BACKGROUND
nephrogenic arterial hypertension comprising renovascular and renoparenchymal aetiologies is increasingly prevalent  <cit> .
hypertensive patients with chronic kidney disease  are three times more likely to die within 8 years than hypertensive counterparts without ckd  <cit> .
regarding renovascular hypertension, revascularization strategies do not convey any benefit when compared to the best conservative therapy  <cit> .
angiotensin ii-subtype- <dig>  receptor blockers  <cit>  or angiotensin-converting enzyme  inhibitors  <cit>  slow the progression of ckd, yet they are contraindicated in bilateral renal artery stenosis or in unilateral renal artery stenosis and  single kidney situation.
given the constraints inherent to medical and interventional therapies of renovascular hypertension, novel therapeutic targets are still needed.
the autonomic nervous system is such a potential target.
baroreflex function is attenuated in renovascular disease, regardless of residual kidney mass  <cit> .
on the basis of the effect of propranolol and atropine methyl nitrate on resting heart rate, an elevated sympathetic tone in models of renovascular hypertension with  and without kidney-mass reduction  was identified  <cit> .
aside from heart-rate changes, muscle sympathetic nerve activity  <cit>  and functional data like cold-pressor test  <cit>  were not affected by propranolol.
therefore, additional surrogates of sympathetic tone are needed.
the pathomechanism of sympathoactivation in renovascular hypertension is unclear.
in experimental renovascular hypertension with preserved kidney mass , the renin-angiotensin-aldosterone system  is found to be activated  <cit> , and central nervous system effects of angiotensin ii probably are the driving force of sympathoactivation  <cit> .
in experimental renovascular hypertension with reduced kidney mass , however, the raas is suppressed  <cit> , and other sympathoactivating pathomechanisms must be operative.
the intracellular rho a/rho kinase system emerges as a novel target for the treatment of cardiovascular disease  <cit> .
rho a, a small gtpase, has numerous functions and is involved in cytoskeletal organization.
upon activation, rho a interacts with and activates the rho a-dependent kinase .
as a consequence endothelial nitric oxide synthase mrna is destabilized and cellular contraction is initiated by means of calcium-sensitization  <cit>  which also increases endothelial cell permeability  <cit> .
established rho a/rock inhibitors are fasudil but also statins, 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors.
statins inhibit the formation of geranyl-geranylpyrophosphate, a prerequisite for rhoa membrane anchoring  <cit> .
we have previously shown that simvastatin lowers sympathetic tone in experimental chronic heart failure, another condition characterized by sympathoexcitation  <cit> .
also rock inhibition  by fasudil was shown to improve baroreflex sensitivity in experimental chronic heart failure when given in to the intracerebroventricular space  <cit> .
this effect was blunted by intracerebroventricular application of l-name, an inhibitor of endothelial nitric oxide synthase, suggesting direct central effects and a contribution of central nitric oxide in this process.
the value of rock inhibition for the treatment of hypertension at large, however, is still unclear.
in the present study, we hypothesize that sympathoactivation is more pronounced in renovascular hypertension with preserved  versus reduced  kidney mass when using heart-rate independent surrogates of sympathetic tone.
sham surgery animals and irbesartan -treated 2k1c animals were used as control groups.
the at1-receptor blocker treatment was used as a positive control for its sympathoinhibitory actions  <cit> .
secondly, we hypothesize that roki enhances baroreflex sensitivity of heart rate in models of renovascular hypertension  in analogy to the chronic-heart failure situation  <cit> .
methods
animals
male c57bl/6 n mice  were housed in individual cages in a separate room under standard conditions , standard chow and drinking water ad libitum.
care was provided daily at the same time, body weight was taken weekly.
all animal procedures and experiments adhered to the aps’s guiding principles in the care and use of vertebrate animals in research and training.
during surgeries, inhalational anaesthesia using a precision vaporizer with isoflurane , and subcutaneous  fentanyl  were used.
following surgeries, pain-relief medication buprenorphine , and antibiotic prophylaxis with ampicillin  were administered.
after observing the animals for 4– <dig> weeks, mice were sacrificed , and heart weight  was determined.
ethical approval was obtained from local animal-care officials and the supervising federal authority .
telemetric monitoring
aortic blood pressure of unrestrained, conscious mice was monitored by telemetry  attached to a femoral-artery catheter.
for catheter placement, a 15 mm skin incision was made, and the left femoral artery and vein were separated using a non-serrated fine-tip forceps .
the left femoral artery was tied off  caudally of the superficial epigastric and superficial circumflex iliac artery.
a second tie was placed  <dig> to 12 mm cranially and kept under tension to stop perfusion.
a 90°-bent  <dig> gauge injection needle serving as a catheter introducer was inserted into the left common femoral artery right above the distal tie.
the telemetry catheter was inserted and advanced to the proximal tie.
after releasing the proximal tie temporarily, the catheter was further advanced into the lower aorta  and secured by two knots.
after freeing a subcutaneous pouch on the right flank, the transmitter unit connected to the intra-arterial catheter was inserted and  <dig>  –  <dig>  g gentamicin solution  was applied before skin suture .
