INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MOST INVESTIGATORS have been using descendants of the normotensive Wistar-Kyoto (WKY) rat strain as the control for the Okamoto and Aoki (20) spontaneously hypertensive rat (SHR). As a result of both highly inbred original stock and standardized breeding practices, virtual uniformity has resulted in the SHR supplied by major commercial vendors (5). However, the rigorous procedures applied to the initial inbreeding and subsequent distribution of the SHR were not used for the WKY rat derived after the SHR. As a result, animals were distributed to commercial suppliers as early as the 10th generation of inbreeding (13). Furthermore, subsequent breeding was not standardized with some suppliers (including Charles River Laboratories) maintaining brother-sister matings (10) to sustain the founder stock, whereas others (including Taconic Farms) used a random breeding (outbred) protocol (26).

Studies from several laboratories, including our own, compared WKY rats from Charles River Laboratories (WKY/cr; Wilmington, MA) and Taconic Farms (WKY/tf; Germantown, NY) and reported pronounced trait differences (9, 13, 14, 24). Our own laboratory (8), as early as 1984, noted differences in 1) consummatory behaviors including an enhanced salt preference in WKY/tf, 2) age-weight relationships, and 3) cardiovascular traits between these WKY substrains. Furthermore, we reported distinct differences in resting systolic (SP) and diastolic (DP) arterial pressures and circadian variations among inbred WKY/cr, outbred WKY/tf, and inbred SHR/cr rats (9). Our colonies were begun in the early 1980s from breeder stock obtained from both Charles River Laboratories and Taconic Farms. After observing cardiovascular and behavioral differences we decided to maintain both of these WKY substrains and in a breeding manner as they were provided to us, namely brother-sister mating to maintain the strains from La Jolla-Charles River (WKY/lj-cr and SHR/lj-cr) as inbred, currently over 50 generations, and random breeding for the Taconic Farms (WKY/tf). Because the unique cardiovascular and behavioral traits of the WKY/tf, evident in the early 1980s were still present in the early 1990s, we began inbreeding of this substrain, currently designated as WKY/lj-tf, and animals used for the present study were in the 33-34th generation of inbreeding.

We (1) previously reported significant differences in cardiovascular responses to airpuff startle between commercially available inbred WKY and our inbred WKY/lj-cr, which have been brother-sister mated from colony inception and will report a more extensive genetic characterization of these strains in a separate report. Present cardiovascular analysis was undertaken to compare our inbred WKY/lj-cr and inbred WKY/lj-tf with each other and with SHR/lj-cr. Cardiovascular analyses during both normal and high NaCl diets were undertaken with radiotelemetry monitoring to ensure direct and minimally stressful arterial pressure and heart rate (HR) measurements in unanesthetized, unrestrained, and undisturbed freely moving rats. Genotyping was conducted to compare the extent of polymorphism among all three strains. Results indicate that the WKY/lj-tf constitutes a new inbred, salt-sensitive WKY substrain with borderline hypertension, relative to WKY/lj-cr, which offers unique opportunities for future genomic studies.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Rat strains. The three inbred colonies of rats studied in this report are designated as WKY/lj-cr, WKY/lj-tf, and SHR/lj-cr. WKY/lj-cr and SHR/lj-cr were reported to be inbred when purchased originally from Charles River Laboratories and have been maintained by brother-sister mating through over 50 generations in La Jolla, CA. The original outbred WKY/tf were obtained from Taconic Farms and were maintained initially by random breeder selection until 1991 when inbreeding began. Postweaning and before instrumentation, all rats were housed in groups of three in clear plastic cages with wood shavings, with food and water ad libitum. The vivarium was temperature (20 ± 1°C) and light (12:12-h light-dark cycles; 6 AM to 6 PM) controlled. Body weights for all animals were determined by sampling groups of rats 3-14 wk of age. Data presented are averages at 4, 8, 12, and 14 wk of age; WKY/lj-cr (n = 72), WKY/lj-tf (n = 48), and SHR/lj-cr (n = 48). All animal studies were reviewed and approved by the University of California San Diego Institutional Animal and Care and Use Committee and were conducted in conformity with the "Guiding Principles for Research Involving Animals and Human Beings."

