Renal Denervation Attenuates the Sodium Retention and Hypertension Associated With Obesity
Abstract Recent studies have indicated that obesity is associated with hypertension, sodium retention, and increased sympathetic nervous system activity. The purpose of this study was to determine the role of renal nerves in mediating the sodium retention and hypertension associated with obesity. We determined the hemodynamic and renal excretory responses to a high-fat diet in control (n=6) and bilaterally renal-denervated (n=7) chronically instrumented dogs. After a control period of 8 days, dogs were placed on a high-fat diet for 5 weeks. In response to a high-fat diet, body weight increased from 19.9±2.2 to 29.9±2.4 kg in the control group and from 21.1±2.0 to 32.4±1.9 kg in the bilaterally renal-denervated group. Heart rate increased from 81±8 to 113±7 beats per minute in the control group and from 79±7 to 103±8 beats per minute in the bilaterally renal-denervated group. Arterial pressure increased significantly from 95±2 to 109±4 mm Hg in the control group. In contrast, 5 weeks of a high-fat diet in the bilaterally renal-denervated group did not significantly increase arterial pressure (which went from 87±3 to 90±4 mm Hg). Furthermore, the decrease in sodium excretion in response to the high-fat diet was significantly greater in the control group than in the bilaterally renal-denervated group. After 5 weeks of a high-fat diet, cumulative sodium retention was 455±85 mmol in the control group and only 252±47 mmol in the bilaterally renal-denervated group. Similar increases in glomerular filtration rate and renal plasma flow occurred in both groups in response to the high-fat diet. The results of this study indicate that the renal nerves play an important role in mediating the sodium retention and hypertension associated with obesity in dogs.
Although the relationship between body weight and arterial pressure is well established, the mechanisms involved in the pathogenesis of obesity-induced hypertension are still unclear.1 2 Results from recent studies, however, indicate that abnormalities in the renal excretion of sodium may be involved.3 4 5 6 We and others have previously reported that feeding dogs a high-fat diet for 5 weeks caused significant increases in arterial pressure associated with sodium retention.3 4 5 We have also recently reported a hypertensive shift in the pressure-natriuresis relationship in obese dogs, further indicating an abnormality in renal excretory function in obesity.6 Although various mechanisms such as hyperinsulinemia and activation of the sympathetic nervous and renin-angiotensin systems have been proposed to be involved, the quantitative importance of each of these factors in mediating the sodium retention and hypertension associated with obesity is unknown.1 7 8
A role of renal nerves in obesity-induced hypertension is supported by studies indicating that obesity is associated with enhanced sympathetic nervous activity. Plasma and urinary catecholamines are increased in many obese animal models as well as in obese humans.3 9 10 In addition, the plasma norepinephrine response to stimuli such as upright posture and isometric handgrip is elevated in obese subjects.9 Finally, results from recent studies indicate that acute ganglionic or adrenergic blockade causes much greater reductions in blood pressure in fat-fed dogs and rats than in their lean controls.11 12 Although the results of these studies are indicative of enhanced sympathetic activity in obesity, the role of the renal sympathetic nerves in mediating sodium retention and hypertension during the development of obesity is unknown. Thus, the purpose of this study was to determine the role of renal nerves in mediating sodium retention and hypertension in obesity. To achieve this goal, we determined the effect of high-fat diet on systemic and renal hemodynamics and renal excretory function in control dogs and dogs with bilateral renal denervation.
This study was conducted in 13 mongrel dogs that were conditioned prior to the study. Surgical procedures were performed with dogs under pentobarbital anesthesia (30 mg/kg IV) and with aseptic techniques. The experimental procedures were done according to the guidelines of the National Institutes of Health and in conformance with the Animal Welfare Act. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.
All animals underwent surgery for implantation of chronic vascular catheters in both femoral arteries and veins. The catheters were tunneled subcutaneously and exteriorized in the upper back to allow easy sampling and infusions as well as continuous monitoring of arterial pressure. In one group of dogs, renal denervation in both right and left kidneys was performed as previously described.13 In brief, flank incisions were made on both sides and the renal vessels were exposed, stripped of all visible renal nerves, and painted with 10% phenol in absolute ethanol for approximately 20 minutes. This procedure markedly depleted the renal tissue norepinephrine to less than 10% in both kidneys.
