Plasma and Renal Prorenin/Renin, Renin mRNA, and Blood Pressure in Dahl Salt-Sensitive and Salt-Resistant Rats
Abstract We measured plasma prorenin and renin levels, renal renin mRNA, renal anti-renin and anti–prorenin-prosequence immunoreactivity, and blood pressure in maturing Brookhaven Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats during 14 days of low (0%), medium (0.4%), or high (4%) NaCl diets. Blood pressure was higher in Dahl S rats and did not increase with high NaCl. Seven-week-old Dahl R rats had twofold and sixfold higher levels of plasma prorenin and renal prosequence immunoreactivity, respectively, which by 9 weeks were the same as in Dahl S rats. The anti-renin antiserum, BR1-5, was found to detect prorenin better than renin; Dahl S rats had suppressed renal anti-renin immunoreactivity relative to Dahl-R rats. Dahl R rats were unresponsive to high NaCl, whereas in Dahl S rats, plasma renin and renal prosequence immunoreactivity fell by 90% (P<.01), renal anti-renin immunoreactivity and renal renin mRNA fell by 35% (P<.05 for both), and plasma prorenin fell by 30% (P=NS). NaCl depletion increased prorenin/renin parameters similarly in both strains. There were direct relationships among all of the prorenin/renin parameters. Between low and high salt diets in Dahl S rats, plasma renin increased 20-fold, plasma total renin (renin plus prorenin) and renal renin mRNA both increased threefold, and plasma prorenin increased twofold. The results indicate that under steady-state conditions, plasma and renal renin/prorenin parameters change concordantly and that plasma total renin (renin plus prorenin) reflects changes in renal renin mRNA. The lower blood pressure of Dahl R rats is associated with later maturation-related declines in plasma and renal prorenin. Suppression of plasma renin may delay the salt-induced blood pressure rise in Dahl S rats. Finally, the renin system and blood pressure of Dahl R rats have remarkable disregard for a high salt diet.
Prorenin is the biosynthetic precursor of renin.1 2 3 Although renin gene expression occurs in many organs, processing of prorenin to renin has been conclusively demonstrated only in renal tissue.4 In rats, both plasma renin and plasma prorenin fall to undetectable levels after nephrectomy, indicating that the kidneys are the primary source of both.5 Plasma prorenin levels are consistently 2 to 10 times higher than those of plasma renin, indicating that the primary product of renal renin gene expression is prorenin,6 since renin and prorenin are cleared at similar rates.7 8 Renal renin mRNA, therefore, is more likely to reflect plasma prorenin than renin levels. To date, however, most studies have related renal renin mRNA levels only to plasma and renal renin.9 10 11 12
The renin system of the Dahl S hypertensive rat model has been extensively studied13 ; mature Dahl S rats are reported to have lower PRA, renal renin activity, and juxtaglomerular granulation than the Dahl R strain.14 15 Isolated perfused kidneys of Dahl S rats secrete less renin than those of Dahl R rats at similar inflow pressure.16 Both strains reportedly suppress plasma renin levels when fed high salt diets and increase it when fed low salt diets.14 However, paradoxical increases in PRA occur in Brookhaven selectively outbred Dahl S rats when fed very high salt (8%) diets for 6 or more weeks.17 Of interest, genetic polymorphism in the renin gene of inbred Dahl rats has been shown to cosegregate with BP.18 Yet genetic transfer of the Dahl S renin gene from the inbred Dahl S rat to the inbred Dahl R rat does not promote NaCl sensitivity and decreases BP,19 20 showing that neither height of BP nor NaCl sensitivity is solely dependent on the presence of the Dahl S rat renin gene.
Few studies of prorenin have been carried out in rats.21 To investigate whether suppression of plasma renin in salt-loaded Dahl S rats is accompanied by commensurate changes in plasma prorenin and renal renin gene expression, we compared changes in plasma prorenin and renin and renal renin mRNA in Dahl S and R rats under conditions in which renin secretion would be suppressed (high salt diet) or increased (low salt diet). We used immunohistochemical techniques with polyclonal antibodies prepared either against the whole prosequence of rat prorenin or against recombinant human renin to explore changes in renal prorenin and renin.
Dietary manipulation was begun at 7 weeks, a time when strain differences in the responses of BP and renin parameters to high salt diet should begin to manifest themselves.13 22 Four percent NaCl, rather than 8%, was fed as the high salt diet to moderate the expected changes in BP and avoid the effect of development of renal damage on the interpretation of the results.17
Fifty Dahl S and 50 Dahl R rats (Brookhaven strain, Hsd:DS and Hsd:DR; Harlan Sprague-Dawley, Indianapolis, Ind) aged 4 to 5 weeks on receipt between November 20, 1991, and January 24, 1992, were housed individually under controlled conditions of light, temperature, and humidity. All procedures performed were in accordance with institutional guidelines for animal research. The rats were allowed water ad libitum and fed a regular rodent chow containing 0.4% NaCl (0.16% sodium expressed as NaCl) and 0.6% KCl (0.31% potassium expressed as KCl) (Zeigler Brothers, Inc) for 13 or 14 days before the beginning of the experiment. On day zero, 19 Dahl S and 19 Dahl R rats were begun on a high NaCl diet containing 4% NaCl (1.6% sodium expressed as NaCl) and 0.75% potassium as potassium citrate (0.30% potassium expressed as tripotassium citrate) (stroke-prone rodent diet, Zeigler Brothers), and a similar number of rats were begun on a low NaCl (no detectable sodium) diet containing 0.7% potassium as potassium citrate (0.28% potassium expressed as tripotassium citrate) (NaCl-deficient rodent chow, Zeigler Brothers). The remaining 12 Dahl S and 12 Dahl R rats were continued on regular rodent chow. Six Dahl S and 6 Dahl R rats on regular chow were killed on days 0 and 14. Six or 7 rats from each of the other four dietary groups (R-high Na, R-low Na, S-high Na, and S-low Na) were killed on each of experimental days 2, 7, and 14.
