Kidney International, Vol. 51(1997), pp. 1548—1552

Distribution of a1-adrenergic receptor mRNA expression alongrat nephron segments

SATOSHI UMEMURA, SATOSHI YAMAGUCHI, KouTcHi TAMURA, KIY0SHI HIBI, NoBuo Nyui,TOMOAKI ISHIGAMI, MIN0Ru KIHARA, MACHIKO YABANA, and MASAO IsHIT

Second Deportment of Internal Medicine, Yokohama City University School of Medicine, Yokohama, Japan

Distribution of a1-adrenergic receptor mRNA expression along ratnephron segments. Although several a-adrenergic receptor genes areexpressed in the rat kidney, little information is available on theirexpression in the renal nephron segments. We investigated the distribu-tion of a1-adrenergic receptor mRNA in rat nephron segments usingreverse transcription and polymerase chain reaction (RT-PCR). Thenephron segments of six- to eight-week-old male Sprague-Dawley ratswere microdissected. Total RNA was prepared by the acid-guanidinium-phenol-chloroform method and used in the following RT-PCR assay. ThePCR products were size-fractionated with electrophoresis, visualized withethidium bromide staining and confirmed by Southern blot analysis.Because the PCR primers spanned an intron, the amplification product ofthe predicted size was considered to be from a10-adrenergic receptoreDNA and not from genomic DNA. The PCR products were detected inglomerulus (GIm), proximal convoluted and straight tubules (PCT, PST)and cortical and medullary thick ascending limbs of Henle (CTAL,MTAL). No signals were detected in cortical or medullary collecting ducts(CCD, MCD). Large signals were detected in the PCT, and PST, whilesmall signals were found in the GIm, CTAL and MTAL. The aIB-adrenergic receptor mRNA was detected for the first time in rat GIm,PCT, PST and TAL using RT-PCR. a1AR mRNA seems to be expressedin the specific sites along the nephron and may play significant roles inrenal functions, although the specific physiological effects of the renala1-adrenergic receptor are unknown.

Renal sympathetic nerve and catecholamines play importantroles in the regulation of renal function, such as sodium and waterreabsorption [1, 2]. Tubular cs1-adrenoceptor activation by sub-pressor renal nerve stimulation, in the absence of renal hemody-namic changes, increases sodium and water reabsorption in thedog [3], rabbit [4] and rat [51, suggesting a direct tubular effect byrenal nerve stimulation. Adrenergic nerves terminate in closeproximity to the basement membranes of proximal and distaltubules, and the loop of Henle, as well as renal blood vessels, thejuxtaglomerular apparatus, and the afferent and efferent arterioles[6, I.

The existence of the cs1-adrenoceptor has been directly demon-strated in the microdisseeted rabbit proximal convoluted tubules(PCT) by radioligand binding studies [81, and in the rat glomer-ules (GIm) and cortical and medullary tubules by autoradiography

Received for publication September 13, 1996and in revised form December 12, 1996Accepted for publication December 12, 1996

© 1997 by the International Society of Nephrology

[91. Three subtypes of a1-adrenoceptors, the alA-, anda0-adrenoeeptors, have recently been identified by molecularcloning [10]. With the use of these probes for an in Situ hybrid-ization method [11], alB-adrenoceptor was observed in rat vesselsin the renal parenchyma and in the ureter. a1-AdrenoceptormRNA was demonstrated in the outer and inner stripes of theouter medulla of the rat kidney. However, autoradiography,ligand binding, and functional studies have limitations in resolu-tion and sensitivity that preclude a systematic evaluation ofreceptor presence. Ligand binding studies also require a largeamount of tissue, which makes nephron segmental analysis ofreceptors both difficult and impractical.

A new method has recently been introduced for measurementof the relative level of specific mRNA in single microdissectedrenal tubules using polymerase chain reaction (PCR) coupledwith reverse transcription (RT) [12, 13]. We have already appliedthis technique to detect the distribution of adenosine A1 receptors[14] and angiotensinogen (S. Yamaguchi et al, unpublished ob-servation) in rat nephron segments. We applied this technique inthe present study to examine the distribution of aIB-adrenocep-tors, which have also been reported to be expressed in rat renalmicrovessels [15], and we performed semiquantitative analysis ofcs113-adrenoceptor mRNA in microdissected rat nephrori segmentsusing RT-PCR.

Methods

Animals

Male Sprague-Dawley rats aged six to eight weeks old, weighing220 to 250 g, were obtained from Charles River Laboratories(Atsugi, Kanagawa, Japan). The rats were fed ad libitum on anormal salt diet and had free access to water until the experiment.