blood pressure readings were transmitted to a receiver placed below the mouse cage, digitized with a sampling rate of 1000 hz and stored and analyzed on a workstation in a separate room.
systolic and diastolic blood pressures, pulse pressure and pulse intervals  were extracted from aortic blood pressure waveforms using art  <dig>  gold software .
one week later, the mice were randomized in a 1: <dig> fashion to sham surgery  or unilateral renal-artery stenosis, i.e.
the 2k1c model of hypertension with preserved kidney mass.
there, a u-shaped metal clip  was implanted around the right renal artery as reported previously  <cit> .
one week later, every second 2k1c mouse was subjected to nephrectomy of the non-clipped kidney yielding the 1k1c model of renovascular hypertension with reduced kidney mass.
autonomic nervous system testing
after a two-week recovery period, a one-hour baseline recording was taken in the morning.
thereafter, intraperitoneal injections of atropine-methyl nitrate   <cit>  or metoprolol  were performed.
atr was used to block the parasympathetic component while met was used to block the sympathetic component of the autonomic nervous system.
after injection of either substance, another hour of continuous blood-pressure recording was performed.
for each one-hour recording, the last 30 minutes were used for analysis.
mean heart rate and blood pressure were determined.
in addition, consecutive, continuous one-minute series of digitized systolic blood pressure and pulse-interval data were linearly interpolated with an equidistant sampling interval of  <dig>  s .
power spectral analysis of those systolic blood pressure and pulse intervals was performed using fourier transformation .
each spectral band obtained was a harmonic of 20/1024 hz .
the power spectral analysis of blood pressure and pulse intervals yielded intensities  for a given spectral bandwidth.
the cumulative intensity of the low-frequency band  of power spectrum of systolic blood pressure  was regarded as a quantitative measure of sympathetic tone, whereas the cumulative intensity of the high-frequency band  of power spectrum of heart rate  was considered as a quantitative measure of parasympathetic tone  <cit> .
power spectrum  of systolic blood pressure   <cit>  and power spectrum  of heart rate  were provided as supplemental data.
with regard to sympathovagal balance, interpretation of hf-sbp data still remains inconclusive for the mouse model.
however, in contrast to humans, lf-hrv is considered to be an alternative quantitative measure of parasympathetic tone in mice  <cit> .
in addition, changes in resting heart rate after administration of metoprolol or atropine were determined.
an overnight recovery was required after injection of either substance.
baroreflex sensitivity
baroreflex sensitivity was determined by the sequence technique  <cit>  of concomitant changes of systolic blood pressure and pulse intervals  utilizing the hemolab software .
concomitant changes of systolic blood pressure  and pulse intervals of at least  <dig> consecutive heart beats were correlated.
for individual baroreflex curves, a correlation coefficient of at least  <dig>  was mandated for analysis.
in addition, a time delay of 0 seconds was chosen for analysis of concomitant blood-pressure and pulse-interval changes according to a previous study with murine models  <cit> .
the average of at least  <dig> individual baroreflex slopes  was considered as baroreflex sensitivity.
urinary catecholamine assay
mice were placed in metabolic cages  for 24-hour urine collection.
urine was collected over 24 hours in a vial containing 30 μl hcl , stored at −20°c.
for analysis urinary norepinephrine, dopamine and epinephrine were determined by a radioimmunoassay method .
medical intervention
the at <dig> receptor blocker irbesartan  was dissolved in water  and given to five 2k1c mice orally ad libitum.
drug uptake was recorded by weighing drinking bottles every 48 h. likewise, the rho-kinase inhibitor sar407899a was given to 2k1c and 1k1c mice with the drinking water .
the actual drug uptake is shown in table  <dig>  experiments were carried out after  <dig> – 9 days on treatment and compared to normal controls and to 2k1c animals without treatment.
irbesartan and the rho-kinase inhibitor sar407899a were kindly provided by sanofi-aventis, frankfurt/main, germany.
statistical analysis was performed between groups.
statistics
results are given as means ± one standard deviation.
for inter-group comparisons with equal variances, one-way anova/newman-keul post-hoc test or one- or two-tailed student’s t-test were used, where appropriate.
if the normality test failed, nonparametric tests  were used, where appropriate.
a p <  <dig>  was considered significant.
asterisks highlight significances .
statistical analysis was carried out with graphpad .
