Am J Physiol Cell Physiol AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 294: C1034-C1045, 2008. First published February 6, 2008; doi:10.1152/ajpcell.00432.2007
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/C1034    most recent
00432.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X. C.
Right arrow Articles by Zhuo, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X. C.
Right arrow Articles by Zhuo, J. L.

RECEPTORS AND SIGNAL TRANSDUCTION

Intracellular ANG II directly induces in vitro transcription of TGF-β1, MCP-1, and NHE-3 mRNAs in isolated rat renal cortical nuclei via activation of nuclear AT1a receptors

Xiao C. Li1 and Jia L. Zhuo1,2

1Laboratory of Receptor and Signal Transduction, Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit; and 2Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan

Submitted 18 September 2007 ; accepted in final form 1 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study tested the hypothesis that intracellular ANG II directly induces transcriptional effects by stimulating AT1a receptors in the nucleus of rat renal cortical cells. Intact nuclei were freshly isolated from the rat renal cortex, and transcriptional responses to ANG II were studied using in vitro RNA transcription assays and semiquantitative RT-PCR. High-power phase-contrast micrographs showed that isolated nuclei were encircled by an intact nuclear envelope and stained strongly by the DNA marker 4',6-diamidino-2-phenylindole, but not by the membrane or endosomal markers. Fluorescein isothiocyanate-labeled ANG II and [125I]Val5-ANG II binding confirmed the presence of ANG II receptors in the nuclei with a predominance of AT1 receptors. RT-PCR showed that AT1a mRNA expression was threefold greater than AT1b receptor mRNAs in these nuclei. In freshly isolated nuclei, ANG II increased in vitro [{alpha}-32P]CTP incorporation in a concentration-dependent manner, and the effect was confirmed by autoradiography and RNA electrophoresis. ANG II markedly increased in vitro transcription of mRNAs for transforming growth factor-β1 by 143% (P < 0.01), macrophage chemoattractant protein-1 by 89% (P < 0.01), and the sodium and hydrogen exchanger-3 by 110% (P < 0.01). These transcriptional effects of ANG II on the nuclei were completely blocked by the AT1 receptor antagonist losartan (P < 0.01). By contrast, ANG II had no effects on transcription of angiotensinogen and glyceraldehyde-3-phosphate dehydrogenase mRNAs. Because these transcriptional effects of ANG II in isolated nuclei were induced by ANG II in the absence of cell surface receptor-mediated signaling and completely blocked by losartan, we concluded that ANG II may directly stimulate nuclear AT1a receptors to induce transcriptional responses that are associated with tubular epithelial sodium transport, cellular growth and hypertrophy, and proinflammatory cytokines.

angiotensin II; in vitro transcription; kidney; nucleus; reverse transcription-polymerase chain reaction; sodium transport

IT IS NOW WELL ACCEPTED that endocrine and paracrine ANG II activate intracellular signaling pathways and induce diverse biological effects via binding to its cell surface G protein-coupled receptors (9, 39, 51). Evidence supporting the endocrine and paracrine roles of ANG II in cardiovascular and renal regulation is overwhelming (9, 39, 51). Indeed, there is little doubt that this mode of ANG II-induced responses plays an essential role in the physiological regulation of blood pressure, cardiovascular homeostasis, and renal function. In physiological settings, however, binding of ANG II to its cell surface receptors evokes two classical responses, namely rapid activation of intracellular signaling pathways and simultaneous initiation of type 1 (AT1) receptor-mediated endocytosis (or internalization) of ANG II (9, 15, 21, 49, 51). The latter response is thought to desensitize the cellular responses to ANG II by moving extracellular ANG II into the cells for degradation, with internalized receptors being recycled back to the cell surface after dissociation from the agonist (15, 21, 51). There is evidence that not all internalized ANG II peptides are directed to lysosomes for degradation, and some of them may be trafficked to other organelles or the nucleus (3, 6, 38, 41).

ANG II is widely implicated in the development of hypertension and several progressive renal diseases by causing salt and fluid retention and inducing growth and proinflammatory responses (23, 42, 45, 54). For example, ANG II has been shown to induce expression of the epithelial sodium channel (1), the sodium and hydrogen exchanger-3 (NHE-3) (12, 34, 40), and protooncogenes, growth factors, and hypertrophic marker genes (28, 42, 50, 54). These growth-promoting and proliferative effects of ANG II are thought to be primarily due to activation of cell surface receptors by extracellular ANG II. However, extracellular ANG II is well recognized to internalize with its receptors as a source of intracellular ANG II in vascular smooth muscle cells (VSMCs) (18, 46, 49) and renal proximal tubule cells after binding to cell surface AT1 receptors (21, 32, 34, 46, 48, 51). Moreover, endosomal and/or nuclear ANG II receptors have been identified in the liver and kidney cells (2, 36, 43, 47). This suggests that internalized ANG II may be partly involved in ANG II-induced growth and proinflammatory responses in these tissues by activating cytoplasmic and nuclear ANG II receptors.

In the present study, we used freshly isolated, intact rat renal cortical nuclei to test the hypothesis that internalized or intracellular ANG II directly stimulates AT1a receptors to induce RNA synthesis and in vitro transcription of mRNAs for growth-promoting and proinflammatory cytokines, including angiotensinogen, transforming growth factor-β1 (TGF-β1), macrophage chemoattractant protein-1 (MCP-1), and NHE-3. We demonstrated that freshly isolated and intact nuclei bound predominantly AT1 (AT1a) receptors and that ANG II directly induced transcriptional responses of mRNAs for TGF-β1, MCP-1, and NHE-3, but not angiotensinogen, via stimulation of the AT1a receptor in the nucleus. Our results are consistent with an important role of internalized and/or intracellular ANG II and nuclear AT1a receptors in mediating growth and proinflammatory responses and sodium-retaining effects of ANG II in ANG II-dependent hypertensive renal diseases.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials Animals. Adult male Sprague-Dawley rats (200–250 g) were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a temperature-controlled room with a 12:12-h light-dark cycle. The animals were fed on a standard rodent chow and had free access to tap water (33, 52, 55). This study was approved by the Institutional Animal Care and Use Committee of Henry Ford Health System.

Chemicals. Heat-inactivated FBS was purchased from ATCC. Val5-ANG II and ANG II enzyme immunoassay kits were obtained from Biochem (Peninsula, CA) (32). Bicinchoninic acid (BCA) protein assay kits were bought from Pierce. The AT1 receptor antagonist losartan was a gift from Merck Pharmaceuticals, and the AT2 receptor antagonist PD-123319 was donated by Pfizer. All fluorescence-labeled peptides or markers, including fluorescein isothiocyanate (FITC)-labeled ANG II, the fluorescent nuclear marker 4',6-diamidino-2-phenylindole (DAPI), the fluorescent membrane-specific marker Oregon green 488 conjugate-labeled wheat germ agglutinin (WGA), and the fluorescent endosomal marker Alexa Fluor 594-labeled transferrin were obtained from Invitrogen (Molecular Probes). [{alpha}-32P]CTP was purchased from Amersham (specificity: 3,000 Ci/mmol). [125I]Val5-ANG II was radioiodinated and HPLC-purified by Dr. Robert Speth of The University of Mississippi Peptide Radioiodination Service Center (specificity: 2,175 Ci/mmol) (31, 33). The in vitro SP6/T7 RNA transcription system (transcription optimized buffer, dithiothreitol, rATP, rGTP, rUTP, rCTP, recombinant RNasin ribonuclease inhibitor, SP6 RNA polymerase, and T7 RNA polymerase) was purchased from Promega. Tri-Reagent was purchased from Molecular Research Center. Forward (or sense) and reverse (or antisense) primers for RT-PCR of mRNAs for AT1a and AT1b receptors (24), angiotensinogen (27), TGF-β1 (25, 50), MCP-1 (5), NHE-3 (29), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5) were synthesized by Invitrogen.