Renal tubule microdissection and RNA preparation

The methods used for the microdissection of renal tubules, theRNA preparation and reverse transcription in this study wereessentially the same as previously reported [14, 16]. Animals wereanesthetized with pentobarbital (50 mg/kg, i.p.), following whichthe abdomen of each animal was opened and the aorta wascannulated with polyethylene tubing below the left kidney. Theleft kidney was perfused initially with 10 ml of ice-cold dissectionsolution containing the following: 135 mtvi NaC1, 1 mM Na2SO4,1.2 m MgSO4, 5 mi KCI, 2 mt CaCI2, 5.5 mvt glucose, and 5mMN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES,

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Umemura et al: aJB-Adrenoceptor mRNA in rat nephron segments 1549

pH 7.4). The kidney was then perfused again with 10 ml of thesame solution containing 1 mg/mi coilagenase (type I, 400 U/mg)and 1 mg/mI bovine serum albumin (BSA). The kidney wasremoved and decapsulated and slices of kidney tissue weredissected on the corticomedullary plane. The sections were trans-ferred into tubes containing 10 ml of the same collagenasesolution, and incubated with 95% 02 and 5% CU2, bubbling for40 minutes at 37°C. Then the sections were transferred to petridishes on ice filled with dissection solution containing 10 mmol/liter vanadyl ribonucleotide complex.

Tubule dissection was performed using dissecting forceps(sharpened Dumont No. 5; A. Dumont and Fils, Switzerland),under a dissection microscope with dark-field illumination. Tu-bule segments were identified based on previously describedcriteria [16]. We microdissected the following structures: Glm,PCT, proximal straight tubules (PST), cortical collecting ducts(CCD), medullary collecting ducts (MCD), cortical thick ascend-ing limbs (CTAL), and medullary thick ascending limbs (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), transferred using pipettes coated with 0.1% BSA(RNase-free) to clean dissection buffer, and washed free ofcontaminating debris, The segments were then transferred intoindividual tubes containing I ml of RNAzo1B (Cinna/BiotecxLaboratories, Houston, TX, USA), and immediately homoge-nized. Total RNA was precipitated from the extract of eachsegment with an equal volume of isopropanol in the presence of25 g of glycogen. The total RNA was collected by centrifugation(12000 >< g for 15 mm) and the resultant pellets were washed with75% ethanol. The final RNA pellets were resuspended in 20 il ofdiethylpyrocarbonate (DEPC)-treated water and quantified byabsorbance at 260 nm. As a control for possible contamination, 10jil of the final wash buffer was carried through the RNA prepa-ration, reverse transcription and PCR steps.

Reverse transcription

Total RNA (9 sal; 0.2 tg) containing 100 M random hexanucle-otide primer were heated to 94°C for two minutes, and 37°C forfive minutes. RT reaction mixture (11 p1) containing 20 U RNaseinhibitor, 10 mvt DTr, 2 m'vi dNTP, 5X reaction buffer, and 100U Moloney murine leukemia virus reverse transcriptase wasadded. The reaction mixture was incubated at 37°C for 60minutes, and at the end of the incubation period heated to 98°Cfor 10 minutes to inactivate the reverse transcriptase activity andto denature RNA-cDNA hybrids. Negative control reactionscontaining all the reagents except the reverse transcriptase wereperformed in parallel.

Polymerase c/lain reaction

PCR was performed with rat alB-adrenoceptor specific primers5 '-CTTGAACTCCTTGCTGGAGC-3' (antisense representingnucleic acids 1289-1308) and 5'-TGAGCClTATGTGCCATC-TCC-3' (sense representing nucleic acids 641-660). These primerswere designed to localize to separate exons, and yielded a productof 668 bases. Simultaneously, we performed RT and PCR for thehousekeeping gene 13-actin in the renal structures as a positivecontrol. The primers for /3-actin were defined by the followingcDNA base sequences: 5 '-GGCCATCTCTTGCTCGAAGT-3'(antisense, corresponding to nucleic acids 2457-2476) and 5'-AAGAGAGGCATCCTGACCCT-3' (sense, corresponding to

nucleic acids 1509-1528), which spanned an intron and resulted ina 504 bp product. After RT, we divided 20 tl samples into 15 j.dfor analysis of alB-adrenoceptor and 5 p1 for 13-actin. The volumewas adjusted to 20 p1 with sterile water, and parallel PCRreactions were run with each set of primers. To each tube wasadded 80 d of a PCR master mix containing 100 picomoles ofeach primer, 10 p1 of lOx reaction buffer (a final composition was10 mM Tris-HC1, pH 8.3, 50 mrvi KC1, 1.5 mrvi MgCl2 and 0.001%gelatin), 1 p1 of 10 mrvi dNTP and 2.5 units of Taq DNApolymerase. The reaction mixture (100 jtl) was overlaid with 50 Llof mineral oil, and the tubes were placed in a DNA thermal cycler(Perkin Elmer Cetus, Norwalk, CT, USA) programmed as fol-lows: incubation at 94°C for three minutes (initial melt); then 26cycles of 94°C for one minute (melt), 60°C for one minute(anneal), and 72°C for three minutes (extension). Final incubationwas performed at 72°C for seven minutes. The samples were thenkept at 4°C until analysis.

