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(Hypertension. 1995;26:1181-1185.)
© 1995 American Heart Association, Inc.

Articles

Adenosine A₁ Receptor mRNA in Microdissected Rat Nephron Segments

Satoshi Yamaguchi; Satoshi Umemura; Kouichi Tamura; Tamio Iwamoto; Nobuo Nyui; Tomoaki Ishigami; Masao Ishii

From the Second Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama, Japan.

	Abstract

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Abstract Adenosine plays several roles in the kidney mediated by the specific receptors A₁, A₂, and possibly A₃. We studied the localization of adenosine A₁ receptor mRNA in rat nephron segments using reverse transcription and polymerase chain reaction (RT-PCR). The nephron segments of male Sprague-Dawley rats (6 to 8 weeks old) were microdissected. Total RNA was prepared by the acid-guanidinium–phenol–chloroform method and used in the following RT-PCR assay. Because the PCR primers spanned no intron, samples reacted in the absence of RT were used as controls for amplification of genomic DNA. The PCR products were size-fractionated by electrophoresis, visualized with ethidium bromide staining, and confirmed by Southern blot analysis. PCR products were detected in all of the nephron segments examined. No signals were detected in samples reacted in the absence of RT. Strong signals were detected in glomeruli, medullary collecting duct, cortical thick ascending limb, and medullary thick ascending limb, while weak signals were found in proximal convoluted and straight tubules. Previously, the presence of A₁ receptors has been demonstrated in glomeruli, collecting duct, and thick ascending limb in the rat kidney by autoradiography and binding studies. In addition to these segments, we further detected A₁ receptor mRNA in proximal convoluted and straight tubules. Thus, A₁ receptor mRNA seems to be broadly expressed along the nephron.

Key Words: adenosine • purinergic receptors • reverse transcription • polymerase chain reaction • renal tubule

	Introduction

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Adenosine plays an important regulatory role in various functions in the kidney, including renal blood flow, glomerular filtration rate, renin secretion, tubuloglomerular feedback, tubular reabsorption of sodium and water, sympathetic neurotransmitter release, and erythropoietin secretion.^{1 2 3} Adenosine produces its diverse physiological effects by interacting with at least two membrane receptors, termed A₁ and A₂, which can be differentiated on the basis of their affinity for various adenosine analogues and their effects on adenylate cyclase. These receptors couple to the inhibition and stimulation of adenylate cyclase through G_i and G_s proteins, respectively.^{4 5} The A₁ and A₂ adenosine receptor subtypes also have distinct pharmacological specificities and different distributions within the central nervous system and peripheral tissues.⁶

Recently, the A₁, A₂, and A₃ receptors have been cloned from cDNA libraries, sequenced, and expressed in a variety of cellular expression systems.^{7 8 9 10} Furthermore, the A₂ receptor subtype has been subdivided into distinct A_2a and A_2b subtypes.¹¹

Many studies using molecular biological techniques have been performed to detect the localization of the adenosine receptors. To identify target sites of adenosine within the kidney, it is important to clarify the localization of these receptors. By using autoradiography, the intrarenal expression of A₁ receptor has been demonstrated,^{12 13 14} and the intrarenal tissue sites of A₁ receptor mRNA synthesis have been localized by Northern blot analysis and in situ hybridization.¹⁵

Recently, Moriyama et al¹⁶ and Terada et al¹⁷ introduced a new method for measurement of relative levels of specific mRNA in single microdissected renal tubules using PCR coupled to RT (RT-PCR). Using this technique, they performed relative quantification of mRNA using only 2-mm lengths of renal tubules.¹⁷ Furthermore, Makino et al¹⁸ demonstrated a new approach for the relative quantification of mRNA by RT-PCR.

Therefore, in this study we examined the localization and performed semiquantitative analysis of A₁ receptor mRNA in microdissected rat nephron segments using RT-PCR.

	Methods

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Animals
Male Sprague-Dawley rats 6 to 8 weeks old, weighing 220 to 250 g, were obtained from Charles River Laboratories, Inc (Atsugi, Kanagawa, Japan). These rats were fed ad libitum on a normal salt diet and had free access to water until the experiment.

