(Hypertension. 1995;26:656-664.)
© 1995 American Heart Association, Inc.
Articles |
Renin mRNA, Quantified by Polymerase Chain Reaction, in Renal Hypertensive Rat Tissues
From the Molecular Biology and Hypertension Laboratory, Department of Physiology, The University of Sydney (Y.-k.L., B.J.M.), and Department of Medicine, St George Hospital (D.T.L., J.A.W.), Sydney, Australia.
Correspondence to Brian J. Morris, DSc, Molecular Biology and Hypertension Laboratory, Department of Physiology, Building F13, The University of Sydney, NSW 2006, Australia.
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Abstract To determine responses in renin gene expression in different tissues of two-kidney, one clip hypertensive Sprague-Dawley rats and the effect of NaCl loading, we developed a novel, highly sensitive quantitative polymerase chain reaction technique and measured renin mRNA at 19 and 40 days after clipping. Basal renin mRNA concentrations were 1575±127 fg/µg total RNA in kidney, 52±7 in hypothalamus, 7.9±0.7 in adrenal, and 4.7±0.5 in atria. In two-kidney, one clip rats, renin mRNA in the clipped kidney was increased 5.4-fold (P=.00001) and 2.3-fold (P=.001) on each respective day after clipping and in the unclipped kidney was decreased by 27% (P=.01) and 38% (P=.04). In adrenal, 3.9-fold (P=.004) and 1.7-fold (P=.02) increases were seen on days 19 and 40, respectively, and a decrease of 57% (P=.02) was found in a hypothalamic block at day 19 but not at day 40. The decrease in hypothalamus was abolished by 1% oral NaCl, which reduced renin mRNA by 37% in the clipped kidney and by 30% in the adrenal but did not lead to any change in the unclipped kidney or hypothalamus at day 40. Hypothalamic renin mRNA was also decreased by enalapril compared with increases of sixfold to ninefold in other tissues. In conclusion, we have quantified a decrease in hypothalamic renin mRNA in two-kidney, one clip rats 19 days after clipping that can be abolished by NaCl loading, whereas in the adrenal, renin mRNA was increased. Similar relative tissue-specific changes were also seen in enalapril-treated rats.
Key Words: renin • RNA, messenger • blood pressure • hypertension, renovascular • hypothalamus • adrenal glands • heart • kidney • polymerase chain reaction
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Renal artery constriction increases renin secretion into the bloodstream, and in the 2K1C Goldblatt model of human renovascular hypertension, such renin most likely contributes to the early rise in BP. The plasma renin level shows a strong correlation with renin mRNA in the clipped kidney,1 suggesting that elevated renin gene expression in this kidney but not in the contralateral kidney, in which renin mRNA is suppressed,2 3 4 5 6 7 maintains the early elevation in circulating renin. The early rise in plasma renin in 2K1C rats8 9 is followed by a return of renin toward control values in the chronic phase, whereas BP remains elevated.10 Despite this, blockade of the renin-angiotensin system in the chronic phase can lower the BP of 2K1C rats, consistent with renin-angiotensin system involvement (reviewed in Reference 1111 ). Renin mRNA exists in extrarenal tissues,12 13 14 15 16 17 18 19 20 21 22 and semiquantitative changes have been detected in extrarenal renin mRNA levels in the 2K1C model.23 24 However, its concentration is low, so results from older, less sensitive techniques such as RNase protection/solution hybridization23 and Northern blotting24 require confirmation by more sensitive methods. Also, actual quantification would assist in the evaluation of its role in this model.
The aim of the present study was to accurately measure renin mRNA in tissues that may be relevant to BP control in 2K1C rats at both early and late stages of hypertension and to test the effect of NaCl loading. To this end we developed a sensitive, quantitative, competitive RT-PCR technique amenable to the relatively rapid analysis of large numbers of samples. We then applied the method to the measurement of renin mRNA in kidneys, adrenal, and hypothalamus at 19 and 40 days after clipping in 2K1C rats on regular and high NaCl intakes.