RESULTS
characteristics of models of renovascular arterial hypertension
pulse pressure, systolic and diastolic blood pressure were significantly elevated in both models  of renovascular hypertension as compared to controls .
treatment with the rho-kinase inhibitor sar407899a did not affect blood pressure .
in comparison to controls, 1k1c animals also exhibited an increase in both absolute and relative heart weight.
this difference in heart weight was not observed after roki  treatment .
as the increase in heart weight indicates left-ventricular hypertrophy, this finding suggests that roki treatment attenuates 1k1c-associated left-ventricular hypertrophy.
heart rate was not different among groups at baseline or following selective autonomic-nervous-system blockade with atropine  or metoprolol  .
however, resting heart rate significantly increased in response to atr in all groups.
in the control group, atr also increased the diastolic blood pressure.
neither pulse pressure nor systolic blood pressure were affected by atr or met in any group.
to gauge autonomic nervous system effects of an at <dig> blockade, irb treatment was performed in 2k1c animals.
hemodynamic effects of irb included a decrease of both systolic blood pressure and heart rate in 2k1c animals .
statistical analysis was performed among groups.
baroreflex sensitivity of heart rate is attenuated in models of renovascular hypertension, irrespective of residual renal mass
in comparison to controls, baroreflex sensitivity of heart rate was significantly attenuated in both models of renovascular hypertension.
beta-1-adrenergic blockade with met did not alter this difference .
following atr, baroreflex sensitivity of heart rate was blunted in all groups.
at1-receptor blockade with irb restored baroreflex sensitivity of heart rate in 2k1c animals .
elevated sympathetic tone in both models of renovascular arterial hypertension
in comparison to controls, both models of renovascular hypertension  presented with an elevated sympathetic nervous system tone as determined by power spectral analysis of systolic blood pressure.
in 2k1c as well as in 1k1c animals, sympathoactivation persisted after atr application .
in the 2k1c model, sympathetic tone was significantly higher without irb and returned to normal levels with irb treatment.
this effect was maintained after atr injection .
parasympathetic tone is reduced in the model of renovascular hypertension with preserved kidney mass
parasympathetic tone, as determined by the cumulative intensity of hf-hrv, was significantly attenuated in both models of renovascular hypertension.
beta-adrenergic blockade with met confirmed the significant attenuation of parasympathetic tone in 2k1c animals.
an at1-receptor blockade with irb prevented the significant attenuation of parasympathetic tone in comparison to controls .
as an alternative assessment of parasympathetic tone, lv-hrv was determined .
following metoprolol, lf-hrv data confirmed the suppression of parasympathetic tone in 2k1c animals in comparison to controls .
however, when using lf-hrv as a surrogate for parasympathetic tone, there was no significant change in parasympathetic tone following at1-receptor blockade in 2k1c animals .
in addition to the lf-hrv data, hf-sbp data is provided for all groups in table  <dig> and table  <dig>
statistical analysis was performed among groups.
statistical analysis was performed among groups.
collectively, renovascular hypertension with preserved kidney mass  associates with a lower parasympathetic tone when compared to normal controls, which is reversed, at least partly, by at <dig> receptor blocker treatment.
urinary catecholamines do not reflect sympathoexcitation in renovascular hypertension
urinary norepinephrine excretion was significantly reduced in renovascular hypertension with reduced kidney mass  when compared to normal controls .
in renovascular hypertension with preserved kidney mass, norepinephrine excretion was not different when compared to 1k1c animals or normal controls.
this finding contrasts telemetric power spectral data of systolic blood pressure suggesting a state of sympathoactivation in both models of hypertension , irrespective of residual kidney mass.
rho-kinase inhibition improves baroreflex sensitivity of heart rate in renovascular hypertension with reduced kidney mass
as demonstrated in figure  <dig>  baroreflex sensitivity of heart rate was improved by roki treatment in renovascular hypertension with reduced renal mass .
in contrast, roki treatment led to an attenuation of baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass .
these differential treatment effects of sar407899a did, however, not translate into alterations of sympathetic or parasympathetic tone .
discussion
in this study, power spectral analysis of systolic blood pressure  <cit>  and urinary catecholamines  <cit>  were determined to gauge sympathetic tone.
in addition, vagal tone was assessed using power spectral analysis of heart rate  <cit> .
slope data of concomitant, spontaneous pulse-interval and blood-pressure changes were gathered to estimate baroreflex function  <cit> .
the results suggest that therapeutic interventions in renovascular hypertension may depend on residual renal mass.
as shown for rho-kinase inhibition, a beneficial effect on baroreflex function only emerged in the 1k1c model, but not in the 2k1c model of renovascular hypertension.
this finding may potentially be due to the oxidative stress in the 1k1c model which leads to a more profound rho a/rock activation  <cit> .
in addition, rho-kinase inhibition may increase nitric oxide availability in hypothalamic centres of baroreflex regulation similar to the heart failure situation  <cit>  which, in turn, improves baroreflex sensitivity of heart rate.
although baroreflex sensitivity of heart rate improved upon rho-kinase inhibition in the 1k1c model of hypertension, this change did not translate into a reduction of the sympathetic or an increase in parasympathetic tone.
this observation was unexpected given that carotid baroreflex function and/or baroreflex-dependent central nervous system regulations affect both sympathetic and parasympathetic tone  <cit> .
in renovascular hypertension with preserved kidney mass , at1-receptor blockade improved baroreflex sensitivity of heart rate.
these data are supported by observations in humans with “essential” hypertension  <cit> .
in addition, in response to at <dig> receptor blockade, sympathetic tone normalized in the 2k1c model of hypertension which is in line with previous observations  <cit> .
however, rho-kinase inhibition was not shown to improve baroreflex sensitivity of heart rate in the 2k1c model.
this difference to the 1k1c model may be due to lower nitric oxide availability in hypothalamic centres of baroreflex regulation in the 2k1c model of hypertension.
additional studies on the effect of rho kinase inhibition are therefore needed to dissect the specific regulations of autonomic nervous system tone in different models of renovascular hypertension.
concerning the parasympathetic tone, different results have been obtained for the renovascular hypertension models with and without reduced kidney mass  as well.