Isolation of Fresh Intact Cortical Nuclei from the Rat Kidney Fresh intact nuclei were isolated and purified from the cortex of the rat kidneys using a modification of the method as described by others for preparing the nuclei or nuclear extracts from the rat livers (2, 47). Briefly, Sprague-Dawley rats were killed by decapitation, and the kidneys were removed quickly. Unless specified elsewhere, all isolation procedures were performed on ice or at 4°C. After the capsule was removed, the kidneys were rinsed with ice-cold isolation buffer A containing 320 mM sucrose, 3 mM MgCl2, and 20 mM Tris, pH 7.4. The kidneys were cut longitudinally in half with a scalpel blade, and the entire medulla (inner and outer) was removed with a fine scissor so that intact nuclei were isolated only from the renal cortex. The renal cortex of each kidney was then chopped into fine pieces, scooped into a Sorvall centrifuge tube in 15 ml buffer A, and incubated on ice for 15 min. The tissues were then homogenized using a glass homogenizer and motor-driven Teflon pestle. The homogenates were filtered through a 50-µm metal sieve, washed with buffer A if necessary, and centrifuged for 15 min at 1,000 g using a Beckman benchtop centrifuge at 4°C. The supernatants were removed, and the pellets were resuspended by gentle homogenization with a P5000 pipette in ~30 ml of buffer B (2.2 M sucrose, 1 mM MgCl2, and 10 mM Tris, pH 7.4). The resuspended homogenates were differentially centrifuged for 60 min at 64,000 g using a Beckman swing out rotor at 4°C. After centrifugation, the supernatants were removed and cleared before isolated nuclei were suspended and washed two times by centrifugation in 2 ml of buffer A (2, 47).

Measurements of Rat Renal Cortical Nuclear Protein and DNA Contents Protein contents in isolated rat renal cortical nuclei were measured using a BCA protein assay kit (Pierce), as described (13). Nuclear DNA concentrations were determined by a fluorospectrometer (13). Furthermore, isolated nuclei were also stained with the fluorescent nuclear acid marker DAPI (300 nM in PBS), placed on glass cover slips, and examined using a Nikon Eclipse TE2000-U inverted fluorescence microscope coupled with a Lambda DG4 illumination system (Sutter Instruments) to confirm the purity of the nuclei, as described (32, 34).

Visualization of the Integrity of Freshly Isolated Rat Renal Cortical Nuclei The integrity of the nuclear structural and functional machinery is important for studying nuclear binding of [125I]Val5-ANG II or FITC-ANG II and the transcriptional responses to ANG II as described below. Immediately after isolation and purification procedures, the nuclei were visualized without fixation on a phase-contrast microscope at low (x10) and high (x100) magnifications, as described (47). Special attention was focused on whether the nuclear envelopes or membranes remain intact in isolated nuclei after purification. To exclude the possibility of potential contamination by plasma membranes or endosomal organelles, isolated nuclei were stained with Oregon green 488 conjugate-labeled WGA (100 µg/ml) or Alexa Fluor 594-labeled transferrin (100 µg/ml) for 30 min at 22°C. Oregon green 488 conjugate-labeled WGA primarily stains plasma membranes, whereas Alexa Fluor 594-labeled transferrin stains endosomal organelles (32, 55). Unstained fluorescent markers were removed by two washes with PBS, and the nuclei were placed on glass cover slips and examined using a Nikon Eclipse TE2000-U inverted fluorescence microscope (32, 34).

Characterization of AT1 and AT2 Receptors in Isolated Rat Renal Cortical Nuclei Localization of AT1 and AT2 receptor binding in isolated nuclei using FITC-labeled ANG II. To colocalize ANG II receptor binding in freshly isolated rat renal cortical nuclei, 10 µg of intact nuclei were placed in each well of four-well Lab-Tek II chamber slides (Nalge Nunc, Naperville, IL) and incubated in 500 µl of 50 mM Tris buffer containing 1 nM FITC-ANG II (Molecular Probes), 120 mM NaCl, 4 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 10 µg/ml bacitracin, and 2 mg/ml D-glucose, pH 7.5, for 60 min at 22°C (32, 34). Nonspecific binding, AT1 receptor binding, and AT2 receptor binding were determined as described below for [125I]Val5-ANG II receptor binding (31, 32, 53). After incubation, the nuclei were gently washed in a fresh volume of the same buffer without FITC-ANG II two times before being counterstained with DAPI (300 nM) for 5 min at 22°C for nuclear localization. The FITC- and ANG II-labeled nuclei were examined by a Nikon Eclipse TE 2000-U fluorescence microscope. Fluorescent images of FITC-labeled ANG II (green) and DAPI (blue) in the same nuclei were captured and analyzed using the MetaMorph image system (Universal Image) with filters suitable for FITC (488-nm wavelength) or DAPI (~500 nm) (32, 34, 56).

Characterization of AT1 and AT2 receptor binding in isolated nuclei using [125I]Val5-ANG II radioreceptor binding assays. To characterize pharmacological properties of AT1 and AT2 receptors in isolated renal cortical nuclei, we used [125I]Val1-ANG II as the radioligand for ANG II receptor binding assays as described previously (31, 32, 52, 53). Briefly, ANG II receptor binding affinity constant (Kd) and maximal binding sites (Bmax) were determined from saturation binding curves and Scatchard plot by incubating 100 µg of freshly isolated nuclei in 10 mM sodium phosphate buffer with increasing concentrations of [125I]Val5-ANG II alone (0–100 nM) (31, 32, 52, 53). Kd and Bmax were calculated using GraphPad Prism 4.0 (31–33). The specificities of AT1 and AT2 receptor binding in freshly isolated nuclei were determined by incubating 100 µg of isolated nuclei with [125I]Val5-ANG II (~100 pmol) for 60 min at 22°C in the presence, or absence, of increasing concentrations (0–100 µM) of unlabeled competing ANG II, the AT1 receptor-selective antagonist losartan, or the AT2 receptor-selective antagonist PD-123319, as described (31, 32, 52, 53). Total ANG II receptor binding was calculated as the binding in the absence of competing unlabeled ANG II or its receptor subtype-selective antagonists in the incubation. Nonspecific binding was determined as the binding in the presence of 10 µM competing unlabeled ANG II. AT1 receptor binding was determined in the presence of 10 µM competing unlabeled AT2 receptor blocker PD-123319, whereas AT2 receptor binding was calculated as the binding in the presence of 10 µM of the competing unlabeled AT1 blocker losartan (31, 32, 52, 53).

RT-PCR of AT1a and AT1b receptor mRNAs. Because the rat kidney expresses two subtypes of AT1 receptors, AT1a and AT1b, and neither [125I]Val5-ANG II nor FITC-labeled ANG II receptor binding assays can distinguish these two receptors, we used semiquantitative RT-PCR to determine AT1a and AT1b receptor mRNAs in isolated rat renal cortical nuclei, as described (24, 37). Briefly, total RNA was extracted from ~200 µg isolated rat renal cortical nuclei using the Tri-Reagent RNA isolation protocol (Molecular Research Center, Cincinnati, OH), and the quality of total RNA was verified by spectrophotometry with a ratio of the optical density at 260 to 280 nm of 1.9 or above for RT-PCR analyses. First-strand cDNA was synthesized from 1 µg total RNA using the SuperScript III-First Strand Synthesis System for RT-PCR kit (Invitrogen) in a final volume of 25 µl containing 25 mM MgCl2, 10 mM dNTP mixture, 10 µM sense and antisense primer mixers (AT1a or AT1b), 5 U/µl Tag DNA polymerase, and 2 µl cDNA. The sense and antisense primers for AT1a or AT1b receptor mRNA were described by Johren et al. (24) (see Table 1). RT-PCR amplification was performed as follows: 35 cycles of denaturation for 2 min at 94°C, annealing for 1 min at 58°C, and extension for 1 min and 15 s at 72°C. The PCR products were size fractionated on a 2% agarose gel and visualized with ethidium bromide staining and ultraviolet (UV) transillumination. RT-PCR of GAPDH mRNA was used as a housekeeping gene control (24, 37).


