the fact that ACE expression in the kidney is significantly increased in obese rats, thus converting Ang I to more Ang II and thereby compensating for reduced renin expression. Moreover, it is likely that proangiotensinogen (Ang 1-12) is converted to Ang II which is independent of renin. Contrary to our results, Sharma et al.,52 reported an increase in renal Ang II levels in obese compared to lean rats. The basis of this discrepancy is not clear; although it must be noted that different methods (HPLC/ELISA vs LC/MS) were used in these studies. The AT2R antagonist PD treatment caused an increased cortical expression of renin in obese rats16 suggesting a role of AT2R regulating renin in obesity. In the present study, treatment of HK-2 cells with CGP decreased renin expression. These findings suggest that similar to AT1R, AT2R also negatively regulates renin levels. In light of these reports, we expected that AT2R agonist treatment will lower the renal renin expression in obese rats but we observed no changes. Since the renal renin expression in obese rat kidney is already low, it is likely that renin could not be further reduced by the AT2R agonist treatment in obese rats. Nevertheless, the role of AT2R in keeping low renin in obese rat kidney highlights the importance of AT2R as a multifaceted regulator of the kidney RAS.

Obesity is reported to be associated with higher NO generation,53 which could be a compensatory mechanism that contributes to vasodilatation in obesity.54 Since AT2R coupled with NO pathway is functional in obese and not in lean rats, AT2R activation by CGP was expected to cause an increase in urinary nitrates in response to CGP-treatment. However, this increase was not sustained throughout the 14 day treatment period. Although AT2R is classified as a G-protein coupled receptor (GPCR), unlike most GPCRs the AT2R does not undergo endocytosis in the presence of the agonist, 55, 56 and thereby could be resistant to agonist-induced cellular degradation. This notion is supported by our observation that there was no reduction in AT2R expression in CGP-treated rats. Whether AT2Rs are desensitized without leading to endocytosis and degradation upon long-term agonist exposure, and thereby could not produce the sustained increase in nitrates over the agonist treatment period is not known. We have earlier demonstrated that AT2R inhibits Na-pump via NO/cGMP pathway.29, 57 The increased natriuresis observed in obese CGP-treated rats could be the direct effect of AT2R on Na-pump via NO pathway. Pharmacological activation of AT2R reduced BP in conscious animals which were measured by tail-cuff method. This decrease in BP is in agreement with our earlier studies, 30 whereby arterial pressure was measured in anesthetized animals. It is likely that increased urinary Na excretion shifted the BP to a lower level in AT2R agonist-treated obese rats. This is further supported by our observation that there were no changes in GFR or renal blood flow, which were already higher in obese rats compared to lean rats. Since obesity is associated with enhanced GFR and vasodilatation, 25, 58 our findings support the notion that obesity-related hypertension is caused by excessive renal Na reabsorption and not by changes in GFR and/or vascular tone. Consistent with this notion, AT1R antagonist losartan treatment has been shown to reduce renal Na reabsorption and BP without causing any changes in GFR and renal blood flow in obese rats. 26

Numerous studies linked abnormal renal sodium handling (excessive Na reabsorption) with the pathogenesis of hypertension in obesity.25 Since obesity is also associated with low grade inflammation59 and oxidative stress60 as well as enhanced renal AT1R functions28, which are inter-regulatory in a positive manner,60 all of them are likely to contribute to Na reabsorption and blood pressure increase in obesity. The anti-natriuresis and blood pressure increase in obesity may be further contributed by a reduced ACE2 expression41, 61 and consequently reduced levels of Ang (1-7),41 which recently has been implicated in promoting natriuresis17, 45, 62, 63 and blood pressure regulation.64-66 We have earlier reported that the AT2R agonist CGP treatment in obese rats caused a reduction in oxidative stress and inflammation.30 Advancing our understanding of the AT2R functions, present study shows that lesser known arm of the RAS, ACE2-Ang-(1-7) levels were increased in response to AT2R agonist treatment in obese rats. Role of these parameters as independent regulators of renal Na handling and blood pressure increase in obesity has not been tested, and it may be difficult due to their inter-regulation. However, it is reasonable to suggest that the changes in these parameters including reduced AT1R function and enhanced ACE2-Ang-(1-7)/MasR expression might have contributed to the AT2R agonist-induced changes in Na excretion and blood pressure in obese rats. These studies provide a new dimension to the AT2R function and amplification of the net response. To ascertain the role of ACE2-Ang-(1-7)/MasR axis in renal Na handing and lowering blood pressure in obesity warrants a further investigation.