To minimize stress, rats were handled regularly. Body weight and SBP were measured weekly and/or just before death. SBP was recorded in awake rats with tail-cuff sphygmomanometry (PE-300, Narco BioSystems, Inc). At the time of death, subsets were selected randomly from each dietary group and were decapitated without anesthesia. Trunk blood was collected in beakers coated with porcine heparin (Sigma Chemical Co) and transferred to tubes containing potassium-EDTA chilled in ice (time to collect, 30 to 60 seconds). Plasma was harvested immediately by centrifugation at 4°C and stored at −40°C.
The rats were autopsied immediately after decapitation and blood collection. Hearts, kidneys, and brains were rapidly removed, chilled in saline at 4°C, and weighed. Three standardized sections of kidney were immersion-fixed as outlined below. The remainder of the kidneys were snap-frozen at −70°C. A central transverse section of the left kidney was fixed in Carnoy’s fixative (Polyscientific Research and Development Corp). A similar section of the right kidney was fixed in 4% paraformaldehyde (Fisher Scientific) in phosphate-buffered saline buffer (FA buffer, Difco Laboratories) at a final concentration of 0.85% NaCl and 0.1 mol/L phosphate, pH 7.2. An adjacent paracentral section of right kidney was fixed in activated Bouin’s solution, pH 1.4 (Polyscientific). After overnight fixation, tissues were transferred to 70% ethanol. Subsequently, fixed tissues were dehydrated and all three separately fixed pieces were embedded in a single paraffin block. Six micron sections were cut and were picked up on previously cleaned and baked glass slides coated with polylysine (Sigma).
Microhematocrit was determined on a sample of whole potassium-EDTA–treated blood from each rat.
Plasma total renin was measured by an enzyme kinetic assay23 after enzymatic cleavage of the prosequence of prorenin by limited proteolysis with trypsin.24 In brief, rat plasma was incubated at 0°C for 30 minutes with 4 mg/mL trypsin (treated with N-tosyl-l-phenylalanine chloromethyl ketone [TPCK], Sigma) in 500 mmol/L Tris buffer, pH 7.5, containing 5 mmol/L CaCl2, 0.1% NaCl azide, and 1% bovine serum albumin. Soybean trypsin inhibitor (Sigma, 8 mg/mL final concentration) was added to stop the reaction. After the plasma was passed over Dowex resin (Sigma) for removal of angiotensinogen fragments, it was added to nephrectomized rat plasma (used as a source of angiotensinogen) and incubated at 37°C for 0 and 3 hours in the presence of 3 mmol/L EDTA, phenylmethylsulfonyl fluoride (0.04%), and 8(OH) quinoline (0.05%). The Ang I generated was measured by radioimmunoassay.23
PRC was measured as described above except trypsin was replaced by buffer. Total renin and PRC were calculated by subtracting the zero-time (blank) from the 3-hour Ang I levels and dividing by 3 (hours). Results are expressed as nanograms Ang I per milliliter per hour (multiply by 0.2778 to express as nanograms Ang I per liter per second). Plasma prorenin equals plasma total renin minus PRC.
A polyclonal rabbit antiserum directed against recombinant human renin (Upjohn) was prepared. The renin had been derived from recombinant prorenin after cleavage by limited proteolysis with trypsin, pH 8.0, and affinity-column purification.25 This antiserum (BR1-5) neutralized more than 50% of recombinant human renin (0.67 to 2.7 ng/mL) at a dilution of 1:10 000 in vitro and has been shown in separate studies to reveal immunoreactivity with rat renal JGA. In vitro absorption with recombinant human renin and prorenin indicated that this antiserum identifies both reagents, as indicated by marked reduction of immunoreactivity in human placenta and cultured placental cells.26 For the current immunohistochemical studies, it was used at a dilution of 1:1000.
A polyclonal rabbit antiserum directed against the entire free (unconjugated) rat synthetic prosequence polypeptide (46 amino acids plus cystine) was commercially prepared (Immuno-Dynamics). This antiserum (IR 657-2) neutralized 80% of crude recombinant rat prorenin (0.63 ng/mL) at a 1:10 dilution in vitro and 50% at a 1:100 dilution. For immunohistochemical studies, it was used at a dilution of 1:500. Absorption of this reagent with whole rat prosequence coupled to a Protrano Affinity Disk (ICN Biochemical) at neutral pH markedly reduced staining of juxtaglomerular structures of kidneys from rats fed a low (0%) salt diet and usually abolished staining of kidneys from rats fed a high (4%) salt diet.
Staining was performed with the aid of a Vectastain ABC kit for use with polyclonal rabbit antisera (Vector Laboratories). Sections were deparaffinized and equilibrated with isotonic FA buffer, pH 7.2. Separate incubations followed with avidin D and biotin blocking solutions. The slides were then washed with FA buffer and quenched for endogenous peroxidase activity with multiple applications of 6% H2O2. After incubation with a 10% dilution of normal goat serum, an appropriate dilution of the specific antiserum was applied. After incubation at 4°C for 16 hours and washing in FA buffer, a biotinylated goat anti-rabbit IgG (dilution 1:80) was applied for 0.5 hour. After washing, an appropriately diluted Vectastain biotinylated ABC solution (containing avidin DH and biotinylated horseradish peroxidase H) was applied for 30 minutes. Peroxidase was detected with 3,3′-diaminobenzidine tetrachloride (Aldrich Chemical Co) (0.01%), Ni(NH4)2(SO4)2 (0.02%), and H2O2 (0.003%) dissolved in phosphate buffer. Sections were counterstained with Lerner’s hematoxylin.