A major objective in the development of quantitative RT-PCR methods is the control of tube-to-tube variations in amplification efficiency, which can lead to marked deviations because of the exponential nature of DNA amplification that is a feature of PCR. Some groups have achieved this by using a synthetic cRNA as an internal competitive template25 26 27 28 that can be differentiated from wild-type products on the basis of size by introduction of a deletion-mutant.26 27 This can then be added to each sample in a dilution series. However, this approach becomes cumbersome when large numbers of samples are analyzed. Our approach was to use a modified competitive PCR based on standardization of interpolation from a calibration curve in which a single set of standards contains known amounts of synthetic wild-type template.28 Standards and samples then are spiked with a deletion-mutant renin cRNA that is not only shorter than the wild-type but contains a 15-bp unique "foreign" DNA fragment, so that renin and competitor can be distinguished by different specific probes during slot-blot hybridization. In our technique, unlike other renin mRNA PCRs that have been developed29 30 31 but in common with a recent study that used RNase protection,1 mRNA for a constantly expressed (housekeeping) gene, namely, that for ß-actin, in samples was also monitored with PCR. This helped us to ascertain the quality of the extracted sample RNA, gauge the efficiency of the reverse transcriptase step, and control for sample-to-sample variation in total mRNA. The method also did not suffer from deficiencies of other renin mRNA PCR techniques, such as lack of interassay reproducibility31 or generation of results for kidney at least an order of magnitude higher29 or lower31 than theoretical expectations.
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Rats
Male Sprague-Dawley rats aged 6 to 7 weeks (150 to 250 g) were from Animal Resources Centre, Perth, Western Australia, and were kept in the St George Hospital Animal House, with separate rooms for surgical procedures, BP monitoring, and metabolic studies. All procedures were approved by the St George Hospital and University of Sydney animal ethics review committees.
Determination of Physiological
Parameters
Rats subjected to different NaCl regimens were housed in
separate 23x23x23-cm metabolic cages (Mascot Wire Works
Pty Ltd) for determination of 24-hour urine volume and excreted sodium
measured by flame photometry (Autoanalyzer, Technicon
Instrument Co). Body weight was measured with an animal balance
(BB2400, Mettler-Toledo). While rats were restrained for 10 minutes in
a plastic holder on a 38°C plate, indirect systolic BP was determined
with the use of the tail-cuff method, involving a pneumatic pulse
transducer and PE-300 Programmed Electro-Sphygmomanometer (Narco
Biosystems Inc), together with a data acquisition system (MP100, Biopac
System Inc) and Apple Macintosh computer. The mean of the last three
readings that differed by less than 10 mm Hg was taken as systolic BP.
A 1-mL sample of blood was collected by cardiac puncture of
anesthetized rats before death and was used for determination
of plasma renin activity by generation rate of Ang I as measured by
radioimmunoassay.32
Surgical Procedures
Rats were anesthetized with 60 mg/kg IP sodium
pentobarbital (Nembutal, Boehringer Ingelheim GmbH), and with
the use of sterile conditions a 5- to 7-cm incision was made on the
left dorsal flank for placement of a 0.22-mm-gap silver clip around
the left renal artery. In sham-operated rats the clip was placed
next to the renal artery. After closure with 3-0 coated vicryl sutural
thread for abdominal muscles and stainless steel clips for skin, rats
were injected with buprenorphine (0.1 to 0.5 mg/kg) repeated at 12 and
24 hours. The rats were observed hourly for the first 6 hours, twice
daily for 5 days, and daily thereafter.
2K1C Rats on Normal and High NaCl
There were four groups of 2K1C rats: 12 rats were given a
regular diet and killed by cervical dislocation at 19 and 40 days after
clipping, and 15 were given 1% NaCl in place of drinking water and
killed at these times. Kidney cortex, adrenal, and hypothalamic blocks
taken from brain and both atria, after separation from heart, were
removed quickly and placed in liquid nitrogen, and tissues were stored
at -80°C for up to 2 weeks before RNA extraction.
Low NaCl and Enalapril Treatment
Groups of six rats were used: one was a control group; one group
was maintained for 7 days on a low NaCl (0.04 g NaCl/100 g dry chow)
diet and given tap water to drink; another group was given 0.25 mg/mL
enalapril maleate (Amprace, Amrad Pharmaceuticals) in drinking water
(daily dose, 5 mg/kg body wt) and standard rat chow (0.37 g NaCl/100 g
rat chow); and a final group was given a low NaCl diet plus oral
enalapril.