Materials and Methods

Chronic Study

Male lean and obese Zucker rats (10-11 weeks) were purchased from Harlan, Indianapolis, IN. The detailed protocol is provided in the supplemental file.

Hemodynamic parameters

BP was measured by tail-cuff method using CODA system which provides 99% correlation with telemetry and direct BP measurements. 67, 68 The detailed protocol is provided in the supplemental file.

Renal functions

The urine samples were analyzed for Na and urinary Na volume (UNaV) mmol/day was calculated. GFR was measured as described by Qi et al.69 The detailed protocol is provided in the supplemental file.

RAS Components

The detailed protocol is provided in the supplemental file.

LC/MS Quantification of angiotensin peptides

The angiotensin peptides were analyzed as described in our publication.61 The detailed protocol is provided in the supplemental file.

Acute Study Candesartan-induced natriuresis as AT1R function in CGP-treated obese rats

This protocol is represented in figure 1 and the details are provided in the supplemental file

Fig. 1.

Fig. 1

Open in a new tab

Schematic representation of the protocol used in the renal function study.

Cell culture

HK2 cells expresses RAS components70, 71 and they were cultured as described earlier.70 The detailed protocol is provided in the supplemental file.

Chemicals

The details are provided in the supplemental file

Statistical analysis

Data are presented as mean±SEM and were analyzed using GraphPad Prism 4 and subjected to one-way ANOVA with Newman-Keuls post hoc test. N= 5 to 7 in each group, as detailed in figure legends. A p value of less than 0.05 was considered statistically significant.

An expanded version of the methods is given here as supplemental file

Chronic Study

All the animal experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Houston. The lean and obese-control groups (N=5-7 per group) were treated with normal saline as vehicle and the lean and obese-CGP42112A (CGP) groups were treated with CGP (1 μg/kg/min) for 2-weeks using Alzet osmotic pumps (Model 2ML-2, CA) implanted subcutaneously. In one set of treatment groups, another micro-osmotic pump (Alzet, model 1002) filled with FITC-inulin (5%) was placed at the same time in the peritoneal cavity for the measurement of glomerular filtration rate (GFR) in these rats. The animals were placed singly in metabolic cages. Normal rat chow and water were provided ad libitum. Daily 24-hour food and water intake and urine volume were recorded during the treatment period.

Hemodynamic parameters

BP was measured by tail-cuff method using CODA system which is clinically validated and provides 99% correlation with telemetry and direct BP measurements. The CODA tail-cuff blood pressure system utilizes Volume Pressure Recording (VPR) sensor technology to measure the rat tail blood pressure. At the end of the blood pressure measurements, the kidneys were removed and stored frozen at −80°C for measuring the cortical expression of RAS components.

Renal functions

The urine samples were analyzed for Na by the AAnalyst 400 atomic absorption spectrometer (Perkin Elmer, Waltham, MA) and urinary Na volume (UNaV) mmol/day was calculated. To measure GFR, blood (80 μl) from tail vein on multiple days (days 6, 9 and 14) during drug treatment was collected. The FITC-inulin fluorescent count in plasma samples and in urine collected on the same day was determined using spectrofluorometer (Cytofluor Series 4000, Applied Biosystem). The GFR was calculated using the formula GFR=(Urinary fluorescence counts/24h) / (Plasma fluorescence counts/μl), as described by Qi et al. 1 On the last of the treatment (day 14), renal blood flow was determined under anesthesia using a transit time blood flowmeter (Model T402, Transonic Ststems Inc, NY, USA). Briefly, a midline incision was made in the abdominal region and an ultrasonic flow probe was carefully placed around the left renal artery. The blood volume flow (ml/minute) was recorded and analyzed by using Powerlab 4/35 data acquisition system (PL3504, ADI Instruments, Colorado Springs, CO, USA). Total nitrates/nitrites excreted at two time points (day 3 as early and day 14 as late) of the treatment were measured using EIA kits (R&D System, Minneapolis, MN).