2k1c animals showed a significantly depressed parasympathetic tone which persisted after beta-adrenergic blockade.
as novel findings, at1-receptor blockade with irb significantly increased parasympathetic tone, decreased resting heart rate, and restored baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass .
baroreflex and heart-rate data are in line with published evidence from experimental renoparenchymal hypertension  <cit> .
given the tremendous effect of baroreflex activating therapies in refractory hypertension  <cit> , the beneficial role of at1-receptor blockade with regard to baroreflex function deserves further attention in studies.
in the present study, at <dig> blockade was not applied to 1k1c animals because of the risk of kidney failure.
previous ultra-short term studies with losartan have not shown an improved baroreflex function in this volume-dependent model of renovascular hypertension   <cit> .
as another main result, both models of renovascular arterial hypertension exhibited a state of sympathoactivation as detected by power spectral analysis of blood pressure.
thus, previous results obtained with selective blockade  experiments on heart rate were confirmed  <cit> .
for renovascular hypertension with preserved kidney function , a prevalent sympathoactivation was also reported in a recent study using the same methodology in rats as used in the present study  <cit> .
interestingly, in that study, baroreflex depression occurred almost instantaneously upon induction of goldblatt hypertension .
in the present study, the level of sympathoactivation was similar between 1k1c and 2k1c animals, despite the different hormonal cause of hypertension  <cit>  and the different volume state  <cit> .
apart from renovascular hypertension, a state of sympathoactivation was found in patients with “essential” arterial hypertension  <cit>  and in chronic heart failure.
in the latter, effects on the central nervous system by angiotensin ii were postulated  <cit> .
for the 1k1c model, the detailed mechanism of sympathoactivation, however, is still unclear.
in 1k1c animals, renin release is known to be similar to normal controls  <cit> .
therefore, systemic raas activation cannot be a cause of sympathoactivation under this condition.
however, the brain “ouabain” and/or the brain renin-angiotensin system may become pertinent for sympathoactivation in renovascular hypertension with reduced kidney mass possibly through a sodium-dependent mechanism  <cit> .
in addition, kidney mass reduction and renal artery stenosis may reduce urinary norepinephrine excretion as shown here .
diminished excretion and/or attenuated catecholamine degradation in the kidney  <cit>  may lead to catecholamine accumulation and, potentially, sympathoactivation.
as a limitation of the present study, plasma catecholamines were not determined.
uremic toxins are unlikely to play a sympathoactivating role in the 1k1c model because glomerular filtration was shown to be reduced by only 30%  <cit> .
CONCLUSIONS
as main results, sympathetic tone  was found to be increased, whereas baroreflex sensitivity of heart rate was depressed in models of renovascular hypertension, irrespective of residual renal mass.
differential results relate to parasympathetic tone  that was depressed in the 2k1c model only.
in addition, left ventricular hypertrophy was present in experimental renovascular hypertension with reduced renal mass  only.
renal norepinephrine excretion was reduced in the 1k1c model exclusively.
hypothetically, renal reduced catecholamine excretion and/or impaired renal catecholamine degradation may be considered as mechanisms of sympathoactivation in the 1k1c model.
finally, rho-kinase inhibition improved baroreflex function solely in experimental renovascular hypertension with reduced renal mass , whereas at <dig> blockade improved baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass .
taken together, rho-kinase inhibition might be an additive strategy to improve survival in renovascular hypertension with reduced renal mass, whereas low-dose at <dig> blockade might be a therapeutic choice in all other cases of renovascular hypertension.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
rup made substantial contributions in study conception and design and the acquisition of data.
he performed experiments and drafted the manuscript.
sb and oj collected data, performed experiments and provided substantial input in data interpretation and analysis.
rpb conceived the study, contributed to study design and was involved in drafting the manuscript.
all authors gave final approval to the version to be published.