Renal Tubule Microdissection and RNA Preparation
Animals were anesthetized with pentobarbital; then the abdomen of each animal was opened, and the aorta was cannulated with polyethylene tubing below the left kidney. The left kidney was perfused initially with 10 mL ice-cold dissection solution containing the following (mmol/L): NaCl 135, Na₂SO₄ 1, MgSO₄ 1.2, KCl 5, CaCl₂ 2, glucose 5.5, and HEPES 5, pH 7.4. The kidney was then perfused again with 10 mL of the same solution containing 1 mg/mL collagenase (type I, 400 U/mg) and 1 mg/mL bovine serum albumin. The kidney was removed and decapsulated, and thin coronal sections were cut. The sections were transferred into tubes containing 10 mL of the same collagenase solution and incubated with 95% O₂ and 5% CO₂ bubbling for 40 minutes at 37°C. Then these sections were transferred to Petri dishes on ice filled with dissection solution containing 10 mmol/L vanadyl ribonucleotide complex. At the end of the experiment the rats were killed by decapitation under anesthesia.

Tubule dissection was performed using dissecting forceps (sharpened Dumont No. 5, A. Dumont and Fils) under a dissection microscope with dark-field illumination. Tubule segments were identified on the basis of previously described criteria.¹⁹ We microdissected the following structures: Glm, PCT, PST, CCD, MCD, CTAL, and MTAL. Dissected segments were measured with an ocular micrometer. Each segment was collected to a sum of 20 to 30 mm in length (200 glomeruli) and transferred using pipettes coated with 0.1% bovine serum albumin (RNase-free) to clean dissection buffer and washed free of contaminating debris. These segments were transferred into individual tubes containing 1 mL of RNAzolB (Cinna/Biotecx Laboratories, Inc) and immediately homogenized. Total RNA was precipitated from the extract of each segment with an equal volume of isopropanol in the presence of 25 µg glycogen. The total RNA was collected by centrifugation, and the resultant pellets were washed with 75% ethanol. The final RNA pellets were resuspended in 20 µL of diethylpyrocarbonate-treated water and quantified by absorbance at 260 nm. As a control for possible contamination, 10 µL of the final wash buffer was carried through RNA preparation, RT, and PCR steps.

Reverse Transcription
Total RNA (9 µL; 0.3 µg) containing 100 pmol/L random hexanucleotide primer was heated to 94°C for 2 minutes and 37°C for 5 minutes. RT reaction mixture (11 µL) containing 20 U RNase inhibitor, 10 mmol/L dithiothreitol, 2 mmol/L dNTP, 5x reaction buffer, and 100 U Moloney Murine Leukemia Virus Reverse Transcriptase was added. The reaction mixture was incubated at 37°C for 60 minutes and at the end of the incubation period heated to 98°C for 10 minutes to inactivate the reverse transcriptase activity and to denature RNA-cDNA hybrids. Negative control reactions containing all the reagents except the reverse transcriptase were performed in parallel.

Polymerase Chain Reaction
PCR was performed with rat adenosine A₁ receptor specific primers 5'-TGGGAGGTCTTCATCGATGGG-3' (antisense, corresponding to nucleic acids 1293 to 1313) and 5'-GAGCTGAAGATCGCCAAGTCG-3' (sense, corresponding to nucleic acids 1038 to 1058). These primers yielded a product of 276 bases and spanned no intron. Simultaneously, we performed RT and PCR for the housekeeping gene ß-actin in the renal structures as a positive control. The primers for ß-actin were defined by the following cDNA base sequences: 5'-GGCCATCTCTTGCTCGAAGT-3' (antisense, corresponding to nucleic acids 2457 to 2476) and 5'-AAGAGAGGCATCCTGACCCT-3' (sense, corresponding to nucleic acids 1509 to 1528), which spanned an intron and resulted in a 504-bp product. After RT, we divided 20-µL samples into 15 µL for analysis of adenosine A₁ receptor and 5 µL for ß-actin. The volume was adjusted to 20 µL with sterile water, and parallel PCR reactions were run with each set of primers. To each tube was added 80 µL of a PCR master mix containing 100 picomoles of each primer, 10 µL of 10x reaction buffer (a final composition was 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, and 0.001% gelatin), 1 µL of 10 mmol/L dNTP, and 2.5 U of Taq DNA polymerase. The reaction mixture (100 µL) was overlaid with 50 µL mineral oil, and the tubes were placed in a DNA thermal cycler (Perkin Elmer Cetus) programmed as follows: incubation at 94°C for 3 minutes (initial melt); then 30 cycles of 94°C for 1 minute (melt), 60°C for 1 minute (anneal), and 72°C for 3 minutes (extension). Final incubation was performed at 72°C for 7 minutes. Then samples were kept at 4°C until analysis.