Genotyping. To obtain DNA samples, rats were briefly anesthetized with halothane-oxygen at 16 wk of age, and a 10-mm tip from the tail was removed, snap-frozen, and stored at 70°C. Genomic DNA was isolated from tail snips by phenol-chloroform extraction followed by ethanol precipitation. Genotyping was performed by PCR amplification of DNA microsatellites using forward and reverse primer pairs (Research Genetics; Huntsville, AL). Primers were selected on the basis of their map locations (formerly available at http://www.informatics.jax.org/rat and now at http://www.rgd.mcw.edu) and initially tested to be polymorphic between WKY/lj-cr and SHR/lj-cr. The PCR reaction volume was 25 µl and contained 1.5 mM MgCl₂, 50 µM of each 2-deoxynucleotide 5'-triphosphate (Boehringer-Mannheim), 0.264 µM of each primer, 1 U Taq polymerase (Promega), and 0.5-1.5 µg rat genomic DNA. PCR cycling consisted of an initial 94°C denaturation for 3 min, followed by 34 cycles of 93°C (40 s), 55°C (40 s) and 72°C (90 s), using an Ericomp TwinBlock System (Ericomp; San Diego, CA). PCR products were analyzed on a 7% polyacrylamide gel at 25 mA of constant current, and amplified DNA products visualized with ethidium bromide. Gels were photographed and each gel and photograph was scored independently by two readers. The genotype status of WKY/lj-tf was analyzed using 49 microsatellite markers (Table 1) scattered throughout the genome.

Cardiovascular phenotyping. Data Sciences radiotelemetry monitoring, collection, and analysis system (Data Sciences International; St. Paul, MN) was used for measurement of SP, mean arterial pressure (MAP), DP, HR, and locomotor activity. Rats at 11 wk of age were anesthetized with halothane-oxygen anesthesia, a midline abdominal incision was made, and the descending abdominal aorta was exposed. Two sutures were passed around the abdominal aorta distal to the renal arteries. The telemetry probe (TA11PA-C40) catheter was inserted into the aorta and advanced rostrally to lie just below the renal arteries. The catheter was secured in place with tissue glue, and the probe was secured and tested before closure of the incision. Rats were then housed individually in a vivarium room dedicated for radiotelemetry acquisition with restricted human access. Each rat cage was assigned a designated telemetry receiver (RPC-1). One week after surgery, implanted telemetry probes were turned on, and collection of data began. SP, DP, and HR were recorded in unanesthetized, unrestrained, and undisturbed rats as 5-s averages every 5 min, day and night, for a period of >50 days. All data were then off-loaded from the acquisition computers for subsequent analyses. Probe drift was checked and corrected as recommended by the manufacturer. In addition, independent studies were conducted using simultaneous direct arterial pulsatile blood pressure measurements with a Statham transducer and Gould pressure processor to ensure accuracy of probe measurements. To test the effect of dietary NaCl, rats were sequentially monitored for at least 10 days while on the following defined rat chow diets (Harlan Teklad, Madison, WI): breeder colony chow regular diet (0.7% NaCl with 24.79 and 6.5% protein and fat, respectively); a control diet to match the high NaCl diets, except for the NaCl (0.7% NaCl with 19.7 and 5.1% protein and fat, respectively); 3% NaCl diet (3% NaCl in the control diet composition); 8% NaCl diet (8% NaCl in the control diet); and the 0.7% NaCl control diet. We measured pulsatile arterial pressures and HRs continuously in a separate group of male WKY/lj-cr (n = 11) and WKY/lj-tf (n = 9). We also measured SHR/lj-cr (n = 9) under both regular diet (0.7% NaCl) and the high NaCl (8%) diet.

