Differential expression of nuclear AT 1 receptors and angiotensin II within the - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:42 |只看该作者 |倒序浏览 |打印

作者：Karl D. Pendergrass, David B. Averill, Carlos M. Ferrario, Debra I. Diz, and Mark C. Chappell作者单位：Hypertension and Vascular Disease Center and Wake Forest University Health Sciences, Winston-Salem, North Carolina * [& K9 b; i% V  P% L

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      【摘要】
   We established a new congenic model of hypertension, the mRen2.Lewis rat and assessed the intracellular expression of angiotensin peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)27 transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: 195 ± 2 vs. 107 ± 2 mmHg, P < 0.01). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(1-7) were 270% higher and ANG I concentrations were 40% lower in the mRen2.Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2.Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(1-7) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand 125 I-sarthran. Isolated CORT nuclei exhibited a high density (B max 200 fmol/mg protein) and affinity for the sarthran ligand ( K D <0.5 95%) were the AT 1 receptor subtype. CORT ANG II receptor B max and K D values in nuclei were 75 and 50% lower, respectively, for the mRen2.Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 ± 4 vs. mRen2.Lewis: 21 ± 5 fmol/mg protein) and affinity (Lewis: 0.31 ± 0.1 vs. 0.69 ± 0.1 nM) were lower in the mRen2.Lewis rats. In summary, the hypertensive mRen2.Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM AT 1 sites in the mRen2.Lewis. The downregulation of AT 1 sites in the mRen2.Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II. 6 x) d1 J$ J; n* A9 e' k1 B5 f# f* W
      【关键词】 ANG II type receptor hypertension intrarenal angiotensin renal cortex renal medulla ANG II
   THE INFLUENCE of the renin-angiotensin system (RAS) on the development and progression of hypertension and renal injury is without dispute. It is also well accepted that the major active components of the RAS that contribute to increased blood pressure and tissue injury are the sustained or enhanced expression of angiotension II (ANG II) and aldosterone. Numerous studies confirm that the AT 1 receptor mediates the majority of the actions of ANG II and that blockade of this receptor ameliorates the deleterious effects of the peptide. The angiotensin type 1 receptor (AT 1 ) is regarded as a typical seven-transmembrane, G-coupled protein residing on the cellular membrane that binds circulating or extracellular ANG II; however, several studies suggest that intracellular ANG II has significant effects ( 3, 18, 23, 25 ). These actions of ANG II are entirely consistent with evidence of intracellular and/or nuclear ANG II binding sites in various cell types including hepatocytes, vascular smooth muscles cells, and neonatal neurons, as well as several tissues ( 6, 7, 23, 25, 28, 34, 44, 46, 60 ).& Q2 m1 ], `5 w$ }

To our knowledge, only one study by Licea and colleagues ( 34 ) has characterized the presence of nuclear ANG II receptors in the kidney and the extent that these receptors are altered in ANG II-dependent hypertension. Although their data revealed a significant concentration of AT 1 receptors in the nuclear fraction of the renal cortex, infusion of ANG II at a dose that markedly increased blood pressure did not influence the density of these nuclear sites ( 34 ). Therefore, in the present study, we determined the density and pharmacological characteristics of nuclear and plasma membrane ANG II receptors in the renal cortex and medulla from adult hypertensive mRen2.Lewis rats, a new congenic strain developed from the backcross of the original Ren27( 2 ) transgenic (that overexpresses renin) and the normotensive Lewis. Similar to the original transgenic strain, mRen2.Lewis rats exhibit significant hypertension, gender differences in the degree of elevated blood pressure and renal injury, as well as the normalization of blood pressure by RAS blockade ( 11, 16 ). However, studies are lacking in this new strain on the characterization of the intrarenal RAS. In addition to the characterization of renal ANG II receptors, we also assessed tissue expression of ANG II, ANG I, and ANG-(1-7) in both the cortical and medullary regions of the kidneys from both strains. These studies are the first to document differential expression of both receptor and peptide levels in the renal cortex and medulla of the male mRen2.Lewis hypertensive strain.