After at least 1 week of recovery from surgery, all dogs were housed in individual metabolic cages in an air-conditioned room with an adjusted temperature and humidity and a 12-hour dark/light cycle. Isotonic saline was continuously infused intravenously by a roller pump (model 375A, Sage Instruments) that delivered a fixed amount of saline at a rate of approximately 450 mL/d. Intravenous lines were mounted with disposable filters (0.22 μm; Cathivex, Millipore Corp) to prevent contaminants, microorganisms, and air bubbles from entering the infusion lines. These filters were frequently changed throughout the study. Arterial pressure was recorded through a pressure transducer that was mounted at the level of the heart. Transducer cables and intravenous lines were protected by a flexible vacuum hose attached to a harness that was covered by a jacket worn by the dog. Blood pressure signals were continuously recorded, and the analog signals were sent to a digital computer to be analyzed. The computer was adjusted to take samples each minute and calculate the average mean arterial pressure and heart rate during the period from 2 pm to 8 am. Daily care of the dogs was performed between 8 am and 2 pm.
During the entire period of the study, dogs were on a sodium-deficient diet (H/D Hill’s Pet Products) that provided approximately 6 to 7 mmol of sodium and 65 mmol of potassium. In addition, dogs were supplemented with 10 mL of a multivitamin preparation (VAL syrup, Ft Dodge Laboratories) each day. Sodium intake was fixed at approximately 75 to 80 mmol/d; this included the saline infused and the sodium provided in the food.
A period of 1 week was allowed for training of the dogs to lie and sit quietly in cages and for hemodynamic measurements to stabilize and a state of sodium balance to be reached. After a 1-week period of stable control measurements, dogs were placed for 5 weeks on a high-fat diet that consisted of 0.7 kg cooked beef fat per day in addition to their regular food. Glomerular filtration rate (GFR) and renal plasma flow were determined from the clearances of 125I-iothalamate (Glofil, Isotex Diagnostics) and 131I-iodohippurate (Hippuran, Syntex), respectively, by use of the single-injection technique as previously described.14 On the same day renal hemodynamics were measured, blood samples were withdrawn for measurement of plasma renin activity (PRA), plasma aldosterone and insulin as well as serum electrolytes. Renal hemodynamics were determined during the control period as well as during the first, third, and fifth weeks of the high-fat diet.
Urinary and serum sodium and potassium were determined by flame photometry (IL-943, Instrumentation Laboratories). PRA, aldosterone, and insulin were measured by radioimmunoassay. Concentrations of 125I and 131I in plasma were determined by a gamma counter (model 1185, Searle). At the end of the experiment, the dogs were euthanatized with intravenous KCl under pentobarbital anesthesia, and the kidneys were examined for any gross pathological changes. The kidneys were then immediately removed, homogenized with 0.1 mol/L perchloric acid, and centrifuged, and the supernatant was stored at −70°C until it was assayed. Renal tissue norepinephrine concentration was determined by high-performance liquid chromatography with electrochemical detection according to the method of Moyer.15
Data are expressed as mean±SEM. Comparisons of control data with the period after fat feeding were analyzed by one-way ANOVA for repeated measures and subsequent Dunnett’s t test for simultaneous comparisons within groups and subsequent use of Bonferroni’s t test for nonsimultaneous comparisons between groups. A value of P<.05 was accepted as statistically significant.
Consuming a high-fat diet for 5 weeks increased the body weight and heart rate significantly in both groups of dogs (Fig 1⇓). Body weight increased from 19.9±2.2 to 29.9±2.4 kg (an increase of 50%) in the control (INN) group and from 21.1±1.2 to 32.4±1.9 kg (54%) in the bilaterally renal-denervated (DNX) group. Heart rate increased from 81±8 to 113±7 beats per minute (39%) in the INN group and from 79±7 to 103±8 beats per minute (30%) in the DNX group. Although the body weight and heart rate responses to the high-fat diet were similar in both groups, the arterial pressure response was markedly different. Arterial pressure in the INN group increased significantly from 95±2 to 109±4 mm Hg (15%). In contrast, arterial pressure in the DNX group after 5 weeks of the high-fat diet was not statistically different from baseline (increasing from 87±3 to 90±4 mm Hg) (Fig 1⇓). Although blood pressure tended to be slightly lower in the DNX group than in the INN group under basal conditions, this difference was not statistically significant (P=.1).