Data analysis. We (9) previously reported that these strains exhibit circadian variation over a 24-h period and confirmed this over an extended period of time of monitoring in the present study (Fig. 1). SP, DP, MAP, HR, and locomotor activity exhibited circadian variation with sustained peak values during the dark (active) period and minimal values during the light (resting) period. Because the onset and offset transitions would confound averaging, for arterial pressure and HR analyses we used data from a window in the middle of the 12-h rhythms between 9 AM and 3 PM (resting) and 9 PM and 3 AM (active). From the 24-h data collected every day (288 data points/day) we extracted the 72 data points from each window and averaged for each rat for each day. The averaged light or dark period values were then taken as repeated measurements. Likewise, because changes in high NaCl diet caused lags in the cardiovascular measurements, we averaged values obtained between the 4th and 10th day of each diet. Diurnal variation was defined as the difference between the average resting (light period) and active (dark period) values. Standard deviation and coefficients of variation of 24-h resting and active period values were calculated to assess arterial pressure and HR variability. Coefficients of variation were calculated by the formula [(SD/Mean)100%] (4). A repeated-measure ANOVA and one-way ANOVA with a Duncan's multiple-comparison test was performed to compare the value obtained between and within groups. P < 0.05 was considered significant.

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Line graph for diurnal rhythm of locomotor activity (A), heart rate (HR) (B), pulse pressure (PP) (C), diastolic arterial pressure (DP) (D), mean arterial pressure (MAP) (E), and systolic arterial pressure (SP) (F) for spontaneously hypertensive rats (SHR)/lj-cr (), Charles River Wistar-Kyoto (WKY/lj-cr) rats (), and Taconic Farms (WKY/lj-tf) rats (). Dark period (lights off) is from 6 PM to 6 AM. Each data point represents the mean ± SE of the 5-s average, every 5 min for 9-11 rats per group. Data are from 10 days of continuous radiotelemetry data collection under regular salt diet. lj, La Jolla.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Genotypes of WKY/lj-cr, WKY/lj-tf, and SHR/lj-cr were compared (Table 1) using 49 polymorphic markers between WKY/lj-cr and SHR/lj-cr. It was found that 14/49 (28.6%) were polymorphic across all three strains and were located on chromosomes 1, 2, 4, 5, 6, 9, 14, 16, 18, 19, and 20. In comparing WKY/lj-tf with WKY/lj-cr, 37 markers (75.5%) were polymorphic, whereas between WKY/lj-tf and SHR/lj-cr, 26 markers (53.1%) were polymorphic. It was found that WKY/lj-tf contain markers (designated tt) not found in either WKY/lj-cr or SHR/lj-cr. This is consistent with observations made shortly after the colony was started in 1982 that WKY/tf were markedly different from either WKY/cr or SHR/cr, although their blood pressures appeared "normotensive" when measured by direct arterial catheterization.

Body weights of the three strains were compared at 4, 8, 12, and 14 wk of age (Table 2). At 4 wk of age there was no difference in weight among the strains, while at 8 wk strains separated in weight with SHR/lj-cr > WKY/lj-tf > WKY/lj-cr. However, after 8 wk, WKY/lj-tf rats gained weight faster and surpassed both SHR/lj-cr and WKY/lj-cr rats (WKY/lj-tf > SHR/lj-cr > WKY/lj-cr).

The 24-h diurnal rhythm of locomotor activity, HR, arterial pulse pressure (PP), DP, MAP, and SP are shown in Fig. 1. In all groups, there were marked increases in locomotor activity, HR, and arterial pressures associated with the awake and active dark period. However, before the onset of the dark period, increases in these variables varied among the groups and reflected anticipatory responses similar to what we (9) previously reported. SHR/lj-cr exhibited a significant enhancement of locomotor activity nearly 3 h before the onset of the dark period (lights off), and also exhibited the reverse trend before lights on. Both WKY/lj-cr and WKY/lj-tf rats also exhibited an anticipatory HR response ~2 h before lights off, while SHR/lj-cr exhibited increases coincident with the increase in locomotor activity. Anticipatory changes in arterial pressures were not as evident, although there was an increasing trend approximately 2 h before lights off. Arterial pressures did change abruptly with lights off in all three strains, whereas SHR/lj-cr and WKY/lj-tf rats exhibited marked variability throughout the night. PP did not exhibit significant circadian variation, but variability of PP was greater in SHR/lj-cr. WKY/lj-tf rats exhibited steepest descents in HR, locomotor activity, DP, MAP, and SP 1 h before lights on; however, all parameters, except PP in all groups, reached the lowest values within 1 h.