Histology was performed on the Bouin’s-fixed section of each slide in a blind fashion, ie, the observer (W.G.C.) was unaware of the experimental group to which the rats had been assigned. Duplicate estimations of staining in all epithelioid cell–containing structures of juxtaglomerular structures, ie, JGAs plus arterioles, were carried out on the rat kidneys of the 14-day groups. For the slides stained with anti-renin serum (BR1-5), a least-squares regression of the two analyses yielded an r38 value of .954 (P<.0001). The SD of residuals for the regression line was ±4.7. The deviation from the mean of the two analyses of each slide was between +1.9 and −0.55. The variation from the mean of the two analyses for each experimental group ranged from 0 to 1.7. For the slides stained for prosequence (IR 657-2), a least-squares regression of the two analyses yielded an r38 value of .950 (P<.0001). The SD of residuals for the regression line was ±3.2. The deviation from the mean of the two analyses of each slide was between +1.2 and −0.26. The variation from the mean of the two analyses for each experimental group ranged from 0.5 to 1.1.
Quantitation and Semiquantitation of Renal Anti-Renin and Anti–Prorenin- Prosequence Immunoreactivity
The numbers of immunostained JGAs and hilar arterioles on Bouin’s-fixed sections were counted and assigned an intensity value of 0.5, 1, 2, or 3. These values were totaled, normalized to 100 glomeruli, and reported as a weighted score.
For the absorption studies and comparison of tissue fixatives, slides were graded as trace, 0.5, 1, 2, or 3 on the basis of both the number of staining sites and their intensity.
mRNA Content of the Kidneys
Total cellular RNA was prepared by the method of Chomczynski and Sacchi.27 Renin mRNA levels were estimated with a ribonuclease protection assay.28 For probe synthesis, renin cDNA coding sequences29 were inserted between the HindIII and BamHI sites of a pBluescript II vector (Stratagene) in an orientation such that the cRNA sequence was transcribed by T7 polymerase. Cleavage of this plasmid (prRen6) with Aval before transcription yielded a 377-base cRNA containing 331 bases of the renin 3′-cRNA. To control for RNA recovery, a 100-base GAPDH cRNA probe,30 which yielded a 70-base protected fragment, was included in the ribonuclease protection assay. Each determination used 50 μg RNA and 250 000 cpm of both renin and GAPDH probes. The intensities of protected bands were quantified with a phosphorimager, and individual renin mRNA values were normalized to the GAPDH mRNA (which differed <10% among samples). Final results were normalized to the mean level of renin mRNA in the 0-day 0.4%-NaCl (regular rodent chow) dietary group of Dahl S rats.
Results are expressed as mean±SE. Because many of the variables were not normally distributed, we used a nonparametric approach to assess differences among the rats on different diets. Specifically, for each strain, overall differences among the rats on the three diets over a given time were assessed with the Kruskal-Wallis test. Pairwise differences between the dietary groups were further examined with the Mann-Whitney test. For those variables whose distributions were not different from the standard normal, one-way ANOVA models were also examined for assessment of the overall differences among the various diets. Paired comparisons in this parametric analysis were made with Student’s t tests, which were adjusted (where appropriate) with the Bonferroni method. The probability values presented with the tabulated data were calculated from the Mann-Whitney tests unless otherwise noted. Reported correlations between variables were determined from linear regression models and where appropriate were compared with log/log regression models. All associations and pairwise comparisons were considered significant at a value of P<.05.
All rats were fed the 0.4% NaCl diet for at least 13 days before the beginning of the study.
SBP rose gradually in rats of all groups by 10% to 27% throughout the 14-day experiment (Fig 1⇓). SBP of Dahl S rats rose on average more than 20 mm Hg, whereas that of Dahl R rats rose about 16 mm Hg. The mean SBP of each of the groups never exceeded the generally accepted upper limit of normal of 140 mm Hg.
On average, SBP tended to be higher in Dahl S than in Dahl R rats and was significantly higher (P<.05) in Dahl S rats on all three diets after 14 days, ie, at 9 weeks of age. The high salt diet did not significantly increase SBP in either Dahl S or R rats compared with rats of the same strain on regular chow.
Hematocrits rose slightly but progressively throughout the experiment irrespective of strain and diet (Table 1⇓). Dahl R rats consistently had slightly lower hematocrits than Dahl S rats, but a statistically significant difference occurred between Dahl S and R rats only after 14 days of the low salt diet.
Rats of all groups gained weight throughout the study (Table 1⇑). On regular chow, Dahl S and R rats gained weight similarly (71 versus 86 g). On low and high salt diets, Dahl R rats had similar weight gains (60 and 91 g, respectively), but Dahl S rats gained less weight on both low and high salt diets (11 and 43 g, respectively).
Kidney weight indexes (KWI×100), ie, the ratio of kidney to body weight, were higher in Dahl S rats after 14 days of the high salt diet than in Dahl S rats on regular chow or Dahl R rats on the high salt diet (Table 1⇑). In both Dahl S and R rats, the indexes were lower during low salt than during high salt.
Heart weight indexes (HWI×100) tended to be higher in Dahl S than R rats throughout the experiment. After 14 days, the significant differences were as follows: high salt: Dahl S versus R, 4.22±0.11 versus 3.64±0.09, P<.05; low salt: Dahl S versus R, 3.87±0.04 versus 3.63±0.09, P<.05.
Immunohistochemical Studies With Antisera to Renin and Prorenin Prosequence
Only the results from Bouin’s-fixed tissues are reported because tissue fixed otherwise (see “Methods”) gave internally variable results.
The morphological features of the tissues are illustrated in Figs 2⇓ and 3⇓. The total number of epithelioid cell–containing juxtaglomerular structures, ie, hilar arterioles and JGAs, immunostained for renin in 14-day high salt–fed Dahl S rats was about half that of rats on low salt diet (Fig 2⇓). In Dahl S rats on high salt, staining was largely limited to the hilar juxtaglomerular regions (Fig 2B⇓) and rarely occurred upstream toward the intralobular arteries or downstream into the efferent arterioles; most structures that stained were in the outer cortex (Fig 2⇓). In Dahl S rats on low salt, JGA regions stained intensely, and staining often extended upstream toward intralobular arteries (Fig 2D⇓) and sometimes downstream. In addition, outer and inner cortical JGAs were more evenly stained. Staining patterns of Dahl S rats on normal chow were intermediate. Staining in Dahl R rats was overall qualitatively more similar to that in Dahl S rats on low salt diet than to Dahl S rats on the other two diets, which showed perceptibly less staining.
