Renal Sympathetic Neural Mechanisms as Intermediate Phenotype in Spontaneously Hypertensive Rats
Abstract The borderline hypertensive rat, the F1 of a cross between a hypertensive spontaneously hypertensive rat (SHR) and a normotensive Wistar-Kyoto (WKY) rat, is a NaCl-sensitive model of genetic hypertension. In addition to hypertension, borderline hypertensive rats fed 8% NaCl develop characteristic alterations in the regulation of efferent renal sympathetic nerve activity and the neural control of renal function that are similar to those observed in the SHR parent. Like the normotensive WKY rat parent, borderline hypertensive rats fed 1% NaCl remain normotensive and do not exhibit these alterations in renal sympathetic neural mechanisms. These renal sympathetic neural mechanisms constitute a complex quantitative trait that may represent an intermediate phenotype. They have a plausible pathogenetic role in hypertension and are different between SHR and WKY rats. This study evaluated two aspects of this complex quantitative trait, enhanced renal sympathoexcitation with air-jet stress and enhanced renal sympathoinhibition with guanabenz, as a candidate intermediate phenotype. As neither of these aspects was observed in two-kidney, one clip Goldblatt-hypertensive rats, this suggests that the trait is not secondary to hypertension from an acquired cause. In a backcross population (F1×WKY) fed 8% NaCl for 12 weeks, both enhanced renal sympathoexcitation with air-jet stress and enhanced renal sympathoinhibition with guanabenz cosegregated with the hypertension. These results support renal sympathetic neural mechanisms as an intermediate phenotype in SHR.
As described by Sanders and Lawler,1 the BHR is the F1 of a cross between a hypertensive SHR and a normotensive WKY rat. Exposure of BHRs to either environmental stress or increased dietary NaCl intake (8% NaCl) produces sustained hypertension (BHR-8%); in the absence of these interventions, the BHR remains normotensive (BHR-1%). BHRs subjected to prior renal denervation exhibit an attenuated increase in arterial pressure compared with BHRs with intact renal innervation.2 This finding suggests an important role for ERSNA in the development of hypertension in BHR.
We have examined the influence of increased dietary NaCl intake on aspects of the regulation of ERSNA and the neural control of renal function in BHR.3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 For comparison, the hypertensive (SHR) and the normotensive (WKY) parents were studied concurrently. The findings, ie, phenotypic features, are observed in both the hypertensive SHR parent and the hypertensive BHR-8% but are not seen in the normotensive WKY parent and the normotensive BHR-1%. In addition to the development of hypertension, there is an exaggerated natriuresis to volume expansion that is dependent on the concurrent exaggerated inhibition of ERSNA.3 6 14 Arterial baroreflex control of ERSNA is reset to the higher level of arterial pressure.17 Cardiac baroreflex control of ERSNA is augmented, and this accounts for the exaggerated inhibition of ERSNA during volume expansion.3 18 AJS responses, in terms of increases in MAP, HR, and ERSNA and decreases in urinary flow rate and sodium excretion, are increased.4 5 7 8 10 12 13 14 15 19 The inhibition of ERSNA after ICV administration of the α2-adrenoceptor agonist guanabenz is enhanced.11 16 These phenotypic features are seen in SHR and BHR-8% but not in WKY rats or BHR-1%.
The possibility that the features that deal with renal sympathetic neural mechanisms represent a complex quantitative trait that may serve as an intermediate phenotype was considered. In the current study, two of these features were chosen for initial analysis: augmented increases in ERSNA in response to AJS and enhanced decreases in ERSNA in response to ICV guanabenz.
Rapp20 set forth criteria for a complex quantitative trait as an intermediate phenotype: (1) the trait should have a plausible pathophysiological role in hypertension; (2) there should be evidence for a difference in the trait in progenitor hypertensive and normotensive strains that would implicate the trait in the pathogenesis of hypertension; (3) the difference in the trait should not be secondary to the hypertension; and (4) the difference in the trait should cosegregate with arterial pressure in F2 or backcross populations. For the first criterion, alterations in ERSNA, with their well-established effects on renal vascular resistance, sodium handling, and renin release have a plausible pathophysiological role in hypertension.21 Second, the differences in the renal sympathetic neural mechanisms between the hypertensive SHR parent and the normotensive WKY parent noted above would implicate alterations in ERSNA in the pathogenesis of hypertension.