Analysis of productsThe identity of PCR products was confirmed by Southern

hybridization, following size-fractionation by 1.4% agarose gelelectrophoresis. After electrophoresis and ethidium bromidestaining, DNA bands were photographed using an ultraviolettransilluminator (UVP, Inc., San Gabriel, USA) and Polaroidtype 667 positive-negative film (Polaroid Corp, Cambridge, USA).PCR products were transferred onto Gene Screen Plus nylonmembranes (DuPont-New England Nuclear, Boston, USA) asdescribed previously. Hybridization was at 65°C for 16 hours in 1mM NaCI, 1% SDS, 10% dextran sulfate, 100 mglml denaturedsalmon sperm DNA, and 1 X 106 cpm/ml labeled probe; the probewas obtained from the 2.1 kbp restriction fragment of rat aiu-adrenergic receptor cDNA [171 using a Random Primed DNALabeling Kit (Boehringer Mannheim, GmbH Mannheim, Germa-ny). Filters were washed twice with 2 X SSC at room temperaturefor five minutes each time, twice with 2 x SSC and 1% SDS at60°C for 30 minutes, and twice with 0.1 X SSC at room temper-ature. The filters were then subjected to autoradiography at roomtemperature for six hours with BAS 2000 imaging plates (FujiFilm, Tokyo, Japan).

Relative quantification of mRNA level from autoradiographsThe relative amounts of PCR products were determined by

densitometric scanning of autoradiographs using a BAS 2000 laserimage analyzer (Fuji Film, Tokyo, Japan). For normalizing thevariability of each assay, data were expressed as percentage of thedensitometric values obtained in PST in the same expertimentsince PST gave the largest signal consistently. In order to seewhether RT-PCR of o1-adrenoceptors mRNA was performedsuccessfully, we used 1 j.g of the same total RNA of whole ratkidney (reference RNA) in every RT-PCR assay.

Preliminary experiments were performed to obtain appropriatecycles numbers of PCR and amounts of sample RNA for thesemiquantitative analysis. We chose 0.1, 0.3, 0.5, and 1.0 g oftotal RNA of rat whole kidney and 20, 26, 30, and 35 cycles ofPCR.

Statistical analysisThe results are given as mean SEM. Where appropriate, the

data were analyzed for significance by Student's t-test for unpaireddata and accepted at a value of P < 0.05.

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1550 Umemura et al: cE/B-Adrenoceptor mRNA in rat nephron segments

Results

Preliminary experiments showed that at 26 cycles, PCR prod-ucts as reflected by densitometry values increased in a linearfashion with increasing amounts of total RNA between 0.1 jig and0.5 jig. At other cycles, however, attenuation was observed by aplateau effect above 0.5 jig. Figure 1 shows that the densitometryvalues of PCR products at 26 cycles, when the 1.0 jig sample wasassigned as an arbitrary unit of 100, were 29.5 (0.1 jig), 61.8 (0.3jig), and 99.0 (0.5 jig). Therefore, we set the number of amplifi-cation cycles in this experiment at 26 cycles, and the initial amountof total RNA of each segment at 0.2 jig (0.15 jig was used for thePCR reaction of 1-adrenoceptor and 0.05 jig for -actin).

Figure 2 shows representative photographs of ethidium bro-mide-stained agarose gels and corresponding Southern blots forthe PCR products of a1-adrenoceptor and f3-actin mRNA inglomeruli and tubules. The expected size of each PCR productwas apparent: a-adrenoceptor (668 bp) and -actin (504 bp).

As seen in Figure 2A, by agarose gel staining, a single 668 bpband of the expected size amplified from the alB-adrenoceptorprimers was found in Glm, PCT, PST, CTAL and MTAL.

As can he seen in Figure 2B, Southern hybridization usingspecific probes confirmed the identity of each of these PCRproducts. The radioactive intensity of the signal of each segmentwas almost the same as ethidium bromide staining: The strongestsignal was consistently found in PST. A strong signal was found inPCT. Weak but detectable signals were found in Gim, CTAL andMTAL.

