Altered Vascular Resistance Properties and Acute Pressure-Natriuresis Mechanism in Neonatal and Weaning Spontaneously Hypertensive Rats
Although it has been extensively scrutinized, the factor(s) involved in the initiation and development of hypertension in spontaneously hypertensive rats (SHRs) remains unresolved. The objective of the present study was to determine whether, early in development, the causal mechanism(s) for the development of hypertension in young SHRs involves an integration of 2 processes, specifically an upregulation of structurally based vascular resistance properties and a rightward shift in the hemodynamic component of pressure-natriuresis. Mean arterial pressure was determined in conscious 4-week–old SHRs and Wistar-Kyoto rats via previously implanted aortic catheters. Structurally based hindlimb vascular resistance properties were assessed in 2- and 4-week–old SHRs and Wistar-Kyoto rats. Renal interstitial hydrostatic pressure was measured after short-term manipulations of renal arterial pressure (RAP) in 4-week–old, anesthetized rats. Although mean arterial pressure in conscious SHRs (113±5 mm Hg) and Wistar-Kyoto rats (110±6 mm Hg) was not significantly different at 4 weeks of age, SHRs at 2 and 4 weeks of age already had increases in structurally based vascular resistance properties of ≈30% above age- and weight-matched Wistar-Kyoto rats. Furthermore, the acute RAP-renal interstitial hydrostatic pressure relationship was found to be linear in both strains, and the temporal coupling of the stimulus to response was rapid; that is, renal interstitial hydrostatic pressure responses to changes in RAP were <2 s. Although the slope of the RAP-renal interstitial hydrostatic pressure relationship was not significantly different between strains, the relationship was significantly shifted (18%) to higher RAPs in SHRs. These results suggest that alterations in both vascular structure and renal function in young SHRs occur before elevations in mean arterial pressure.
- vascular resistance properties
- renal hemodynamics
- hydrostatic pressure
- arterial pressure
Although the factors responsible for the initiation and development of genetic hypertension have been extensively studied in the spontaneously hypertensive rat (SHR), the basis and time course for blood pressure elevation remain enigmatic. Specifically, what remains controversial is whether the 2 most widely studied processes, vascular abnormalities and renal dysfunction, lead to increases in arterial pressure or vice versa.
Since the 1970s,1 it has been well established that vascular abnormalities, such as increases in vascular resistance, vascular reactivity to vasoconstrictors, and media:lumen ratio, are associated with hypertension in the adult SHR.1–3 Despite the number of studies describing vascular morphometric and histological differences in young SHRs,4–9 evidence linking blood pressure elevations with vascular structural and functional changes remains unresolved in the neonatal and weaning SHRs. The hypothesis that vascular structural changes antecede and initiate a rise in arterial pressure has been disputed for 2 main reasons, the presence8,10–13 or lack4–7,9,14,15 of blood pressure elevation in studies of young SHRs and substantial differences between body weights of SHRs and Wistar-Kyoto (WKY) rats4,15 or body weights not being reported.12,14,16 Since then, we reported longitudinally consistent differences in vascular resistance properties between weight-matched SHRs and WKY rats, where there was a 30% to 40% increase in SHRs throughout a broad interval of the postweaning period (ie, 4–50 weeks).9 However, it remains unknown whether similar differences in vascular resistance properties occur during the suckling neonatal (2 weeks) and weaning (≈3–4 weeks) periods, which represent a time of intensive vascular structural maturation17 and fall within the putative prehypertensive stage of SHRs.7,9,18,19
Furthermore, given that the kidneys serve a critical role in the regulation of arterial pressure,20,21 it is not surprising that abnormalities specific to renal vascular resistance properties are associated with blood pressure elevations in young SHRs.22–26 Roman and Kaldunski27,28 demonstrated that components of the pressure-natriuresis mechanism (ie, medullary blood flow, urine flow, and sodium excretion) are shifted rightward toward greater renal perfusion pressures in 3- to 5-week–old SHRs. These findings suggest that renal medullary vascular resistance properties are elevated early in the development of hypertension and that alterations in medullary hemodynamics may participate in resetting pressure-natriuresis in young SHRs.27,28 Because transmission of renal arterial pressure (RAP) into the vasa recta capillaries in the renal