An average of 6 h of light and dark periods for SP, DP, PP, and HR is illustrated across the 51 days of diet and radiotelemetry measurements in Fig. 2. WKY/lj-cr SP and DP were essentially unchanged over the regular and control diet periods, increased slightly during the 3% NaCl diet, and increased more significantly during the 8% NaCl. PP increased significantly (P < 0.05) in WKY/lj-cr rats over the control diet period and even more during the 3 and 8% diets. In contrast, SP, DP, and PP of WKY/lj-tf rats were all significantly (P < 0.05) greater, and HR was lower than WKY/lj-cr rats at all time points regardless of the type of diet used (Fig. 2). Circadian variation in SP, DP, and HR was evident in both strains but was especially pronounced in WKY/lj-tf values of SP and DP during the 8% NaCl diet. PP variation over the 24-h period was minimal but was consistently lower during the active (dark) period. The 8% NaCl diet further increased values in both light and dark periods of SP, DP, and PP and decreased HR in WKY/lj-tf and WKY/lj-cr (Fig. 2).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Line graph showing arterial SP (A), DP (B), and PP (C) in mmHg, and HR (D) in beats/min for WKY/lj-cr and WKY/lj-tf rats, abstracted from radiotelemetry measurements over a total study period of 51 days. Data represent means ± SE of 6-h blocks of either light ( and ) or dark ( and ). Dietary content of NaCl noted.

On replacement of the 8% NaCl diet with the control diet, arterial pressures decreased and HR increased in both WKY/lj-tf and WKY/lj-cr rats but failed to attain corresponding pre-high-NaCl control values (Fig. 2). In contrast to WKY/lj-cr rats, WKY/lj-tf rats exhibited a biphasic response on replacement of the 8% NaCl with the control diet. Initially, SP, DP, and PP decreased and HR increased after replacement and plateaued by day 2; however, this was followed by a gradual increase in SP, DP, and PP and a decrease in HR. All the post-high-NaCl control diet SP, DP, PP, and HR values are significantly (P < 0.05) different from the corresponding pre-high-NaCl control diet values in both WKY/lj-tf and WKY/lj-cr rats except for the DP value in WKY/lj-cr rats.

Results under each diet are provided in Table 3, which also includes corresponding values for SHR/lj-cr obtained from a separate radiotelemetry study. As shown, basal SP, DP, MAP, and PP values for the WKY/lj-tf rats were significantly (P < 0.05) lower and HR was higher than corresponding SHR/lj-cr values. On introduction of the 8% NaCl diet, the SP of both SHR/lj-cr and WKY/lj-tf rats exhibited a twofold greater increase than WKY/lj-cr rats during both dark and light periods, e.g., dark period SHR/lj-cr: 20 ± 1 mmHg increase, WKY/lj-tf: 21 ± 1 mmHg; WKY/lj-cr: 10 ± 1 mmHg (Fig. 3, A-C). Correspondingly, lower increases were seen in DP, whereas the rank order of change remained the same. In contrast to the rank order of baseline SP, DP, and PP, the order of HR values among the groups was reversed with WKY/ lj-cr > WKY/lj-tf > SHR/lj-cr. The daytime or the nighttime differences between WKY/lj-cr and SHR/lj-cr versus WKY/lj-tf and SHR/lj-cr in SP, DP, MAP, PP, and HR were significantly (P < 0.05) smaller in the latter.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Bar graph showing 8% NaCl-induced changes (A-C) and diurnal variations (D-F) in cardiovascular parameters for WKY/lj-cr, WKY/lj-tf, and SHR/lj-cr rats. A-C: effects of 8% NaCl diet-induced changes in HR (A), DP (B) and SP (C), during 6-h of light (open bar) and dark (filled bar) periods. D-F: diurnal differences defined as averages of the active (dark) period minus averages of the rest (light) period. Shown are changes in HR (D), DP (E), and SP (F) during 0.7% NaCl diet (open bar) and 8% NaCl diet (filled bar). Values are means ± SE of 9-11 rats per group. A-C: P < 0.05; * dark period values vs. light; values vs. corresponding values for WKY/lj-cr; values vs. corresponding values for WKY/lj-tf. D-F: P < 0.05; * values vs. 0.7% NaCl; values vs. WKY/lj-cr values; values vs. WKY/lj-tf values.