RNA Extraction
Tissue was cut into small pieces with a sterile blade while
frozen on dry ice, and then 100 mg was transferred into a
glass-polytetrafluoroethylene homogenizer
containing 2 mL RNAzol B solution (Biotex Laboratories Inc) and
homogenized with several strokes. One-tenth volume of
chloroform was added, and after vigorous shaking for 15 seconds the
sample was cooled on ice for 5 minutes before
centrifugation in a microfuge for 5 minutes at 4°C.
Approximately 80% of the upper (colorless) aqueous phase, which
contains the RNA, was transferred to a fresh tube, with care taken not
to disturb the DNA-protein–containing interface and the lower
(blue) organic phase. An equal volume of isopropanol was added to the
aqueous phase, and after 15 minutes of chilling on ice a
white-yellow pellet of RNA was obtained by
centrifugation in a microfuge for 15 minutes at 4°C.
After the pellet was washed in 400 µL of 75% ethanol and
centrifuged again, the RNA was dried under vacuum and
resuspended in diethyl pyrocarbonate-treated Milli-Q deionized
distilled water. RNA concentration was determined
spectrophotometrically at 260 nm, after dilution if necessary to give a
reading in the range of 0.2 to 0.8 OD260 units, and
aliquots were stored at -80°C. An OD unit of 1.0 was taken as
40 µg/mL RNA. Purity was assessed according to an
A260/A280 ratio (absorbances at 260 and
280 nm) of 2.0,33 and quality was gauged by
electrophoresis on a 1.2% neutral agarose gel after denaturation with
formaldehyde.
Oligonucleotides
Wild-Type
Oligonucleotide primers for use in PCR were made
to amplify a 421-bp segment corresponding to nucleotides
709 to 1129 of rat renin cDNA, a region that contains four intron
sites, therefore enabling genomic amplicons, if produced, to be
distinguished as a 3.6-kb band. The upstream primer (RR1) spanning the
exon 5/exon 6 border was 5'-ACA GCA GGG AGT CCC ACC TGC T-3'
(nucleotides 709 to 730), and the downstream primer (RR2)
directed at sequences in exon 9 was 5'-TCC AGG CCT TGG AGA GCC AGT-3'
(nucleotides 1109 to 1129). Oligonucleotide
probe for wild-type renin mRNA PCR products corresponded to
cDNA nucleotides 825 to 842 spanning the exon 6/exon 7
region and had the sequence 5'-ACC ATG AGA GGG GTC TCT-3'. The
uniqueness of these and the sequences below was supported by
computer-assisted comparisons with related sequences, such as
cathepsin D, and the GenBank database with the use of the Australian
National Genomic Information Service.
Deletion-Mutant
Oligonucleotide primer sequences synthesized
were P2, 5'-CCC CCC AGA TCT CAG TAA TGT TGA GGG TCA CT-3',
and P3, 5'-TGA GAT CTG GGG GGC CTG CCA CCT TGT GT-3',
where underlined nucleotides are "foreign." An
oligonucleotide probe for the deletion-mutant PCR
product was also made. Its sequence, 5'-TGA GAT CTG GGG GGC CT-3',
corresponded to the "foreign" segment, and it contained a
Bgl II site.
ß-Actin
These primers were designed to amplify a 240-bp region of the
ß-actin cDNA. Sense primer (RB1) corresponding to
nucleotides 2733 to 2752 in exon 5 of the 4098-bp
ß-actin gene34 was 5'-AGT GTG ACG TTG ACA TCC GT-3',
and anti-sense primer (RB2), nucleotides 3081 to 3100
in exon 6, was 5'-GAC TGA TCG TAC TCC TGC TT-3'.