Prosequence immunochemical staining was generally localized to the same areas as renin, although the intensity was typically noticeably less (Fig 3⇑).
Changes in Prorenin/Renin Parameters
Differences Between Dahl S and R Rats on Regular Chow
Intrinsic differences in prorenin/renin parameters occurred between the two rat strains (Table 2⇓; Fig 4⇑, baseline versus 14-day control). In 7-week-old rats, plasma prorenin concentration and renal prosequence immunostaining were 77% and more than 600% higher, respectively, in Dahl R than in S rats (plasma prorenin concentration, P<.01; renal prosequence, P<.01). These differences disappeared by 9 weeks of age as both parameters declined dramatically in Dahl R rats (Fig 4⇑, baseline versus 14-day control). These prorenin parameters did not change in Dahl S rats.
Renal anti-renin immunostaining was lower in Dahl S than in R rats. It also fell between 7 and 9 weeks in Dahl S rats (P<.05). Although at both 7 and 9 weeks of age anti-renal renin immunostaining was 40% lower in Dahl S than in R rats (P<.01 at both times), renal renin mRNA was not different between the strains.
PRC did not change in Dahl S or R rats between 7 and 9 weeks of age. Although mean PRC was consistently higher in Dahl R rats, the differences were not statistically significant in this study.
Plasma total renin in Dahl R rats was 45% higher than in Dahl S rats at 7 (P<.01) but not at 9 (P=NS) weeks of age.
Effect of Diets on Prorenin/Renin Parameters in Both Strains
Fig 4⇑ illustrates prorenin/renin parameters at 2, 7, and 14 days of the high and low salt diets. Fig 5⇑ compares the 14-day values. Since there were maturation-related changes in some of the parameters in the Dahl S and R rats fed normal chow (see above), the data in Fig 4⇑ are used to illustrate the timing of the responses to high and low salt diets, respectively. The data in Fig 5⇑ summarize the new steady-state (14-day) differences in the prorenin/renin parameters among the diets.
High sodium diet. In Dahl S rats, high salt diet suppressed renal renin mRNA by a maximum of about 50% within 2 days of initiation of the high salt diet (Fig 4⇑, left), whereas PRC and renal prosequence immunostaining fell gradually over 14 days by 90% (P<.01) to nearly undetectable levels (Fig 4⇑, left; Fig 5⇑, top). Renal anti-renin immunostaining fell by 38% (P<.03) and renal mRNA by 30% (P<.05) over the 14 days. Although plasma prorenin concentration fell by 25%, this was not statistically significant. Plasma total renin was significantly reduced (P<.01), reflecting the change in PRC.
In contrast, in Dahl R rats on the high salt diet, prorenin/renin parameters were essentially unchanged (Fig 4⇑, left; Fig 5⇑, bottom). Significant declines in plasma prorenin concentration, plasma total renin concentration, and renal prosequence immunohistochemical content over the 14-day experiment (Fig 4⇑, left) merely mirrored changes in the time controls on regular chow. PRC was transiently significantly lower than control values only at 7 days (P<.01). After 14 days of salt loading, all parameters measured were lower in Dahl S than in R rats (Fig 4⇑, left).
Low sodium diet. No differences were apparent in the responses of Dahl S and R rats during salt depletion (Fig 4⇑, right; Fig 5⇑, top and bottom). Both strains exhibited marked and significant increases relative to time controls in PRC (more than threefold), plasma total renin concentration (more than twofold), renal anti-prosequence immunoreactivity (more than fourfold), and renal renin mRNA (more than twofold). Plasma prorenin increased in Dahl S rats at 7 and 14 days (P<.01, P<.05) compared with the time controls (Fig 4⇑, right) but was not changed in Dahl R rats. Fig 5⇑ shows that renal anti-renin immunoreactivity increased nearly twofold (P<.01) after 14 days in Dahl S rats and 28% (P<.05) in Dahl R rats. In Dahl R rats, this parameter was higher than in Dahl S rats (P<.05) after 14 days of low NaCl.
Relative Changes and Relationships Among Parameters After 14 Days of Low, Medium, or High Salt Diets
Values were analyzed after 14 experimental days when a new steady state was assumed to exist. Direct and highly significant relationships were observed among all of the renin system parameters (Table 3⇓, P<.05 or better for all).
Fig 5⇑ illustrates the different parameters after 14 days of each diet. Fig 6⇓ illustrates the percent changes from medium to high and medium to low salt diets. PRC and renal anti-prosequence immunoreactivity exhibited the largest changes in response to both high and low salt diets. For Dahl S rats, PRC exhibited a near 20-fold difference and anti-prosequence immunoreactivity a more than 50-fold difference between high and low salt (Fig 5⇑, top). For Dahl R rats, the ranges were more than fivefold and threefold, respectively (Fig 5⇑, bottom). The linear regression between PRC and prosequence immunostaining was highly significant (P<.00005).
Changes in plasma total renin and renal renin mRNA were also quantitatively similar to each other. For Dahl S rats, plasma total renin and renin mRNA both increased threefold to fourfold overall (Fig 5⇑, top). For Dahl R rats, both parameters increased twofold to threefold between medium and low salt and exhibited no significant change between medium and high salt diets. The linear regression between renal renin mRNA and plasma total renin (P<.00005) is illustrated in Fig 7⇓.
Plasma prorenin and renal anti-renin immunostaining changed the least (Fig 6⇑). Plasma prorenin did not fall in either strain as a result of the high salt diet (Fig 5⇑) and increased less than twofold only in the Dahl S rats on the low salt diet (P<.05).
In the Dahl S rats, renal anti-renin immunostaining changed in proportion to renal renin mRNA and plasma prorenin, ie, twofold to threefold. In the Dahl R rats, however, anti-renin immunostaining did not increase as much as mRNA (28% versus twofold, P<.01) but increased to a degree similar to that of plasma prorenin (about 30%) (Fig 6⇑).
Although plasma prorenin concentration and PRC correlated directly (r=.67, P<.00005; Table 3⇑), PRC changed much more than prorenin in both Dahl S and R rats (Figs 5⇑ and 6⇑). PRC also changed more than renal renin mRNA (Figs 5⇑ and 6⇑).
Further Characterization of Antisera to Renin and Prorenin Prosequence
Since anti-renin immunoreactivity changed proportionally to plasma prorenin (Fig 6⇑), antiserum BR1-5 was preabsorbed with either recombinant rat prorenin or renin, and anti-renin immunostaining was studied in one representative kidney from each strain collected after 14 days of each diet. At an antibody dilution of 1:1000, 5 μL/mL renin produced little change in the staining of JGAs, but 5 μg/mL prorenin reduced staining intensity (Fig 8⇓). At an antiserum dilution of 1:10 000, staining of JGAs was obliterated or markedly reduced by the same concentration of both antigens.
Absorption of the antiprorenin antibody (IR 657-2, 1:500 dilution) with 500 ng/mL prorenin markedly reduced both the number and intensity of JGA structures that stained. Absorption with the same renin concentration had no effect.
These antibodies were further characterized by their ability to detect recombinant rat renin and prorenin by Western blotting of a 10% sodium dodecyl sulfate–polyacrylamide gel. The anti-renin antiserum stained recombinant rat prorenin about 10 times more intensely than renin (Fig 9⇓). As expected, the anti-prosequence antiserum detected prorenin but not renin.
The results of this study have general relevance to the regulation of BP, sodium handling, and the renin-angiotensin system. They also have relevance to the interpretation of plasma prorenin, plasma total renin, and renin mRNA measurements and the meaning of immunohistochemical staining with the use of antirenin and antiprorenin prosequence antibodies.
St. Lezin et al31 and Lewis et al32 have documented recently that inbred salt-sensitive rats developed by J. Rapp33 (Dahl SS/Jr) and supplied by Harlan Sprague Dawley have become genetically contaminated. The rats used in the present study were not inbred; they were of the Brookhaven strain, rats of which are selectively bred according to the principles developed by Dahl et al34 to maintain a salt-sensitive phenotype. For the present study, 4- to 5-week-old rats were received from Harlan Sprague Dawley between November 20, 1991, and January 24, 1992, ie, before genetic contamination of the Dahl SS/Jr rat was documented.31
Although contamination of the Dahl SS/Jr rats is not an issue in the present study, the source of the rats and their phenotypic responses in experimental circumstances are relevant because the BPs of Dahl S rats during 14 days of the 4% NaCl diet were not different from the BPs of their time controls fed either 0.4% or 0% NaCl. These results are consistent with the early work of Dahl et al,35 who showed that BP does not increase significantly in the salt-sensitive Brookhaven strain after 2 weeks of even 8% NaCl intake. Therefore, the results of the current study (Fig 1⇑) conform to Dahl’s characterization of the strain.34 They also conform to our previous unpublished (1988-1989) studies using Brookhaven Dahl S rats in which we found it took at least 4 weeks for SBP to increase significantly during a 4% NaCl diet. It is therefore likely that BP would have increased in the current study had the rats been maintained on 4% NaCl for a longer period, and this consideration was implicit in the experimental design.
Other characteristics of the Brookhaven selectively bred Dahl S rats studied herein are similar to those previously published13 22 34 ; their SBP averaged 10 to 20 mm Hg higher than SBP of the Dahl R rat irrespective of dietary salt intake (Fig 1⇑), and the ratio of heart weight to body weight was consistently higher in the Dahl S than R rat fed high salt.
Maturation-Related Falls in Plasma Prorenin and Renal Prosequence Immunostaining in the Dahl R Rat: Relationship to BP
The higher BP of the 9-week-old Dahl S rat13 22 was preceded by differences in prorenin parameters between the two strains. Seven-week-old Dahl R rats fed normal chow had twofold higher plasma prorenin levels than Dahl S rats. They also had sixfold greater renal arteriolar prosequence immunostaining, as evidenced by a weighted immunoreactivity that reflects primarily the number of renal arteriolar epithelioid cells that stained positively for prorenin.36 However, by 9 weeks, plasma prorenin and renal prosequence immunostaining were not different between the strains, as plasma prorenin fell 77% and the prosequence weighted scores fell sixfold in the Dahl R rats on normal chow, ie, from 18 per 100 glomeruli to 3 per 100 glomeruli, between 7 and 9 weeks. Renal anti-renin weighted score also fell during the same time but to a much lesser degree (about 15%, ie, from 75 to 65 per 100 glomeruli). However, anti-renin immunostaining was much more intense than prosequence immunostaining and thus may be a less-sensitive indicator of change.
The changes in prorenin in the Dahl R rat between 7 and 9 weeks most likely reflected a finalization of the maturation-related decline in renin gene expression in the renal arterioles that has been previously described.37 Although there was no concurrent fall in mRNA, mRNA is likely to be the first parameter to turn off, preceding declines in stored renin and prorenin.
Whether there is a cause-and-effect relationship between the intrinsically higher BP of the Dahl S rat and the earlier falls in prorenin parameters is worthy of exploration. Although BP was not significantly higher in the Dahl S rat at 7 weeks, we cannot be sure from our relatively imprecise indirect tail-cuff measurements whether the higher BP of the Dahl S rat actually preceded the fall in prorenin parameters. Thus, it is not possible to tell whether the higher BP of the Dahl S rat may have caused the earlier decline in renin gene expression or whether the fall in renin gene expression contributed in some way to the higher BP. However, it should be noted that the average BP of the 7-week-old Dahl S rats was the same as the BP of the 9-week-old Dahl R rats, giving some credence to the latter point of view. Our data do not strongly support the concept that the earlier decline in prorenin in the Dahl S rat is the result of intrinsically lower renin gene expression because renal renin mRNA was usually not different in Dahl S and R rats. Also, translational or posttranslational differences are unlikely because total plasma renin (renin plus prorenin) was often identical in the two strains.
Irrespective of sequence, plasma prorenin levels were significantly lower in the Dahl S than R rats at 7 weeks of age. They were also lower in the 9-week-old Dahl S rats after 14 days of the high salt diet. These results complement our studies in hypertensive patients38 which showed that salt-sensitive patients have lower plasma prorenin levels than salt-resistant patients. Whether the lower plasma prorenin levels of salt-sensitive hypertensive patients and rats contribute to the increased BP and to the salt sensitivity of BP remains to be established, but this interpretation is consistent with our working hypothesis that plasma prorenin causes vasodilation, most specifically in the renal afferent arterioles.39
The idea that the earlier decline in renin gene expression in the Dahl S rat was a consequence of the higher BP is consistent with the reported timing of the fall in renin gene expression in afferent arterioles and interlobular arteries of very young rats, since BP rises as the Dahl rats mature.22 It is also consistent with a greater degree of renin gene expression in subcapsular or outer cortical than in juxtamedullary nephrons36 40 since juxtamedullary nephrons have generally higher levels of perfusion pressure than outer cortical nephrons.
Comparison of the Effects of High and Low Salt Diets on Prorenin/Renin Parameters in Dahl S and R Rats
The responses of prorenin/renin parameters to the 4% NaCl diet were remarkably different in Dahl S and R rats. Unlike the normotensive human subject41 and the inbred Dahl R rat,13 42 the Brookhaven strain Dahl R rats in the present experiment exhibited almost no response to the high salt diet. PRC, plasma prorenin, renal renin immunoreactivity, and renal renin mRNA were slightly but not significantly lower than in Dahl R rats of the same age fed normal chow for 14 days; PRC was significantly lower only at 7 days. These results suggest that the macula densa signal to suppress renin release is insensitive in the Dahl R rat to a 10-fold increase in the NaCl load; that a macula densa–induced fall in renin release is counterbalanced by a commensurate, non–macula densa–mediated signal for increased renin release41 ; or that the regulation of renin gene expression may be inherently different in the Dahl R rat. The absence of any concurrent increase in BP suggests that either the Dahl R rat did not retain NaCl or its BP was insensitive to NaCl retention.
In marked contrast, plasma renin concentration and renal prosequence immunostaining were 10-fold lower in Dahl S rats fed 4% NaCl compared with control rats fed normal (0.4% NaCl) chow. This was accompanied by 35% lower levels of renal renin mRNA and renal anti-renin immunoreactivity, but plasma prorenin was not significantly lower. The lack of any significant salt-induced increase in the BP of the 2-week 4% NaCl–fed Dahl S rat suggests that its suppression of renin-mediated vasoconstrictor activity, and perhaps also its lack of suppression of plasma prorenin vasodilator activity, was able to offset whatever proclivity for a salt-mediated rise in BP that might have occurred. With additional time these rats most likely overwhelm this buffering capacity of the renin system and suppress plasma prorenin, at which time BP rises.
Relationship of mRNA to PRC, Plasma Prorenin, and Total Renin
Although there were highly significant positive relationships among all of the prorenin/renin parameters (Table 3⇑), there were quite marked quantitative differences (Fig 6⇑). For example, between low and high salt diets for the Dahl S rats, PRC changed 20-fold, plasma prorenin changed twofold, and renal renin mRNA and plasma total renin (prorenin plus renin) both changed threefold (Fig 6⇑). These results are in keeping with the premise that changes in total plasma renin reflect changes in renin gene expression. These results may also mean that renin gene expression is determined by the demand for both renin and prorenin, ie, not for renin alone, as has been reasoned previously.
Meaning of Prosequence Immunoreactivity Measurements
Strikingly similar quantitative changes in PRC and renal prosequence immunoreactivity occurred in this study (Figs 5⇑ and 6⇑). Renal prosequence immunoreactivity did not change in proportion to plasma prorenin except during the maturation decline between 7 and 9 weeks in the Dahl R rat (Fig 4⇑). These findings may indicate that under steady-state conditions, our prosequence antibodies detect primarily that portion of synthesized prorenin destined for processing to renin, even though it is only a small fraction of the total prorenin synthesized. This interpretation is consistent with the findings of Taugner et al,43 44 who identified prosequence immunostaining in paracrystalline inclusions of protogranules that are thought to transform into renin secretory granules. The larger amounts of prorenin destined for constitutive secretion may pass rapidly through the cell, largely avoiding detection, or the epitope or epitopes detected by IR 657 may be inaccessible in prorenin during constitutive secretion. In contrast, the maturation-related proportional changes in plasma prorenin and prosequence immunoreactivity may mean that cells in the process of turning off renin gene expression, which are not under steady-state conditions, no longer process prorenin to renin in proportion to the rate they secrete renin.
Our findings and interpretation of the prosequence immunostaining results are at variance with those of Berka et al,45 who observed parallel changes in anti-renin and anti-prosequence immunostaining during converting enzyme inhibition. It is possible that the polyclonal antibody used by Berka et al, which was made against a 12–amino acid fragment of the prosequence, in contrast to the entire 46–amino acid prosequence used in our experiment, detected a different pool of prorenin and/or prosequence fragments. Alternatively, the lower intensity of staining with our prosequence antiserum may have exaggerated the discrepancy in staining incidence, especially with high salt.
Meaning of Renin Immunoreactivity Measurements
The renin immunoreactivity results are difficult to interpret because anti-renin antibodies should be able to detect prorenin and renin. If they detect both parameters with equal sensitivity, the antirenin immunostaining could be interpreted as showing changes in total renin (renin plus prorenin). However, since current dogma states that renin is stored in JGAs and prorenin is secreted constitutively,46 one might then presume (as have others47 48 49 50 51 ) that antirenin immunostaining primarily detects stored renin. However, several observations suggest a different interpretation. First, our anti–human renin antibodies stained recombinant rat prorenin about 10 times more intensely than renin on sodium dodecyl sulfate–polyacrylamide gels (Fig 9⇑). Second, prorenin quenched JGA anti-renin immunostaining better than renin, although both were able to quench staining completely when the antibody titer and antigen concentrations were increased (Fig 8⇑). Third, anti-renin immunoreactivity changed in proportion to plasma prorenin not plasma renin between low and high salt diets (Fig 6⇑). Altogether, these results support the premise that renal anti-renin immunoreactivity detects large amounts of prorenin, primarily prorenin that is in the constitutive pathway. If this is correct, since renal renin immunoreactivity was usually lower in Dahl S than R rats (Fig 4⇑, left), the data suggest that constitutive secretion of prorenin is lower in Dahl S than R rats.
Regulation of Renin Gene Expression
The results of these studies are generally consistent with a view that changes in renin mRNA are primarily accomplished by changing the number of cells that express the renin gene rather than by increasing the level of mRNA per cell. For further investigation of this premise, additional studies of the distribution of intrarenal immunoreactivity of renin, prorenin, and angiotensins are being performed on the kidneys of these rats and will form the basis of a separate report (unpublished data, 1992-1995).
Comparison With Other Reports
Unlike our PRC results, early studies showed unequivocally that Dahl S rats had lower PRA than Dahl R rats.52 53 54 There are several possible explanations for this discrepancy. First, we used a normal chow diet with a lower NaCl content (0.4% NaCl) than had been used by some earlier investigators (1% NaCl).13 Second, the blood for renin measurement in the present studies was collected from rats that were rapidly decapitated in circumstances that minimized stress and renin release. It is possible that in certain of the earlier studies, blood may have been drawn under more stressful conditions. Third, we measured PRC rather than PRA, which integrates changes in both renin and angiotensinogen.
Bouhnik and coworkers42 performed a somewhat similar study of salt loading (8% NaCl) and depletion for 4 weeks in young inbred Dahl SS/Jr and SR/Jr rats (Mollgaard Breeding Center, Ejby, Denmark). Although the results of their salt depletion studies are consistent with our findings in Brookhaven selectively outbred Dahl S and R rats, several aspects of their study of salt loading differ markedly from ours. Most notably, PRA was suppressed by high salt diet in Dahl R rats but not in Dahl S rats, even though renal renin immunoreactivity was suppressed in both. In our studies of selectively outbred Dahl R rats, all renin parameters were insensitive to high NaCl diets, whereas Dahl S rats suppressed all renin parameters. These discrepancies can be explained in part by the timing of the hormonal measurements. In our earlier reported study of Brookhaven Dahl S rats fed 8% salt, PRA was suppressed for 2 to 4 weeks, but between 4 and 6 weeks, a paradoxical rise in PRA was observed.17 These findings suggest that early suppression of PRA by the 8% salt diet was missed by Bouhnik et al in SS/Jr rats because they studied these parameters only at 4 weeks.
The suppression of PRA by the high salt diet in the SR/Jr rats studied by Bouhnik et al42 is clearly different from our results. Whether this discrepancy is due to a fundamental difference between inbred and outbred strains, is related to feeding of an 8% NaCl diet for 4 weeks, or is due to the fact that we measured PRC rather than PRA remains to be elucidated.
The rise in hematocrits that occurred in all groups of the present study was unexpected and is difficult to explain. However, a lack of a fall in hematocrit (hemodilution) with salt loading or rise (hemoconcentration) with salt depletion is consistent with other studies utilizing the Brookhaven strain, suggesting that no significant long-term retention of NaCl or volume expansion occurs in Dahl S or R rats on high salt diets.55 Therefore, it appears that the changes in hematocrits observed in the present study may be another manifestation of maturation.
In summary, the present results indicate that changes in plasma total renin (renin plus prorenin) directly and quantitatively reflect changes in renin gene expression. They also suggest that under steady-state conditions, anti-prosequence immunostaining (using our polyclonal antibodies against the entire prosequence of rat prorenin) may reflect primarily that portion of synthesized prorenin destined for intracellular processing to renin. In addition, the results show that renal anti-renin immunoreactivity changes in proportion to plasma prorenin and is lower in Dahl S than R rats. They also demonstrate that with salt loading, the intrinsically higher BP of the Dahl S rat is associated with an earlier maturation-related decline in plasma prorenin and renal prosequence immunoreactivity and with lower renal renin immunoreactivity. They confirm that salt loading in the Dahl S rat is accompanied by marked suppression of renin system parameters, that the fall in renin precedes any rise in BP, and that with salt loading Dahl S rats have lower plasma prorenin levels than Dahl R rats. Finally, the results confirm that the Dahl R rat exhibits a remarkable disregard of both BP and the renin system to high salt diet.
Selected Abbreviations and Acronyms
|Ang I||=||angiotensin I|
|Dahl R||=||Dahl salt-resistant|
|Dahl S||=||Dahl salt-sensitive|
|PRA||=||plasma renin activity|
|PRC||=||plasma renin concentration|
|SBP||=||systolic blood pressure|
This work was supported in part by grant HL-18323-SCR from the National Heart, Lung, and Blood Institute, National Institutes of Health, and from the Wallace, Starr and Maxwell Foundations. The histological support of Eva Kraus, Rebecca Bethea, Scott Gerber, and Louis Alcarese; the technical assistance of Charles Robinson, Tina Pitarresi, Henrietta Manapat, Carmen Merali, and Matthew Gilbert; the photographic assistance of Sarah Carson; and the secretarial support of Frances Thomas are gratefully acknowledged.
Reprint requests to Jean E. Sealey, Cardiovascular Center, A 863, New York Hospital–Cornell Medical Center, 525 E 68th St, New York, NY 10021.
- Received June 20, 1995.
- Revision received August 21, 1995.
- Accepted February 1, 1996.
Lutterotti NV, Catanzaro DF, Sealey JE, Laragh JH. Renin is not synthesized by cardiac and extrarenal vascular tissues: a review of experimental evidence. Circulation. 1994;89:458-470.
Kim S, Hosoi M, Nakajima K, Yamamoto K. Immunological evidence that kidney is primary source of circulating inactive prorenin in rats. Am J Physiol. 1991;260:E526-E536.