Night-day differences in SP, DP, and HR during the 0.7% NaCl control diet and the 8% NaCl diet are illustrated (Fig. 3, D-F). In all groups, there was significant diurnal variations in HR, DP, and SP, whereas WKY/lj-tf and SHR/lj-cr rats showed significantly (P < 0.05) greater variation than WKY/lj-cr. In the WKY/lj-tf rats, 8% NaCl potentiated diurnal variation in DP (+5.1 ± 0.7 vs. +8.6 ± 0.6 mmHg) and SP (+4.4 ± 0.8 vs. +7.3 ± 0.7 mmHg). In WKY/lj-cr and WKY/lj-tf rats, but not SHR/lj-cr, diurnal variation in HR was significantly (P < 0.05) attenuated by high NaCl.

Analysis of variability index (standard deviation and coefficients of variation) over the 24-h period and over the two averaging windows (light period 9 AM-3 PM and dark period 9 PM-3 AM) are provided in Table 4. During both the control and 8% NaCl diets, WKY/lj-tf exhibited highest variability (P < 0.05) in HR over 24 h compared with WKY/lj-cr rats and SHR/lj-cr. When analyzed over the light period only, differences among the strains in HR variability were minimal so that the major strain differences became evident during the dark (active) periods. In contrast, under the control and high NaCl diet SHR/lj-cr and WKY/lj-tf exhibited the greatest variability in SP. Furthermore, compared with normal NaCl diet, the high NaCl diet increased all period SP variability in SHR/lj-cr, whether it is expressed as SD or coefficient of variation, whereas it increased dark period and decreased light-period SP variability in the WKY/lj-cr and WKY/lj-tf rats only when it was expressed as coefficient of variation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The first major finding of the present study is that there is marked genetic diversity between inbred WKY/lj-tf and inbred WKY/lj-cr rats, and we have documented the extent of polymorphism among fully inbred WKY/lj-tf, WKY/lj-cr, and SHR/lj-cr strains. Earlier studies (13, 24) reported that DNA fingerprint analysis of Taconic Farms WKY rats had shown them to be different from Charles River WKY rats, a finding not unexpected, because the former was randomly bred, whereas the latter was inbred. Likewise, those studies (13, 24) reported greater genetic diversity between individual Taconic Farms WKY rats, whereas Charles River rats exhibited genetic consistency. No evidence of differentiation is found between Taconic Farms and Charles River SHR regarding blood pressure; however, a difference in stress-induced body temperature responses have been reported (18), suggesting that allelic variations as a result of a genetic drift and/or minor genotypic differences may have been fixed in the Taconic Farms and Charles River SHR. We plan to study SHR from Taconic Farms in the future. Our present findings indicate that the new inbred WKY/lj-tf rats have unique allelic variants and differ from both WKY/lj-cr and SHR/lj-cr rats. Interestingly, we also find that WKY/lj-tf rats exhibit less polymorphism with SHR/lj-cr (53.1%) than with WKY/lj-cr (75.5%) rats, potentially implying that the inbred WKY/lj-tf may contain more alleles in common with SHR/lj-cr than with WKY/lj-cr rats. This preliminary conclusion is also supported by the results of our cardiovascular studies.