medulla is a critical mediator of the pressure-natriuretic response and renal interstitial hydrostatic pressure (RIHP) is coupled to medullary blood flow, then a similar rightward shift is expected in the RIHP response to changes in RAP.29,30 Interestingly, we demonstrated recently, using a different approach, that the acute RAP-RIHP relationship has an underlying vascular basis.31 Specifically, the coupling between changes in RAP and RIHP was found to occur on a moment-to-moment basis and independent of neurohumoral control (ie, renin-angiotensin and autonomic nervous systems).31 Therefore, in addition to characterizing the pressure-natriuresis mechanism, this moment-to-moment relationship provides a hemodynamic assessment of renal medullary vascular resistance properties, where a rightward shift along the RAP operating point is indicative of increased renal vascular resistance.31–33 However, it remains unknown whether increases in renal medullary vascular resistance properties, and thereby pressure-natriuresis, occur in weaning SHRs (ie, 4 weeks old), at a time when renal organogenesis is complete.34
We hypothesized that young SHRs are programmed to have increased vascular resistance properties and a rightward shift in the acute pressure-natriuresis mechanism before elevations in arterial pressure. Thus, the objective of the present study was to assess vascular resistance properties and the moment-to-moment RAP-RIHP relationship in weight-matched SHRs and WKY rats at 2 and/or 4 weeks of age.
Male WKY rats and SHRs aged 2 and 4 weeks old were used. Rats were housed individually (22±1°C; 12-hour light/dark cycle), with food and water provided ad libitum. All of the procedures followed the guidelines of the Canadian Council on Animal Care and were approved by the Queen's University Animal Care Committee. Details of the methods can be found in the online-only Data Supplement available at http://www.hypertensionaha.org .
Conscious Mean Arterial Pressure and Heart Rate Assessments
Mean arterial pressure (MAP) was measured directly in 4-week–old WKY rats (n=6) and SHRs (n=7) surgically implanted previously (ie, 2–4 days) with aortic cannulas under ketamine (70 mg/kg IP)/xylazine (5–10 mg/kg IP) anesthesia.35 MAP and heart rate were calculated from average values recorded every 15 minutes for 3 hours after an hour acclimatization period starting at 9:00 am.
Hemodynamic Analysis of Hindlimb Vascular Resistance Properties
Hindlimb vascular resistance assessments were performed in 2- and 4-week–old WKY rats and SHRs according to well-established methods that have been described previously.9 Briefly, perfusion pressures at maximum dilatation and maximum constriction were determined at a flow rate of 4 mL/min per 100 g of body weight. In addition, graded flow-pressure relationships were constructed at flow rates of 1, 2, 4, 6, and 8 mL/min per 100 g of body weight.
In Vivo Assessments of Renal Medullary Vascular Resistance Properties
In vivo assessments of renal medullary vascular resistance properties, specifically the RAP-RIHP relationship, were performed in anesthetized 4-week–old WKY rats (n=11) and SHRs (n=9), based on methodology described previously.31
All of the statistical calculations were performed and graphs constructed using Prism 5 (GraphPad Software). Linear regression analysis was used to calculate the slopes and y intercepts of the hindlimb flow-pressure and RAP-RIHP relationships.31–33 The Grubb test was conducted on all of the data sets to determine statistical outliers. All of the data are presented as mean±SEM. Statistical significance between WKY rats and SHRs was determined using a Student t test, and P<0.05 was considered statistically significant.
Hindlimb Vascular Resistance Assessments in 2- and 4-Week–Old Rats
Physical and Hemodynamic Characteristics
The growth curves of the SHRs and WKY rats used have been shown previously to be almost identical.9 Corroborating previous findings, in the present study, rats had similar body weights at the predetermined ages of 2 weeks (SHR, 24.0±0.2 g; WKY, 24.7±0.3 g) and 4 weeks (SHR, 66.9±0.8 g; WKY, 68.0±1.0 g) without any special selection of animals. In addition, we determined that SHRs and WKY rats of 1 to 3 weeks of age have similar hindlimb weights. At 7 to 10 days, hindlimb weights were 18% of body weight and at 28 days, 26% of body weight (data not shown). Previous data has shown that SHRs, in comparison with WKY rats, obtained from this colony have similar proportional hindlimb weights throughout their life span.9 Furthermore, at 4 weeks of age, there was no significant difference in conscious MAPs (SHR, 113.0±5.0 mm Hg; WKY, 110.2±5.9 mm Hg), although heart rate was elevated by 16% in the SHRs (459.1±9.5 bpm) versus WKY rats (395.2±7.8 bpm; P<0.05).