Lenz T, Sealey JE, Maack T, James GD, Heinrikson RL, Marion D, Laragh JH. Half-life, hemodynamic, renal and hormonal effects of prorenin in cynomolgus monkeys. Am J Physiol. 1991;29:R804-R810.
Hiruma M, Kim S, Ikemoto F, Murakami K, Yamamoto K. Fate of recombinant human renin administered exogenously to anesthetized monkeys. Hypertension. 1988;12:317-323.
Makrides SC, Mulinari R, Zannis VI, Gavras H. Regulation of renin gene expression in hypertensive rats. Hypertension. 1988;12:405-410.
Dene H, McIlwani C, Rapp JP. Quantitation of renal renin and renin mRNA in Dahl rats in response to provocative stimuli. Clin Exp Hypertens. 1989;11:1585-1594.
Rapp JP. Dahl salt-sensitive and salt-resistant rats. Hypertension. 1982;4:753-763.
Iwai J, Dahl LK, Knudsen KD. Genetic influence on the renin-angiotensin system. Circ Res. 1973;32:678-684.
Tobian L, Lange J, Azar S, Iwai J, Koop D, Coffee K, Johnson M. Reduction of natriuretic capacity and renin release in isolated, blood-perfused kidneys of Dahl hypertension-prone rats. Circ Res. 1978;43:192-198.
Rapp JP, Wang SM, Dene H. A genetic polymorphism in the renin gene of Dahl rats cosegregates with blood pressure. Science. 1989;243:p542-p544.
St Lezin EM, Wong AL, Liu W, Wang J, Pravenec M, Reid IA, Kurtz TW. Transfer of the renin gene from the Dahl S rat into the Dahl R rat reduces renin gene expression, plasma renin concentration, and blood pressure. Hypertension. 1994;24:387. Abstract.
St Lezin EM, Wong A, Wang J, Pravence M, Stec D, Jacob H, Roman R, Kurtz TW. Transfer of the Dahl S renin gene into the Dahl R strain does not promote NaCl-sensitivity and may actually cause a decrease in blood pressure. Hypertension. 1993;22:421. Abstract.
Kurtz TW, Morris RCJ. Hypertension in the recently weaned Dahl salt-sensitive rat despite a diet deficient in sodium chloride. Science. 1985;230:808-810.
Sealey JE. Plasma renin activity and plasma prorenin assays. Clin Chem. 1991;37:1811-1819.
Sun J, Oddoux C, Gilbert MT, Yan Y, Lazarus A, Campbell WGJ, Catanzaro DF. Pituitary-specific transcription factor (Pit-1) binding site in the human renin gene 5′-flanking DNA stimulates promoter activity in placental cell primary cultures and pituitary lactosomatotropic cell lines. Circ Res. 1994;75:624-629.
Gilman M. Ribonuclease protection assay: current protocols in molecular biology. In: Ausubel FM, ed. Current Protocols in Molecular Biology. New York, NY: John Wiley & Sons; 1991:1-6.
Burnham CE, Hawelu-Johnson CL, Frank BM, Lynch KR. Molecular cloning of rat renin cDNA and its gene. Proc Natl Acad Sci U S A. 1987;84:5605-5609.
Fort P, Piechaczyk M, El Sabrouty S, Dani C. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate dehydrogenase multigenic family. Nucleic Acids Res. 1985;13:1431-1442.
Lewis JL, Russell RJ, Warnock DG. Analysis of the genetic contamination of salt-sensitive Dahl/Rapp rats. Hypertension. 1994;24:255-259.
Dahl LK, Heine M, Tassinari L. Effects of chronic excess salt ingestion: evidence that genetic factors play an important role in susceptibility of experimental hypertension. J Exp Med. 1962;115:1173-1190.
Dahl LK, Knudsen KD, Heine MA, Leitl GJ. Modification of experimental hypertension in the rat by variations in the diet. Circ Res. 1968;22:11-18.
Taugner R, Hackenthal E, Helmchen U, Ganten D, Kugler P, Marin-Grez M, Nobiling R, Unger T, Lockwald I, Keilbach R. The intrarenal renin-angiotensin system: an immunocytochemical study on the localization of renin, angiotensin converting enzyme and the angiotensins in the kidney of mouse and rat. Klin Wochenschr. 1982;60:1218-1222.
Gomez RA, Lynch KR, Sturgill BC, Elwood JP, Chevalier RL, Carey RM, Peach MJ. Distribution of renin mRNA and its protein in the developing kidney. Am J Physiol. 1989;257:F850-F858.
Pecker MS, James GD, Sealey JE, Jackson S, DiFabio B, Carroll L, Orlic S, Blake M, Alderman M, Pickering TG, Schnall P, Atlas SA, Laragh JH. Predictors of sodium sensitivity in mild hypertension. Am J Hypertens. 1989;2:85a. Abstract.
Sealey JE, Laragh JH. The renin-angiotensin-aldosterone system for normal regulation of blood pressure and sodium and potassium homeostasis. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Publishers; 1990:1287-1317.
Galen FX, Devaux C, Houot AM, Ménard J, Corvol P, Corvol MT, Gubler MC, Mounier F, Camilleri JP. Renin biosynthesis by human tumoral juxtaglomerular cells: evidences for a renin precursor. J Clin Invest. 1984;73:1144-1155.
Gomez RA, Lynch R, Chevalier RL, Everett AD, Johns DW, Wilfong N, Peach MJ, Carey RM. Renin and angiotensinogen gene expression and intrarenal renin distribution during ACE inhibition. Am J Physiol. 1988;254:F900-F906.
Orstavvik TB, Inagami T. Localization of kallikrein in the rat kidney and its anatomical relationship to renin. J Histochem Cytochem. 1982;30:385-390.
Rodriguez-Sargent C, Cangiano JL, Opava-Stitzer S, Martinez-Maldonado M. Renal Na K ATPase in Okamoto and Dahl hypertensive rats. Hypertension. 1981;3(suppl II):II-86-II-91.