A second major finding of this study is that the basal SP, DP, PP, and HR values for WKY/lj-tf rats lie between WKY/lj-cr and SHR/lj-cr. This is not a reflection of a new animal resulting from inbreeding, because the SP and DP findings in the present study are similar to those in our earlier report (9) that utilized outbred rats and where a similar rank order of arterial pressures was observed. Thus we would conclude that both the original and inbred WKY/lj-tf exhibit elevated arterial pressures. Yet there are also clear differences in cardiovascular traits between the present study and our earlier findings (9). For example, unlike the previous report (9) where WKY/lj-tf had the highest HR, here we find that WKY/lj-cr rats exhibit the highest basal HR. Additionally, in the present study, the absolute values of SP are lower both during the day and at night, and the strain pattern of diurnal variation in SP, DP, and HR exhibited a different rank order. The reasons for these differences might relate to measurement technique. In our previous study, arterial pressure and HR were recorded continuously for a period of 24 h from an indwelling femoral arterial catheter, in conscious, unrestrained rats 4 days after surgery. Whereas in the present study we utilized radiotelemetry monitoring that is direct, minimally stressful, and permits measurement in unrestrained and undisturbed freely moving rats for an extended period, here 50 days starting 7 days after surgery. Thus rats in the present study are less stressed and likely reflect more closely their natural colony environment. Furthermore, circadian rhythm has been reported to be disrupted after surgery and requires up to a week to return back to normal rhythm (2).

It has been shown that rhythms of arterial pressure and HR are controlled by an endogenous circadian oscillating system (27, 30) and that the suprachiasmatic nucleus is responsible for their rhythmicities in rats (11, 23, 29); however, the final pathway from the suprachiasmatic nucleus to the 24-h rhythms of arterial pressure and HR remains unknown. The 24-h diurnal variations in arterial pressure and HR are believed to be related mainly to behavioral changes with elevated levels of sympathetic activity during the active period and a dominance of parasympathetic influence during the rest period in rats (7, 16, 22, 28) as in humans (17, 19). Thus the baroreflex and autonomic nervous system appear to have central roles in the control of cardiovascular circadian rhythmicity over the 24-h period. On the basis of the observed differences in diurnal variation in HR, SP, and DP among the strains in the present study, we would conclude that this reflects their allelic differences, and such differences may directly control autonomic nervous system and baroreflex activity among the strains. Additional genomic studies are necessary to test this hypothesis.

A third major finding of our study is that WKY/lj-tf appear to exhibit a pronounced salt sensitivity in arterial pressure relative to WKY/lj-cr rats. We (A. Alemayehu et al., unpublished observations), and others (3), have shown that the arterial pressure of inbred SHR is markedly elevated by a high NaCl intake, and this has been confirmed again in the present study. Both WKY/lj-tf and SHR/lj-cr exhibited a significant and equivalent pressor response to a high NaCl diet, especially during the dark (active) period. Interestingly, during the rest (light) period, the pressor effects of high NaCl were lower in WKY/lj-tf (relative to the dark period), whereas the effects remained the same in SHR/lj-cr. Our results are in agreement with those of Calhoun et al. (3), who reported significant increases in both daytime and nighttime MAP during high NaCl exposure in SHR, whereas WKY rats had an increased nighttime, but not daytime, MAP during high NaCl intake. Our findings also pattern studies of patients with essential hypertension where blood pressure fails to fall during rest when the subject is on a high NaCl diet and exhibits a salt-sensitive form of hypertension. This finding in humans was associated with a loss of the circadian pattern of plasma norepinephrine (21), which may imply a failure of circadian variation in sympathetic nervous system activity. Because the present WKY/lj-tf and WKY/lj-cr rats both exhibit decreases in daytime arterial pressure and SHR/lj-cr lack the ability to attenuate the pressor effects of high NaCl during rest, the allelic differences among the strains may facilitate identifying those genes that contribute to this failure in regulation. Such genes may also relate to baroreflex activity because, like arterial pressure, high NaCl diet resulted in a significant decrease in HR in WKY/lj-cr and WKY/lj-tf rats. This decrease may be due to baroreflex activation secondary to increases in arterial pressure, and if correct, may indicate that the rank order of efficiency of the baroreflex system varies as WKY/lj-cr > WkY/lj-tf > SHR/lj-cr.