Analysis of Products
The identity of PCR products was confirmed by Southern hybridization, following size-fractionation by 1.4% agarose gel electrophoresis. After electrophoresis and ethidium bromide staining, DNA bands were photographed by using an ultraviolet transilluminator (UVP, Inc) and Polaroid type 667 positive-negative film (Polaroid Corp). PCR products were transferred onto Gene Screen Plus nylon membranes (DuPont–New England Nuclear) as described previously.²⁰ Hybridization was conducted at 65°C for 16 hours in 1 mol/L NaCl, 1% SDS, 10% dextran sulfate, 100 mg/mL denatured salmon sperm DNA, and 1x10⁶ cpm/mL labeled probe; the probe was obtained from the 1.8-Kbp restriction fragment of rat A₁ adenosine receptor cDNA⁸ using a Random Primed DNA Labeling Kit (Boehringer Mannheim GmbH). Filters were washed twice with 2xSSC at room temperature for 5 minutes each time, twice with 2xSSC and 1% SDS at 60°C for 30 minutes, and twice with 0.1xSSC at room temperature. The filters were then subjected to autoradiography at room temperature for 6 hours with BAS 2000 imaging plates (Fuji Film Corp).

Relative Quantification of mRNA Level From Autoradiographs
The relative amounts of PCR products were determined by densitometric scanning of autoradiographs using a BAS 2000 laser image analyzer (Fuji Film Corp). For normalizing the variability of each assay, we used 1 µg of the same total RNA of whole rat kidney (reference RNA) in every RT-PCR assay and calculated the percentage of the reference RNA densitometry value for each segment.

Preliminary experiments were performed to obtain appropriate cycle numbers of PCR and amounts of sample RNA for the semiquantitative analysis. We chose 0.1, 0.3, and 0.5 µg of total RNA of rat whole kidney and 25, 30, 35, and 40 cycles of PCR.

Statistical Analysis
The results are given as mean±SEM. Where appropriate, the data were analyzed for significance by Student's t test for unpaired data and accepted at a value of P<.05.

	Results

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Preliminary experiments showed that at 25 and 30 cycles, PCR products as reflected by densitometry values increased in a linear fashion with increasing amounts of total RNA between 0.1 µg and 0.5 µg. However, at 35 and 40 cycles, attenuation was observed by a plateau effect above 0.3 µg. Fig 1 shows that the densitometry values of PCR products at 30 cycles, when the 0.5-µg sample is assigned as an arbitrary unit of 100, were 24.2 (0.1 µg) and 61.6 (0.3 µg). Therefore, we set the number of amplification cycles in this experiment as 30 cycles and the initial amount of total RNA of each segment as 0.3 µg (0.225 µg was used for PCR reaction of A₁ receptor and 0.075 µg for ß-actin).

View larger version (13K): [in this window] [in a new window]   Figure 1. Plot shows the relation between quantity of total RNA and resulting amplification products by RT-PCR. The reference value of 100 was assigned to the densitometry value representing 0.5 µg of total RNA.

Fig 2 shows representative photographs of ethidium bromide–stained agarose gels and corresponding Southern blots for the PCR products of A₁ receptor and ß-actin mRNA in rat glomeruli and tubules. The expected size of each PCR product was apparent: A₁ receptor (276 bp) and ß-actin (504 bp).

View larger version (72K): [in this window] [in a new window]   Figure 2. Blots show RT-PCR products of adenosine A1 receptor mRNA in microdissected nephron segments detected by ethidium bromide–stained agarose gels (A) and autoradiograms of the corresponding Southern blots (B). RT-PCR products of ß-actin as a positive control were detected by ethidium bromide–stained agarose gels (C). The arrow indicates the PCR product of the predicted size (adenosine A1 receptor mRNA: 276 bp; ß-actin: 504 bp). PCR was carried out with (+) and without (-) RT. The right lane shows the amplification product from 1 µg of total RNA of whole kidney.

As seen in Fig 2A by agarose gel staining, a single 276-bp band of the expected size amplified from the A₁ receptor primers was found from all nephron segments. Among renal nephron segments, the densest band was consistently found in MTAL, and relatively dense bands were found in CCD, MCD, and CTAL. Weak but detectable bands were observed in PST, PCT, and Glm.

As can be seen in Fig 2B, Southern hybridization using specific probes confirmed the identity of each of these PCR products. The radioactive intensity of the signal of each segment was almost the same as the ethidium bromide staining: the strongest signal was detected in MTAL; strong signals in CTAL, MCD, and Glm; and a weak signal in CCD. Very weak and almost undetectable signals were found in PCT and PST.

When PCR was carried out in the absence of reverse transcriptase, the bands were not seen, indicating that each band was derived from mRNA and not from genomic DNA contamination. Furthermore, the RT-PCR product from the final wash buffer produced no reaction product, which indicated that the samples were not contaminated by other structures at the steps of microdissection and RNA isolation.

Fig 2C shows that the amplification product of ß-actin was detected from all renal structures at the predicted size (504 bp), indicating that RT-PCR was successful in each nephron segment. Similarly, in the case of ß-actin, the bands of PCR products were not seen without RT reaction, confirming that there was no genomic DNA contamination in the samples.

Fig 3 graphically summarizes the relative levels of the A₁ receptor amplification products among the nephron segments. The densitometric value from each segment was normalized by dividing by the densitometric value from reference RNA (1 µg of total RNA of whole rat kidney). Data points represent results from three independent experiments and are expressed as percentages of the densitometric values obtained in MTAL in the same experiment. The MTAL consistently gave the largest signal that was relatively invariant from experiment to experiment (MTAL arbitrary values of 100±2.5%). In Glm, the signal was 88.5±4.0%. In CTAL and MCD, the signals were 81.3±10.4% and 70.5±8.4%, respectively; in CCD the signal was 32.0±2.8%, and in proximal convoluted tubules and straight tubules, signals were 2.8±1.0% and 13.5±2.1%, respectively, of that in MTAL.

View larger version (34K): [in this window] [in a new window]   Figure 3. Bar graph shows relative quantification of adenosine A1 receptor mRNA in microdissected nephron segments. Values for each segment are expressed as a percentage of the value from the products of MTAL obtained in the same amplification run.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrated for the first time the localization of adenosine A₁ receptor mRNA in the rat kidney by a combination of microdissection and RT-PCR. Adenosine A₁ receptor mRNA was expressed in all nephron segments studied. In Glm, PCT, PST, CCD, MCD, CTAL, and MTAL, the PCR products were detected, and no signals were detected in the samples reacted in the absence of RT. Strong signals were detected in Glm, MCD, CTAL, and MTAL, while weak signals were found in PCT and PST.

Previous studies using ligand binding and autoradiography have shown the presence of adenosine receptors in such gross anatomic structures as papillae,^{21 22} cortex,^{13 23} and medulla.^{12 13} A₁ receptors have been shown to be present in rabbit renal Glm in a radioligand binding study using [¹²⁵I]HPIA.²³ We have also demonstrated the existence of A₁ receptors in isolated human Glm by [³H]CCPA binding.²⁴ In addition, A₁ receptors have been detected in both human and guinea pig Glm and in the inner and outer medulla of guinea pig kidney by the specific localization of the A₁ ligand [³H]CHA by using autoradiography.¹³ Studies in the rat kidney using [¹²⁵I]HPIA autoradiography and [³H]DPCPX binding analysis demonstrated specific binding in crude membranes from the inner stripe of the outer medulla and the inner papilla as well as isolated MTAL, suggesting the presence of A₁ receptors in the MTAL and papillary collecting duct.^{14 25} It is generally concluded that A₁ receptors are widely distributed throughout the nephron and renal vasculature.