Construction of Deletion-Mutant Renin cDNA
Deletion-mutant renin cDNA was made with the use of 5 ng
plasmid RR-4 as template, which contained full-length 1434-bp rat
renin cDNA inserted between the HindIII and BamHI
sites of pGEM-4,35 and a previously described
approach.27 RR1 and P2 primers and RR2 and P3 primers were
used in separate PCRs, and the PCR products were separated by
electrophoresis on a 3% low-melting-point NuSieve GTG agarose
minigel (FMC Bioproducts). After excision from the gel and
elution into 100 µL Tris-EDTA buffer (10 mmol/L Tris-HCl and 1 mmol/L
EDTA, pH 7.5) at 50°C, 10 µL was used for further PCR with RR1 and
RR2 primers in 40 µL PCR mixture. This involved initial incubation at
55°C for 90 seconds and 70°C for 90 seconds before amplification
for 25 cycles in a DNA Thermal Cycler (Perkin-Elmer), with the
step-cycle program set at 94°C for 45 seconds, 55°C for 60
seconds, and 72°C for 60 seconds. The PCR product was digested
with Bgl II to confirm the presence of the extra sequences
and was then ligated into pGEM-3Z. To do this, 5 µL of PCR
product was "filled in" and "polished" with Klenow
fragment of DNA polymerase, and 2 µg pGEM-3Z was digested with
Sma I and 5'-termini dephosphorylated with
calf intestinal phosphatase to prevent
self-ligation.36 After electrophoresis of each on a
3% low-melting-point NuSieve minigel, insert and vector bands
were excised quickly, quantified, and mixed in a ratio of 3:1,
respectively. After the agarose slices were remelted at 68°C for 10
minutes and maintained at 37°C, 5 µL of 10x T4 DNA ligase buffer
was added to 50 µL together with 2 U T4 DNA ligase, and ligation was
allowed to proceed at room temperature overnight. The construct
produced was named pGD362 and was used for synthesis of
deletion-mutant renin cRNA.
Synthesis of Deletion-Mutant and Wild-Type Renin cRNA
pGD362 was linearized with EcoRI, and rat renin cDNA
in pGEM-4 was linearized with Bal I and then purified by
phenol/chloroform extraction and ethanol precipitation.
Deletion-mutant cRNA was transcribed by SP6, and the wild-type
cRNA was transcribed by T7 RNA polymerase with a Riboprobe
Transcription System kit (Promega) in accordance with the
manufacturer's recommendations. After digestion of DNA templates by
incubation at 40°C for 20 minutes with 0.3 µg RNase-free DNase
RQ1 (Promega), transcription products were separated by
electrophoresis, in which, after the cRNA band had migrated two thirds
of the gel length, a slot was made just in front of the band and the
gel was run at 40 V for 4 to 8 minutes. The cRNA was collected by
aspirating buffer from the slot well and was precipitated with 2.5
mol/L ammonium acetate in ethanol. The pellet was dried and then
dissolved in 20 µL diethyl pyrocarbonate water. The cRNA
concentration was estimated spectrophotometrically with the use of 2
µL, and the remainder was stored for no more than 3 days at
-80°C with yeast tRNA carrier before use in the RT-PCR
procedure.
Renin mRNA Reverse Transcription
Six micrograms of total RNA sample was mixed with the following
amounts of deletion-mutant renin cRNA competitor: kidney, 9000 fg;
whole brain and hypothalamus, 180 fg; and adrenal and heart, 36 fg. The
proportion of the mixture used for PCR in each case was 3.3%, 16.7%,
and 83%, respectively. Standards consisted of a dilution series of
wild-type renin cRNA containing a constant concentration of
deletion-mutant renin cRNA competitor, as follows: for kidney, 15,
7.5, 3.8, 1.9, and 0.94 pg wild-type and 7.5 pg competitor; for
whole brain and hypothalamus, 1000, 500, 250, and 125 fg wild-type
and 150 fg competitor; and for adrenal and heart, 100, 50, 25, 12.5,
and 6.3 fg wild-type and 30 fg competitor. Each reaction mixture
also contained 5 µg SMG total RNA to allow for any nonspecific
background effects. Also added were 200 U M-MLV Superscript reverse
transcriptase (BRL), 50 mmol/L each dNTP (Promega), 0.1 µmol/L random
hexamers (Promega), and 20 U RNase inhibitor (Promega). The
total reaction volume was made up to 20 µL in 10 mmol/L Tris-HCl (pH
8.3), 50 mmol/L KCl, 0.1 mg/mL bovine serum albumin, and 4
mmol/L MgCl2 and was incubated at 42°C for 1 hour and
then heated to 76°C for 10 minutes. After rapid cooling to 4°C on
ice, 10 µL double-distilled water was added, and cRNA was
purified by extraction with 40 µL phenol/chloroform.
Renin mRNA Competitive PCR
The volume of purified and resuspended cRNA used for PCR was
equivalent to an amount of starting total RNA of 0.2 µg for kidney, 1
µg for whole brain and hypothalamus, and 5 µg for adrenal and
heart. The PCR mixture contained 0.1 µmol/L of each primer (RR1 and
RR2) and 1.25 U AmpliTaq DNA polymerase in a final volume of 50 µL,
with 20 µL liquid wax (Bresatac) added to reduce evaporation. PCR was
performed with the Thermal Cycler set to 94°, 60°, and 72°C for
90 seconds each for 32 cycles. Since the Taq polymerase
extension rate is 6000 to 9000 bases per minute per
template,37 extension time was adequate.
Analysis of PCR Products
Size of PCR products was checked by comparison with a size
marker with the use of electrophoresis and ethidium bromide staining.
Expected sizes were 421 bp for wild-type renin cRNA PCR
product, 362 bp for deletion-mutant renin cRNA PCR product,
and 240 bp for ß-actin cRNA PCR product. Slot-blot
hybridization38 was then performed by denaturation of 5
µL PCR product in 500 µL of NaOH (0.4 mol/L)/EDTA (10 mmol/L)
in duplicate for 30 minutes; after twofold serial dilutions were made,
the two sets of samples were transferred onto separate
Hybond-N+ nylon membranes (Amersham) with the use of a
slot-blot apparatus (Bio-Rad) or, in the case of
ß-actin mRNA PCR products, to a single filter with the use of
a dot-blot apparatus (Bio-Rad). The filters were dried
for 1 hour at 85°C in a vacuum oven to immobilize DNA and
were hybridized for 4 hours at 42°C with
oligonucleotide probes that were labeled to 2 to
5x105 cpm/0.1 µg with the use of
[
-32P]ATP, with a specific activity of 4000 Ci/mmol
(Amersham), and polynucleotide kinase (New England
BioLabs). Membranes were rinsed once in 2x SSC (1x SSC is 15 mmol/L
sodium citrate and 150 mmol/L NaCl, pH 7.0) at room temperature for 2
minutes, twice in 2x SSC and 0.1% sodium dodecyl sulfate at
42°C for 10 minutes each wash, and then in 2x SSC at room
temperature for 20 minutes. The membranes were then exposed to Kodak
X-Omat AR film at room temperature for 4 to 8 hours.
ß-Actin RT-PCR
ß-Actin RT-PCR was carried out to monitor nonspecific
influences of experimental treatment, the quality of sample RNA and its
semiquantification, and the efficiency of RT-PCR. From the same reverse
transcriptase mixture as above, 5 µL was transferred to 45 µL
double-distilled water, and then 10 µL (0.2 µg original total
RNA) was transferred to 40 µL PCR buffer and constituted as above
except that primers were those for ß-actin (0.1 µmol/L). After
denaturation at 95°C for 5 minutes, 20 cycles of PCR at 94°, 60°,
and 72°C for 1 minute each were performed.
Quantification of Signals Detected
Signal intensity measurements were made in triplicate with a
Personal Densitometer (Molecular Dynamics Corp). The sample dilutions
stated above had been chosen after preliminary experiments so that
comparisons could be made on the linear portion of the
dose-response curve. The standard curve of the ratio of
slot-blot hybridization signal for PCR of wild-type renin cRNA
to competitor signal was plotted. A ratio of 1.0 was obtained when the
amounts of wild-type and competitor were equal.27 28
The amount of renin mRNA in a sample was interpolated from the standard
curve. The assay involved duplicate or triplicate samples from each rat
tissue.
Statistical Analyses
The significance of differences between groups was tested by
one-way ANOVA with the use of STATWORKS (Abacus
Concepts); if a probability value of .05 or less was obtained,
Student's t test was then used for comparison of each
individual group with the appropriate control.
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ß-Actin mRNA
A similar hybridization signal intensity for the 240-bp ß-actin mRNA PCR product indicated similar total mRNA for different tissue samples (Fig 1). Interassay coefficients of variation for 0.2 and 0.02 µg total RNA were 3.2% and 16.8%, respectively (n=5) and were less than 5% among averaged values for triplicate samples. In treated rats ß-actin mRNA was measured concomitantly with renin mRNA. In the unclipped kidney of 2K1C rats ß-actin mRNA was 3717±182 arbitrary OD units per 0.02 µg total RNA compared with 2629±132 (n=6) in the clipped kidney (P=.00003, n=6); electrophoretic analysis of RNA showed a size decrease (Fig 1C). Results were similar for sham and unclipped kidneys, as were those for the different tissues between each treatment group.
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Renin mRNA RT-PCR
Fig 2A shows the RT-PCR products. The
deletion-mutant cRNA was free of renin cDNA, and this was verified
by the absence of a 362-bp product after PCR of an
RNase-treated sample of this cRNA. Also, DNase treatment of RNA
samples confirmed that any band seen after PCR did not arise from PCR
product carry-over contamination. Since the wild-type renin
cRNA differed from renin mRNA in vivo insofar as it lacked a
poly(A) tail and 5'-cap structure, the efficiency of RT-PCR of
each was compared and found to be identical. Hybridization probing
yielded greater sensitivity than ethidium bromide staining. Also, slot
blotting gave sharper signals than Southern blotting, with signal
intensity showing approximately 60% lower sensitivity of the latter
(Fig 2B through 2D). Standard curves of the quantity of
native renin mRNA or wild-type renin cRNA versus optical density
were always linear.
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An example of an actual assay in which separate membranes were probed for either wild-type or deletion-mutant PCR product is shown in Fig 3. No signal was seen for rat SMG or HeLa cell RNA. The assay was sensitive to at least 5 fg (6500 molecules) of tissue renin mRNA. The 95% confidence intervals for within- and between-assay basal values obtained for tissue with highest expression (kidney) were ±21% and ±31%, respectively, and for tissue with lowest expression (heart), ±24% and ±16%, respectively.
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Only during the exponential phase of PCR did amplification reflect the initial amount of template. We therefore carried out preliminary experiments to optimize conditions for each tissue source of renin mRNA. For 32 PCR cycles these were 0.2 µg total RNA and 300 fg competitor for kidney, 1 µg total RNA and 30 fg competitor for whole brain and hypothalamus, and 5 µg total RNA and 30 fg competitor for adrenal and heart. Slot blot results using these are shown in Fig 4.
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In untreated rats renin mRNA concentrations (femtograms per microgram total RNA) were found to be 1575±127 (±SEM) (n=6) for kidney, 52±6.5 (n=6) for hypothalamus, 38±2.8 (n=6) for whole brain, 7.9±0.7 (n=12) for adrenal, and 4.7±0.5 (n=6) for cardiac atria. In the case of heart and adrenal no band was seen on electrophoretic gels, but a faint signal was detected after slot-blot hybridization. No signal was ever detected for SMG.
Physiological Parameters in
2K1C Rats
Comparison of 2K1C and sham rats at 19 and 40 days after clipping
showed anticipated differences in BP and other parameters
(Table 1). A high NaCl diet increased
urine volume and urinary sodium excretion and was associated with the
death of 7 of 15 rats compared with 2 of 12 on a regular diet. At day
40, although BP of the high NaCl rats was greater than that of shams,
this did not reach statistical significance. Plasma renin activity at
day 40 in 2K1C high NaCl rats was suppressed (3±1 [±SEM] compared
with 14±4 pmol Ang I/hour per ml for high NaCl versus regular diet
groups, respectively; P<.05). As a comparison, plasma renin
activities in unclipped rats on regular, high NaCl, and low NaCl
regimens were 9±3, 2±1, and 14±2 pmol Ang I/hour per ml,
respectively.
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Renin mRNA in 2K1C Rats
Kidney
At day 19 renin mRNA in clipped kidney was 5.4 times that of sham,
but by day 40 it was only 2.3 times higher (Table 2). In the unclipped kidney renin mRNA
was suppressed by 39% and 42% at days 19 and 40, respectively, versus
sham. Although renin mRNA in the clipped kidney was 37% lower in
NaCl-loaded versus regular-diet rats at day 40, this did not
reach statistical significance.
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Adrenal
At day 19 2K1C values were 3.9 times those of sham but by day 40
were only 1.7-fold higher (Table 2). Concentrations in
each adrenal were similar. High NaCl suppressed renin mRNA by 31% and
32% at days 19 and 40, respectively, but values remained greater than
in shams.
Hypothalamus
At day 19 renin mRNA was reduced by 57% versus sham, but by day
40 it was not significantly lower (Table 2). With high
NaCl, however, values were similar to those in shams.
Atria
Values for sham rats were similar to those stated above for
untreated rats, but in 2K1C rats PCR products were low and not
accurately quantifiable.
Renin mRNA in Low NaCl– and Enalapril-Treated
Rats
This experiment served as a comparison of renin mRNA
responsiveness. Such rats had reduced urinary sodium (0.3±0.1 mmol/d
compared with 1.3±0.2 for controls; P<.0001). In kidney,
renin mRNA was increased 1.7-, 3.9-, and 7.0-fold by low NaCl,
enalapril, and low NaCl plus enalapril, respectively (Table 3). In adrenal, increases were 4.1-,
2.2-, and 6.2-fold, respectively; in the heart there was no change with
low NaCl but increases of 5.5- and 8.9-fold with enalapril and
enalapril plus low NaCl, respectively. In the hypothalamus, enalapril
caused a decrease of 28% by itself and of 36% with a low NaCl
diet.
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We have developed a quantitative renin mRNA RT-PCR and used it to measure changes in different tissues of 2K1C rats. In kidney of untreated rats the renin mRNA concentration of 1.6 pg/µg total RNA was comparable to values in mouse by dot-blot (1 to 5 pg/µg total RNA12 13 ) or solution hybridization (5 pg/µg total RNA39 ), Dahl rat by dot-blot (0.1 pg/µg total RNA40 ), male WKY by solution hybridization (0.4 pg/µg total RNA41 ), female Wistar rat by the present PCR (0.9 pg/µg total RNA; Y.-k.L., B.J.M., unpublished data, 1994), and humans by PCR (1.7 pg/µg total RNA42 ) but differed from a value by PCR of 56 pg/µg total RNA in WKY by other researchers.29 Moreover, the increases we saw in the clipped kidney of 2K1C rats resemble those reported by others using older techniques,1 2 4 5 24 namely, sixfold at 4 weeks and fourfold at 20 weeks by dot-blot in female Wistar rats with a 0.2-mm-gap clip,4 2.6-fold at 4 weeks and 2.2-fold at 16 weeks by slot-blot in male Wistar rats with a 0.2-mm-gap clip,5 and 3.0-fold at day 1 and 4.3-fold at day 2 by RNase protection in male Sprague-Dawley rats with a 0.2-mm-gap clip.1 The present study has also shown that the increase in renin mRNA occurs in the face of partial degradation of RNA in the ischemic kidney. In the contralateral kidney the suppression in renin mRNA we saw was not as great as seen by others, namely, 92% at 4 and 20 weeks,4 48% at day 1, and 74% at day 2.1 The fact that a 0.3-mm-gap clip can decrease renin mRNA with no rise in BP,6 together with other evidence,1 has suggested a role for a humoral factor released from the clipped kidney in the suppression of renin mRNA and, in the clipped kidney, its later return toward normal. Moreover, since BP correlates only weakly with plasma renin in 2K1C rats, secreted renin is not the only cause of the BP increase.6
In adrenal the rises we saw were earlier than in a solution hybridization study of 2K1C Wistar rats with a 0.2-mm-gap clip, in which an increase of 3.5-fold was not seen until 20 weeks.23 Such increases occurred in the face of elevated plasma renin and therefore Ang II. Adrenal renin activity is increased in 2K1C hypertensive rats,43 as is Ang II, which changes in parallel with alterations in plasma Ang II.24 Since we found that enalapril was also able to increase adrenal renin mRNA, the increase in 2K1C rats could have been even greater in the absence of circulating Ang II. Furthermore, the increase contravenes servoregulatory expectations, consistent with a role in 2K1C hypertension. Adrenal renin mRNA and renin are increased by corticotropin and K+,31 but the trigger in 2K1C rats is not known. Basal adrenal values approximated estimates by RNase protection41 and one30 but not another29 PCR study.
The reduction seen in hypothalamus at day 19 but not day 40 compares with 50% lower levels in brain at 4 weeks but not 16 weeks by Northern blotting.24 Since brain Ang II was increased at 4 weeks,24 it might cause such suppression, as Ang II does normally in kidney.44 45 The only other study found no change in brain renin mRNA at either 4 or 20 weeks.23 Our basal hypothalamic values resembled values obtained by other researchers for several rat strains by PCR (approximately 10 to 30 fg/µg30 ) or RNase protection for WKY (approximately 60 fg/µg41 ) or CD-1 mice (approximately 30 fg/µg39 ).
In the heart, renin mRNA is mainly in atria.21 In the only previous study of 2K1C heart by RNase protection, results were similar to negative control. In our study levels were low, possibly as a response to the high BP, and not accurately determined. Although the levels seen normally may be so low as to preclude a function, the marked response to low NaCl and enalapril points to a physiological role under such circumstances. The responsiveness also argues against the low levels in heart being errors caused by low fidelity of the PCR.
High NaCl, the effect of which on 2K1C renin mRNA has not previously been studied, caused suppression in the clipped but not the unclipped kidney at day 40. Little decrease was seen in sham rats, whereas other researchers saw 50% suppression in rats on a 1% to 4% NaCl diet for 2 weeks or longer.3 46 47 48 In contrast, high NaCl markedly suppressed renin in plasma in 2K1C rats, as has been seen by others.49 This might point to greater posttranslational compared with pretranslational control by high NaCl. In adrenal, renin mRNA was decreased on each day, and in hypothalamus, high NaCl reversed the suppression at day 19. The mechanisms of these effects are unclear.
Curiously, many of the changes in renin mRNA seen in the 2K1C rat, which has high renin and Ang II, resembled those elicited in each tissue by low NaCl and an angiotensin-converting enzyme inhibitor. Our results were consistent with previous findings for kidney of a twofold to sixfold (average, fourfold) increase for low NaCl13 48 50 51 and 3- to 17-fold (average, 7-fold) for low NaCl plus an angiotensin-converting enzyme inhibitor.1 3 13 40 41 46 50 52 53 54 55 56 Furthermore, the increases in renin mRNA and plasma renin with low NaCl occur in parallel48 and might not involve the macula densa, baroreceptor, or sympathetic nerves6 48 54 57 but rather a humoral mechanism, possibly resembling that from the stenosed kidney of 2K1C rats. In adrenal, increases of twofold58 or greater48 have been seen by dot-blot and RNase protection, respectively, in low NaCl rats and involve the zona glomerulosa,58 59 where renin is also increased.60
The decrease seen for renin mRNA in hypothalamus after enalapril has been seen in brain after captopril with the use of nonquantitative RNase protection.53 However, another group using PCR reported a twofold increase.30 Angiotensin-converting enzyme is inhibited in circumventricular organs,61 but the extent of penetration of the blood-brain barrier, which is more permeable in hypertensive rats,62 has been the subject of controversy.63 It is not known whether enalapril might be followed by increases in Ang II in central nervous system sites that it does not access, thereby explaining the suppression observed.
In conclusion, we have quantified an increase in adrenal renin mRNA in 2K1C rats and have found that a decrease in hypothalamus levels can be ameliorated by high NaCl.
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This study was supported by a grant from the National Heart Foundation of Australia. We thank Dr Kevin R. Lynch, University of Virginia, for kindly providing the rat renin cDNA clone and Dr Robert Y.L. Zee for technical assistance.
Received January 16, 1995; first decision February 20, 1995; accepted July 10, 1995.
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