Criteria three and four are the subject of the current investigation. The responses of ERSNA to AJS and ICV guanabenz were investigated in a nongenetic acquired model of hypertension, the 2K,1C Goldblatt rat, to examine whether these ERSNA alterations are secondary to hypertension. A backcross population (F1×WKY) was created to see whether the ERSNA alterations cosegregated with hypertension.
Adult male Sprague-Dawley rats, female SHR, and male WKY rats were purchased from Taconic Farms (Germantown, NY). Female SHR were bred with male WKY rats to produce BHR. BHRs of both sexes were bred with WKY rats of the opposite sex to produce the backcross population. Backcross-population rats of both sexes were weaned at 4 weeks of age and fed 8% NaCl with tap water drinking solution ad libitum until age 16 weeks, when they were studied. Sprague-Dawley rats were fed standard laboratory rat chow (1% NaCl) with tap water drinking solution ad libitum. All animal procedures were in accordance with the guidelines of the University of Iowa Animal Care and Use Committee.
The rats were anesthetized with methohexital (Brevital, 20 mg/kg IP supplemented with 10 mg/kg IV as needed; Eli Lilly).
2K,1C Goldblatt Hypertension
Via a right-flank incision, a 0.2-mm silver clip was placed on the right renal artery of 6-week-old Sprague-Dawley rats. The rats were studied 4 weeks later. Control rats were subjected to similar surgery, but a clip was not placed on the right renal artery.
The rats were instrumented with polyethylene catheters in the right jugular vein and right carotid artery for infusion of isotonic saline (0.05 mL/min maintenance) and the measurement of MAP and HR. The catheters were tunneled to the dorsum of the neck, where they were exteriorized and plugged.
Renal Sympathetic Nerve Activity Recording Electrode19
The left kidney was exposed through a left-flank incision via a retroperitoneal approach. With the use of a dissecting microscope (25×), a renal nerve branch from the aorticorenal ganglion was isolated and carefully dissected free. The renal nerve branch was then placed on a bipolar platinum wire (Cooner Wire Company) electrode. ERSNA was amplified (50 000×) and filtered (low, 30 Hz; high, 3000 Hz) with a high-impedance probe and preamplifier (Grass HIP 511 and P511, respectively; Grass Instrument Co). The output of the amplifier was channeled to a Tektronix 5113 oscilloscope for visual evaluation and an audio amplifier/loudspeaker (Grass Instrument Co model AM 8 audio monitor) for auditory evaluation. The quality of the ERSNA signal was assessed by its pulse-synchronous rhythmicity and by examining the magnitude of decrease in recorded ERSNA during sinoaortic baroreceptor loading with an IV bolus injection of norepinephrine (3 μg). The ERSNA remaining after maximum inhibition pursuant to norepinephrine administration was within 5% of the background noise observed approximately 30 minutes postmortem or pursuant to hexamethonium 30 mg/kg IV (ICN Pharmaceuticals)22 ; this background noise value was subtracted from all experimental values of ERSNA. When an optimal ERSNA signal (pulse-synchronous rhythmicity, abolition by norepinephrine-induced arterial pressure increase) was observed, the recording electrode was fixed to the renal nerve branch with a silicone cement (Wacker Sil-Gel 604, Wacker-Chemie). The electrode cable was then secured in position by suturing it to the abdominal trunk muscles and further tunneled to the dorsum of the neck, where it was exteriorized. The flank incision was closed in layers.
To avoid potential carryover effects, the ERSNA responses to AJS and ICV guanabenz were studied in separate groups of 2K,1C Goldblatt-hypertensive rats and backcross-population rats. All rats were studied on the day after the above-described chronic instrumentation in the conscious, unrestrained state. In the backcross-population rats, a 1-hour period of continuous recording of basal arterial pressure was made before beginning either the AJS or ICV guanabenz protocols.
During two consecutive 10-minute control periods, continuous recordings of arterial pressure, HR, and ERSNA were made. Then acute environmental stress (AJS, continuous) was begun; 5 minutes thereafter, two consecutive 10-minute experimental periods were conducted, during which continuous recordings of MAP, HR, and ERSNA were made. The AJS was stopped; 5 minutes thereafter, two consecutive 10-minute recovery periods were conducted, during which continuous recordings of MAP, HR, and ERSNA were made. Then hexamethonium (30 mg/kg IV) was administered, and the ERSNA remaining 15 minutes thereafter was taken as the background noise.