View this table:
[in this window]
[in a new window]

  		Table 1. Nucleotide sequences of forward (or sense) and reverse (or antisense) primers for semiquantitative RT-PCR of target gene mRNAs

 
Effects of ANG II on [{alpha}-32P]CTP Incorporation into RNA in Isolated Rat Renal Cortical Nuclei The effects of ANG II on total RNA synthesis in isolated rat renal cortical nuclei were studied using a standard in vitro [{alpha}-32P]CTP RNA transcription system (Promega), as described (13, 14). To determine the concentration and response relationship, 100 µg of freshly isolated nuclei were first stimulated by increasing concentrations of ANG II (0, 1.0, 10, and 100 nM) in the absence of the AT1 receptor antagonist losartan for 30 min at 37°C. ANG II-pretreated nuclei were then incubated with an in vitro SP6/T7 RNA transcription system consisting of 500 µM of ATP, GTP, and UTP; 250 µM CTP; and 2 U/µl RNasin at 37°C for 1 h, with 50 µCi [{alpha}-32P]CTP added as an index of in vitro CTP incorporation into RNA during RNA synthesis. To determine the role of AT1 receptors in mediating [{alpha}-32P]CTP RNA transcription responses to ANG II in isolated nuclei, 100 µg of nuclei were pretreated with ANG II (10 nM) in the presence of losartan for 30 min at 37°C, which was followed by incubations with the in vitro SP6/T7 RNA transcription system, as described above. The effect of PD-123319 was not examined because ANG II receptor binding assays detected little AT2 receptor binding in isolated nuclei. After incubation, RNA was extracted using TRI reagent according to the manufacturer's instructions (Molecular Research Center). The extracted RNA was revealed by 2% agarose gel electrophoresis and stained by ethidium bromide. To quantify newly transcribed RNA, RNA in the nuclear extract was precipitated with 10% TCA on glass fiber filters, washed, and counted in a scintillation counter (Beckman). The newly transcribed RNA on the filters was also exposed to X-ray film for 2 wk, and resultant autoradiographs were analyzed using a microcomputer imaging device (Imaging Research).

Effects of ANG II on In Vitro Transcription of mRNAs for Angiotensinogen, TGF-β1, MCP-1, and NHE-3 in Freshly Isolated Rat Renal Cortical Nuclei To determine whether intracellular ANG II stimulates nuclear ANG II receptors to specifically induce expression of mRNAs for angiotensinogen, TGF-β1, MCP-1, and NHE-3, the same in vitro SP6/T7 RNA transcription system (Promega) and RNA isolation procedures were followed, with the exception that 500 µM unlabeled CTP were used in place of [{alpha}-32P]CTP (13, 14). We first examined the concentration-dependent responses of TGF-β1 mRNA expression to ANG II (0, 1.0, 10, and 100 nM) in isolated nuclei to assist with determining the optimal ANG II concentration, which was used for studying the responses of other target genes (10 nM). Specifically, first-strand cDNA for each of above target genes was synthesized from 1 µg total RNA using the SuperScript III-First Strand Synthesis System for the RT-PCR kit (Invitrogen) in a final volume of 25 µl containing 25 mM MgCl2, 10 mM dNTP mixture, 10 µM of forward (or sense) and reverse (or antisense) primer mixers, 5 U/µl Taq DNA polymerase, and 1 µl cDNA. The sequences of forward (or sense) and reverse (or antisense) primers for angiotensinogen (27), TGF-β1 (25), MCP-1 (5), and NHE-3 (29) were chosen from the published literature (see Table 1) and synthesized by Invitrogen. RT-PCR amplification was performed as follows: 35 cycles of denaturation for 2 min at 94°C, annealing for 1 min at 58°C, and extension for 1 min and 15 s at 72°C. The PCR products were size fractionated on a 1.5% agarose gel and visualized with ethidium bromide staining and UV transillumination. mRNA levels were quantified by a micromputerized imaging system (Imaging Research) and normalized to the housekeeping GAPDH mRNA (5, 13).

Statistical Analysis All data are presented as means ± SE. Differences in the same parameters between groups of isolated nuclei or between different treatments were compared using one-way ANOVA or unpaired Student's t-test. The significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of Freshly Isolated Rat Renal Cortical Nuclei Although the presence of ANG II receptors in nuclear extracts or nuclear fractions of the rat kidney was reported previously (36, 43), the morphological and biochemical properties of freshly isolated rat renal cortical nuclei have not been characterized. Figure 1 shows some unique characteristics of freshly isolated nuclei from the rat renal cortex. At low magnification (x10), light microscopic imaging shows dense homogeneous round black particles, spreading throughout the microscopic field (Fig. 1A). At high magnification (x100), phase-contrast microscopic imaging reveals the integrity of the nuclei, with a fine layer of nuclear envelope encircling each nucleus (Fig. 1B). Furthermore, these freshly isolated nuclei were readily stained by DAPI, a standard marker of cellular nuclear acids (Fig. 1C; x40), but not by the membrane-specific fluorescent marker Oregon green 488 conjugate-labeled WGA (Fig. 1D; x40) or the endosomal fluorescent marker Alexa Fluor 594-labeled transferrin (Fig. 1E; x40). Finally, high concentrations of DNA were found in these freshly isolated nuclei, as expected (nucleus: 1,029 ± 88 µg/ml DNA vs. cytoplasmic supernatant: 30 ± 5 µg/ml DNA, P < 0.001) (Fig. 1F). Taken together, these data confirm that freshly isolated nuclei bear characteristic morphological and biochemical properties of intact rat renal cortical nuclei.


Figure 1
View larger version (59K):
[in this window]
[in a new window]

  		Fig. 1. Morphological and biochemical characteristics of freshly isolated rat renal cortical nuclei. A: light microscopic image of the nuclei, magnification x10. B: high-power phase-contrast image (x100) showing that the nuclei are encircled by an intact nuclear envelope (arrows). C: 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei (x40). D: Oregon green 488 conjugate-labeled wheat germ agglutinin (WGA) to stain contaminated plasma membranes (x40). E: Alexa Fluor 594-labeled transferrin to stain endosomal organelles (x40). F: high DNA concentrations indicate the purity of isolated nuclei. These are representative images from 3–5 different experiments. **P < 0.01 vs. the cytoplasm.