Resistance Properties of the Neonatal Hindlimb Vasculature
Perfusion pressure at maximum dilatation was elevated by ≈30% in SHRs at both 2 and 4 weeks of age (P≤0.05; Figure 1A). A similar increase in absolute perfusion pressures at maximum dilatation between 2 and 4 weeks was found in both SHRs and WKY rats, in accordance with the rapid normal maturation during this time (Figure 1A). The pressure responses to acute changes in flow in the vasculature of SHRs at 2 and 4 weeks of age were significantly elevated in comparison with age-matched WKY rats at all of the flow rates tested (P<0.05; Figure 1B). A comparison of the y intercepts of the lines representing the flow-pressure relationships indicated that there was a ≈35% increase in SHRs (P<0.05; Figure 1B). Furthermore, the slope was ≈1.2-fold greater in SHRs (Figure 1B). At both 2 and 4 weeks of age, the hindlimb vasculature of SHRs generated a greater maximum contractile force using the constrictor mixture, as indicated by the 30% to 40% greater perfusion pressure at maximum constriction than that of WKY rats vasculature (P<0.05; Figure 1C). Comparison of rats aged 2 and 4 weeks demonstrated that a rapid maturation (ie, ≈2-fold increase) of the maximal vasoconstrictor capacity of this vascular bed occurs in both SHRs and WKY rats. Although the data are not presented, the maximum pressure response using methoxamine alone in both SHRs and WKY rats was 80% to 90% of the total maximum pressure response observed with the supramaximal constrictor mixture. The inability to produce a maximum pressure response with ∝1-adrenoceptor activation alone was similar at both ages.
In Vivo Assessments of Renal Medullary Vascular Resistance Properties of 4-Week–Old Rats
Physical and Hemodynamic Characteristics
Body weights of 4-week–old SHRs and WKY rats used were not significantly different (Table) and did not significantly differ from age-matched rats used in the hindlimb vascular resistance assessments. Consistent with the development of hypertension, kidney weights, kidney:body weight ratios, and the left ventricular:body weight ratios were significantly higher in SHRs than in WKY rats (P<0.05; Table).
In Vivo Renal Hemodynamic Properties of Neonatal Rats
Mean RAP was ≈18% greater in anesthetized SHRs compared with age-matched WKY rats (P<0.05), whereas RIHP was not different (Table). As a result, there was a rightward shift in the acute RAP-RIHP relationship of SHRs in comparison with WKY rats (P<0.05; Figure 2). Furthermore, there were no differences in the slopes of the overall acute RAP-RIHP relationship between the 2 strains of rats (SHR, 0.09±0.01; WKY, 0.07±0.01). However, there was a significant difference in the y intercepts (P<0.05), and calculating RAP at 0-mm Hg RIHP also revealed a rightward shift of ≈20 mm Hg in the SHR (P<0.05). Comparison of the pressor versus depressor slopes revealed that, in both SHRs and WKY rats, the pressor slope is ≈2.5-fold higher than the depressor slope (P<0.05; Figure 2). Similar to the overall assessment, there was a significant difference in the y intercepts of the pressor and depressor relationships between SHRs and WKY rats, as well as pressor versus depressor y intercepts in both SHRs and WKY rats (P<0.05). Calculating RAP at 0-mm Hg RIHP for the depressor relationships revealed that again there was a rightward shift in the RAP-RIHP relationship in the SHR (SHR, 20.2 mm Hg; WKY, 3.6 mm Hg). The overall time for RIHP to respond to any type of change in RAP in the SHR was 2 times quicker than in the WKY rat (P<0.05). A similar trend between the 2 strains was present when comparing pressor versus depressor times. Interestingly, pressor manipulations in RAP resulted in quicker RIHP response times than depressor manipulations in both strains (P<0.05; Figure 3).