 nephrogenic arterial hypertension comprising renovascular and renoparenchymal aetiologies is increasingly prevalent. hypertensive patients with chronic kidney disease are three times more likely to die within 8 years than hypertensive counterparts without ckd. regarding renovascular hypertension, revascularization strategies do not convey any benefit when compared to the best conservative therapy. angiotensin ii-subtype- receptor blockers or angiotensin-converting enzyme inhibitors slow the progression of ckd, yet they are contraindicated in bilateral renal artery stenosis or in unilateral renal artery stenosis and single kidney situation. given the constraints inherent to medical and interventional therapies of renovascular hypertension, novel therapeutic targets are still needed. the autonomic nervous system is such a potential target. baroreflex function is attenuated in renovascular disease, regardless of residual kidney mass. on the basis of the effect of propranolol and atropine methyl nitrate on resting heart rate, an elevated sympathetic tone in models of renovascular hypertension with and without kidney-mass reduction was identified. aside from heart-rate changes, muscle sympathetic nerve activity and functional data like cold-pressor test were not affected by propranolol. therefore, additional surrogates of sympathetic tone are needed. the pathomechanism of sympathoactivation in renovascular hypertension is unclear. in experimental renovascular hypertension with preserved kidney mass, the renin-angiotensin-aldosterone system is found to be activated, and central nervous system effects of angiotensin ii probably are the driving force of sympathoactivation. in experimental renovascular hypertension with reduced kidney mass, however, the raas is suppressed, and other sympathoactivating pathomechanisms must be operative. the intracellular rho a/rho kinase system emerges as a novel target for the treatment of cardiovascular disease. rho a, a small gtpase, has numerous functions and is involved in cytoskeletal organization. upon activation, rho a interacts with and activates the rho a-dependent kinase. as a consequence endothelial nitric oxide synthase mrna is destabilized and cellular contraction is initiated by means of calcium-sensitization which also increases endothelial cell permeability. established rho a/rock inhibitors are fasudil but also statins, 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors. statins inhibit the formation of geranyl-geranylpyrophosphate, a prerequisite for rhoa membrane anchoring. we have previously shown that simvastatin lowers sympathetic tone in experimental chronic heart failure, another condition characterized by sympathoexcitation. also rock inhibition by fasudil was shown to improve baroreflex sensitivity in experimental chronic heart failure when given in to the intracerebroventricular space. this effect was blunted by intracerebroventricular application of l-name, an inhibitor of endothelial nitric oxide synthase, suggesting direct central effects and a contribution of central nitric oxide in this process. the value of rock inhibition for the treatment of hypertension at large, however, is still unclear. in the present study, we hypothesize that sympathoactivation is more pronounced in renovascular hypertension with preserved versus reduced kidney mass when using heart-rate independent surrogates of sympathetic tone. sham surgery animals and irbesartan -treated 2k1c animals were used as control groups. the at1-receptor blocker treatment was used as a positive control for its sympathoinhibitory actions. secondly, we hypothesize that roki enhances baroreflex sensitivity of heart rate in models of renovascular hypertension in analogy to the chronic-heart failure situation. methods animals male c57bl/6 n mice were housed in individual cages in a separate room under standard conditions, standard chow and drinking water ad libitum. care was provided daily at the same time, body weight was taken weekly. all animal procedures and experiments adhered to the aps’s guiding principles in the care and use of vertebrate animals in research and training. during surgeries, inhalational anaesthesia using a precision vaporizer with isoflurane, and subcutaneous fentanyl were used. following surgeries, pain-relief medication buprenorphine, and antibiotic prophylaxis with ampicillin were administered. after observing the animals for 4– weeks, mice were sacrificed, and heart weight was determined. ethical approval was obtained from local animal-care officials and the supervising federal authority. telemetric monitoring aortic blood pressure of unrestrained, conscious mice was monitored by telemetry attached to a femoral-artery catheter. for catheter placement, a 15 mm skin incision was made, and the left femoral artery and vein were separated using a non-serrated fine-tip forceps. the left femoral artery was tied off caudally of the superficial epigastric and superficial circumflex iliac artery. a second tie was placed to 12 mm cranially and kept under tension to stop perfusion. a 90°-bent gauge injection needle serving as a catheter introducer was inserted into the left common femoral artery right above the distal tie. the telemetry catheter was inserted and advanced to the proximal tie. after releasing the proximal tie temporarily, the catheter was further advanced into the lower aorta and secured by two knots. after freeing a subcutaneous pouch on the right flank, the transmitter unit connected to the intra-arterial catheter was inserted and –  g gentamicin solution was applied before skin suture. blood pressure readings were transmitted to a receiver placed below the mouse cage, digitized with a sampling rate of 1000 hz and stored and analyzed on a workstation in a separate room. systolic and diastolic blood pressures, pulse pressure and pulse intervals were extracted from aortic blood pressure waveforms using art gold software. one week later, the mice were randomized in a 1: fashion to sham surgery or unilateral renal-artery stenosis, i.e. the 2k1c model of hypertension with preserved kidney mass. there, a u-shaped metal clip was implanted around the right renal artery as reported previously. one week later, every second 2k1c mouse was subjected to nephrectomy of the non-clipped kidney yielding the 1k1c model of renovascular hypertension with reduced kidney mass. autonomic nervous