Western blotting

The expression of AT1R, AT2R, and renin, ACE, ACE2 and MasR in the idney cortex was determined by Western blotting and ACE2 activity was measured by Sensolyte 390 ACE2 activity kit (Anaspec Inc, CA) using spectrofluorometer (Cytofluor Series 4000, Applied Biosystem).For western blotting, the kidney cortices were homogenized in the buffer containing (in mM) Tris 50, EDTA 10, PMSF 1, cocktail of protease inhibitors (aprotinin, calpain inhibitors, leupeptin, pepstatin and trypsin inhibitor). Proteins in the homogenates were determined by BCA method using a kit (Pierce, Rockford, IL). Equal amounts of protein (30 μg for AT1 receptor, 60 μg for AT2 receptors, 30 μg for renin, 60 μg for ACE, 65 μg for ACE2 and 40 μg for Mas receptor) from various rat groups were subjected to SDS-PAGE and were transferred onto immobilon P (blot). The blots were incubated with primary polyclonal antibodies for the AT1 receptor, AT2 receptor, renin, ACE, ACE2 and Mas receptor. Following the incubation with the primary antibodies, the blots were incubated with appropriate HRP-conjugated anti rabbit/anti-goat IgGs. The signal was detected by ECL system, recorded and analyzed by Fluorchem 8800 (Alpha Innotech Imaging System, San Leandro, CA) for the densitometric analysis. For loading control, the blots were stripped, and re-probed with β-actin mouse monoclonal antibody.

Determination of angiotensin peptides

To determine angiotensin peptide levels quantitatively in the kidney cortex, tissue was homogenized in lysis buffer (10 mM Tris, pH 7.4) with and without protease inhibitors. Since the presence or absence of protease inhibitors did not affect the Ang II and Ang-(1-7) levels measured in the tissues, we presented data for only with protease inhibitors. After homogenization the samples were centrifuged for 15 min at 4°C at 1,600g, and resulting supernatant was loaded onto an C18-E (55μm, 70A) cartridge, equilibrated with 60% acetonitrile, 1% TFA and 39% distilled water. After sample application C18-E cartridge was washed twice with 3ml of 1% TFA. The column was eluted with the equilibrium buffer and collected in a 15 ml tube. The eluent was dried under vacuum centrifuge, and reconstituted in 80% acetonitrile and 0.1 % formic acid prior to LC/MS analysis. Protein samples were analyzed using an Ultra Performance LC Systems (ACQUITY, Waters Corp., Milford, MA, USA) coupled with a quadrupole time-of-flight mass spectrometer (Q-TOF Premier, Waters) with electrospray ionization (ESI) in both ESI+-MS and ESI+-MS/MS (SetMass without fragmentation) mode operated by the Masslynx software (V4.1) as discussed in our earlier publication by Samuel et al., 2012.

Candesartan-induced natriuresis as AT1R function in CGP-treated obese rats

After tracheotomy the carotid artery, jugular vein and ureter were cannulated for BP measurement, drug infusion and urine collection, respectively. For studying the AT1R function in response to candesartan, the AT2R were blocked by infusing AT2R antagonist PD (50 μg/kg/min) in both the control and CGP-treated obese rats. Basal urine was collected twice for 30 min each time. After that, the AT1R antagonist candesartan (100 μg/kg bolus) was infused and urine was again collected twice for thirty minutes each time (figure1).

Cell Culture

We performed in-vitro experiments using HK2 cells which is human kidney proximal tubule cell line and routinely used in our laboratory and expresss all the components of RAS.2 HK2 cells were cultured using K-SFM supplemented with 5% FBS, epidermal growth factor (EGF) and bovine pituitary extract (BPE). The cells used in the experiment were between passages 5-9. Cells were seeded at 1 × 106 in 100 × 20 mm culture dish. At ~65% confluency, the HK-2 cells were treated with CGP42212A (10nM), PD 123319 (1μM) and CGP (10nM) + PD (1μM) for 24 hrs. After treatment with the drugs, the cell were lysed and processed for ACE2 and renin activity and western blotting for AT1R and MasR. ACE2 and renin activity was measured using Varioskan plate reader, Thermo Scientific, IL.