AJS consisted of an air jet delivered continuously to the dorsum of the rat’s head through a tube located 4 to 5 cm behind the rat. Repeated applications of AJS in the same rat resulted in similar increases in HR, MAP, and ERSNA, indicating reproducibility and lack of adaptation.4 5 7 8 10 12 13 14 15 19
During a 10-minute control period, continuous recordings of MAP, HR, and ERSNA were made. Then 5 μg guanabenz in 1 μL isotonic saline was injected ICV. Fifteen minutes was allowed for equilibration, followed by a 10-minute experimental period. Then 25 μg guanabenz in 1 μL isotonic saline was injected ICV. Fifteen minutes was allowed for equilibration, followed by a 10-minute experimental period. Next 125 μg guanabenz in 5 μL isotonic saline vehicle was injected ICV. Fifteen minutes was allowed for equilibration, followed by a 10-minute experimental period. Continuous recordings of MAP, HR, and ERSNA were made during each of the experimental periods. It has been demonstrated previously11 that the ICV injection of up to 7 μL isotonic saline alone has no effect on MAP, urinary sodium excretion, or renal sympathetic nerve activity. Finally, 30 mg/kg hexamethonium IV was administered, and the ERSNA that remained 15 minutes after hexamethonium administration was taken as the background noise.
The amplified and filtered renal neurogram was full-wave rectified and integrated (Grass 7P3 resistance-capacitance integrator, 20 ms time constant) and stored as ERSNA on videotape (Vetter 4000A PCM; A.R. Vetter Co) along with the neurogram, MAP (Statham 23Db pressure transducer; Statham-Gould), and HR (Grass 7P4 tachograph) signals for later off-line analysis.
With the use of an analog-to-digital converter (Data Translation 2801) and standard data acquisition software (LABTECH Notebook 7.3), the steady state ERSNA, MAP, and HR were sampled at 5 Hz and averaged over the duration of the control, experimental, and recovery periods in the AJS protocol and over the duration of the control and experimental periods in the ICV guanabenz protocol.
Statistical analysis was conducted with one-way ANOVA and Scheffé’s test for pairwise comparisons among means for the comparison of MAP and ERSNA responses to AJS and guanabenz (125 μg ICV dose) among 2K,1C Goldblatt-hypertensive rats, SHR, WKY rats, BHR-1%, and BHR-8%. One-way ANOVA with repeated measures and Scheffé’s test for pairwise comparisons among means were used for comparison of MAP, HR, and ERSNA responses to AJS and guanabenz (5, 25, and 125 μg ICV) in the backcross population. In the backcross population, the correlation coefficient values (r) between basal MAP and MAP, HR, and ERSNA responses to AJS and guanabenz (5, 25, and 125 μg ICV) were calculated. A value of P<.05 was considered significant. Data in text and figures are mean±SE.
2K,1C Goldblatt-Hypertensive Rats
Fig 1⇓ displays the baseline MAP and Fig 2⇓ displays the ERSNA responses to AJS and ICV guanabenz of 2K,1C Goldblatt-hypertensive rats. For comparison, data are shown for SHR, BHR-8%, BHR-1%, and WKY rats previously studied in our laboratory under identical conditions.4 14 Baseline MAP in 2K,1C Goldblatt-hypertensive rats was 191±5 mm Hg; baseline MAP in control rats was 109±5 mm Hg. MAP in 2K,1C Goldblatt-hypertensive rats was similar to that in SHRs and BHR-8% and higher than that in BHR-1% and WKY rats (P<.01). However, despite a similar magnitude of hypertension, 2K,1C Goldblatt-hypertensive rats exhibited only an 18±3% increase in ERSNA with AJS; the increase in ERSNA with AJS in control rats was 15±4%. In 2K,1C Goldblatt-hypertensive rats, the renal sympathoexcitatory response to AJS was lower than those responses observed in SHR (77±6%) and BHR-8% (62±6%) (P<.01 for both) and was in the range of those observed in WKY (12±2%) and BHR-1% (17±5%). In 2K,1C Goldblatt-hypertensive rats, AJS produced no significant change in MAP (from 191±5 to 189±6 mm Hg) or HR (430±22 to 443±24 beats per minute). Similarly, 2K,1C Goldblatt-hypertensive rats exhibited only a 45±6% inhibition of ERSNA with the 125 μg dose of ICV guanabenz; the response in control rats was 51±4%. This inhibitory response is lower than those responses observed in SHR (80±4%) and BHR-8% (86±4%) (P<.05 for both) and is in the range of those observed in WKY (52±3%) and BHR-1% (48±2%). In 2K,1C Goldblatt-hypertensive rats, this dose of ICV guanabenz did not significantly affect MAP (−3.2±3.7%) or HR (−11.0±4.6%). Therefore, despite similar hypertensive levels of MAP, 2K,1C Goldblatt-hypertensive rats did not exhibit augmented ERSNA responses to either AJS or ICV guanabenz.