The major findings of the present study were as follows: (1) vascular resistance properties are higher in weight-matched SHRs compared with WKY rats at both 2 and 4 weeks of age, suggesting that vascular abnormalities are present before differences in conscious MAP between both strains; (2) the acute RAP-RIHP relationship is shifted toward greater pressures in 4-week–old, weight-matched SHRs than WKY rats; and (3) the time delay for RIHP responses to acute changes in RAP is <2 s, indicating that there is an underlying vascular basis for this component of the pressure-natriuresis mechanism. Together, these findings suggest that neonatal and weaning SHRs have phenotypic differences in their vasculature and renal hemodynamics compared with age-matched WKY rats. Consequently, these phenotypic differences could, in part, render them susceptible to hypertension later in adulthood.
There has been a long-standing debate about whether elevations in arterial pressure precede alterations in vascular structure or vice versa.4–8,10,11,15,18 In our colony, conscious MAP was not different between SHRs and WKY rats at 4 weeks of age; however, structurally based vascular resistance properties were higher in both 2- and 4-week–old SHRs versus WKY rats. These findings extend our previous results in rats from the same colony and suggest that vascular resistance properties differ to the same extent (ie, ≈30%) from neonatal to adult SHRs (≤50 weeks), whereas the full expression of hypertension in SHRs only appears at ≈14 weeks of age.9 That is, because the full magnitude of the difference in SHR versus WKY vasculature already exists at 2 weeks of age and there does not appear to be a critical period between 2 and 50 weeks during which SHR vessels function differently, these data do not support the concept that elevated arterial pressure is a predominant factor in inducing further vascular structural and functional adaptation.
It is important that evidence for a prevailing difference in the configuration of blood vessel architecture in these neonatal SHRs and WKY rats comes from 3 assessments. First, differences in perfusion pressures at maximum dilatation in hindlimb vasculature of SHRs elevate pressure ≈30% more than the normotensive rat. According to the Poiseuille law, which states that resistance to flow is inversely proportional to radius to the fourth power, it can be calculated that, in the average hindlimb blood vessel of 2-, 4-, or even 50-week–old SHRs,9 the average lumen is ≈7% smaller than in WKY rats either when there is no vasoconstrictor tone or at the same level of vessel contraction, that is, structurally determined. Second, assessments of perfusion pressure at different flow rates extend this concept further, where at all flow rates between 1 and 8 mL/min per 100 g of body weight, resistance to flow is elevated such that there is a narrowing of the vessel lumen that is not dependent on the distending pressure. Last, perfusion pressures maximum constriction in hindlimb vascular beds of young SHRs demonstrate a greater contractile capacity compared with WKY rats. Mulvany et al2 have demonstrated differences in the contractile capacity of small isolated segments of resistance vessels in SHRs that can be wholly accounted for by an increased muscle mass within the blood vessel wall. Accordingly, the increase in resistance/vasoconstrictor capacity of ≈30% suggests that the average hindlimb blood vessel of the neonatal SHRs relative to WKY rats has a smaller lumen and/or a thicker wall.
Similar to vascular resistance properties of peripheral resistance vessels (ie, hindlimb arteries), previous findings demonstrated that renal vascular resistance is elevated in young “prehypertensive” SHRs (ie, ≤4 weeks of age)22–26 and continues to be elevated into adulthood (ie, 16 weeks of age).22,23,25,26 Given that transmission of RAP to vasa recta capillaries in the renal medulla is a critical mediator of the pressure-natriuretic response, an elevated renal vascular resistance would require a greater RAP to induce homeostatically appropriate changes in sodium excretion.21,36 Corroborating previous findings,27,28 the present study demonstrated that, although the overall, pressor, and depressor slopes of the acute RAP-RIHP relationship are not different between strains, there is a rightward shift toward a greater RAP in 4-week–old SHRs relative to WKY rats. This finding not only supports the proposed cascade of players in pressure-natriuresis (ie, medullary blood flow, RIHP, urine flow, and sodium excretion)37 but also, more importantly, the hypothesis that renal medullary vascular resistance properties are elevated in young SHRs28,34 after completion of postnatal renal organogenesis (ie, postnatal day 30).28,34 This notion is further supported by the moment-to-moment nature of RIHP responses (ie, <2 s) to changes in RAP in 4-week–old rats. Specifically, it appears that there is an underlying vascular basis for this component of the pressure-natriuresis mechanism, because it is unlikely that slower-acting neurohumoral mechanisms play a role in altering the acute functioning of this relationship.31 Thus, renal medullary vascular resistance properties appear to be elevated in weaning SHRs and may participate in the development of hypertension by inducing a rightward shift in the acute pressure-natriuresis mechanism. It should, however, be noted that other factors (eg, renal capsule or pressor versus depressor stimuli) may also, in part, influence this acute relationship, as well as the full expression of MAP, and thereby warrant further investigation (a post hoc assessment and detailed discussion on these factors can be found in the online-only Data Supplement).