Chemicals

CGP was synthesized (21st Century Biochem, MA). PD was a gift from Pfizer Inc and candesartan was a gift from AstraZeneca. Antibody for MasR (Alomone Labs Ltd) and for renin, ACE, ACE2, AT1 and β-actin were purchased from Santa Cruz, CA. AT2R antibody was custom raised (EZ Biolab). K-SFM was purchased from Life Technologies, Grand Island, NY.

Acknowledgment

The study is supported by National Institutes of Health grant R01-DK61578. PD123319 was a generous gift from Pfizer Inc, USA. The authors would like to acknowledge Dr. Rifat Sabuhi, Dr. Preethi Samuel and Ms. Fatima Qamar for reviewing the manuscript and for the help in the animal study.

Footnotes

Disclosures: None

References

  • 1.Vickers C, Hales P, Kaushik V, et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem. 2002;277:14838–14843. doi: 10.1074/jbc.M200581200. [DOI] [PubMed] [Google Scholar]
  • 2.Rice GI, Thomas DA, Grant PJ, et al. Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism. Biochem J. 2004;383:45–51. doi: 10.1042/BJ20040634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.de Gasparo M, Catt KJ, Inagami T, et al. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev. 2000;52:415–472. [PubMed] [Google Scholar]
  • 4.Akishita M, Ito M, Lehtonen JY, et al. Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth. J Clin Invest. 1999;103:63–71. doi: 10.1172/JCI5182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Masaki H, Kurihara T, Yamaki A, et al. Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects. J Clin Invest. 1998;101:527–535. doi: 10.1172/JCI1885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ Res. 1998;83:1182–1191. doi: 10.1161/01.res.83.12.1182. [DOI] [PubMed] [Google Scholar]
  • 7.Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA. 1996;93:156–160. doi: 10.1073/pnas.93.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Meffert S, Stoll M, Steckelings UM, et al. The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells. Mol Ccell Endocrionol. 1996;;122:59–67. doi: 10.1016/0303-7207(96)03873-7. [DOI] [PubMed] [Google Scholar]
  • 9.Hakam AC, Hussain T. Renal angiotensin II type-2 receptors are upregulated and mediate the candesartan-induced natriuresis/diuresis in obese Zucker rats. Hypertension. 2005;45:270–275. doi: 10.1161/01.HYP.0000151622.47814.6f. [DOI] [PubMed] [Google Scholar]
  • 10.Hakam AC, Siddiqui AH, Hussain T. Renal angiotensin II AT2 receptors promote natriuresis in streptozotocin-induced diabetic rats. Am J Physiol Renal Physiol. 2006;290:F503–508. doi: 10.1152/ajprenal.00092.2005. [DOI] [PubMed] [Google Scholar]
  • 11.Hilliard LM, Jones ES, Steckelings UM, et al. Sex-specific influence of angiotensin type 2 receptor stimulation on renal function: a novel therapeutic target for hypertension. Hypertension. 2012;59:409–414. doi: 10.1161/HYPERTENSIONAHA.111.184986. [DOI] [PubMed] [Google Scholar]
  • 12.Ali Q, Hussain T. AT(2) receptor non-peptide agonist C21 promotes natriuresis in obese Zucker rats. Hypertens Res. 2012;35:654–660. doi: 10.1038/hr.2012.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Carey RM. Update on the role of the AT2 receptor. Curr Opin Nephrol Hypertens. 2005;14:67–71. doi: 10.1097/00041552-200501000-00011. [DOI] [PubMed] [Google Scholar]
  • 14.Duke LM, Evans RG, Widdop RE. AT2 receptors contribute to acute blood pressure-lowering and vasodilator effects of AT1 receptor antagonism in conscious normotensive but not hypertensive rats. Am J Physiol Heart Circ Physiol. 2005;288:H2289–2297. doi: 10.1152/ajpheart.01096.2004. [DOI] [PubMed] [Google Scholar]