system testing after a two-week recovery period, a one-hour baseline recording was taken in the morning. thereafter, intraperitoneal injections of atropine-methyl nitrate or metoprolol were performed. atr was used to block the parasympathetic component while met was used to block the sympathetic component of the autonomic nervous system. after injection of either substance, another hour of continuous blood-pressure recording was performed. for each one-hour recording, the last 30 minutes were used for analysis. mean heart rate and blood pressure were determined. in addition, consecutive, continuous one-minute series of digitized systolic blood pressure and pulse-interval data were linearly interpolated with an equidistant sampling interval of  s. power spectral analysis of those systolic blood pressure and pulse intervals was performed using fourier transformation. each spectral band obtained was a harmonic of 20/1024 hz. the power spectral analysis of blood pressure and pulse intervals yielded intensities for a given spectral bandwidth. the cumulative intensity of the low-frequency band of power spectrum of systolic blood pressure was regarded as a quantitative measure of sympathetic tone, whereas the cumulative intensity of the high-frequency band of power spectrum of heart rate was considered as a quantitative measure of parasympathetic tone. power spectrum of systolic blood pressure and power spectrum of heart rate were provided as supplemental data. with regard to sympathovagal balance, interpretation of hf-sbp data still remains inconclusive for the mouse model. however, in contrast to humans, lf-hrv is considered to be an alternative quantitative measure of parasympathetic tone in mice. in addition, changes in resting heart rate after administration of metoprolol or atropine were determined. an overnight recovery was required after injection of either substance. baroreflex sensitivity baroreflex sensitivity was determined by the sequence technique of concomitant changes of systolic blood pressure and pulse intervals utilizing the hemolab software. concomitant changes of systolic blood pressure and pulse intervals of at least consecutive heart beats were correlated. for individual baroreflex curves, a correlation coefficient of at least was mandated for analysis. in addition, a time delay of 0 seconds was chosen for analysis of concomitant blood-pressure and pulse-interval changes according to a previous study with murine models. the average of at least individual baroreflex slopes was considered as baroreflex sensitivity. urinary catecholamine assay mice were placed in metabolic cages for 24-hour urine collection. urine was collected over 24 hours in a vial containing 30 μl hcl, stored at −20°c. for analysis urinary norepinephrine, dopamine and epinephrine were determined by a radioimmunoassay method. medical intervention the at receptor blocker irbesartan was dissolved in water and given to five 2k1c mice orally ad libitum. drug uptake was recorded by weighing drinking bottles every 48 h. likewise, the rho-kinase inhibitor sar407899a was given to 2k1c and 1k1c mice with the drinking water. the actual drug uptake is shown in table  experiments were carried out after – 9 days on treatment and compared to normal controls and to 2k1c animals without treatment. irbesartan and the rho-kinase inhibitor sar407899a were kindly provided by sanofi-aventis, frankfurt/main, germany. statistical analysis was performed between groups. statistics results are given as means ± one standard deviation. for inter-group comparisons with equal variances, one-way anova/newman-keul post-hoc test or one- or two-tailed student’s t-test were used, where appropriate. if the normality test failed, nonparametric tests were used, where appropriate. a p <  was considered significant. asterisks highlight significances. statistical analysis was carried out with graphpad.  characteristics of models of renovascular arterial hypertension pulse pressure, systolic and diastolic blood pressure were significantly elevated in both models of renovascular hypertension as compared to controls. treatment with the rho-kinase inhibitor sar407899a did not affect blood pressure. in comparison to controls, 1k1c animals also exhibited an increase in both absolute and relative heart weight. this difference in heart weight was not observed after roki treatment. as the increase in heart weight indicates left-ventricular hypertrophy, this finding suggests that roki treatment attenuates 1k1c-associated left-ventricular hypertrophy. heart rate was not different among groups at baseline or following selective autonomic-nervous-system blockade with atropine or metoprolol. however, resting heart rate significantly increased in response to atr in all groups. in the control group, atr also increased the diastolic blood pressure. neither pulse pressure nor systolic blood pressure were affected by atr or met in any group. to gauge autonomic nervous system effects of an at blockade, irb treatment was performed in 2k1c animals. hemodynamic effects of irb included a decrease of both systolic blood pressure and heart rate in 2k1c animals. statistical analysis was performed among groups. baroreflex sensitivity of heart rate is attenuated in models of renovascular hypertension, irrespective of residual renal mass in comparison to controls, baroreflex sensitivity of heart rate was significantly attenuated in both models of renovascular hypertension. beta-1-adrenergic blockade with met did not alter this difference. following atr, baroreflex sensitivity of heart rate was blunted in all groups. at1-receptor blockade with irb restored baroreflex sensitivity of heart rate in 2k1c animals. elevated sympathetic tone in both models of renovascular arterial hypertension in comparison to controls, both models of renovascular hypertension presented with an elevated sympathetic nervous system tone as determined by power spectral analysis of systolic blood pressure. in 2k1c as well as in 1k1c animals, sympathoactivation persisted after atr application. in the 2k1c model, sympathetic tone was significantly higher without irb and returned to normal levels with irb treatment. this effect was maintained after atr injection. parasympathetic tone is reduced in the model of renovascular hypertension with preserved kidney mass parasympathetic tone, as determined by the cumulative intensity of hf-hrv, was significantly attenuated in both models of renovascular hypertension. beta-adrenergic blockade with met confirmed the significant attenuation of parasympathetic tone in 2k1c animals. an at1-receptor blockade with irb prevented the significant attenuation of parasympathetic tone in comparison to controls. as an alternative assessment of parasympathetic tone, lv-hrv was determined. following metoprolol, lf-hrv data confirmed the suppression of parasympathetic tone in 2k1c animals in comparison to controls. however, when using lf-hrv as a surrogate for parasympathetic tone, there was no significant change in parasympathetic tone following at1-receptor blockade in 2k1c animals. in addition to the lf-hrv data, hf-sbp data is provided for all groups in table  and table  statistical analysis was performed among groups. statistical analysis was performed among groups. collectively, renovascular hypertension with preserved kidney mass associates with a lower parasympathetic tone when compared to normal controls, which is reversed, at least partly, by at receptor blocker treatment. urinary catecholamines do not reflect sympathoexcitation in renovascular hypertension urinary norepinephrine excretion was significantly reduced in renovascular hypertension with reduced kidney mass when compared to normal controls. in renovascular hypertension with preserved kidney mass, norepinephrine excretion was not different when compared to 1k1c animals or normal controls. this finding contrasts telemetric power spectral data of systolic blood pressure suggesting a state of sympathoactivation in both models of hypertension, irrespective of residual kidney mass. rho-kinase inhibition improves baroreflex sensitivity of heart rate in renovascular hypertension with reduced kidney mass as demonstrated in figure  baroreflex sensitivity of heart rate was improved by roki treatment in renovascular hypertension with reduced renal mass. in contrast, roki treatment led to an attenuation of baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass. these differential treatment effects of sar407899a did, however, not translate into alterations of sympathetic or parasympathetic tone. discussion in this study, power spectral analysis of systolic blood pressure and urinary catecholamines were determined to gauge sympathetic tone. in addition, vagal tone was assessed using power spectral analysis of heart rate. slope data of concomitant, spontaneous pulse-interval and blood-pressure changes were gathered to estimate baroreflex function. the results suggest that therapeutic interventions in renovascular hypertension may depend on residual renal mass. as shown for rho-kinase inhibition, a beneficial effect on baroreflex function only emerged in the 1k1c model, but not in the 2k1c model of renovascular hypertension. this finding may potentially be due to the oxidative stress in the 1k1c model which leads to a more profound rho a/rock activation. in addition, rho-kinase inhibition may increase nitric oxide availability in hypothalamic centres of baroreflex regulation similar to the heart failure situation which, in turn, improves baroreflex sensitivity of heart rate. although baroreflex sensitivity of heart rate improved upon rho-kinase inhibition in the 1k1c model of hypertension, this change did not translate into a reduction of the sympathetic or an increase in parasympathetic tone. this observation was unexpected given that carotid baroreflex function and/or baroreflex-dependent central nervous system regulations affect both sympathetic and parasympathetic tone. in renovascular hypertension with preserved kidney mass, at1-receptor blockade improved baroreflex sensitivity of heart rate. these data are supported by observations in humans with “essential” hypertension. in addition, in response to at receptor blockade, sympathetic tone normalized in the 2k1c model of hypertension which is in line with previous observations. however, rho-kinase inhibition was not shown to improve baroreflex sensitivity of heart rate in the 2k1c model. this difference to the 1k1c model may be due to lower nitric oxide availability in hypothalamic centres of baroreflex regulation in the 2k1c model of hypertension. additional studies on the effect of rho kinase inhibition are therefore needed to dissect the specific regulations of autonomic nervous system tone in different models of renovascular hypertension. concerning the parasympathetic tone, different results have been obtained for the renovascular hypertension models with and without reduced kidney mass as well. 2k1c animals showed a significantly depressed parasympathetic tone which persisted after beta-adrenergic blockade. as novel findings, at1-receptor blockade with irb significantly increased parasympathetic tone, decreased resting heart rate, and restored baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass. baroreflex and heart-rate data are in line with published evidence from experimental renoparenchymal hypertension. given the tremendous effect of baroreflex activating therapies in refractory hypertension, the beneficial role of at1-receptor blockade with regard to baroreflex function deserves further attention in studies. in the present study, at blockade was not applied to 1k1c animals because of the risk of kidney failure. previous ultra-short term studies with losartan have not shown an improved baroreflex function in this volume-dependent model of renovascular hypertension. as another main result, both models of renovascular arterial hypertension exhibited a state of sympathoactivation as detected by power spectral analysis of blood pressure. thus, previous results obtained with selective blockade experiments on heart rate were confirmed. for renovascular hypertension with preserved kidney function, a prevalent sympathoactivation was also reported in a recent study using the same methodology in rats as used in the present study. interestingly, in that study, baroreflex depression occurred almost instantaneously upon induction of goldblatt hypertension. in the present study, the level of sympathoactivation was similar between 1k1c and 2k1c animals, despite the different hormonal cause of hypertension and the different volume state. apart from renovascular hypertension, a state of sympathoactivation was found in patients with “essential” arterial hypertension and in chronic heart failure. in the latter, effects on the central nervous system by angiotensin ii were postulated. for the 1k1c model, the detailed mechanism of sympathoactivation, however, is still unclear. in 1k1c animals, renin release is known to be similar to normal controls. therefore, systemic raas activation cannot be a cause of sympathoactivation under this condition. however, the brain “ouabain” and/or the brain renin-angiotensin system may become pertinent for sympathoactivation in renovascular hypertension with reduced kidney mass possibly through a sodium-dependent mechanism. in addition, kidney mass reduction and renal artery stenosis may reduce urinary norepinephrine excretion as shown here. diminished excretion and/or attenuated catecholamine degradation in the kidney may lead to catecholamine accumulation and, potentially, sympathoactivation. as a limitation of the present study, plasma catecholamines were not determined. uremic toxins are unlikely to play a sympathoactivating role in the 1k1c model because glomerular filtration was shown to be reduced by only 30%.  as main results, sympathetic tone was found to be increased, whereas baroreflex sensitivity of heart rate was depressed in models of renovascular hypertension, irrespective of residual renal mass. differential results relate to parasympathetic tone that was depressed in the 2k1c model only. in addition, left ventricular hypertrophy was present in experimental renovascular hypertension with reduced renal mass only. renal norepinephrine excretion was reduced in the 1k1c model exclusively. hypothetically, renal reduced catecholamine excretion and/or impaired renal catecholamine degradation may be considered as mechanisms of sympathoactivation in the 1k1c model. finally, rho-kinase inhibition improved baroreflex function solely in experimental renovascular hypertension with reduced renal mass, whereas at blockade improved baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass. taken together, rho-kinase inhibition might be an additive strategy to improve survival in renovascular hypertension with reduced renal mass, whereas low-dose at blockade might be a therapeutic choice in all other cases of renovascular hypertension. competing interests the authors declare that they have no competing interests. authors’ contributions rup made substantial contributions in study conception and design and the acquisition of data. he performed experiments and drafted the manuscript. sb and oj collected data, performed experiments and provided substantial input in data interpretation and analysis. rpb conceived the study, contributed to study design and was involved in drafting the manuscript. all authors gave final approval to the version to be published. 
regardless of residual renal mass, blood pressure and sympathetic tone are increased, whereas baroreflex sensitivity is depressed in murine models of renovascular hypertension. here, murine models of renovascular hypertension with preserved and reduced kidney mass were studied with regard to autonomic nervous system regulation and baroreflex sensitivity of heart rate by spontaneous, concomitant changes of systolic blood pressure and pulse interval. in comparison to control mice, both models presented with a significantly increased sympathetic tone and lower baroreflex sensitivity of heart rate. rho kinase inhibition given to a subset of 1k1c and 2k1c animals improved baroreflex sensitivity of heart rate selectively in the 1k1c model. blockade of at receptors normalized the sympathetic tone, decreased resting heart rate, improved baroreflex sensitivity of heart rate and parasympathetic tone. however, only 2k1c animals had a lower parasympathetic tone, whereas urinary norepinephrine excretion was reduced in the 1k1c model. rho kinase inhibition had no additional effects on autonomic nervous system in either model of renovascular hypertension and did not change the blood pressure. dysregulation of the autonomic nervous system is frequent in subjects with cardiovascular disease. the contribution of different forms of renovascular hypertension and the mechanisms contributing to autonomic dysfunction in hypertension are incompletely understood. reduced norepinephrine excretion and/or degradation might contribute to sympathoactivation in renovascular hypertension with reduced renal mass. c57bl6n mice with reduced kidney mass or with preserved kidney mass developed a similar degree of hypertension.
 dysregulation of the autonomic nervous system is frequent in subjects with cardiovascular disease. the contribution of different forms of renovascular hypertension and the mechanisms contributing to autonomic dysfunction in hypertension are incompletely understood. here, murine models of renovascular hypertension with preserved and reduced kidney mass were studied with regard to autonomic nervous system regulation and baroreflex sensitivity of heart rate by spontaneous, concomitant changes of systolic blood pressure and pulse interval. involvement of the renin-angiotensin system and the rho-kinase pathway were determined by application of inhibitors.  c57bl6n mice with reduced kidney mass or with preserved kidney mass developed a similar degree of hypertension. in comparison to control mice, both models presented with a significantly increased sympathetic tone and lower baroreflex sensitivity of heart rate. however, only 2k1c animals had a lower parasympathetic tone, whereas urinary norepinephrine excretion was reduced in the 1k1c model. rho kinase inhibition given to a subset of 1k1c and 2k1c animals improved baroreflex sensitivity of heart rate selectively in the 1k1c model. rho kinase inhibition had no additional effects on autonomic nervous system in either model of renovascular hypertension and did not change the blood pressure. blockade of at receptors normalized the sympathetic tone, decreased resting heart rate, improved baroreflex sensitivity of heart rate and parasympathetic tone.  regardless of residual renal mass, blood pressure and sympathetic tone are increased, whereas baroreflex sensitivity is depressed in murine models of renovascular hypertension. reduced norepinephrine excretion and/or degradation might contribute to sympathoactivation in renovascular hypertension with reduced renal mass. overall, the study helps to direct research to optimize medical therapy of hypertension. arterial hypertensionsympathetic nervous systembaroreflexirbesartan 