In the backcross-population rats, the 1-hour period of continuous recording of arterial pressure before beginning the AJS or ICV guanabenz protocols gave values of MAP that were not significantly different from those derived from the shorter-duration control periods in each of the respective protocols. Of the 163 backcross-population rats studied, there were 83 male rats whose MAP was 138±2 mm Hg and 80 female rats whose MAP was 141±2 mm Hg.
For each backcross rat, the MAP from the preprotocol 1-hour continuous monitoring period was plotted against the respective values for the percent increase in ERSNA with AJS (Fig 3⇓) and the percent inhibition in ERSNA with 125 μg guanabenz ICV (Fig 4⇓). In the 81 backcross rats in the AJS protocol, the percent increase in ERSNA with AJS was correlated with the basal level of MAP (r=.76, P<.001). Significant correlations at lower correlation coefficient values were found with both the MAP (r=.59, P<.001) and HR (r=.50, P<.001) responses to AJS. In the 82 backcross rats in the ICV guanabenz protocol, the percent inhibition in ERSNA with 125 μg guanabenz ICV was correlated with the basal level of MAP (r=.70, P<.001). Significant correlations at lower correlation coefficient values were found with the ERSNA responses to either the 5 (r=.49, P<.001) or 25 (r=.53, P<.001) μg ICV dose of guanabenz or the MAP (range of r, .51 to .62; P<.001) and HR (range of r, .48 to .57; P<.001) responses to all three doses of ICV guanabenz.
The BHR (F1) inherits genetic information from a hypertensive SHR parent and a normotensive WKY parent. When BHRs ingest an 8% NaCl diet, they develop hypertension and exhibit aspects of regulation of ERSNA and the neural control of renal function that are similar to the hypertensive SHR parent. When BHRs ingest a 1% NaCl diet, they remain normotensive and exhibit aspects of regulation of ERSNA and the neural control of renal function that are similar to the normotensive WKY parent. These results suggest that increased dietary NaCl intake is able to induce or unmask the capabilities for these responses that are genetically conveyed to the BHR by the hypertensive SHR parent in latent forms.
The current study evaluated renal sympathetic neural mechanisms as a complex quantitative trait for suitability as an intermediate phenotype. Complex traits refer to phenotypes or intermediate phenotypes that do not exhibit classic mendelian inheritance attributable to a single gene locus. Variation in these traits may result from variation in multiple genes and environmental influences. Quantitative traits refer to continuous variables such as MAP, in contrast to discrete traits measured by a specific outcome, such as albino versus pigmented.
With regard to the criteria for complex quantitative traits as intermediate phenotypes, renal sympathetic neural mechanisms, through their multiple actions on various aspects of renal function,25 have a plausible pathophysiological role in the pathogenesis of hypertension (criterion 1). Increased ERSNA is known to increase renal vascular resistance, which, in view of the fraction of cardiac output delivered to the kidneys, may represent a substantial contribution to total peripheral vascular resistance. ERSNA at levels below the threshold for effects on renal blood flow or glomerular filtration rate directly increases renal tubular sodium and water reabsorption throughout the nephron. In this way, increased ERSNA opposes pressure diuresis and natriuresis, resulting in the need for a higher level of MAP to achieve the same level of water and sodium excretion (ie, a rightward shift of the renal function curve along the MAP axis).26 Graded increases in ERSNA produce graded increases in renin release, beginning at levels of ERSNA that are subthreshold for effects on renal blood flow, glomerular filtration rate, or renal tubular sodium and water reabsorption.