  • 15.Gao J, Zhang H, Le KD, et al. Activation of central angiotensin type 2 receptors suppresses norepinephrine excretion and blood pressure in conscious rats. Am J Hypertens. 2011;24:724–730. doi: 10.1038/ajh.2011.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Siddiqui AH, Ali Q, Hussain T. Protective role of angiotensin II subtype 2 receptor in blood pressure increase in obese Zucker rats. Hypertension. 2009;53:256–261. doi: 10.1161/HYPERTENSIONAHA.108.126086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ferrario CM, Varagic J. The ANG-(1-7)/ACE2/mas axis in the regulation of nephron function. Am J Physiol Renal Physiol. 2010;298:F1297–1305. doi: 10.1152/ajprenal.00110.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Caruso-Neves C, Lara LS, Rangel LB, et al. Angiotensin-(1-7) modulates the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule. Biochim Biophys Acta. 2000;1467:189–197. doi: 10.1016/s0005-2736(00)00219-4. [DOI] [PubMed] [Google Scholar]
  • 19.De Souza AM, Lopes AG, Pizzino CP, et al. Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+-ATPase activity through AT2 receptor. Regul Pept. 2004;120:167–175. doi: 10.1016/j.regpep.2004.03.005. [DOI] [PubMed] [Google Scholar]
  • 20.Santos RA, Simoes e Silva AC, Maric C, et al. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc Natl Acad Sci USA. 2003;100:8258–8263. doi: 10.1073/pnas.1432869100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Garcia NH, Garvin JL. Angiotensin 1-7 has a biphasic effect on fluid absorption in the proximal straight tubule. J Am Soc Nephrol. 1994;5:1133–1138. doi: 10.1681/ASN.V541133. [DOI] [PubMed] [Google Scholar]
  • 22.Zimpelmann J, Burns KD. Angiotensin-(1-7) activates growth-stimulatory pathways in human mesangial cells. Am J Physiol Renal Physiol. 2009;296:F337–346. doi: 10.1152/ajprenal.90437.2008. [DOI] [PubMed] [Google Scholar]
  • 23.Su Z, Zimpelmann J, Burns KD. Angiotensin-(1-7) inhibits angiotensin II-stimulated phosphorylation of MAP kinases in proximal tubular cells. Kidney Int. 2006;69:2212–2218. doi: 10.1038/sj.ki.5001509. [DOI] [PubMed] [Google Scholar]
  • 24.Welches WR, Brosnihan KB, Ferrario CM. A comparison of the properties and enzymatic activities of three angiotensin processing enzymes: angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11. Life Sci. 1993;52:1461–1480. doi: 10.1016/0024-3205(93)90108-f. [DOI] [PubMed] [Google Scholar]
  • 25.Hall JE, Jones DW, Kuo JJ, et al. Impact of the obesity epidemic on hypertension and renal disease. Curr Hypertens Rep. 2003;5:386–392. doi: 10.1007/s11906-003-0084-z. [DOI] [PubMed] [Google Scholar]
  • 26.Alonso-Galicia M, Brands MW, Zappe DH, et al. Hypertension in obese Zucker rats. Role of angiotensin II and adrenergic activity. Hypertension. 1996;28:1047–1054. doi: 10.1161/01.hyp.28.6.1047. [DOI] [PubMed] [Google Scholar]
  • 27.Shah S, Hussain T. Enhanced angiotensin II-induced activation of Na+, K+-ATPase in the proximal tubules of obese Zucker rats. Clin Exp Hypertens. 2006;28:29–40. doi: 10.1080/10641960500386650. [DOI] [PubMed] [Google Scholar]
  • 28.Becker M, Umrani D, Lokhandwala MF, et al. Increased renal angiotensin II AT1 receptor function in obese Zucker rat. Clin Exp Hypertens. 2003;25:35–47. doi: 10.1081/ceh-120017739. [DOI] [PubMed] [Google Scholar]