Despite the rightward shift in the moment-to-moment RAP-RIHP relationship, conscious MAP was found to not be different between weight-matched SHRs and WKY rats at 4 weeks of age. One explanation for this paradoxical finding may be that young SHRs are in a state of sodium and/or volume retention. It may be that, during development, these young animals are in a dynamic state of cardiovascular flux, where various sodium/fluid body homeostatic mechanisms modulate the overall pressure-natriuretic response to maintain a positive sodium balance necessary for growth.38,39 Indeed, it has been reported previously that young SHRs retain 4-fold more sodium (ie, at 4–5 weeks of age)38 and have lower fractional sodium and water excretion levels (ie, during the second postnatal month)40 than WKY rats despite having similar intakes. Although not assessed in the present study, the rightward shift in the moment-to-moment RAP-RIHP relationship coupled with no differences in MAP support the notion that young SHRs may be in a relatively greater state of sodium retention than WKY rats. The exact reasons for this are not completely known; however, it can be speculated that disorders in sodium homeostatic mechanisms (eg, renin-angiotensin and sympathetic nervous systems),41 changes in renal interstitial compliance,42 and a relatively higher positive sodium balance requirement for growth than that of WKY38,39 may influence the overall pressure-natriuretic response in young SHRs without influencing MAP. Interestingly, as the animals mature and a positive sodium balance is no longer necessary for growth, the differences in sodium balance diminish between SHRs and WKY rats, where by postnatal week 9 through adulthood (ie, at the time hypertension becomes evident in the SHR), no differences in sodium balance exist.40,41
Although the outcomes of these studies provide a better understanding of vascular structure and renal hemodynamics in young SHRs versus WKY rats, there are some limitations that need to be addressed. First, the presence of potential unanticipated or uncontrollable environmental factors during critical periods of the perinatal development may have influenced the programming of the phenotypes expressed in these rats. Furthermore, comparisons of hindlimb and renal hemodynamic assessments might be complicated, because these studies were conducted in 2 separate colonies; however, the rats had similar body weights, as well as left ventricular: body weight ratios, suggesting that the growth rates of these 2 colonies are comparable.9 Lastly, rats had to be anesthetized for the renal hemodynamic assessments. Given that it has been demonstrated previously that SHRs may have altered sensitivity to certain anesthetics compared with WKY rats, this may have affected the outcome.43 However, despite this caveat, the results of the present study are comparable to those of others.27,28
Taken together, these data indicate that the SHR may be programmed susceptible to developing hypertension via a combination of increased structurally based vascular resistance properties and a rightward shift in acute renal vascular properties after critical periods of perinatal development, that is, vasculogenesis and renal organogenesis, respectively.
The present studies suggest that there is a probable mechanistic and temporal link between alterations in vascular structure and renal function in SHRs, which make these animals susceptible to developing hypertension in adult life. Future studies are warranted to investigate the genetic and environmental mechanisms “programming” these vasculogenic alterations in young and growing SHRs. Such knowledge could lead to the development of interventions during critical periods of perinatal programming aimed at improving blood pressure outcomes in adult life.
Sources of Funding
Study funded by Canadian Institutes of Health Research. Marina Komolova was funded by the Canadian Institutes of Health Research Canada Graduate Doctoral Scholarship.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.178194/-/DC1.
- Received June 15, 2011.
- Revision received July 10, 2011.
- Accepted February 28, 2012.
- © 2012 American Heart Association, Inc.
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