As noted above, the various renal sympathetic neural mechanisms differ substantially between SHR and WKY (criterion 2). Furthermore, the influence of dietary NaCl intake in the BHR (F1) is such that 8% NaCl intake results in the BHR resembling the hypertensive SHR parent in becoming hypertensive and exhibiting these features, whereas 1% dietary NaCl intake results in the BHR resembling the normotensive parent and not exhibiting these features.
In the current study, we have provided evidence that two aspects of this complex quantitative trait are not secondary to hypertension (criterion 3). Using an acquired or secondary model of hypertension, 2K,1C Goldblatt hypertension, we showed that despite hypertension of a magnitude similar to that seen in SHR and BHR-8%, the ERSNA responses to AJS and ICV guanabenz were not enhanced. The ERSNA responses of 2K,1C Goldblatt-hypertensive rats to AJS and ICV guanabenz were not different from control rats, BHR-1%, or WKY rats.
Using a backcross population (F1×WKY) fed 8% NaCl, we have demonstrated that the ERSNA responses to AJS and ICV guanabenz cosegregate with MAP (criterion 4). Higher levels of MAP were accompanied by greater renal sympathoexcitatory responses to AJS and greater renal sympathoinhibitory responses to ICV guanabenz.
Therefore, with respect to the complex quantitative trait of renal sympathetic neural mechanisms as reflected by enhanced increases in ERSNA with AJS and enhanced decreases in ERSNA with ICV guanabenz, these results support the view that this trait represents an intermediate phenotype for SHR.
It should be emphasized that cosegregation of a phenotypic defect with arterial pressure in an F2 or backcross population is evidence of an association of the defect with the pathogenesis of hypertension. However, used in this manner, such studies cannot determine directly whether the association is due to cause or effect (ie, secondary to the hypertension). There are several ways of overcoming this problem. One way is to use a longitudinal approach,27 ie, to look for the phenotypic defect before the onset of hypertension and determine whether it correlates with later development of hypertension. Harrap and Doyle28 studied groups of F2 WKY and SHR rats at different ages and demonstrated that although MAP was negatively correlated with glomerular filtration rate in 11-week-old rats, there was no such correlation in 16-week-old rats. These results were interpreted as indicating that MAP increased from 11 to 16 weeks to normalize glomerular filtration rate. Nørrelund et al29 used a longitudinal approach to follow individual F2 WKY and SHR rats and demonstrated that a narrowed lumen of distal afferent arterioles at 7 weeks (when MAP was not increased) was negatively correlated with MAP at 23 weeks. Another approach is to demonstrate that the phenotypic defect or trait occurs in a genetic model of hypertension but not in acquired (secondary) models of hypertension. In the current study, the two aspects of the complex quantitative trait of renal sympathetic neural mechanisms studied, enhanced ERSNA responses to AJS and ICV guanabenz, were observed in SHR but not in 2K,1C Goldblatt hypertension. Thus, if the trait were simply a secondary consequence of hypertension from any cause, it should have been present in the 2K,1C Goldblatt-hypertensive rats, whose level of MAP was similar to that observed in SHR.
These results suggest that the complex quantitative trait of renal sympathetic neural mechanisms is causally involved in hypertension and that it lies on the pathogenetic pathway at some point between the level of the gene(s) and the expression of hypertension in SHR. Thus, the complex quantitative trait of renal sympathetic neural mechanisms is an intermediate phenotype for SHR.
Selected Abbreviations and Acronyms
|2K,1C||=||two-kidney, one clip|
|BHR||=||borderline hypertensive rat|
|BHR-8%||=||borderline hypertensive rats with sustained hypertension|
|BHR-1%||=||normotensive borderline hypertensive rats|
|ERSNA||=||efferent renal sympathetic nerve activity|
|MAP||=||mean arterial pressure|
|SHR||=||spontaneously hypertensive rat(s)|
This study was supported by grants DK-15843 and HL-44546 from the National Institutes of Health and grants from the Department of Veterans Affairs.
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