  • 29.Hakam AC, Hussain T. Angiotensin II type 2 receptor agonist directly inhibits proximal tubule sodium pump activity in obese but not in lean Zucker rats. Hypertension. 2006;47:1117–1124. doi: 10.1161/01.HYP.0000220112.91724.fc. [DOI] [PubMed] [Google Scholar]
  • 30.Sabuhi R, Ali Q, Asghar M, et al. Role of the angiotensin II AT2 receptor in inflammation and oxidative stress: opposing effects in lean and obese Zucker rats. Am J Physiol Renal Physiol. 2011;300:F700–706. doi: 10.1152/ajprenal.00616.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Vaziri ND, Rodriguez-Iturbe B. Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Cin Pract Nephrol. 2006;2:582–593. doi: 10.1038/ncpneph0283. [DOI] [PubMed] [Google Scholar]
  • 32.Kaneto H, Katakami N, Matsuhisa M, et al. Role of reactive oxygen species in the progression of type 2 diabetes and atherosclerosis. Mediators Inflamm. 2010;2010:453892. doi: 10.1155/2010/453892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Horiuchi M, Akishita M, Dzau VJ. Recent progress in angiotensin II type 2 receptor research in the cardiovascular system. Hypertension. 1999;33:613–621. doi: 10.1161/01.hyp.33.2.613. [DOI] [PubMed] [Google Scholar]
  • 34.Ichihara A, Hayashi M, Hirota N, et al. Angiotensin II type 2 receptor inhibits prorenin processing in juxtaglomerular cells. Hypertens Res. 2003;26:915–921. doi: 10.1291/hypres.26.915. [DOI] [PubMed] [Google Scholar]
  • 35.Gallagher PE, Ferrario CM, Tallant EA. Regulation of ACE2 in cardiac myocytes and fibroblasts. Am J Physiol Heart Circ Physiol. 2008;295:H2373–2379. doi: 10.1152/ajpheart.00426.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Morrison RG, Mills C, Moran AL, et al. A moderately high fat diet promotes salt-sensitive hypertension in obese zucker rats by impairing nitric oxide production. Clin Exp Hypertens. 2007;29:369–381. doi: 10.1080/10641960701578360. [DOI] [PubMed] [Google Scholar]
  • 37.Tallam LS, Jandhyala BS. Significance of exaggerated natriuresis after angiotensin AT1 receptor blockade or angiotensin-converting enzyme inhibition in obese Zucker rats. Clin Exp Pharmacol Physiol. 2001;28:433–440. doi: 10.1046/j.1440-1681.2001.03457.x. [DOI] [PubMed] [Google Scholar]
  • 38.Ferrario CM, Chappell MC, Tallant EA, et al. Counterregulatory actions of angiotensin-(1-7) Hypertension. 1997;30:535–541. doi: 10.1161/01.hyp.30.3.535. [DOI] [PubMed] [Google Scholar]
  • 39.Pinheiro SV, Simoes ESAC. Angiotensin converting enzyme 2, Angiotensin-(1-7), and receptor MAS axis in the kidney. Int J Hypertens. 2012;2012:414128. doi: 10.1155/2012/414128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Santos RA, Ferreira AJ, Pinheiro SV, et al. Angiotensin-(1-7) and its receptor as a potential targets for new cardiovascular drugs. Expert Opin Investig Drugs. 2005;14:1019–1031. doi: 10.1517/13543784.14.8.1019. [DOI] [PubMed] [Google Scholar]
  • 41.Gupte M, Thatcher SE, Boustany-Kari CM, et al. Angiotensin converting enzyme 2 contributes to sex differences in the development of obesity hypertension in C57BL/6 mice. Arterioscler Thromb Vasc Biol. 2012;32:1392–1399. doi: 10.1161/ATVBAHA.112.248559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Porsti I, Bara AT, Busse R, et al. Release of nitric oxide by angiotensin-(1-7) from porcine coronary endothelium: implications for a novel angiotensin receptor. Br J Pharmacol. 1994;111:652–654. doi: 10.1111/j.1476-5381.1994.tb14787.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Grobe JL, Mecca AP, Mao H, et al. Chronic angiotensin-(1-7) prevents cardiac fibrosis in DOCA-salt model of hypertension. Am J Physiol Heart Circ Physiol. 2006;290:H2417–2423. doi: 10.1152/ajpheart.01170.2005. [DOI] [PubMed] [Google Scholar]