The present study found that SP variability was highest in SHR/lj-cr followed by WKY/lj-tf and WKY/lj-cr rats during both light and dark periods and these strain relationships were preserved during the 8% NaCl diet (Table 4). However, HR variability, when expressed either over 24 h or a dark period only, was highest in WKY/lj-tf rats compared with WKY/lj-cr rats and SHR/lj-cr, but not when analyzed over a light period only, and this was true during both control and 8% NaCl diets. This finding is similar to other studies (7) where HR variability was shown to be reduced in SHR compared with normotensive WKY. It has been suggested that HR variability is partly under autonomic nervous system control (6, 7), and if correct, may implicate allelic differences among the strains as determining aspects of autonomic control.

A fourth major finding of this study is the apparent upward drift of blood pressure in WKY/lj-tf before the high NaCl diet and the failure to reverse to pre-NaCl values after cessation of the NaCl diet. This observation complicates the interpretation of the blood pressure/salt sensitivity relationship as temporal drift could be argued as the source of change rather than the high NaCl. We cannot dismiss this argument and greater temporal drift may be a consequence of increased blood pressure variability. However, in an independent series of studies (A. Alemayehu et al., unpublished data) where blood pressure was monitored for a period of 6 wk under the regular diet, we observed a minimal drift of blood pressure over time in normotensive rat strains (WKY/lj-cr, Brown Norway congenic-BN.Lx, and polydactylous-PD/Cub), whereas hypertensive strains SHR/lj-cr and SHR/Ola showed gradual increases similar to that observed in WKY/ lj-tf. Additionally, such temporal changes may indicate the presence of pressure-independent alleles present in WKY/lj-tf that determine irreversible structural changes in the blood vessels or kidney.

It is also clear that in both WKY/lj-tf and SHR/lj-cr, after removal of the 8% NaCl diet, neither SP nor DP returned to values before high NaCl control diet, implying a permanent and/or sustained elevation of arterial pressure after removal of the high NaCl diet. In contrast, WKY/lj-cr rats exhibited a very small post-high-NaCl elevation in SP with no change in DP. Whereas high salt sensitivity of arterial pressure reflects, in part, a failure of pressure-natriuresis to adjust the set point of arterial pressure, a secondary phenomenon could be vascular and kidney remodeling leading to secondary contributions to vascular resistance and NaCl retention. Because the new WKY/lj-tf strain exhibits both salt sensitivity and, like SHR, a failure to compensate post-NaCl, this could imply that genetic determinants of salt susceptibility and organ system remodeling to high NaCl and high pressure may be the same. This possibility should be examined in future studies with WKY/lj-tf.

A positive correlation between body mass index and blood pressure has been reported for lean as well as for obese human subjects (12). In our study, body weights were moderately elevated (~10%) in WKY/lj-tf rats and SHR/lj-cr compared with WKY/lj-cr rats. The contribution of the increased body weight to the altered blood pressure in the present study is not known from our study design; however, it is possible that the increase in body weight might partly have contributed to the altered blood pressure. This possibility should also be examined in the future.

Properties of the new inbred WKY/lj-tf are similar to those documented for the borderline hypertensive rat (BHR) (15, 25), which is an F1 derived from a cross between female SHR and male WKY rats. The BHR was shown to exhibit a resting arterial pressure slightly greater than the inbred WKY, to be salt sensitive (15), and to be particularly sensitive to environmental stressors (25). Early studies (unpublished) suggested that our outbred WKY/tf were abnormally reactive to stressors; however, we have not yet examined stress responses in the new inbred WKY/lj-tf.

In conclusion, this study documents the properties of a new inbred rat strain with a genotype differing both from its normotensive and hypertensive counterparts. The new inbred WKY/lj-tf rats are heavier than either WKY/lj-cr or SHR/lj-cr, have unique allelic variants, exhibit a higher level of arterial pressure than our inbred WKY/lj-cr rats, are intermediate in HR between SHR/lj-cr and WKY/lj-cr rats, exhibit greater circadian variation especially during a high NaCl diet, and show salt sensitivity for arterial pressures. Taken together, the traits of this new WKY substrain mimic a number of features observed in the early development of human genetic hypertension and also reported for the BHR, and make this new strain of interest for future genomic studies particularly relevant to functional abnormalities that precede the onset of genetic hypertension.