Recently, the direct actions of adenosine and adenosine analogues have been reported in renal epithelial cells and were summarized.²⁶ For example, adenosine and adenosine analogues stimulate cAMP production in human and rat isolated Glm, resulting in a decrease in GFR through changes in glomerular filtration coefficient (K_f).^{23 27} We have also reported the existence of the A₁ receptor–cAMP system in human Glm.²⁴ In the isolated perfused rabbit collecting tubule, it was reported that the hydraulic conductivity was stimulated by 5'-N-ethylcarboxamidoadenosine (NECA).²⁸ In addition, in the isolated segments of dog MTAL and collecting tubule, NECA increased adenylyl cyclase activity.²⁹ Furthermore, A₁ receptor activation was reported to result in a decrease of transtubular voltage in isolated rat CTAL.³⁰ The presence of adenosine receptors was also reported in the rabbit renal CCD and CTAL.^{31 32} These findings confirmed the results of this study that A₁ receptor mRNA was expressed at high levels in CTAL, MCD, and CCD, although there might be species differences in receptor distribution.^{12 13}

However, autoradiography, ligand binding, and functional studies suffer limitations in resolution and sensitivity that preclude a systematic evaluation of receptor presence. Furthermore, ligand binding studies require a large amount of tissue, which makes nephron segmental analysis of receptors both difficult and impractical.

With the development of molecular biology, the cloning of adenosine receptors from a variety of tissues has been achieved, and new approaches have been applied to determine the segmental localization and relative abundance of adenosine receptors in the kidney. Weaver and Reppert¹⁵ demonstrated that A₁ receptor mRNA was most abundant in the collecting ducts of the papilla and inner medulla and could be detected in collecting ducts in the outermost portion of the inner stripe of the outer medulla and cells of the juxtaglomerular apparatus using Northern blot analysis and in situ hybridization. The significant signals of A₁ receptor mRNA were not observed in other nephron segments.

The discrepancies between the results of these studies and the present study might have been due to the methods used to evaluate the localization of A₁ receptor mRNA. Although our method is a semiquantitive assessment of mRNA abundance and not strictly quantitative, RT-PCR is the most sensitive method for detecting mRNA. Thus, in our study we could detect the presence of A₁ receptor mRNA in segments where no expression was observed by in situ hybridization. Furthermore, the different posttranscriptional regulation and translational control of mRNA among the nephron segments might have been responsible for the discrepancies between the results from the binding studies and our results.

In this study we detected A₁ receptor mRNA along all the dissected nephron segments for the first time. A₁ receptor mRNA was most abundant in MTAL, a result that is in good agreement with those of preliminary studies.^{14 25} We also detected A₁ receptor mRNA in PCT and PST for the first time. Previous studies demonstrated that ecto-5' nucleotidase, by which adenosine is formed through the availability of the substrate 5'-AMP, was abundant in the proximal tubule brush border.³³ Furthermore, Takeda et al³⁴ demonstrated that A₁ antagonists had an inhibitory effect on Na⁺-3HCO₃^- cotransporter in the rabbit PCT via A₁ receptor by microperfused study. LeVier et al³⁵ also demonstrated the functional localization of A₁ receptor–mediated pathways in the LLC-PK₁ renal cell line thought to be from proximal tubules. Although there may be species differences in receptor distribution, our results are consistent with these findings. Further studies are required to determine the physiological significance of A₁ receptor mRNA in PCT and PST.

In summary, this is the first study to detect the localization of the adenosine A₁ receptors in rat nephron segments using RT-PCR. We observed the expression of adenosine A₁ receptor mRNA along all nephron segments studied, indicating that A₁ receptor in these nephron segments may be involved in the regulation of various renal functions induced by adenosine. Further studies will be required to investigate the physiological role of the A₁ receptor and regulation of the expression of A₁ receptor mRNA.

	Selected Abbreviations and Acronyms

 

CCD = cortical collecting duct CTAL = cortical thick ascending limb Glm = glomeruli MCD = medullary collecting duct MTAL = medullary thick ascending limb PCR = polymerase chain reaction PCT = proximal convoluted tubule PST = proximal straight tubule RT = reverse transcription

	Acknowledgments

 
This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan (Nos. 05454275, 07266221, 05670956, and 4512). We would like to express our thanks to Dr Steven M. Reppert (Laboratory of Developmental Chronobiology, Children's Service, Massachusetts General Hospital and Harvard Medical School, Boston, Mass) for providing us with the rat adenosine A₁ receptor cDNA (pRc/A1).

	Footnotes

 
Reprint requests to Satoshi Umemura, MD, Second Department of Internal Medicine, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-Ku, Yokohama 236, Japan.

Received June 18, 1995; first decision August 1, 1995; accepted September 5, 1995.

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