  • 44.Ferrario CM, Trask AJ, Jessup JA. Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function. Am J Physiol Heart Circ Physiol. 2005;289:H2281–2290. doi: 10.1152/ajpheart.00618.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.DelliPizzi AM, Hilchey SD, Bell-Quilley CP. Natriuretic action of angiotensin(1-7) Br J Pharmacol. 1994;111:1–3. doi: 10.1111/j.1476-5381.1994.tb14014.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Cerrato BD, Frasch AP, Nakagawa P, et al. Angiotensin-(1-7) upregulates central nitric oxide synthase in spontaneously hypertensive rats. Brain Res. 2012;1453:1–7. doi: 10.1016/j.brainres.2012.03.022. [DOI] [PubMed] [Google Scholar]
  • 47.Nagata S, Kato J, Sasaki K, et al. Isolation and identification of proangiotensin-12, a possible component of the renin-angiotensin system. Biochem Biophys Res Commun. 2006;350:1026–1031. doi: 10.1016/j.bbrc.2006.09.146. [DOI] [PubMed] [Google Scholar]
  • 48.AbdAlla S, Lother H, Abdel-tawab AM, et al. The angiotensin II AT2 receptor is an AT1 receptor antagonist. J Biol Chem. 2001;276:39721–39726. doi: 10.1074/jbc.M105253200. [DOI] [PubMed] [Google Scholar]
  • 49.Xu ZG, Lanting L, Vaziri ND, et al. Upregulation of angiotensin II type 1 receptor, inflammatory mediators, and enzymes of arachidonate metabolism in obese Zucker rat kidney: reversal by angiotensin II type 1 receptor blockade. Circulation. 2005;111:1962–1969. doi: 10.1161/01.CIR.0000161831.07637.63. [DOI] [PubMed] [Google Scholar]
  • 50.Yang J, Chen C, Ren H, et al. Angiotensin II AT2 receptor decreases AT1 receptor expression and function via nitric oxide/cGMP/Sp1 in renal proximal tubule cells from Wistar-Kyoto rats. J Hypertens. 2012;30:1176–1184. doi: 10.1097/HJH.0b013e3283532099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Siragy HM, Inagami T, Carey RM. NO and cGMP mediate angiotensin AT2 receptor-induced renal renin inhibition in young rats. Am J Physiol Regul Integr Comp Physiol. 2007;293:R1461–1467. doi: 10.1152/ajpregu.00014.2007. [DOI] [PubMed] [Google Scholar]
  • 52.Sharma R, Sharma M, Reddy S, et al. Chronically increased intrarenal angiotensin II causes nephropathy in an animal model of type 2 diabetes. Front Biosci. 2006;11:968–976. doi: 10.2741/1853. [DOI] [PubMed] [Google Scholar]
  • 53.Choi JW, Pai SH, Kim SK, et al. Increases in nitric oxide concentrations correlate strongly with body fat in obese humans. Clin Chem. 2001;47:1106–1109. [PubMed] [Google Scholar]
  • 54.Tsutsumi Y, Matsubara H, Masaki H, et al. Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation. J Clin Invest. 1999;104:925–935. doi: 10.1172/JCI7886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Hein L, Meinel L, Pratt RE, et al. Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand. Mol Endocrinol. 1997;11:1266–1277. doi: 10.1210/mend.11.9.9975. [DOI] [PubMed] [Google Scholar]
  • 56.Hunyady L. Molecular mechanisms of angiotensin II receptor internalization. J Am Soc Nephrol. 1999;10(Suppl 11):S47–56. [PubMed] [Google Scholar]
  • 57.Hakam AC, Hussain T. Angiotensin II AT2 receptors inhibit proximal tubular Na+-K+-ATPase activity via a NO/cGMP-dependent pathway. Am J Physiol Renal Physiol. 2006;290:F1430–1436. doi: 10.1152/ajprenal.00218.2005. [DOI] [PubMed] [Google Scholar]