 
Localization and Characterization of AT1 and AT2 Receptors in Freshly Isolated Rat Renal Cortical Nuclei To determine whether AT1 and AT2 receptors are present in isolated rat renal cortical nuclei, three different approaches were used. Figure 2 shows in vitro fluorescence imaging of ANG II receptor subtypes in these freshly isolated rat renal cortical nuclei. The nuclei were strongly stained by FITC-labeled ANG II (Fig. 2A; total), which was completely colocalized with the nuclear acid marker DAPI (Fig. 2B), thus confirming the presence of ANG II receptor binding in isolated rat renal cortical nuclei (Fig. 2C). FITC-labeled ANG II receptor binding was largely displaced by the AT1 receptor antagonist losartan (10 µM; Fig. 2, D-F). In contrast, FITC-ANG II receptor binding was not blocked by the AT2 receptor antagonist PD-123319 (10 µM; Fig. 2, G-I). Further radioreceptor binding assays show that [125I]Val5-ANG II bound intact nuclei in a saturable manner (Fig. 3A). Scatchard analysis of [125I]Val5-ANG II binding revealed a single class of ANG II receptor binding sites with a Bmax of 708.5 ± 46.2 fmol/mg nuclear proteins and a Kd of 19.1 ± 3.3 nM (Fig. 3A). Unlabeled ANG II and losartan largely inhibited [125I]Val5-ANG II receptor binding, whereas PD-123319 had no significant effect (Fig. 3B). These data suggest that ANG II receptors in freshly isolated rat renal cortical nuclei are predominantly of the AT1 subtype.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 2. Localization and subtype specificity of ANG II receptors in freshly isolated rat renal cortical nuclei using fluorescein isothiocyanate (FITC)-labeled ANG II (see METHODS for details). A, D, and G: total FITC-ANG II receptor binding. B, E, and H: DAPI-labeled nucleic acids in the same isolated nuclei. C, F, and I: merged images of FITC-ANG II binding and DAPI-labeled nucleic acids in the same nucleus. J: levels of AT1 (> 90%) and AT2 receptor binding (< 10%) as %total FITC-ANG II binding. Magnification: x40. AU, arbitrary fluorescence intensity unit. **P < 0.01 vs. AT1 binding. These are representative images from 3 different experiments.

 

Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. [125I]Val5-ANG II receptor binding characteristics in freshly isolated rat renal cortical nuclei. A: the maximal binding capacity (Bmax) and binding dissociation constant (Kd) with the inset showing Scatchard analysis. B: ANG II receptor subtype specificity showing the predominance of AT1 receptor binding in isolated nuclei. **P < 0.01 vs. total binding. These data were determined from 3 different experiments.

 
RT-PCR of AT1a and AT1b Receptor mRNAs in Isolated Rat Renal Cortical Nuclei Figure 4 shows semiquantitative RT-PCR analyses of AT1a and AT1b receptor mRNA expression in four different nuclear samples, with GAPDH mRNA expression used as a housekeeping control gene. Based on the sense and antisense primers of AT1a and AT1b receptors used, 300 base pairs (bp) of PCR product were detected for AT1a receptor mRNA, whereas ~800 bp of PCR product were detected for AT1b receptor mRNA. The sizes of AT1a and AT1b PCR products were consistent with what were predicted from the sense and antisense primers (24, 37). AT1a receptor mRNA expression was about threefold higher than that of AT1b receptor mRNA when identical amplification cycles and exposure conditions were used (Fig. 4).


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Identification of AT1a and AT1b receptor mRNAs in isolated rat renal cortical nuclei using semiquantitative RT-PCR (see METHODS for details). M, DNA ladder. The levels of AT1a and AT1b receptor mRNAs were normalized by the housekeeping gene GAPDH mRNA (bottom). **P < 0.01 vs. AT1a mRNA. These are representative images from 3 different experiments.

 
Effects of ANG II on In Vitro [{alpha}-32P]CTP Incorporation into RNA in Freshly Isolated Rat Renal Cortical Nuclei The concentration-dependent responses of in vitro [{alpha}-32P]CTP incorporation in isolated nuclei are shown in Fig. 5A. ANG II induced [{alpha}-32P]CTP incorporation into RNA in a concentration-related manner. At the lower concentration (1.0 nM), ANG II increased [{alpha}-32P]CTP incorporation by >30% [control: 20,720 ± 1,712 counts·min–1 (cpm)·mg nuclear protein–1 vs. 1.0 nM: 28,840 ± 2,649 cpm/mg protein; not significant (NS)]. Higher concentrations of ANG II significantly increased [{alpha}-32P]CTP incorporation by ~150% at 10 nM (51,800 ± 10,596 cpm/mg protein; P < 0.05 vs. control) and ~90% at 100 nM (38,067 ± 6,661 cpm/mg protein; P < 0.05 vs. control). Figure 5B shows representative autoradiographic images of the effects of ANG II (10 nM) on in vitro [{alpha}-32P]CTP incorporation (middle, n = 4) compared with unstimulated nuclei (left, n = 4). The effects of ANG II on [{alpha}-32P]CTP incorporation were completely blocked by the AT1 receptor antagonist losartan (10 µM; right; n = 4). The quantitative results of these experiments were shown in Fig. 5C. [{alpha}-32P]CTP incorporation in nonstimulated nuclei was 32,250 ± 2,835 cpm/mg nuclear proteins. ANG II stimulation increased [{alpha}-32P]CTP incorporation by ~106% (66,440 ± 7,211 cpm/mg proteins, P < 0.01 vs. control nuclei). Blockade of nuclear AT1 receptors with losartan significantly attenuated ANG II-induced increases in [{alpha}-32P]CTP incorporation in the nuclei (39,000 ± 3,946 cpm/mg proteins; P < 0.01 vs. ANG II). Figure 6 further shows the quality of isolated RNA from control (lane 1), ANG II-stimulated (lane 2), and ANG II- plus losartan-treated nuclei (lane 3). As revealed by 2% agarose gel electrophoresis and stained by ethidium bromide, ANG II stimulation increased total RNA synthesis in the nuclei, and the effect was attenuated by losartan (Fig. 6).


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 5. AT1 receptors mediated intracellular ANG II-induced in vitro [{alpha}-32P]CTP incorporation into RNA in freshly isolated rat renal cortical nuclei. A: the concentration and effect relationship with a peak response at 10 nM of ANG II (n = 6–8 samples for each concentration). B: representative autoradiographs with transcriptional responses of four different nuclear samples to ANG II (10 nM) in the absence (middle) or presence (right) of losartan (10 µM). C: semiquantitative results on radioactivity from three different groups of nuclear samples (n = 6–8) of two repeated experiments. *P < 0.05 and **P < 0.01 vs. control nuclei. ++P < 0.01 vs. ANG II-treated nuclei.

 

Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 6. AT1 receptors mediated intracellular ANG II-induced RNA synthesis in freshly isolated rat renal cortical nuclei. A: representative agarose gel electrophoresis from three different experiments shows ethidium bromide-stained RNA extracted from control nuclei (lane 1), ANG II-stimulated nuclei (lane 2), and ANG II- plus losartan-treated nuclei (lane 3). Equal amounts of RNA (5 µg) from each sample or treatment were used. M, RNA ladder of 0.24–9.5 kb. B: semiquantitative results as arbitrary optical density (OD) units from 3 different experiments. **P < 0.01 vs. control nuclei. ++P < 0.01 vs. ANG II-treated nuclei.

 
Effects of ANG II on TGF-β1 mRNA Expression in Freshly Isolated Rat Renal Cortical Nuclei Stimulation of the growth factor such as TGF-β1 mRNA expression by ANG II plays an important role in ANG II-induced cellular growth and hypertrophy, and tissue fibrosis in the kidney (25, 50). Yet whether intracellular ANG II induces TGF-β1 mRNA expression via activation of nuclear AT1 receptors is not known. Figure 7 shows the concentration-related responses of TGF-β1 mRNA expression to ANG II in freshly isolated rat renal cortical nuclei. Figure 7, top, shows RT-PCR images of TGF-β1 mRNA and GAPDH mRNA, and Fig. 7, bottom, shows the semiquantitative results from three experiments. ANG II induced TGF-β1 mRNA expression in a concentration-dependent manner, with the maximal response observed at 10 nM of ANG II (control: 0.02 ± 0.01 vs. 10 nM ANG II: 0.62 ± 0.08 TGF-β1 mRNA/GAPDH mRNA ratio; P < 0.001). Although ANG II also significantly stimulated TGF-β1 mRNA expression in isolated nuclei at 1.0 nM (0.22 ± 0.04 TGF-β1 mRNA/GAPDH mRNA ratio; P < 0.05) or 100 nM (0.35 ± 0.04 TGF-β1 mRNA/GAPDH mRNA ratio; P < 0.01), the responses were not as great as those seen at 10 nM (Fig. 7). The role of nuclear AT1 receptors in mediating ANG II-induced TGF-β1 mRNA expression in isolated nuclei is shown in Fig. 8. At 10 nM, ANG II markedly increased TGF-β1 mRNA expression from 0.06 ± 0.01 to 0.56 ± 0.10 TGF-β1 mRNA/GAPDH mRNA ratio (P < 0.01). Losartan significantly blocked ANG II-increased TGF-β1 mRNA expression in the nuclei (0.18 ± 0.04 TGF-β1 mRNA/GAPDH mRNA ratio, P < 0.01 vs. ANG II) (Fig. 8).


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 7. Concentration-dependent transcriptional responses of transforming growth factor-β1 (TGF-β1) mRNA to intracellular ANG II in freshly isolated rat renal cortical nuclei. TGF-β1 mRNA is the expected size, based on the sense and antisense primers used (Table 1). *P < 0.05 or **P < 0.01 vs. control nuclei. +P < 0.05 vs. previous ANG II concentration(s); n = 3 different experiments.

 

Figure 8
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 8. AT1 receptors mediated intracellular ANG II-induced in vitro transcription of TGF-β1 mRNA in freshly isolated rat renal cortical nuclei. ANG II significantly increased transcription of TGF-β1 mRNA in isolated nuclei, and the responses were blocked by losartan. **P < 0.01 vs. control nuclei. ++P < 0.01 vs. ANG II-treated nuclei; n = 3 different experiments.

 
Effects of ANG II on Angiotensinogen mRNA Expression in Freshly Isolated Rat Renal Cortical Nuclei Because ANG II has been described to stimulate angiotensinogen mRNA expression in rat hepatic nuclei (13, 14), the rat kidney (26, 27.42), or in cultured proximal tubule cells (22), the present study determined whether intracellular ANG II induced expression of angiotensinogen mRNA in freshly isolated rat renal cortical nuclei. Figure 9 shows that, at 10 nM, ANG II had no effect on angiotensinogen mRNA expression (control: 0.45 ± 0.06 vs. 0.48 ± 0.06 angiotensinogen mRNA/GAPDH mRNA ratio; NS). At 10 µM, losartan also did not affect angiotensinogen mRNA expression (+losartan: 0.43 ± 0.10 angiotensinogen mRNA/GAPDH mRNA ratio; NS vs. ANG II or control) (Fig. 9).


Figure 9
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 9. Effects of intracellular ANG II on in vitro transcription of angiotensinogen mRNA in freshly isolated rat renal cortical nuclei. Angiotensinogen mRNA is the expected size, as predicted from sense and antisense primers used (Table 1). ANG II and/or losartan had no effects on angiotensinogen mRNA transcription in the nuclei; n = 3 different experiments.

 
Effects of ANG II on MCP-1 mRNA Expression in Freshly Isolated Rat Renal Cortical Nuclei ANG II not only acts as a growth factor but also a pro-inflammatory cytokine by inducing MCP-1 expression (4, 5, 54). In the present study, incubation of freshly isolated rat renal cortical nuclei with ANG II (10 nM) significantly increased MCP-1 mRNA expression by 89% (control: 0.36 ± 0.07 vs. 0.68 ± 0.10 MCP-1 mRNA/GAPDH mRNA ratio, P < 0.01) (Fig. 10). This stimulatory effect of ANG II on MCP-1 mRNA expression was effectively blocked by losartan (0.23 ± 0.06 MCP-1 mRNA/GAPDH mRNA ratio, P < 0.01), indicating that the effect was mediated by nuclear AT1 receptors (Fig. 10).


Figure 10
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 10. AT1 receptors mediated intracellular ANG II-induced in vitro transcription of macrophage chemoattractant protein-1 (MCP-1) in freshly isolated rat renal cortical nuclei. MCP-1 mRNA is the expected size, as predicted from the sense and antisense primers used (Table 1). ANG II significantly increased transcription of MCP-1 mRNA, and the responses were blocked by losartan. **P < 0.01 vs. control nuclei. ++P < 0.01 vs. ANG II-treated nuclei; n = 3 different experiments.

 
Effects of ANG II on NHE-3 mRNA Expression in Freshly Isolated Rat Renal Cortical Nuclei ANG II plays a critical role in the regulation of sodium transport in proximal tubules by stimulating NHE-3 expression via actions on apical membrane AT1 receptors (12, 30, 34, 40). It is not know whether, following receptor-mediated internalization, ANG II may stimulate nuclear AT1 receptors to induce NHE-3 expression. As shown in Fig. 11, activation of nuclear AT1 receptors by ANG II induced 110% increases in NHE-3 mRNA expression (control: 0.21 ± 0.03 vs. ANG II: 0.42 ± 0.05 NHE-3 mRNA/GAPDH mRNA ratio, P < 0.01). Coadministration of ANG II with losartan significantly reduced NHE-3 mRNA expression to control (0.23 ± 0.05 NHE-3 mRNA/GAPDH mRNA ratio, P < 0.01 vs. ANG II).


Figure 11
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 11. AT1 receptors mediated intracellular ANG II-induced in vitro transcription of the sodium and hydrogen exchanger-3 (NHE-3) mRNA in freshly isolated rat renal cortical nuclei. NHE-3 mRNA is the expected size, as predicted from the sense and antisense primers used (Table 1). ANG II significantly increased transcription of NHE-3 mRNA, and the responses were blocked by losartan. **P < 0.01 vs. control nuclei. ++P < 0.01 vs. ANG II-treated nuclei; n = 3 different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major goal of the present study was to test the hypothesis that intracellular ANG II, whether synthesized intracellularly or internalized from extracellular fluid, stimulates AT1 receptors in the nucleus to induce in vitro transcription of the growth factor TGF-β1, angiotensinogen, the proinflammatory cytokine MCP-1, and the Na+/H+ exchanger NHE-3. The present study demonstrates three key findings: 1) that freshly isolated nuclei from the rat renal cortex expressed ANG II receptors of primarily the AT1a subtype; 2) intracellular ANG II induced in vitro RNA synthesis by the nucleus by increasing [{alpha}-32P]CTP incorporation into RNA via activating AT1 receptors; and 3) ANG II specifically stimulated transcription or expression of TGF-β1, MCP-1, and NHE-3 mRNAs, but not angiotensinogen mRNA, in the nucleus, and these responses were mediated by AT1 receptors. Because only freshly isolated nuclei were used without significant contaminations by plasma membranes or endosomal organelles, these transcriptional responses to ANG II in the nucleus were independent of cell surface AT1 receptor- or cytoplasmic AT1 receptor-mediated signaling cascades. Our results provide evidence that at least in freshly isolated intact nuclei of the rat renal cortex, intracellular ANG II can directly stimulate nuclear AT1a receptors to induce transcription and/or expression of mRNAs for TGF-β1, MCP-1, and NHE-3.

Whether intracellular ANG II induces biological and/or physiological responses has been debated for decades. The difficulty in arguing for a role of intracellular ANG II is due to the lack of approaches that can definitely separate the responses induced by intracellular ANG II via intracellular or nuclear receptors from those evoked by extracellular ANG II via activation of cell surface receptors. To overcome the technical difficulty, Haller et al. (19) microinjected ANG II directly in rat VSMCs to induce intracellular and nuclear calcium responses to ANG II (19). We recently confirmed these intracellular calcium responses to microinjected ANG II in single rabbit proximal tubule cells (56). De Mello (10, 11) dialyzed ANG II directly in hamster cardiomyocytes and demonstrated that ANG II enhanced the L-type calcium currents. Alternatively, Cook et al. reported that overexpression of a nonsecreted form of angiotensinogen or intracellular ANG II (ECFP/AII) and AT1 receptor fusion proteins (AT1R/EYFP) in rat hepatoma cells stimulated hepatic cell or VSMC growth and proliferation (6–8). ANG II has also been shown to stimulate RNA synthesis (44) and increase transcription of mRNAs for renin, angiotensinogen, and growth-related factors in rat hepatic nuclei (13, 14), but there is no evidence that intracellular ANG II may exert nuclear effects in renal cortical cells.

In the present study, we went to a great length to confirm the identity and quality of our freshly isolated rat renal cortical nuclei and the presence of AT1 (AT1a) receptors before in vitro mRNA transcription studies were performed. The purity and integrity of isolated nuclei were characterized by 1) high-power phase-contrast images showing intact nuclear envelops (Fig. 1B); 2) the specific staining with the nuclear acid marker DAPI (Fig. 1C); 3) the absence of potential contaminations by plasma membranes (Fig. 1D) or endosomal organelles (Fig. 1E); and 4) the presence of high concentrations of DNA contents (Fig. 1F). Because the presence of ANG II receptors in the nucleus is critical for studying the nuclear role of intracellular ANG II, we used three different approaches to confirm whether freshly isolated rat renal cortical nuclei have AT1 receptors. FITC-labeled ANG II (Fig. 2) and [125I]Val5-ANG II binding assays (Fig. 3) demonstrated that the nuclei bound both FITC-labeled ANG II or [125I]Val5-ANG II, which were largely displaced by unlabeled competing ANG II (10 µM) and the AT1 antagonist losartan (10 µM), but not by the AT2 antagonist PD-123319 (10 µM). Thus we conclude that the majority of ANG II receptor binding in isolated rat renal cortical nuclei belongs to AT1. Using semiquantitative RT-PCR, we further demonstrated for the first time that the AT1a receptor dominates the AT1b receptor in a ratio of 3:1 in the nucleus of the rat renal cortex (Fig. 4). These data are consistent with those previously reported in nuclear extracts of the rat renal cortex (36, 43), proximal tubule cells (32, 34, 46, 57), and the rat kidney (52, 53). However, the Bmax (~708.5 fmol/mg nuclear proteins) and Kd (~19.1 nM) for ANG II receptors in freshly isolated intact nuclei, as determined by [125I]Sar1-ANG II binding assays, are somewhat higher than those reported in nuclear extracts of the rat renal cortex (36, 43) or rat hepatocytes (2, 47). The differences in AT1 receptor Bmax and Kd in these studies may reflect the differences in the purity and integrity of nuclear preparations and the conditions of binding assays. For example, Booz et al. (2) estimated that, when normalized to 5'-nucleotidase activity, the maximal capacity for specific ANG II binding to the liver nuclei reached 2,875 fmol·min–1·µg PO4–1, seven times higher than plasma membranes. Pendergrass et al. (43) showed that rat renal cortical nuclear fractions had three times higher Bmax and two times higher Kd than plasma membranes. Alternatively, a higher Kd value for nuclear ANG II receptor binding in the present study suggests that nuclear ANG II receptors may not be as sensitive to ANG II as cell surface receptors. Higher intracellular ANG II concentrations may be required to stimulate nuclear AT1 receptors, such as in ANG II-induced hypertension and renal target organ injury (23, 42). Indeed, at picomolar concentrations, ANG II was shown to physiologically stimulate proximal tubule sodium transport in the rat kidney (20), whereas high picomolar to lower nanomolar ANG II were generally required to induce effects in cultured mesangial cells (17, 31) or proximal tubule cells (34, 46, 57). In the present study, although ANG II induced RNA transcriptional responses in isolated nuclei in a concentration-dependent manner, a peak response was observed when 10 nM of ANG II was used (Figs. 5A and 7). Thus ANG II receptor binding characteristics in isolated nuclei are consistent with the concentration and response relationship of ANG II on in vitro transcription of RNA and/or target gene mRNA expression.

The effects of intracellular ANG II on the transcription of mRNAs for TGF-β1, MCP-1, and NHE-3 in freshly isolated rat renal cortical nuclei suggest that intracellular ANG II may play an important role in mediating ANG II-induced sodium retention and inflammation and fibrosis in the kidney. TGF-β1 is a potent growth factor (25, 45, 50), whereas MCP-1 is an important proinflammatory cytokine (4, 5, 54). In vitro, ANG II directly stimulated TGF-β1 and MCP-1 mRNA expression in mesangial cells and tubular epithelial cells via activation of AT1 receptors (4, 5, 25, 54). In animal models of ANG II-induced tubulointerstitial inflammation, TGF-β1 and MCP-1 mRNA expression was increased substantially (42). With respect to sodium retention in hypertensive diseases, ANG II is well recognized to stimulate NHE-3 synthesis and trafficking to apical membranes (12, 30, 34). Increased NHE-3 expression and function in proximal tubules in the renal cortex by ANG II may contribute to sodium retention and the development of hypertension. Although it is widely thought that these effects may be primarily caused by extracellular ANG II acting on cell surface AT1 receptors, these mechanisms are probably not involved in ANG II-induced target gene mRNA transcriptional responses observed in isolated nuclei for the following reasons. First, cell surface and cytoplasmic ANG II receptors were removed along with plasma membranes and endosomal organelles, and only intact nuclei were used in the present study. Second, ANG II-induced nuclear transcriptional responses could be blocked by the AT1 receptor antagonist losartan. Third, not all target genes in the nucleus were affected by ANG II, since ANG II had no effects on mRNAs for angiotensinogen and GAPDH. Our results therefore support a potential role of intracellular ANG II and nuclear AT1a receptors in mediating these transcriptional responses in the kidney.

However, the signaling mechanisms by which intracellular ANG II activates nuclear AT1 receptors to induce the expression of TGF-β1, MCP-1, and NHE-3 mRNAs in isolated nuclei are currently unknown. One of the possibilities may involve nuclear intracellular calcium concentration ([Ca2+]i), because intracellular ANG II has been shown to increase [Ca2+]i in the nucleus of VSMCs and proximal tubule cells (19, 56), and [Ca2+]i may play an important role in the regulation of RNA transcription and target gene mRNA expression (28). Cook et al. (6, 7) showed that expression of an intracellular ANG II fusion protein in VSMCs induced nuclear accumulation of the AT1 receptor and activation of cAMP response element-binding protein, leading to cell proliferation. Finally, translocation and/or activation of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 or nuclear factor-{kappa}B to/in the nucleus is well recognized to play an important role in ANG II-induced cell growth and proliferation (16, 17, 35, 42). Further studies are required to determine whether intracellular ANG II can stimulate AT1 receptors in the nucleus to activate these transcriptional factors or kinases.