MATERIALS AND METHODS1 [1 t$ J7 q) Z1 X
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Experimental animals. Heterozygous male mRen2.Lewis rats were obtained from the Hypertension and Vascular Disease Center Transgenic colony at 20 wk of age. Normotensive male Lewis rats were purchased from Charles River (Raleigh, NC) and utilized at the same age as the congenic rats. Animals were fed a powdered rat chow (Purina Mills, Richmond, VA) to provide a daily intake of 17 and 28 meq/100 g of body wt of sodium and potassium, respectively, had full access to water, and were housed in an AALAC-approved facility in rooms maintained on a 12:12-h light-dark cycle (lights on 6:00 A.M. to 6:00 P.M.). Systolic blood pressure was measured in trained rats (mean of 5 determinations/data point) with a Narco Biosystems device (Houston, TX). Rats were administered heparin (1,000 U, ip) 20 min before anesthesia with halothane, and a catheter (Angiocath, Sandy, UT) was placed in the abdominal aorta (5 ml) for aortic blood collection. Following blood collection, the rats were decapitated and tissues were collected. The cortex and medulla regions of the kidney were then dissected on an ice-filled plastic petri dish, frozen on dry ice, and stored at -80°C. These procedures were approved by the Wake Forest University School of Medicine Institutional Animal Care and Use Committee.
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Plasma and renal tissue angiotensins. Blood was collected into chilled Vacutainer blood collection tubes (Becton Dickinson, Sandy, UT) containing peptidase inhibitors and processed for direct RIA of angiotensin peptides ( 1 ). Frozen renal tissue was homogenized in an acidic ethanol (80% vol/vol 0.1 N HCl) solution containing the peptidase inhibitors described above and processed for RIA analysis as described by Allred et al. ( 1 ). A sample of homogenate was taken to determine total protein content (Bio-Rad Protein Assay Reagent, Bio-Rad Laboratories, Hercules, CA). Details on the RIAs have been previously described ( 1, 11 ). To verify the identity of ANG II immunoreactivity in the kidney, pooled extracts from the cortex or medulla of the Ren2.Lewis kidney were subjected to HPLC using the heptafluorbutyric acid-acetonitrile solvent system as described ( 10 ). Following HPLC separation, the ANG II content of each fraction was determined by ANG II RIA.

Isolation of nuclei and plasma membrane. The frozen tissue was placed in homogenization buffer (20 mM Tricine-KOH, 25 mM sucrose, 25 mM KCl, 5 mM MgCl 2, pH 7.8) and homogenized with a Polytron Ultraturrax T25 Basic (setting 4) for 40 s on ice followed by a dounce homogenizer (Barnant Mixer Series 10, setting 3) and passed through a 100-µm mesh filter ( 25 ). The homogenate was centrifuged twice at 1,000 g at 4°C for 10 min to obtain the nuclear pellet. The supernatant fraction was centrifuged at 25,000 g for 20 min at 4°C to obtain the plasma membrane fraction.

Density gradient separation. Renal nuclei were also isolated from the cortex by isosmotic density gradient separation ( 21 ). For this procedure, the renal cortex was homogenized in the Tricine buffer described above and centrifuged at 1,000 g for 10 min at 4°C. The pellet was resuspended in 20% OptiPrep media (Accurate Chemical and Scientific, Westbury, NY) and layered on a density gradient medium. The gradient consisted of the 10, 20, 25, 30, and 35% OptiPrep media diluted in buffer B containing 150 mM KCl, 30 mM MgCl 2, 120 mM Tricine-KOH, pH 7.8, in a total volume of 13 ml. The gradient was centrifuged at 10,000 g for 20 min at 4°C, and the isolated nuclei were obtained at the 30-35% interface.
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Receptor binding studies. The ANG II binding assay utilized the radioligand 125 I-[Sar 1,Thre 8 ]ANG II ( 125 I-sarthran), as described by Chappell et al. ( 14 ). Sarthran was iodinated using chloramine T and purified by HPLC to a specific 2,000 Cu/mmol. The binding data were analyzed with the GraphPad Prism 4 statistical and graphics program (San Diego, CA).# X' z, V. E5 D! q3 r3 A1 {7 I

Immunoblot analysis. Cellular fractions were boiled in PBS (pH 7.4), Laemmli with -mercaptoethanol solution. Proteins were separated on 10% SDS polyacrylamide gels for 1 h at 120 V in Tris-glycine SDS, transferred onto polyvinylidene difluoride membranes, and subsequently blocked for 1 h with 5% Bio-Rad Dry Milk and TBS with Tween before incubation with primary antibodies against annexin II (dilution 1:2,500, BD Transduction Laboratories, San Diego, Ca); GMP130 (1:250, BD Transduction Laboratories); nucleoporin (Nup93, 1:1,000, BD PharMingen, San Diego, Ca); and AT 1 (1:1,000, Alpha Diagnostics, San Antonio, TX). Immunoblots were then resolved with Pierce Super Signal West Pico Chemiluminescent substrates as described by the manufacturer and exposed to Amersham Hyperfilm enhanced chemiluminescence (Piscataway, NJ)." Q+ x8 ~9 G& }6 H! ]5 X' T

Statistical analysis. Data are represented as means ± SE. Interstrain comparisons used a paired Student's t -test. Between-strain comparisons utilized an unpaired Student's t -test with GraphPad Prism 4.0 plotting and statistical software. To quantify the percentage of competition for specific receptor subtypes and peptide ratios, one-way ANOVA with Tukey's multiple comparison posttest was used for the data in each renal cellular compartment. The minimum statistical significance was reached at P 1 Y; S# ?3 d5 D; T
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Blood pressure. We determined the systolic blood pressure by tail-cuff methods in separate groups of male Lewis and mRen2.Lewis rats. Systolic blood pressure was markedly higher in the congenic rats compared with the normotensive Lewis rats (194 ± 2.0 vs. 107 ± 1.8 mmHg, P

Plasma angiotensin peptides. We assessed plasma peptide concentrations in the arterial blood of both strains. As shown in Fig. 1 A, there was no difference in plasma ANG II between the mRen2.Lewis and Lewis rats. However, ANG I was lower in the congenic rats (83 ± 14 vs. 139 ± 14 pM, P 0.05, n = 5), whereas the circulating level of ANG-(1-7) was 270% higher in the hypertensive strain (126 ± 10 vs. 46 ± 2 pM, P 0.05, n = 5). In Fig. 1 B, we express these plasma values as the ratio of ANG II or ANG-(1-7) to its potential immediate precursor(s). In this case, the mRen2.Lewis ANG-(1-7)/ANG II and ANG-(1-7)/ANG I were 250 and 300% higher, respectively, compared with those for Lewis rats.
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Fig. 1. Circulating angiotensin peptides and peptide ratios in mRen2.Lewis and Lewis rats. A : plasma angiotensins (ANG II), ANG I, and ANG-(1-7) were measured by separate radioimmunoassays and expressed as pM. B : plasma peptide ratios for ANG II/ANG I (AII/AI), ANG-(1-7)/ANG II (A7/AII), and ANG-(1-7)/ANG I (A7/AI). Values are means ± SE. * P

Renal angiotensin peptides. As shown in Fig. 2 A, ANG II content was 60% higher in the mRen2.Lewis rats than the control group in both cortical ( P 0.05, n = 5-6) and medullary tissue ( P 0.05, n = 5-6). Despite the overall higher levels of ANG II in the congenic rats, the peptide concentration was greater in the renal medulla vs. cortex for both the mRen2.Lewis and Lewis strains (160 and 150%, respectively, P 0.05). ANG II immunoreactivity was verified in the remaining extracts pooled from either the cortical or medullary tissues of the mRen2.Lewis kidney by HPLC on a NovaPak C 18 column with subsequent analysis by ANG II RIA; the chromatographs reveal that ANG II was the sole immunoreactive component in both cortical and medullary tissue extracts ( Fig. 3, B and D). In contrast to ANG II, ANG I was significantly lower in the congenic cortex ( P 0.05, n = 6) and medulla ( P 0.05, n = 6). ANG I content was significantly higher in the medullary vs. cortical tissue (280%, P 0.05) of the Lewis, but not the mRen2.Lewis rats, as ANG I levels were markedly lower in both regions. In contrast to either ANG II or ANG I, ANG-(1-7) levels were not different between strains in either cortex ( n = 6) or medulla ( n = 6). As observed for ANG II and ANG I, the renal medulla exhibited significantly higher levels of ANG-(1-7) than the cortex in the Lewis and congenic strains (360 and 430%, respectively, P 0.05). The tissue data were also expressed for the peptide ratios for each strain ( Fig. 3, A and C ) and ANG II/ANG I values for the congenic strain were 650 and 740% higher in the cortical and medullary tissues, respectively, with a trend toward an increase in the ANG-(1-7)ANG I value in both areas as well.

Fig. 2. Renal cortical and medullary angiotensins from mRen2.Lewis and Lewis rats. ANG II ( A ), ANG I ( B ), and ANG-(1-7) ( C ) were measured by separate radioimmunoassays and expressed as fmol/mg protein of tissue. Values are means ± SE. * P

Fig. 3. Comparison of the cortical ( A ) and medullary ( C ) renal angiotensin peptide ratios in the mRen2.Lewis and Lewis rats and HPLC analysis of ANG II content. Peptide ratios are expressed as in Fig. 1. Pooled extracts of cortex ( B ) or medullary ( D ) tissues from mRen2.Lewis rats were separated on a reverse-phase C 18 column, and the collected fractions were assessed by the ANG II radioimmunoassays. Values are means ± SE. * P % E6 K( ~! ~' a6 E8 U

Receptor binding of purified nuclei. Isolated nuclei from the renal cortex of the Lewis rats exhibited specific ANG II binding with the use of the nonselective antagonist 125 I-sarthran. For these studies, nuclei from renal cortex were isolated by differential centrifugation and density gradient separation with OptiPrep medium ( 21 ). Fractions taken from the gradient were enriched in nuclei (hematoxylin and eosin staining; data not shown) and used for receptor binding and immunoblot studies. In Fig. 4, we show the saturation curves and Scatchard analysis of the plasma membrane ( A and C ) and nuclear ( B and D ) fractions from the renal cortex of the Lewis kidney. Both sets of binding data yielded linear plots, suggesting a single population of binding sites; however, the data revealed a greater density of sites (B max ) for the nuclear fraction ( Fig. 4 D ). Characterization of the nuclear binding sites revealed that the AT 1 antagonist's losartan and candesartan competed to the same extent as ANG II ( Fig. 5 A ). The other antagonists selective for the AT 2 and ANG-(1-7) receptors did not significantly displace 125 I-sarthran binding from the cortical nuclei. Competition curves with ANG II, ANG III, and ANG-(1-7) yielded IC 50 values of 3, 5, and 400 nM, respectively, which are typical for binding to an AT 1 receptor ( Fig. 5 B ). Immunoblots on the nuclear and plasma membrane fractions revealed a single band at 52 kDa for the AT 1 receptor in the purified nuclei and plasma membrane fractions, consistent with the binding results in these fractions ( Fig. 6 A ). The antibody to the specific nuclear marker Nup93, a nuclear pore protein, revealed a 93-kDa band for the nuclei but no immunoreactive band in the plasma membrane fraction. In contrast, immunoreactive bands for annexin II (33 kDa, endosomal marker) and GMP 130 (133 kDa, Golgi marker) were evident in the plasma membrane but not the nuclear fraction.- c! n; k5 A2 t! ~' T& `  N) c
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Fig. 4. Representative saturation binding for ANG II receptors in the plasma membrane ( A and C ) and nuclear fraction ( B and D ) obtained from OptiPrep density gradient separation of renal cortex. Saturation binding and Scatchard analysis were performed with increasing concentrations of the specific receptor antagonist 125 I-[Sar 1,Thr 8 ]-ANG II (sarthran). Nonspecific binding was obtained in the presence of 10 µM unlabeled sarthran. The receptor density or number of binding sites and affinity are defined as B max and K D, respectively. Bd, bound.

Fig. 5. Characterization of the receptor subtype in isolated nuclei by OptiPrep density gradient. The nuclear fraction was obtained from the renal cortex of Lewis rats. Competition studies with antagonists ( A ) or agonists ( B ) utilized 0.5 nM 125 I-sarthran. The antagonists included losartan (LOS), candesartan (CV), PD-123319 (PD), D -[Ala 7 ]ANG-(1-7) ( D -ALA), all used at a final concentration of 10 µM. Values are means ± SE. * P , X' ]5 [/ e9 |" |# o* @

Fig. 6. Immunoblot analysis of the AT 1 receptor and cellular markers from the nuclear fractions obtained by either OptiPrep density gradient or differential centrifugation. Antibodies against the AT 1 receptor (52 kDa), nucleoporin (93 kDa), annexin II (33 kDa), and Golgi membrane protein (GMP130; 133 kDa) characterized the nuclear (N) and plasma membrane (P) fractions. Shown are representative full-length gels from 1 of 3 density gradient preparations (Lewis renal cortex; A ) and from 3 rats (mRen2.Lewis renal medulla; B ) by differential centrifugation.
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Plasma membrane vs. nuclear ANG II receptors. Although use of the OptiPrep density medium yielded an enriched nuclear fraction that exhibited high specific binding, this method proved laborious to determine receptor kinetics in multiple fractions from a large group of animals. Therefore, we determined whether differential centrifugation alone would yield an enriched nuclear fraction with similar binding characteristics as that obtained with the density gradient method. As shown in Fig. 7, the saturation binding for renal plasma membrane ( A and C ) and nuclear ( B and D ) fractions from the congenic renal medulla yielded data consistent with a high affinity binding site. As observed for the nuclei obtained by the density gradient method, both AT 1 antagonists essentially abolished the sarthran binding ( Fig. 8 ). Finally, the AT 1 and Nup93 antibodies yielded predominant immunoreactive bands of 52 and 93 kDa, respectively, from the nuclear fraction obtained by the differential centrifugation (see Fig. 6 B ). As these results were quite similar for the nuclei obtained by OptiPrep density medium, potential differences between the congenic mRen2.Lewis and Lewis rats utilized differential centrifugation to obtain the nuclear fraction. In both strains, comparison of the cortical receptor density revealed that the nuclear fraction exhibited a significantly higher B max than that of the plasma membrane fraction ( Fig. 9 A ). However, the congenic nuclear and plasma membrane B max was markedly less than that of Lewis rats ( P 0.05, n = 4/group). The K D was significantly lower in the congenic nuclei compared with Lewis rats ( P 0.05, n = 4). Differences in the K D in the plasma membrane component between the congenic and Lewis rats did not reach a statistical difference. In contrast to the cortex, the B max data for the nuclei were similar to the plasma membrane fraction in medullary tissue ( Fig. 9 B ). There were no differences in nuclear receptor density between the congenic and Lewis rats; however, the plasma membrane of the congenic exhibited a lower density than that of the Lewis rats ( P 0.05, n = 4). Regarding the K D values, the Lewis rats exhibited a lower K D in the nuclear vs. plasma membrane fraction. The congenic rats exhibited a lower K D for the plasma membrane receptor than the Lewis rats ( P 0.05, n = 4) with no difference in the nuclear K D values between congenic and Lewis animals.

Fig. 7. Representative saturation binding for ANG II receptors in plasma membrane ( A and C ) and nuclear fractions ( B and D ) obtained from the density gradient separation of the renal medulla. Saturation binding and Scatchard analysis were performed with increasing concentrations of the specific receptor antagonist sarthran. Nonspecific binding was obtained in the presence of 10 µM unlabeled sarthran.. t2 z. @+ h2 s1 c/ v$ V* |

Fig. 8. Characterization of renal angiotensin receptor subtype in isolated nuclei and the plasma membrane fractions obtained by differential centrifugation of the mRen2.Lewis rat. Competition studies with receptor antagonists utilized 0.5 nM 125 I-sarthran. The antagonists are as shown in Fig. 5 and were all used at a final concentration of 10 µM. Values are means ± SE. * P - o3 o' \* ?. U. O

Fig. 9. Comparison of receptor density and binding affinity for ANG II receptors in the nuclear and plasma membrane fractions in the renal cortex and renal medulla for mRen2.Lewis and Lewis rats. A and B : B max in the nuclear (Nuc) and plasma membrane (PM) fractions in cortex and medulla obtained by differential centrifugation. C and D : differences in the K D in the Nuc and PM fractions. Values are means ± SE. * P
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DISCUSSION
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In the current study, we characterized a new congenic model of hypertension and demonstrate an altered renal expression of tissue angiotensins and intercellular AT 1 receptors in the cortical and medullary areas of the kidney. Specifically, we show that both cortical and medullary levels of ANG II are markedly higher in adult male mRen2.Lewis rats with established hypertension, whereas the immediate precursor ANG I is substantially lower. We also demonstrate the predominant expression of AT 1 receptors in the nuclear fraction vs. the plasma membrane isolated from the renal cortex of both the hypertensive and normotensive Lewis strains; however, the density of these nuclear sites was significantly lower in the hypertensive strain. Although AT 1 receptor density values in the nuclear and plasma membrane fractions from the renal medulla of both strains were similar, the plasma membrane sites were also lower in the mRen2.Lewis strain. Indeed, the reduced receptor expression in both cortical and medullary areas of kidneys from the mRen2.Lewis rats may reflect a compensatory response to dampen the high intrarenal content of ANG II and the sustained increase in blood pressure.. a5 S5 Z& V9 S2 ]

Circulating angiotensins. The mRen2.Lewis strain was established from the backcross of the outbred (Ren2)27 strain originally developed by Mullins and colleagues ( 40 ) in Sprague-Dawley rats into the inbred Lewis line across nine generations. As previously documented, congenic mRen2.Lewis rats exhibit gender-dependent differences in the extent of hypertension that is at least partially dependent on the expression of ovarian hormones ( 11, 16 ). Moreover, the hypertension in the female mRen2.Lewis rat is corrected by blockade of the RAS or estrogen replacement ( 11 ). In the adult mRen2.Lewis 190 mmHg), the circulating levels of ANG II were similar to the Lewis strain; however, plasma ANG-(1-7) was elevated approximately threefold and ANG I levels were significantly lower. In male heterozygous (Ren2)27 rats, the plasma ANG II levels were either unchanged or reduced ( 31, 32, 39 ), but both male and female homozygous (Ren2)27 rats exhibit higher ANG II compared with the Sprague-Dawley strain ( 8, 51 ). The extent to which plasma ANG II contributes to the mRen2.Lewis rat is not known, although the sustained levels of circulating ANG II are clearly inappropriate given the elevated blood pressure. The increased ANG-(1-7)/ANG I in the mRen2.Lewis strain may reflect the enhanced conversion of circulating ANG I to ANG-(1-7) as a compensatory mechanism for the elevated blood pressures. This pathway may serve to both increase ANG-(1-7) and prevent greater conversion of ANG I to ANG II. The potential enzymes that may contribute to formation of circulating ANG-(1-7) from ANG I include neprilysin and thimet oligopeptidase ( 2, 9, 12 ). Although the greater levels of ANG-(1-7) in the circulation as measured under the present conditions may provide protection in the mRen2.Lewis strain, additional studies that utilize an ANG-(1-7) antagonist or inhibitor to block the peptide's production are required. Our previous studies in (Ren2)27 rats maintained on a salt-restricted diet revealed that blockade with the ANG-(1-7) antagonist D -[ Ala 7 ]ANG-(1-7) or peptide sequestration by a monoclonal antibody leads to an increase in blood pressure, suggesting a modulatory role for ANG-(1-7) in the setting of an activated RAS ( 13 ).& _2 B7 _$ Z5 v# j) f3 w

Intrarenal angiotensins. In contrast to the circulation, we found that tissue ANG II levels were significantly increased in both the cortex and medulla of the mRen2.Lewis kidney. In this case, the marked reduction in cortical ANG I of the hypertensive strain suggests an enhanced pathway for ANG I conversion to increase ANG II and maintain ANG-(1-7) concentrations. Indeed, the ANG II/ANG I value was increased about eightfold in the cortex and medulla of the mRen2.Lewis strain. Our results are consistent with previous studies in both homozygous and heterozygous (Ren2)27 rats that demonstrate increased intrarenal levels of ANG II ( 8, 39, 51 ). Although the mRen2.Lewis rat is a genetic model of enhanced renin gene expression, the present data suggest that enzymatic pathways other than renin may contribute to the sustained increase in renal ANG II. The identity of this pathway is not known, and studies are in progress to determine the status of angiotensin-converting enzyme (ACE) and renin expression in the kidney of the mRen2.Lewis rat, as well as other enzymes such as ACE2 and neprilysin that may participate in the degradation of ANG II ( 9, 15 ). In this regard, other hypertensive models including chronic ANG II infusion and two-kidney, one-clip Goldblatt hypertension also exhibit increased renal ACE activity ( 56 ). That medullary ANG I levels were reduced to a greater extent (5-fold) than the corresponding increase in ANG II (1.5-fold) may suggest alternative routes of ANG I metabolism that do not directly lead to ANG II or ANG-(1-7). Indeed, Li and colleagues ( 33 ) recently demonstrated that ACE2 participates in the formation of ANG-(1-9) from exogenous ANG I in isolated and perfused proximal tubules. Although ANG II levels were increased in both regions of the congenic strain, the overall tissue concentration of ANG II, as well as of ANG I and ANG-(1-7), was significantly higher in the medulla. The findings of a higher medullary angiotensin content differ from the study by Ingert et al. ( 27 ) that demonstrated equivalent levels in Wistar-Kyoto rats but is consistent with higher medullary ANG II content in the Sprague-Dawley strain ( 41 ). Renin and angiotensinogen are primarily found in cortical juxtaglomerular cells and proximal tubules, respectively, which may contribute to cortical ANG II ( 26, 30, 50 ). Their role in the formation of medullary ANG II is not established, although renin is expressed in collecting duct cells ( 42 ). A significant portion of renal ANG II may arise from AT 1 receptor-mediated internalization of the peptide ( 41, 55, 61 ), but this will not account for tissue levels of ANG I and may not for ANG-(1-7). Furthermore, the higher medullary ANG II content is at variance with the present results of an increased density of AT 1 sites in the cortex vs. the medulla. Regardless of the origin, the greater content of ANG II in the medulla may be particularly significant with regard to the peptide's influence on oxidative stress and medullary blood flow ( 21, 43, 57, 58 ). The ANG II-AT 1 axis is a key factor in the regulation of NADPH oxidase, the production of oxidative radicals, and the progression of hypertension, inflammation, and renal injury ( 29, 47, 57 ); the assessment of these indexes in the mRen2.Lewis strain is in progress.

Intrarenal AT 1 receptors. In addition to the expression of tissue angiotensin peptides, equally important components of the intrarenal RAS are the corresponding receptor levels. Characterization of ANG II receptors is not straightforward given the extent of cellular heterogeneity within the kidney that typically necessitates the use of autoradiographic methods. Previous studies have identified nuclear ANG II receptors in the liver; however, the density of nuclear sites constituted a minor population compared with the plasma membrane fraction in hepatocytes ( 7, 28 ). Consistent with the study by Licea et al. ( 34 ), we found a significant population of AT 1 sites in the nuclear vs. plasma membrane fractions. Immunoblots using an AT 1 antibody revealed a single band of 52 kDa in both fractions, suggesting that the nuclear AT 1 receptor is not an immature or nonglycosylated form of the protein. For both the nuclear and plasma membrane fractions, the binding to 125 I-sarthan was essentially abolished by the AT 1 antagonists losartan and candesartan. In general, the cortex exhibited a higher density of nuclear sites vs. the plasma membrane, whereas the receptor density was similar for both fractions, albeit lower in the medulla. Thus the overall trend was for reduced expression of AT 1 sites in the kidney of the mRen2.Lewis strain. Our data contrast with those of Zhou et al. ( 59 ), which demonstrated increased renal AT 1 sites in the glomerular, proximal tubular, and inner stripe regions of the (Ren2)27 strain by in vitro autoradiography; however, homozygous transgenics were studied at an earlier age (12 wk) and the intrarenal status of ANG II was not determined. Furthermore, film autoradiography lacks the resolution to distinguish nuclear vs. plasma membrane receptor sites. Because ANG II exhibits distinct cell-specific regulation of renal AT 1 receptors ( 24 ), we are currently assessing the cellular localization of the nuclear AT 1 receptor in mRen2.Lewis and Lewis kidneys., C$ c4 ~5 X! C2 e
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The functional significance of nuclear AT 1 receptors within the kidney was not specifically addressed in the present study. Following binding, the AT 1 receptor undergoes rapid internalization and a portion of the receptor complex may traffic to the nucleus before recycling back to the plasma membrane or undergo degradation ( 5, 17, 22, 25, 48, 52 - 55 ). Lu et al. ( 36 ) blocked trafficking of the internalized ANG II-AT 1 receptor complex in neuronal cultures with a decoy peptide against the nuclear localization sequence of the receptor and the subsequent phosphorylation of the nuclear pore protein p62. In the kidney, the high density of AT 1 sites in the nuclear fraction may reflect substantial internalization of the receptor complex subsequent to ANG II binding. Several reports suggest that AT 1 internalization is required for activation of ANG II-dependent signaling pathways, particularly in the proximal tubule; however, the participation of the nuclear receptor in these events is not known ( 5, 22, 28, 49, 52, 60 ). In the current study, the AT 1 density in the nuclear fraction of the congenic strain was reduced despite higher tissue levels of ANG II and sustained plasma ANG II. Moreover, the density of the cortical nuclear ANG II sites was unchanged following a chronic infusion of ANG II that markedly increased blood pressure ( 34 ). Preliminary data in tissue ACE knockout mice that have markedly depleted intrarenal ANG II ( 38 ) also revealed no differences in nuclear AT 1 receptor density between the wild-type and knockout mice ( 44 ). Eggena and colleagues ( 20 ) have shown that ANG II stimulates the levels of both renin and angiotensinogen mRNA in isolated nuclei of hepatocytes. Nuclear ANG II receptors in the proximal tubule may be linked to the regulation of angiotensinogen and other RAS components that contribute to the local expression of this system. Thus the downregulation of nuclear AT 1 sites in the kidney may reflect a mechanism to dampen expression of intrarenal RAS in hypertensive mRen2.Lewis rats.0 H; q/ a! Q, K  x

Perspective. Despite numerous investigations of the renal RAS and the development and progression of hypertension, new genetic models are key in revealing novel aspects of this complex peptide system. The mRen2.Lewis strain represents a unique congenic model of monogenetic ANG II-dependent hypertension. The relevance of the mRen2.Lewis strain lies not in the origin of the hypertension in this strain but in the adaptive responses of the RAS components and downstream systems that mediate the sustained increased in blood pressure and tissue injury in adult or aged animals. Our studies in the mRen2.Lewis strain strengthen the concept of an intracellular or "intracrine" RAS ( 45 ) and, perhaps more importantly, suggest that regulation of this system is clearly evident. Blockade of the RAS increasingly constitutes the first line of treatment for hypertension and renal injury; the presence of an intracellular RAS within the kidney raises the issue of whether we are effectively or completely targeting the relevant system with these therapies.4 L1 F& [  Z9 `- R

GRANTS
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This work represents partial fulfillment of the requirements for the degree of Doctorate of Philosophy in the Department of Physiology and Pharmacology at Wake Forest University School of Medicine and is supported by grants from the National Heart Lung and Blood Institute (HL-51952, HL-56973, HL-56973-S1, HL-07790, GM64249) and unrestricted grants from Unifi, Inc., Greensboro, NC, and the Farley-Hudson Foundation, Jacksonville, NC.: Q2 }' x9 v* y2 K

ACKNOWLEDGMENTS  `9 y5 q- w* f% p) e: p  t
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We are grateful to Takeda Chemical Industries for the generous donation of candesartan (CV-11974) and to Merck for losartan.
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Portions of this work were presented at the Experimental Biology 2005 meeting (San Diego, CA, April 2-6, 2005).1 c/ z8 n9 \1 Q0 e( Y3 {
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