When PCR was carried out in the absence of reverse transcrip-tase, the bands were not seen, indicating that each hand wasderived from mRNA and not from contaminating genomic DNA.This observation also confirmed that the sense and antisenseprimers designed for RT-PCR were located on separate exons.Furthermore, the RT-PCR product from the final wash bufferproduced no reaction product, which indicated that the sampleswere not contaminated by other structures at the step of micro-dissection and RNA isolation.

Figure 2C shows that the amplification product of -actin was

504 bp(13-actin)

Fig. 2. Representative photographs of ethidium bromide-stained agarose gelsand corresponding Southern blots for the PCR products of a1-adrenoceptorand j3-actin mRNA in rat glomeruli and tubules. A. Ethidium bromide-stained agarose gels for the PCR products of IB-adrenOceptOr mRNA. Asingle 668 bp band of the expected size amplified from the nlB-adreno-ceptor primers were found in GIm, PCT, PST, CTAL and MTAL. B.Southern hybridization using the specific probes for alB-adrenoceptors.The strongest signal was consistently found in PST. A strong signal wasfound in PCT. Weak, but detectable, signals were found in Glm, CTALand MTAL. C. Ethidium bromide-stained agarose gels for the PCRproducts of j3-actin mRNA. The product of 13-actin was detected from allrenal structures at the predicted size (504 bp), indicating that the RT-PCRwas successfully performed in each nephron segment.

detected from all renal structures at the predicted size (504 bp),indicating that RT-PCR was successful in each nephron segment.

Figure 3 graphically summarizes the relative levels of thecs1-adrenoceptor amplification products among the nephronsegments. Data points represent the results from four indepen-dent experiments and are expressed as percentages of the densi-tometric values obtained in PST in the same experiment. Datafrom each individual experiment are shown in Table 1. The PSTconsistently gave the largest signal that was relatively invariantfrom experiment to experiment (PST arbitrary values of 1002.9%). In the PCT the signal was 71.6 4.0%. In GIm the signalswere 34.3 12.3%, and in CTAL and in MTAL the values were29.6 2.7% and 24.5 3.7%, respectively.

Discussion

The present study demonstrated, for the first time, the distri-bution of r1-adrenoceptor mRNA in the rat nephron by acombination method of microdissection and RT-PCR. crlB-adre-noceptor rnRNA was expressed in Gim, PCT, PST, CTAL andMTAL, but not in CCD or MCD. Strong signals were detected inPCT and PST, while weak signals were found in Glm, MTAL andCTAL,

Since each nephron segment has a different function, it isimportant to use microdissected nephron segments for the study

C

100

80

20

00.2 0.4 0.6 0.8 1.0

Amount of RNA, jig

Fig. 1. Relation between quantity of total RNA and resulting amplificationproducts by RT-PCR. PCR products were increased linearly from 0.1 jig to0.5 jig of the initial amount of total RNA at 26 cycles.

A

B

C

668 bp

668 bp

Umemura et at: a18-Adrenoceptor mRNA in rat nephron segments 1551

100

80

Gim PCT PST CTAL MTAL

Nephron segmentsFig. 3. Relative levels of the a1-adrenoceptor mRNA in each nephronsegments. Each data point represented results from four independentexperiments and was expressed as percent of the densitometer valuesobtained in PST in the same experiments. The relative radioactivity was71.6 4.0% in PCT, 34.3 12.3% in GIm, 29.6 2.7% in CTAL and24.5 3.7% in MTAL.

of the kidney. Few studies, however, have used this method tostudy the a1-adrenergic receptor and its intracellular signaltransduction system. Recent advances in molecular biologicaltechniques have enabled us to examine the distribution of aspecific mRNA using the method of the microdissected singlenephron segment combined with RT-PCT [12, 13], as in thepresent study. To eliminate the variability of the tubule length atthe step of microdissection tubules, we performed RT with a fixedamount of total RNA which was extracted by the acid-guanidiumphenol-chloroform method [18] from each segment collected tothe sum of 20 to 30 mm in length and 200 glomeruli as previouslyreported [14]. The quantitative detection of mRNA by RT-PCRwas described previously by Makino, Sekiya and Hayashi [191. Wechose the number of PCR cycles itt which the amount of productincreased exponentially and was not saturated. We also chose theinitial amount of total RNA for RT-PCR in the range where therewas a linear relationship between the initial amount of RNA andthat of the RT-PCR product. The semiquantitative techniqueused in this study thus allowed us to compare the relative amountof RNA.