  • 58.Chagnac A, Weinstein T, Korzets A, et al. Glomerular hemodynamics in severe obesity. Am J Physiol Renal Physiol. 2000;278:F817–822. doi: 10.1152/ajprenal.2000.278.5.F817. [DOI] [PubMed] [Google Scholar]
  • 59.Herder C, Schneitler S, Rathmann W, et al. Low-grade inflammation, obesity, and insulin resistance in adolescents. J Clin Endocrinol Metab. 2007;92:4569–4574. doi: 10.1210/jc.2007-0955. [DOI] [PubMed] [Google Scholar]
  • 60.Ferder L, Inserra F, Martinez-Maldonado M. Inflammation and the metabolic syndrome: role of angiotensin II and oxidative stress. Curr Hypertens Rep. 2006;8:191–198. doi: 10.1007/s11906-006-0050-7. [DOI] [PubMed] [Google Scholar]
  • 61.Samuel P, Ali Q, Sabuhi R, et al. High Na-intake increases renal angiotensinII levels and reduces the expression of ACE2-AT2R-MasR axis in obese Zucker rats. Am J Physiol Renal Physiol. 2012;303:F412–419. doi: 10.1152/ajprenal.00097.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Dilauro M, Burns KD. Angiotensin-(1-7) and its effects in the kidney. ScientificWorldJournal. 2009;9:522–535. doi: 10.1100/tsw.2009.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Handa RK, Ferrario CM, Strandhoy JW. Renal actions of angiotensin-(1-7): in vivo and in vitro studies. Am J Physiol. 1996;270:F141–147. doi: 10.1152/ajprenal.1996.270.1.F141. [DOI] [PubMed] [Google Scholar]
  • 64.Dharmani M, Mustafa MR, Achike FI, et al. Effects of angiotensin 1-7 on the actions of angiotensin II in the renal and mesenteric vasculature of hypertensive and streptozotocin-induced diabetic rats. Eur J Pharmacol. 2007;561:144–150. doi: 10.1016/j.ejphar.2007.01.037. [DOI] [PubMed] [Google Scholar]
  • 65.Walters PE, Gaspari TA, Widdop RE. Angiotensin-(1-7) acts as a vasodepressor agent via angiotensin II type 2 receptors in conscious rats. Hypertension. 2005;45:960–966. doi: 10.1161/01.HYP.0000160325.59323.b8. [DOI] [PubMed] [Google Scholar]
  • 66.Iyer SN, Ferrario CM, Chappell MC. Angiotensin-(1-7) contributes to the antihypertensive effects of blockade of the renin-angiotensin system. Hypertension. 1998;31:356–361. doi: 10.1161/01.hyp.31.1.356. [DOI] [PubMed] [Google Scholar]
  • 67.Fraser TB, Turner SW, Mangos GJ, et al. Comparison of telemetric and tail-cuff blood pressure monitoring in adrenocorticotrophic hormone-treated rats. Clin Exp Pharmacol Physiol. 2001;28:831–835. doi: 10.1046/j.1440-1681.2001.03531.x. [DOI] [PubMed] [Google Scholar]
  • 68.Feng M, Whitesall S, Zhang Y, et al. Validation of volume-pressure recording tail-cuff blood pressure measurements. Am J Hypertens. 2008;21:1288–1291. doi: 10.1038/ajh.2008.301. [DOI] [PubMed] [Google Scholar]
  • 69.Qi Z, Whitt I, Mehta A, et al. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. Am J Physiol Renal Physiol. 2004;286:F590–596. doi: 10.1152/ajprenal.00324.2003. [DOI] [PubMed] [Google Scholar]
  • 70.Ali Q, Sabuhi R, Hussain T. High glucose up-regulates angiotensin II subtype 2 receptors via interferon regulatory factor-1 in proximal tubule epithelial cells. Mol Cell Biochem. 2010;344:65–71. doi: 10.1007/s11010-010-0529-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Satou R, Gonzalez-Villalobos RA, Miyata K, et al. Costimulation with angiotensin II and interleukin 6 augments angiotensinogen expression in cultured human renal proximal tubular cells. Am J Physiol Renal Physiol. 2008;295:F283–289. doi: 10.1152/ajprenal.00047.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES