Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney

Noriyuki Miyata, Frank Park, Xiao Feng Li, and Allen W. Cowley Jr.

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
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ABSTRACT
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ANG II contributes importantly to the regulation of renal vascular resistance, glomerular filtration, and tubular epithelial transport, yet there remains a paucity of information regarding the localization of the ANG II type 1 and 2 (AT1 and AT2) receptors within the rat kidney particularly within the vasculature. The present study was designed to localize the transcriptional and translational site(s) of AT1 and AT2 receptor (AT1R and AT2R, respectively) expression within the rat kidney. Using immunohistochemistry, we detected the AT1R translational sites throughout the kidney, with the strongest labeling found in the vasculature of the renal cortex and the proximal tubules of the outer medulla. The AT2R protein expression was found throughout the rat kidney, although there was little to no expression found in the glomerulus and medullary thick ascending limbs of Henle (TAL). Gene-specific primers were then designed to distinguish between the receptor subtypes within microdissected renal tubular and vascular segments using RT-PCR. AT1AR, AT1BR, and AT2R mRNA were found within the renal vasculature (afferent arterioles, arcuate artery, and outer medullary descending vasa recta). The mRNA for both the AT1R isoforms was also detected in the glomeruli and the renal tubules (proximal tubules, TAL, and collecting ducts); however, no AT2R mRNA was detected within the glomerulus and was inconsistently found within the medullary TAL (MTAL). Taken together, these data show that mRNA for the AT1R subtypes was located in all of the renal tubular and vascular segments. Evidence for AT2R mRNA was also found in all but two of the vascular and tubular segments, the MTAL, and the glomeruli. These results are consistent with the whole tissue immunohistochemically localized receptors.

reverse transcription-polymerase chain reaction; immunohistochemistry; kidney; microdissection


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TWO TYPES of angiotensin II (ANG II) receptors were pharmacologically described in 1993 and classified as the AT1 and AT2 receptors (AT1R and AT2R, respectively) (40). More recently, the cDNAs that encode the AT1R were cloned and led to the discovery of two distinct AT1R subtypes, which were defined as the AT1AR and AT1BR (12, 14, 33). Interestingly, although it was found that these AT1R subtype genes were located on different chromosomes, they exhibited over 90% homology in the coding region of the cDNA sequence, and 95% homology at the amino acid level (14). In contrast, the coding region of the AT1R and AT2R cDNA sequences exhibit only 30% homology (16, 17).

The predominant biological effects of ANG II are believed to be mediated through the stimulation of AT1R subtypes that mediate vasoconstriction (4), angiogenesis (41), and stimulation of sodium and water reabsorption in the proximal tubules (18). To determine the whole tissue distribution of the AT1R subtypes, several investigators have used RT-PCR (15, 19) and in situ hybridization (22). These studies have demonstrated AT1R mRNA throughout the kidney with the highest abundance in the outer medulla as determined by in situ hybridization (22) and semiquantitative RT-PCR (19).

At the present time, both the localization and the functional role of each of the isoforms of the AT1R subtypes within the rat kidney remain unclear. The AT1AR is believed to play the major role in the renal actions of ANG II, but little is known about the intrarenal role of the AT1BR subtype. Recent studies in the systemic circulation suggest that the AT1BR may play a role similar to the AT1AR (27), but neither renal vascular nor tubular actions of this receptor isoform have been explored. Even the localization of the AT1R isoforms within distinct segments of the renal tubular and vascular system has remained unclear, since it has been difficult to design specific PCR primers for the AT1R isoforms due to the high degree of homology of the AT1AR and AT1BR.

A paucity of information also exists about the localization of the AT2R within the kidney. In situ hybridization studies by Shanmugam et al. (36) initially suggested that the expression of the AT2 mRNA was developmentally regulated within the rat kidney, since AT2R mRNA was detectable only in fetal and immature rats. More recently, Ozono et al. (28) reported AT2R expression in glomeruli and distal tubules of adult rats. In addition, functional studies have provided pharmacological evidence that the AT2R can influence renal function. Lo et al. (20) observed that the pressure-natriuresis relationship in rats was enhanced by AT2R inhibition. Other investigators have reported AT2R involvement in prostaglandin-dependent and -independent vasodilation (13, 38). These studies suggest that AT2R receptors are present within the adult rat kidney and play a role in influencing renal function.

Because of the limited information regarding ANG II receptor distribution within the rat kidney, the aim of the present study was to design specific PCR primers for each of the ANG II receptor isoforms and localize the site(s) of transcription for the AT1R (AT1AR and AT1BR) and AT2R by RT-PCR in isolated renal tubules and blood vessels. In addition, the site(s) of translation for the AT1R and AT2R protein were assessed using immunohistochemical techniques.


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Experimental Animals

Male Sprague-Dawley rats weighing 80-120 g (5-6 wk old) from Sasco (Madison, WI), which were given free access to tap water and fed a standard pellet diet (Purina Mills, St. Louis, MO), were used in all studies. All protocols were approved by the Institutional Animal Care Committee, and rats were maintained in the American Association for Accreditation of Laboratory Animal Care approved Animal Resource Center of the Medical College of Wisconsin.

Protein Isolation and Western Blot Analysis of AT1R and AT2R Antibody

Rat kidney (n = 3) plasma membrane-enriched fractions were isolated as previously described (30). In brief, kidneys were homogenized, and the homogenate was centrifuged at 3,000 g for 5 min and then at 16,000 g for 20 min. The pellet was resuspended and frozen at 80°C for use in Western blot analysis. A 5-µg (for AT2R) or 25-µg (for AT1R) aliquot was placed in sample buffer [2% SDS, 100 mM Tris · HCl, pH 6.8, 5% beta -mercaptoethanol, 12% (vol/vol) glycerol, and 0.02% (wt/vol) bromophenol blue] and denatured at 100°C for 5 min. The protein samples were electrophoresed through a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. Membranes were incubated with the primary antibodies (1:1,000 for the AT1R and 1:2,000 for the AT2R) for 90 min, and then the membranes were washed vigorously. The secondary antibody at a dilution of 1:1,000 was used for 90 min, then the blots were stained for horseradish peroxidase activity using enhanced chemiluminescence (Pierce, Rockford, IL) and placed on film.

Immunohistochemistry for AT1R and AT2R Within the Rat Kidney

Rat kidneys (n = 6) were fixed in 10% Formalin and subsequently embedded in paraffin. Kidney sections (5 µm) were prepared, and sections were incubated with xylene and hydrated through several washes in ethanol and water to remove the paraffin. Endogenous peroxidase activity was quenched by the addition of 0.3% (vol/vol) hydrogen peroxide in methanol for 10 min. Polyclonal anti-rabbit AT1R (Santa Cruz Biotech, Santa Cruz, CA) or a polyclonal anti-rabbit AT2R antibody [generously provided by Dr. A. S. Greene and described previously by Nora et al. (26)] was used for the immunostaining. The antibodies were used at a dilution of 1:100 in 1% BSA in PBS (pH 7.2). The kidney sections were incubated for 45 min with the primary antibody at room temperature in a wet chamber. The specificity of the staining was determined by reacting the antibody with a 10-fold (by weight) excess of control antigenic peptide in PBS, which was incubated with the sections for 2 h at room temperature. Secondary antibody (donkey anti-goat) was incubated with the sections for 30 min followed by a complex of streptavidin and biotinylated peroxidase (DAKO, Bucks, UK). The immunoreactivity was detected by the addition of 0.5 mg/ml diaminobenzidine (Sigma, St. Louis, MO) and 0.01% hydrogen peroxide in PBS. Between each step, the sections were washed three times with 200 ml PBS over a period of 15 min. The sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted.

Preparation of Glomeruli, Renal Tubules, and Renal Microvessels

Rats (n = 6) were injected with 5 mg ip furosemide and 30 min later were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (5 mg/kg) mixture given intramuscularly. Renal tubules and microvessels were isolated as previously described (30, 42). In brief, the left kidney was perfused with 10 ml dissection solution (DS) prewarmed to 37°C. Following kidney perfusion with DS, 1.0 ml of 2.5% latex-coated blue-dyed microparticles (~1-4 µm in diameter; Polysciences, Warrington, PA) were perfused into the left kidneys. The renal pedicle was ligated, and the kidney was excised and cut into coronal slices (~1-2 mm thick). These tissue slices were placed into DS containing 1 mg/ml collagenase (192 U/mg; Worthington Biochemical, Freehold, NJ), and incubated at 37°C for 35-40 min. For the isolation of inner medullary collecting ducts, the tissue was incubated in collagenase for longer periods (up to 60 min). After collagenase treatment, the tissue slices were rinsed with DS, and placed into a microdissection dish for tubules and microvessel isolation using a Leica M3Z stereomicroscope (magnification ×16-100). The following intrarenal blood vessels were dissected from the tissue slices: arcuate artery, afferent arterioles, glomeruli, and outer medullary descending vasa recta (OMDVR) from vascular bundles. For the renal tubules, proximal convoluted tubules, thick ascending limbs of Henle (TAL), and the collecting ducts (cortex and outer and inner medulla) were isolated as previous described (42). All lengths of the vessels were measured by a calibrated eyepiece micrometer, and for each RT reaction, 40 glomeruli, 40 mm of blood vessels, and 40 mm of renal tubules were isolated. Contaminating debris was rinsed from the dissected vessels, tubules, and glomeruli in a separate wash dish before transfer to an RNase-free ultracentrifuge tube containing 100 µl TRIzol reagent (GIBCO-BRL; Life Technologies, Gaithersburg, MD). The total RNA was extracted and treated with DNase solution as previously described (30). As a negative control for the presence of AT1B and AT2 mRNA, liver mRNA was run in parallel with the isolated tubular and microvascular total RNA.

Preparation of Oligonucleotide Primers

All nucleotide primers were purchased from Operon Technologies (Alameda, CA). Oligonucleotide primers were chosen from the published cDNA sequences of angiotensin AT1AR (12), AT1BR (8), and AT2R (17). The primers for the AT2R spanned both introns found in the genomic DNA sequence, which allowed for differentiation of genomic DNA contamination, but the AT1A and AT1B PCR primers were not intron spanning.

The primer sequence for the angiotensin AT1AR corresponded to 5'-CGT CAT CCA TGA CTG TAA AAT TTC-3' (sense; bp 1097-1120) and 5'-GGG CAT TAC ATT GCC AGT GTG-3' (antisense; bp 1381-1402). The final PCR product was 306 bp in size.

The primer sequence for the angiotensin AT1BR corresponded to 5'-CAT TAT CCG TGA CTG TGA AAT TG-3' (sense; bp 1369-1391) and 5'-GCT GCT TAG CCC AAA TGG TCC-3' (antisense; bp 1712-1732). The final PCR product was 344 bp in size.

The primer sequence for the angiotensin AT2R corresponded to 5'-GGA GCG AGC ACA GAA TTG AAA GC-3' (sense; bp 1586-1608) and 5'-TGC CCA GAG AGG AAG GGT TGC C-3' (antisense; bp 3332-3353). The final PCR product was 445 bp in size.

Reverse Transcription-Polymerase Chain Reaction

The RT-PCR assay was performed as previously described (30) with minor modifications to optimize the conditions for the primers in PCR. Briefly, 7-µl aliquots of the RT reaction products were used in the PCR assay, and the reaction mixtures were initially denatured at 94°C for 5 min and then cycled 35 times between 94°C (denaturation) for 1 min, 64°C (annealing), and 72°C (extension) for 1 min. Samples were incubated for an additional 7 min at 72°C after the completion of the final cycle. Three negative controls were used in each of the PCR reactions: RT-PCR of the DS, PCR amplification of sterile water, and PCR amplification of DNase-treated RNA to determine genomic DNA contamination.

PCR Product Analysis

From each PCR reaction, 10-µl aliquots were electrophoretically size-fractionated on a 1.6% agarose gel. After electrophoresis and ethidium bromide staining, DNA bands were visualized with an ultraviolet transilluminator. To verify the authenticity of the PCR products, restriction enzyme analysis of the PCR products was performed, and the RT-PCR products were subsequently ligated into pCR2.1 vector (Invitrogen, San Diego, CA). The cloned plasmid DNA was purified using ion-exchange columns (QIAGEN, Chatsworth, CA), and the authenticity of the DNA insert was determined by sequencing with ThermoSequenase using the dideoxynucleotide-chain termination reaction (Amersham). The samples were resolved on a DNA sequencer 725 (Molecular Dynamics).


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INTRODUCTION
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Angiotensin AT1 Receptor

Immunohistochemistry of AT1R in the rat kidney. The results from the immunohistochemistry for the AT1R are from six different rat kidneys, which were sectioned and examined. The AT1R antibody produced a single band at ~43 kDa on Western blot analysis, which is the expected size of the AT1R and consistent with the results of a previous study by Paxton et al. (31) using an antibody directed against the same receptor sequence. Using this AT1R antibody for immunohistochemistry, we found the protein translational sites of the AT1R to be localized throughout the kidney, with the most intense region being the outer medulla (Fig. 1A). Higher magnification of renal cortex and medulla revealed that the strongest labeling of the AT1R was found within the vasculature of the renal cortex (Fig. 1B) and the proximal straight tubules (S3 segment) of the outer medulla (Fig. 1C). Lesser staining of the AT1R was detected in the cortical and outer medullary collecting ducts and TAL. Some immunolabeling was found within the glomerulus (Fig. 1B), which is consistent with previous studies by Paxton et al. (31) and Harrison-Bernard et al. (9), who observed glomerular staining in mesangial cells and podocytes with dense staining of the macula densa. In addition, there was light labeling associated with the vascular bundles in the outer medulla, which contain both the thin limbs of Henle's loop and vasa recta. The kidney sections that were incubated with the AT1R antibody preadsorbed with the competing antigenic peptide did not have any labeling, demonstrating the specificity of the binding by the primary antibody (Fig. 1D). Within the inner medulla, the labeling by the AT1R antibody was associated with the collecting ducts.


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Fig. 1.   Immunohistochemical sections of the kidney incubated with the AT1 receptor (AT1R) antibody. A: kidney section showing immunoreactivity of AT1R in 3 different regions of the kidney, which include the cortex (CTX), outer medulla (OM), and inner medulla (IM) (magnification ×40; bar = 400 µm). B: renal cortex demonstrating strong labeling within the blood vessels (IA) and lesser labeling throughout the glomeruli (Glm) and renal tubules, specifically the proximal (PT) and distal tubules (DT; arrowhead) (magnification ×100; bar = 160 µm). C: outer medullary section demonstrating the strong labeling of the proximal tubules (S3 segment) and lesser staining within the thick ascending limbs (T), distal tubules (DT), and vascular bundles (VRB) (magnification ×100; bar = 160 µm). D: renal cortical section incubated with the AT1R preadsorbed with the immunizing peptide demonstrates no immunostaining (magnification ×200; bar = 80 µm); arrowhead indicates thick ascending limb (T).

Specificity of the PCR primers for AT1AR and AT1BR. The AT1R antibody used to determine the translational sites in the kidney cannot differentiate between the AT1AR and AT1BR, because the antigenic peptide sequence used to generate the polyclonal antibody is identical to both AT1R subtypes. For this reason, we designed specific PCR primers to allow us to distinguish the AT1AR and AT1BR cDNA sequences by gel electrophoresis. Figure 2 demonstrates the position of the PCR primers for the AT1AR and AT1BR and the restriction enzyme sites (Msp I and Hinc II) that were used to demonstrate the specificity of the AT1AR and AT1BR PCR primers. The antisense primers were designed within the noncoding region of the AT1R cDNA sequence due to the low homology (62%) between each of the AT1R isoforms. The authenticity of the AT1R PCR products was determined by PCR sequencing and restriction enzyme analysis; Msp I was capable of digesting only AT1AR PCR products, whereas Hinc II was capable of only digesting AT1BR. If any remaining band was detected at the native size of the PCR products, then we assumed that the PCR primer pair was not specific for a particular receptor subtype.


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Fig. 2.   Tissue distribution of the AT1R (AT1AR and AT1BR) and AT2R using RT-PCR. Ethidium bromide-stained gels demonstrate RT-PCR products for the AT1AR (A), AT1BR (B), and AT2R (C) in the adrenal gland (ADR), liver, and aorta. Specificities of PCR primers for the AT1AR and AT1BR were demonstrated by restriction enzyme digestion with Hinc II (ADR+H) and Msp I (ADR+M). Arrows denote the molecular size markers (at left). Size of the RT-PCR products for AT1AR, AT1BR, and AT2R are 306, 344, and 445 bp, respectively.

The optimal PCR primer pairs used in the experiment were ~48% homologous between the two isoforms of the AT1R (i.e., AT1BR and its opposing AT1AR cDNA sequence share 10 of 21 nucleotides). Similar homology was observed when comparing the AT1AR primers to the AT1BR cDNA sequence, and to avoid cross-reactivity of the AT1AR to the AT1BR cDNA products generated in the RT reaction, higher annealing temperatures were used in the PCR assay, which were at or slightly above the midpoint temperature, Tm, of the primers. As shown in Fig. 2, the specificity of the AT1AR and AT1BR primers are demonstrated through differential tissue and restriction enzyme analysis. In every experiment performed (n = 6), AT1AR mRNA was found in the adrenal gland, liver, and aorta, but the AT1BR mRNA was found only in the adrenals and aorta and not the liver. The nucleotide differences in the AT1AR compared with the AT1BR allowed for the use of Msp I and Hinc II to demonstrate the specificity of the AT1R subtype PCR products; Msp I could only digest AT1AR, and Hinc II could only digest AT1BR. The native size of the AT1AR PCR product was 306 bp, which could be digested with Msp I (209 bp + 97 bp), whereas digestion with Hinc II could not cut the AT1AR PCR product. For the AT1BR PCR products (344 bp in size), Hinc II digested the adrenal AT1BR PCR product into 184 bp and 160 bp, and Msp I could not digest the AT1BR.

The combined data that showed the lack of AT1BR amplification in the liver along with the restriction enzyme analysis of the PCR products provide evidence of the specificity of the AT1AR and AT1BR gene-specific primers for their respective cDNAs. No cross-amplification occurred with either of the AT1R isoforms, which demonstrated the ability of designing gene-specific primers in highly homologous cDNAs between closely related genes.

Microlocalization of AT1AR and AT1BR within the rat kidney. The RT-PCR analysis of the AT1AR and AT1BR mRNA in microdissected tubular and vascular segments is shown in Fig. 3 and summarized in Table 1. The AT1AR and AT1BR mRNA was detected in all of the renal tubular and vascular segments isolated and studied. In summary, both ANG II type 1 receptor isoforms were found in the proximal convoluted tubules, medullary TAL (MTAL), and cortical, outer, and inner medullary collecting ducts. In the renal vasculature, the AT1AR and AT1BR mRNA was observed in the glomeruli, OMDVR, afferent arterioles, and arcuate arteries. As summarized in Table 1, we found positive results (denoted as a "+") for both AT1R isoforms in the different segments of the kidney. We performed the RT-PCR assay for the AT1AR and AT1BR in six different rat samples for both the renal vasculature and tubules from the renal medulla to demonstrate consistent localization of the AT1R mRNA; we performed three experiments for the cortical collecting duct and afferent arterioles. It is important to note that the AT1AR and AT1BR mRNA was detected in every rat sample studied. Each experiment was run with tissue controls where we used the adrenal gland and liver (100 ng total RNA) to demonstrate the specificity of the AT1R primers, where the AT1AR was present in both tissues, and only the AT1AR was present in the liver. In addition, contamination was not evident, as demonstrated by the absence of detectable bands in the negative controls: 1) RT-PCR amplification of the DS; and 2) PCR amplification of DNase-treated RNA from the MTAL (MTAL -RT) or OMCD (OMCD -RT).


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Fig. 3.   RT-PCR analysis of AT1AR (A) and AT1BR (B) in microdissected tubules and blood vessels from rat kidney. RT-PCR products from microdissected dissected segments from renal medulla are shown in ethidium bromide-stained agarose gels, which include medullary thick ascending limbs (MTAL), outer (OMCD) and inner medullary collecting ducts (IMCD), and outer medullary descending vasa recta (OMDVR). Adrenal gland and liver were used in each experiment as positive and negative controls for the AT1AR and AT1BR. AT1AR and AT1BR expression was also noted in arcuate arteries (Arc). Negative control samples are dissection solution (Diss Soln) and PCR amplification of DNase-treated RNA from the MTAL (MTAL -RT). Small arrows denote the molecular size markers (at left). Sizes of the RT-PCR products for AT1AR and AT1BR were 306 bp and 344 bp, respectively. Six separate experiments were performed for each whole and microdissected tissue studied.


                              
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Table 1.   RT-PCR profile of whole and microdissected renal tissue

Angiotensin AT2 Receptor

Immunohistochemistry of AT2R in rat kidney. The specificity of the AT2R antibody provided by our colleagues Nora et al. was determined in their laboratory by Western blot analysis on membrane-enriched fractions from the kidney (16,000 g pellet), as recently reported (26). Using this AT2R antibody, we found the translational sites of the AT2R to be localized in each of the three regions of the rat kidney, which included the cortex and outer and inner medulla (Fig. 4A). Higher magnification of the renal cortex revealed that the strongest labeling of the AT2R was found within the proximal tubules and distal tubules of the renal cortex, which includes the collecting ducts (Fig. 4B). Little staining was detected in the TAL or in the glomeruli. In the renal medulla, the labeling of the AT2R appeared to be highest in the collecting ducts (Fig. 4C), with very little labeling within the vascular bundles. As a negative control, kidney sections that were incubated with the AT2R antibody preadsorbed with the competing antigenic peptide did not have any labeling, demonstrating the specificity of the binding by the primary antibody (Fig. 4D).


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Fig. 4.   Immunohistochemical sections of kidney incubated with AT2R antibody. A: kidney section showing immunoreactivity of AT2R in renal cortex (CTX) and medulla (OM) (magnification ×40; bar = 400 µm). B: renal cortex demonstrating labeling within the PT and DT (arrow) with very little labeling within the thick ascending limbs (T; arrow) and glomeruli (Glm) (magnification ×100; bar = 160 µm). C: outer medullary section demonstrating labeling of DT and PT (S3 segment) with lesser staining in the vascular bundles (VB) (magnification ×100; bar = 80 µm). D: renal cortical section incubated with the AT2R preadsorbed with the immunizing peptide demonstrates no immunostaining (magnification ×100; bar = 160 µm).

Specificity of the PCR primers for AT2R. The AT2R primers were designed to span both intervening sequences (introns) of the AT2R gene, and the specificity of the AT2R PCR product was verified by PCR sequencing. Moreover, the presence of AT2R mRNA expression in the adrenal gland and aorta and the absence of liver AT2R mRNA is consistent with previously published observations (26, 37), validating the specificity of the AT2R primers (Fig. 2). We performed six experiments for each of the total tissue RNA samples and observed the same qualitative results.

Microlocalization of AT2R within rat kidney. The localization of the AT2R mRNA was determined in microdissected renal tubules and blood vessels from both the renal cortex and medulla. Figure 5 demonstrates the RT-PCR products for the AT2R in the renal tubules and vasculature from the present study, and the results are fully summarized in Table 1. It was determined that the AT2R mRNA was found in the proximal convoluted tubule, outer and inner medullary collecting duct, arcuate artery, and OMDVR. A positive AT2R signal (denoted as a "+" in Table 1) in the whole tissue or microdissected tissue was observed in six different rat kidney samples by gel electrophoresis in which the AT2R PCR product was always detected. We also performed three experiments on the cortical collecting ducts and afferent arterioles, which also detected AT2R mRNA in every sample isolated. On the other hand, AT2R mRNA was not consistently detected in the MTAL (only one positive result out of six different MTAL preparations) or in glomeruli (no detectable PCR product could be observed in any of the six glomerular preparations), so these renal segments were considered negative (denoted with a "-" in Table 1). Contamination was not evident, as shown by the absence of detectable bands in the DS and the PCR amplification of DNase-treated RNA from inner medullary collecting duct. To verify the authenticity of the AT2R PCR product, total tissue RNA from the adrenal (100 ng) and liver (1 µg) were run in each of the assays; adrenals were used as a positive control for each of the ANG II receptors, and the liver was used as a negative control for the AT2R.


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Fig. 5.   RT-PCR analysis of renal tubular and vascular AT2R in microdissected tubules and blood vessels from the rat kidney. Ethidium bromide-stained gels demonstrate the RT-PCR products from the MTAL, OMCD and IMCD, OMDVR, and the arcuate artery (Arc). Adrenal gland and liver were used in each experiment as positive and negative controls for the AT2R. Negative control samples are dissection solution (Diss Soln) and PCR amplification of DNase-treated RNA from the IMCD (IMCD -RT). Small arrows denote the molecular size markers (at left). Size of the RT-PCR products for the AT2R was 445 bp. Six separate experiments were performed for each whole and microdissected tissue studied.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to differentiate and localize the angiotensin AT1R subtypes within the rat kidney. Specific PCR primers were designed to allow for the discrimination of the AT1AR, AT1BR, and AT2R by gel electrophoresis. In addition, specific polyclonal antibodies targeted against the AT1R and AT2R were used in immunohistochemistry to demonstrate the translational sites within the kidney.

Localization of the AT1AR and AT1BR Within the Rat Kidney

It is well known that ANG II has both vascular and tubular effects within the rat kidney (18, 25, 29, 40). The immunohistochemical results from the present study showed that the AT1R protein was associated with tubular and vascular structures throughout the kidney, with the strongest labeling being observed in the cortical blood vessels and S3 segment of the proximal tubules in the outer medulla. The results from the present study are consistent with a study by Meister et al. (22) in which the AT1R mRNA was highly localized to the outer medullary proximal tubules using in situ hybridization. Moreover, binding studies with 125I-ANG II in microdissected nephron segments showed binding to be highest in the proximal tubules, with lesser binding in the TAL and collecting ducts (23). Subsequent studies using AT1R and AT2R agonist-selective analogs have demonstrated that the majority of the ANG II binding (>80%) within the kidney is to the AT1R (4, 7, 32, 43). In addition, AT1R distribution has also been found localized to the interstitial cells of the kidney using ANG II binding techniques (43), but the techniques utilized in this study could not ascertain whether interstitial cells were labeled. In all, neither immunohistochemistry nor ANG II binding studies are able to differentiate between the AT1AR and AT1BR, because of the lack of specific AT1R subtype analogs and antibodies.

To circumvent this problem, investigators have used RT-PCR to demonstrate the localization of AT1R subtypes within microdissected renal tubules. Terada et al. (39) observed that the AT1R mRNA was present throughout the nephron, which included the proximal tubules, TAL, and collecting ducts. However, the PCR primers used in their study were designed within the coding region of the AT1R where the AT1AR and AT1BR have the highest homology (~92% identical) with each other. Subsequent studies by several investigators began to use restriction enzymes following RT-PCR amplification to distinguish the AT1R isoforms (1). These investigators, however, used this technique only to localize the AT1R isoforms in different regions of the kidney using whole tissue samples. Recent studies by Bouby et al. (2) demonstrated AT1AR and AT1BR mRNA distribution in individual nephron segments, but a number of potential problems could arise from restriction enzyme analysis of PCR products having high homology (greater than 90%), as is the case for the AT1AR and AT1BR. Specifically, the nucleotide mutations can occur during PCR product generation so that a particular restriction enzyme site can be added or removed. Additionally, because restriction enzymes recognize unique palindromic sequences, closely homologous mRNAs may not be digested due to the high PCR cycle numbers. This can result in the formation of AT1AR:AT1BR heteroduplexes, which makes these products unable to be digested. Formation of heteroduplexes is probably the greatest limitation of the restriction enzyme digestion approach (10, 21, 35).

For these reasons, one of the novel contributions of the present study was the development of gene-specific primers that would selectively amplify the AT1R subtypes and thus make the restriction enzyme digestion unnecessary to distinguish the AT1AR from that of AT1BR PCR products. The results of the RT-PCR (as shown in Fig. 2) demonstrated the ability of the present study to design gene-specific primers for highly homologous cDNAs. Further support for the specificity of the PCR primers for selective amplification was obtained using different tissue RNAs, which included the adrenal gland, aorta, and liver. It has been previously reported that the adrenal gland has both isoforms of the AT1R (37) as confirmed in our study. The liver, in contrast, yielded only AT1AR PCR amplification RNA products, but not AT1BR, which confirms observations by others in rats (1) and mice (3). The results of the present study, therefore, showed that the PCR primers, which were designed for the AT1AR and AT1BR, were specific as tested by restriction enzyme digestion and tissue RNA analysis.

The results of our study (summarized in Table 1) demonstrate that all of the tubular segments contain both AT1AR and AT1BR receptor isoforms, including the proximal tubules, TAL, and collecting ducts. In the renal vasculature, the AT1AR and AT1BR mRNA was also detected in the glomerulus, arcuate artery, afferent arterioles, and OMDVR. Studies by Terada et al. (39) demonstrated AT1R mRNA in the arcuate artery and vascular bundles, but the AT1R PCR primers used in that study could not distinguish between the AT1R subtypes. The physiological role of each of these receptors in each of these distinct segments of renal tubules and vessels remains to be determined.

Localization of AT2R Within Rat Kidney

The functional role of the AT2R has not been fully elucidated within the kidney. It is believed that the AT2R is involved in organogenesis, because of its abundant presence during development (36, 37). Initial studies by Grady et al. (7) found that PD-123177, a specific AT2R antagonist, blocked the binding of 125I-[Sar1,Ile8]ANG II during embryogenesis and postnatal development of the rat. In situ hybridization studies found that AT2R mRNA was abundantly found in the rat brain, adrenal glands, and lungs throughout development in the rat, but the AT2R mRNA was detected in the rat kidney only for the first 22 days following parturition (36, 37). Studies by others have indicated that less than 20% of the ANG II binding sites in the adult kidney can be attributed to the AT2R (32, 43).

The present study determined that the AT2R mRNA was detected in the adrenal gland and the aorta, but not in the liver. These findings are consistent with previous in situ hybridization and RT-PCR studies (24, 36). In the kidney, we have localized the AT2R mRNA to various tubular and vascular segments from the cortex and medulla, which include the proximal tubules, collecting ducts, arcuate arteries, afferent arterioles, and OMDVR. The presence of AT2R mRNA in afferent arterioles is consistent with the studies of Ruan et al. (32). An absence of AT2R mRNA was observed within the glomerulus and in the MTAL. The absence of glomerular AT2R mRNA in adolescent rats is consistent with previous immunohistochemical (28) and autoradiographic techniques (5) where glomerular AT2R protein was nearly absent in adolescent and adult rats that were fed a normal salt diet. Moreover, the localization of the AT2R mRNA is consistent with the immunohistochemical study performed in the present study, in which we used a specific AT2R antibody. Although the present study demonstrates the presence of the AT2R within the proximal tubules and collecting ducts, further studies need to be performed to address the role(s) of this receptor in the regulation of tubular function.

Functional Roles of the ANG II Receptor Subtypes Within the Renal Vasculature

The role of the ANG II receptor subtypes within the renal vasculature has begun to be elucidated by several investigators. Terada et al. (39) previously demonstrated AT1R mRNA in microdissected arcuate arteries and vasa recta bundles. The transcription of mRNA for both of the AT1R isoforms in the renal vasculature and the supportive immunocytochemical evidence indicates that the vasoconstrictor actions of ANG II on the renal vasculature were related to the stimulation of these receptors. Ikenaga et al. (11), using an isolated split kidney blood perfused juxtamedullary preparation, found that afferent arterioles constricted in the presence of ANG II. The descending vasa recta vessels, which are of particular interest regarding the regulation of medullary blood flow, clearly expressed both isoforms of the AT1R mRNA. The functional significance of these descending vasa recta receptors has been demonstrated by Pallone (29), who has shown that ANG II can cause reductions in luminal diameters of isolated perfused OMDVR through the stimulation of an AT1R. The ability of ANG II to constrict these descending vasa recta could be blocked by the addition of saralasin, an AT1R antagonist. Although isoform-specific AT1R antagonists do not currently exist to determine the functional roles of these receptors, it is evident from the present study that both of these isoforms are widely expressed throughout the renal vasculature and tubules, which may be of functional or pharmacological significance.

With regard to the AT2R, the present study has demonstrated that the AT2R mRNA and protein are present within different segments of the cortical and medullary vasculature, including the arcuate artery, afferent arterioles, and OMDVR. The finding of the AT2R within renal vessels is consistent with the previous observation by Ruan et al. (32). The role of the AT2R in the renal vasculature remains to be determined but on a whole tissue basis may have a role in the stimulation of nitric oxide (38). Future studies will need to be performed to address the role of the AT2R within the kidney, particularly within the renal vasculature.

In summary, the present study has demonstrated that AT1R mRNA and protein is found throughout the kidneys of adult Sprague-Dawley rats. The translation sites for this receptor appeared to be most intense in the outer medulla. The design of specific AT1AR and AT1BR primers allowed for the differentiation of the two isoforms of these receptors within microdissected tubular and vascular segments from the renal medulla, which included the TAL, collecting ducts, and OMDVR. The AT2R mRNA and protein was also found widely distributed in the various tubular and vascular segments of the renal cortex and medulla. AT2R mRNA was localized to the proximal tubules, collecting ducts, arcuate arteries, afferent arterioles, and OMDVR. The results indicated that the AT2R mRNA was not readily detectable in the glomerulus or the TAL. It is important to note that in the present study we used techniques which determined the qualitative distribution of the AT1R and AT2R but that future studies need to be performed to quantitate and demonstrate their functional roles within distinct segments of the renal vasculature and tubules.


    ACKNOWLEDGEMENTS

We thank Dr. David L. Mattson and Meredith Skelton for critical review of this manuscript, Chibuike Anucha for expert assistance, and Dr. Ai-Ping Zou for helpful discussions.


    FOOTNOTES

F. Park was supported by Wisconsin Affiliate American Heart Association Predoctoral Fellowship (96-F-PRE-29). This study was supported by the National Heart, Lung, and Blood Institute Grant HL-29587.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. W. Cowley, Jr., Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53222.

Received 31 July 1998; accepted in final form 30 April 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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