We found a strong signal of a1-adrenoceptor mRNA in thePCT and PST. These results are consistent with those of previousstudies [8, 11] as well as functional studies [20, 21], although thesubtype of a1-adrenergic receptor has not been identified at thatperiod. In the rabbit, infusion of noradrenaline into the kidney, atdose-rates which did not influence renal hemodynamics, causedan decrese in absolute and fractional sodium excretion. Theserenal responses to noradrenaline were abolished by the selectivea1-adrenoceptor antagonist, prazosin, but not by the selectivea2-adrenoceptor antagonist, idazoxan, suggesting that the a1 -adrenoceptor induced sodium reabsorption in the PCT [20].Furthermore, in isolated rat proximal tubules, selective a-adre-noceptor agonists stimulated 22Na uptake into proximal tubulesegments possibly through the Na/H antiporter [21], which isalso regulated by a2-adrenoceptors in proximal tubules [22].Using in situ hybridization, a18-adrenoceptor mRNA has beendemonstrated in the outer and inner stripes of the outer medulla,corresponding to the S3 segment of proximal tubules and the thickascending limb of loop of Henle [11]. However, those investiga-

Gb PCT CTAL MTAL CCD MCD

Expt 1 61.0 82.7 28.1 27.4 1.2 0Expt 2 45.7 71.4 34.9 33.2 0 0Expt 3 26.5 64.4 32.6 16.4 0 0Expt 4 3.9 67.9 22.7 21.0 0 0Means 34.3 71.6 29.6 24.5 0.03 0SE 12.3 4.0 2.7 3.7 0.03

tors could find no hybridization in the renal cortex, where the PCTexisted. This apparent discrepancy concerning PCT has severalpossible explanations. In situ hybridization using an oligonucleo-tide probe may not be a sensitive enough technique to detect thepossibly very low levels of alB-adrenoceptor mRNA in thesestructures, as noted [11]. Radioligand binding studies of thenonselective a1-adrenoceptor agonist, [3H] prazosine, clearlyshowed the presence of a1-adrenoceptors in the PCT [8]. Recentbinding studies further demonstrated the existence of a1-adre-noceptor subtypes in the renal proximal tubules [23].

Radioligand binding studies [24] using membranes preparedfrom the cortex, the outer stripe of the outer medulla (OSOM),the inner stripe of the outer medulla (ISOM), and the innermedulla dissected from rat kidney showed that the a1-adreno-ceptor predominates in the ISOM where the predominant struc-ture is the thick ascending limb of Henle [25, 261. In the presentstudy, we directly demonstrated aIB-adrenoceptor mRNA inmicrodissected CTAL and MTAL. This is also consistent with theabove-mentioned in situ hybridization study [11]. The thick as-cending limb of Henle has been suggested by us [5] and others [3,4] to be one of the possible sites where renal nerve stimulation-induced Na reabsorption occurs. Direct innervation was alsoreported in this nephron segment [27]. The findings of the presentstudy thus further support the hypothesis that thick ascendinglimb of loop of Henle may be involved in Na reabsorptionstimulated by renal nerve stimulation, possibly through the a1-adrenoceptors, although much study remains to be done beforedrawing any conclusion.

We found a significant signal of a1-adrenoceptor mRNAsignal in the GIm. Although the role of a(B-adrenoceptors in GImis not known, an a1-adrenoceptor-linked K-channel has beenreported in rat cultured mesangial cells [28], which is consistentwith the present finding of aIB-adrenoceptor mRNA in Glm.

In conclusion, the present study demonstrates, for the first time,by using RT-PCR the distribution of a8-adrenoceptor mRNA inmicrodissected rat nephron segments. The alB-adrenoceptormRNA seems to be expressed in specific sites along the nephron.

Acknowledgments

This work was supported in part by grants from the Ministry ofEducation, Science, and Culture of Japan (Nos. 02670404 and 05670956)and by a grant from the Uehara Memorial Foundation. We are grateful toDr. M. Voigt (Department of Pharmacology, University College, Dublin,Ireland) for providing the rat a1-adrenergic receptor eDNA. We alsothank Ms. N. Inaba for excellent secretarial assistance.

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60

40

20

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Table 1. Relative levels of a18-adrenoceptor mRNA in each ncphronsegments (% of PST)

Reprint requests to Satoshi Umemura M. D., Second Department ofinternal Medicine, Yokohama City University, School of Medicine, 3-9Fukuura, Kanazawa-ku, Yokohama 236, Japan.

1552 Umemura ci al: cs18-Adrenoceptor mRNA in rat nephron segments

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