The finding that ANG II did not change transcription of angiotensinogen mRNA in isolated rat renal cortical nuclei is unexpected. Angiotensinogen is the sole substrate for ANG II formation, and ANG II is expected to negatively regulate its substrate's production. However, at nanomolar concentrations, ANG II stimulated angiotensinogen mRNA transcription in isolated hepatic nuclei, also via AT1 receptors (13, 14). This response may not be surprising given the fact that angiotensinogen is primarily synthesized by hepatic cells (13, 14). In the rat kidney, angiotensinogen mRNA expression and protein synthesis were significantly increased in rats chronically infused with ANG II (26, 27, 42). ANG II has also been shown to stimulate angiotensinogen mRNA expression in proximal tubule cells (22). The reasons underlying these differences between the present and the afore-mentioned studies are not known. It could be due to different approaches or nuclear preparations used, such as cultured cells vs. nuclear extracts, isolated nuclei vs. the entire kidney. For example, Eggena et al. (14) found that ANG II had no effects on in vitro transcription of mRNAs for TGF and epidermal growth factor in isolated hepatic nuclei. We reason that extracellular ANG II via cell surface receptor-mediated signaling may mediate the effects of ANG II on angiotensinogen mRNA expression in the kidney.

In summary, we have demonstrated for the first time that, in freshly isolated rat renal cortical nuclei, intracellular ANG II bound to nuclear AT1 receptors and stimulated in vitro RNA synthesis and transcription of mRNAs for TGF-β1, MCP-1, and NHE-3. By contrast, ANG II did not affect transcription of mRNA for angiotensinogen in these isolated nuclei. These results suggest that intracellular ANG II, either synthesized intracellularly or internalized from extracellular ANG II, induces novel effects through activation of AT1 receptors in the nucleus. Thus the effects of intracellular ANG II on in vitro transcription of mRNAs for NHE-3, TGF-β1, and MCP-1 in the nucleus may play an important role in the development of sodium and fluid retention in ANG II-induced hypertension and renal tubulointerstitial injury in progressive renal diseases.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5RO1DK-067299, American Heart Association Grant-in-Aid 0355551Z, and by the National Kidney Foundation of Michigan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Zhuo, Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202 (e-mail: jzhuo1{at}hfhs.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Beutler KT, Masilamani S, Turban S, Nielsen J, Brooks HL, Ageloff S, Fenton RA, Packer RK, Knepper MA. Long-term regulation of ENaC expression in kidney by angiotensin II. Hypertension 41: 1143–1150, 2003.[Abstract/Free Full Text]

2. Booz GW, Conrad KM, Hess AL, Singer HA, Baker KM. Angiotensin-II-binding sites on hepatocyte nuclei. Endocrinology 130: 3641–3649, 1992.[Abstract/Free Full Text]

3. Chen R, Mukhin YV, Garnovskaya MN, Thielen TE, Iijima Y, Huang C, Raymond JR, Ullian ME, Paul RV. A functional angiotensin II receptor-GFP fusion protein: evidence for agonist-dependent nuclear translocation. Am J Physiol Renal Physiol 279: F440–F448, 2000.[Abstract/Free Full Text]

4. Chen XL, Tummala PE, Olbrych MT, Alexander RW, Medford RM. Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells. Circ Res 83: 952–959, 1998.[Abstract/Free Full Text]

5. Chipitsyna G, Gong Q, Gray CF, Haroon Y, Kamer E, Arafat HA. Induction of monocyte chemoattractant protein-1 expression by angiotensin II in the pancreatic islets and beta-cells. Endocrinology 148: 2198–2208, 2007.[Abstract/Free Full Text]

6. Cook JL, Mills SJ, Naquin R, Alam J, Re RN. Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation. J Mol Cell Cardiol 40: 696–707, 2006.[CrossRef][Web of Science][Medline]

7. Cook JL, Re R, Alam J, Hart M, Zhang Z. Intracellular angiotensin II fusion protein alters AT1 receptor fusion protein distribution and activates CREB. J Mol Cell Cardiol 36: 75–90, 2004.[CrossRef][Web of Science][Medline]

8. Cook JL, Zhang Z, Re RN. In vitro evidence for an intracellular site of angiotensin action. Cir Res 89: 1138–1146, 2001.[Abstract/Free Full Text]

9. de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52: 415–472, 2000.[Abstract/Free Full Text]

10. De Mello WC. Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes. Hypertension 32: 976–982, 1998.[Abstract/Free Full Text]

11. De Mello WC. Renin increments the inward calcium current in the failing heart. J Hypertens 24: 1181–1186, 2006.[Web of Science][Medline]

12. du CD, Chalumeau C, Defontaine N, Klein C, Kellermann O, Paillard M, Poggioli J. Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: role of PI 3-kinase. Kidney Int 64: 939–949, 2003.[CrossRef][Web of Science][Medline]

13. Eggena P, Zhu JH, Clegg K, Barrett JD. Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA. Hypertension 22: 496–501, 1993.[Abstract/Free Full Text]

14. Eggena P, Zhu JH, Sereevinyayut S, Giordani M, Clegg K, Andersen PC, Hyun P, Barrett JD. Hepatic angiotensin II nuclear receptors and transcription of growth- related factors. J Hypertens 14: 961–968, 1996.[Web of Science][Medline]

15. Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53: 1–24, 2001.[Abstract/Free Full Text]

16. Godeny MD, Sayeski PP. ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKC{zeta}-controlled ERK1/2 within the nucleus. Am J Physiol Cell Physiol 291: C1297–C1307, 2006.[Abstract/Free Full Text]

17. Gorin Y, Ricono JM, Wagner B, Kim NH, Bhandari B, Choudhury GG, Abboud HE. Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells. Biochem J 381: 231–239, 2004.[CrossRef][Web of Science][Medline]

18. Griendling KK, Delafontaine P, Rittenhouse SE, Gimbrone MA Jr, Alexander RW. Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells. J Biol Chem 262: 14555–14562, 1987.[Abstract/Free Full Text]

19. Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellular angiotensin II in vascular smooth muscle cells. Circ Res 79: 765–772, 1996.[Abstract/Free Full Text]

20. Harris PJ, Navar LG. Tubular transport responses to angiotensin II. Am J Physiol Renal Fluid Electrolyte Physiol 248: F621–F630, 1985.[Abstract/Free Full Text]

21. Hein L, Meinel L, Pratt RE, Dzau VJ, Kobilka BK. Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand. Mol Endocrinol 11: 1266–1277, 1997.[Abstract/Free Full Text]

22. Ingelfinger JR, Jung F, Diamant D, Haveran L, Lee E, Brem A, Tang SS. Rat proximal tubule cell line transformed with origin-defective SV40 DNA: autocrine ANG II feedback. Am J Physiol Renal Physiol 276: F218–F227, 1999.[Abstract/Free Full Text]

23. Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl P, Floege J, Schwartz SM. Renal injury from angiotensin II-mediated hypertension. Hypertension 19: 464–474, 1992.[Abstract/Free Full Text]

24. Johren O, Golsch C, Dendorfer A, Qadri F, Hauser W, Dominiak P. Differential expression of AT1 receptors in the pituitary and adrenal gland of SHR and WKY. Hypertension 41: 984–990, 2003.[Abstract/Free Full Text]

25. Kagami S, Border WA, Miller DE, Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells. J Clin Invest 93: 2431–2437, 1994.[Web of Science][Medline]

26. Kobori H, Harrison-Bernard LM, Navar LG. Enhancement of angiotensinogen expression in angiotensin II-dependent hypertension. Hypertension 37: 1329–1335, 2001.[Abstract/Free Full Text]

27. Kobori H, Harrison-Bernard LM, Navar LG. Expression of angiotensinogen mRNA and protein in angiotensin II-dependent hypertension. J Am Soc Nephrol 12: 431–439, 2001.[Abstract/Free Full Text]

28. Kurtz TW, Gardner DG. Transcription-modulating drugs: a new frontier in the treatment of essential hypertension. Hypertension 32: 380–386, 1998.[Abstract/Free Full Text]

29. Laghmani K, Chambrey R, Froissart M, Bichara M, Paillard M, Borensztein P. Adaptation of NHE-3 in the rat thick ascending limb: effects of high sodium intake and metabolic alkalosis. Am J Physiol Renal Physiol 276: F18–F26, 1999.[Abstract/Free Full Text]

30. Leong PK, Yang LE, Holstein-Rathlou NH, McDonough AA. Angiotensin II clamp prevents the second step in renal apical NHE3 internalization during acute hypertension. Am J Physiol Renal Physiol 283: F1142–F1150, 2002.[Abstract/Free Full Text]

31. Li XC, Campbell DJ, Ohishi M, Shao Y, Zhuo JL. AT1 receptor-activated signaling mediates angiotensin IV-induced responses in renal microvasculature and glomerular mesangial cells. Am J Physiol Renal Physiol 290: F1024–F1033, 2006.[Abstract/Free Full Text]

32. Li XC, Carretero OA, Navar LG, Zhuo JL. AT1 receptor-mediated accumulation of extracellular angiotensin II in proximal tubule cells: role of cytoskeleton microtubules and tyrosine phosphatases. Am J Physiol Renal Physiol 291: F375–F383, 2006.[Abstract/Free Full Text]

33. Li XC, Navar LG, Shao Y, Zhuo JL. Genetic deletion of AT1a receptors attenuates intracellular accumulation of angiotensin II in the kidney of AT1a receptor-deficient mice. Am J Physiol Renal Physiol 293: F586–F593, 2007.[Abstract/Free Full Text]

34. Li XC, Zhuo JL. Selective knockdown of AT1 receptors by RNA interference inhibits Val5-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells. Am J Physiol Cell Physiol 293: C367–C378, 2007.[Abstract/Free Full Text]

35. Li XC, Zhuo JL. Nuclear factor-{kappa}B as a hormonal intracellular signaling molecule: focus on angiotensin II-induced cardiovascular and renal injury. Curr Opin Nephrol Hypertens 17: 37–43, 2008.[Web of Science][Medline]

36. Licea H, Walters MR, Navar LG. Renal nuclear angiotensin II receptors in normal and hypertensive rats. Acta Physiol Hung 89: 427–438, 2002.[CrossRef][Medline]

37. Llorens-Cortes C, Greenberg B, Huang H, Corvol P. Tissular expression and regulation of type 1 angiotensin II receptor subtypes by quantitative reverse transcriptase-polymerase chain reaction analysis. Hypertension 24: 538–548, 1994.[Abstract/Free Full Text]

38. Lu D, Yang H, Shaw G, Raizada MK. Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) receptor in brain neurons. Endocrinology 139: 365–375, 1998.[Abstract/Free Full Text]

39. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292: C82–C97, 2007.[Abstract/Free Full Text]

40. Moe OW. Acute regulation of proximal tubule apical membrane Na/H exchanger NHE-3: role of phosphorylation, protein trafficking, and regulatory factors. J Am Soc Nephrol 10: 2412–2425, 1999.[Free Full Text]

41. Morinelli TA, Raymond JR, Baldys A, Yang Q, Lee MH, Luttrell L, Ullian ME. Identification of a putative nuclear localization sequence within the angiotensin II AT1A receptor associated with nuclear activation. Am J Physiol Cell Physiol 292: C1398–C1408, 2007.[Abstract/Free Full Text]

42. Ozawa Y, Kobori H. Crucial role of Rho-nuclear factor-{kappa}B axis in angiotensin II-induced renal injury. Am J Physiol Renal Physiol 293: F100–F109, 2007.[Abstract/Free Full Text]

43. Pendergrass KD, Averill DB, Ferrario CM, Diz DI, Chappell MC. Differential expression of nuclear AT1 receptors and angiotensin II within the kidney of the male congenic mRen2. Lewis rat Am J Physiol Renal Physiol 290: F1497–F1506, 2006.

44. Re R, Parab M. Effect of angiotensin II on RNA synthesis by isolated nuclei. Life Sci 34: 647–651, 1984.[CrossRef][Web of Science][Medline]

45. Ruiz-Ortega M, Lorenzo O, Suzuki Y, Ruperez M, Egido J. Proinflammatory actions of angiotensins. Curr Opin Nephrol Hypertens 10: 321–329, 2001.[CrossRef][Web of Science][Medline]

46. Schelling JR, Hanson AS, Marzec R, Linas SL. Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells. J Clin Invest 90: 2472–2480, 1992.[Web of Science][Medline]

47. Tang SS, Rogg H, Schumacher R, Dzau VJ. Characetrization of nucler angiotensin II binding sites in rat liver and comparison with plasma membrane receptors. Endocrinology 131: 374–380, 1992.[Abstract/Free Full Text]

48. Thekkumkara T, Linas SL. Role of internalization in AT(1A) receptor function in proximal tubule epithelium. Am J Physiol Renal Physiol 282: F623–F629, 2002.[Abstract/Free Full Text]

49. Ullian ME, Linas SL. Role of receptor cycling in the regulation of angiotensin II surface receptor number and angiotensin II uptake in rat vascular smooth muscle cells. J Clin Invest 84: 840–846, 1989.[Web of Science][Medline]

50. Wolf G, Ziyadeh FN, Zahner G, Stahl RA. Angiotensin II-stimulated expression of transforming growth factor β in renal proximal tubular cells: attenuation after stable transfection with the c-mas oncogene. Kidney Int 48: 1818–1827, 1995.[Web of Science][Medline]

51. Zhuo JL, Li XC. Novel roles of intracrine angiotensin II and signalling mechanisms in kidney cells. J Renin Angiotensin Aldosterone Syst 8:23–33, 2007.[Abstract/Free Full Text]

52. Zhuo JL, Ohishi M, Mendelsohn FA. Roles of AT1 and AT2 receptors in the hypertensive Ren-2 gene transgenic rat kidney. Hypertension 33: 347–353, 1999.[Abstract/Free Full Text]

53. Zhuo JL, Song K, Harris PJ, Mendelsohn FA. In vitro autoradiography reveals predominantly AT1 angiotensin II receptors in rat kidney. Ren Physiol Biochem 15: 231–239, 1992.[Web of Science][Medline]

54. Zhuo JL. Monocyte chemoattractant protein-1: a key mediator of angiotensin II-induced target organ damage in hypertensive heart disease? J Hypertens 22: 451–454, 2004.[CrossRef][Web of Science][Medline]

55. Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG. Ang II accumulation in rat renal endosomes during Ang II-induced hypertension: role of AT1 receptor. Hypertension 39: 116–121, 2002.[Abstract/Free Full Text]

56. Zhuo JL, Li XC, Garvin JL, Navar LG, Carretero OA. Intracellular angiotensin II induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal tubule cells. Am J Physiol Renal Physiol 290: F1382–F1390, 2006.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
X. C. Li, U. Hopfer, and J. L. Zhuo
AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway
Am J Physiol Renal Physiol, November 1, 2009; 297(5): F1342 - F1352.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
T. M. Gwathmey, H. A. Shaltout, K. D. Pendergrass, N. T. Pirro, J. P. Figueroa, J. C. Rose, D. I. Diz, and M. C. Chappell
Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production
Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1484 - F1493.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. M. de Cavanagh, M. Ferder, F. Inserra, and L. Ferder
Angiotensin II, mitochondria, cytoskeletal, and extracellular matrix connections: an integrating viewpoint
Am J Physiol Heart Circ Physiol, March 1, 2009; 296(3): H550 - H558.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/C1034    most recent
00432.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X. C.
Right arrow Articles by Zhuo, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X. C.
Right arrow Articles by Zhuo, J. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.