Urinary sodium excretion during the control period was not statistically different between the INN and DNX groups (Fig 2⇓). During the control period, sodium excretion in the INN group averaged 74.8±2.9 mmol/d. However, average sodium excretion during the 35 days of the high-fat diet decreased in the INN group to 61.2±1.8 mmol/d (P<.001). In contrast, sodium excretion in the DNX group was not statistically different between control conditions (72.2±2.5 mmol/d) and the high-fat diet period (68.7±3.7 mmol/d). Cumulative sodium retention in the INN group averaged 455±85 mmol after 5 weeks of the high-fat diet. In contrast, the DNX group retained only 252±47 mmol of sodium after the same period on the diet. The difference in cumulative sodium retention between the INN and DNX groups was significant after the third week of the high-fat diet (Fig 2⇓).
GFR increased significantly from 84.7±5.7 to 95.3±6.2 mL/min in the INN group and from 78.0±6.0 to 87.8±2.7 mL/min in the DNX group (Fig 3⇓). Renal plasma flow also increased, from 220.5±14.1 mL/min to 260.5±21.4 mL/min in the INN group and from 199.2±8.0 to 218.5±12.3 mL/min in the DNX group. There were no significant differences in GFR or renal plasma flow between the INN and DNX groups under basal conditions or in response to the high-fat diet (Fig 3⇓).
During the control period, PRA was lower in the DNX group (0.54±0.16 ng angiotensin I · mL−1 · h−1) compared with the INN group (0.72±0.22 ng angiotensin I · mL−1 · h−1) (Fig 4⇓). The high-fat diet increased PRA significantly in the INN group after the first week of high-fat feeding but decreased toward control during the third and fifth weeks of high-fat feeding. The PRA response to the high-fat diet was markedly attenuated in the DNX group. The PRA in the INN group was significantly higher than in the DNX group during the entire period of the high-fat diet. Plasma aldosterone concentration also increased from 3.1±0.1 to 6.7±1.4 ng/dL in the INN group and from 3.0±0.5 to 5.4±1.1 ng/dL in the DNX group during the 5 weeks of the high-fat diet (Fig 4⇓). The high-fat diet increased plasma aldosterone levels significantly in both groups after the first and third weeks. However, plasma aldosterone levels tended to decrease toward control values during the fifth week of the high-fat diet. There were no differences in plasma aldosterone concentration between the INN and DNX groups under basal conditions or in response to the high-fat diet. Plasma insulin levels averaged 33.3±3.0 and 34.9±5.5 μU/mL in the INN and DNX groups, respectively, during the control period. The high-fat diet increased plasma insulin levels significantly in both groups to an average of 52.5±6.5 μU/mL in the INN dogs and 59.2±7.2 μU/mL in the DNX dogs. There were no differences in plasma insulin levels between the INN and DNX dog groups under basal conditions or in response to the high-fat diet (Fig 4⇓).
In the control period, plasma sodium concentration averaged 140.0±0.8 and 138.2±0.1 mmol/L in the INN and DNX groups, respectively, and plasma potassium concentration averaged 4.5±0.1 and 4.7±0.1 mmol/L, respectively. There were no changes in plasma sodium or potassium during the period of high-fat feeding in either group. Renal tissue norepinephrine concentration averaged 548±66 pg/mg kidney weight in the INN group and 45±6 pg/mg kidney weight in the DNX group.
As previously reported,3 4 16 feeding dogs a high-fat diet for 5 weeks causes significant increases in arterial pressure and marked sodium retention. In the present study, renal denervation markedly attenuated the sodium retention and hypertension that normally occurs in response to a high-fat diet. These findings indicate that the renal nerves play an important role in mediating the sodium retention and hypertension associated with obesity.
A reduction in renal excretory function is thought to play an important role in the pathogenesis of all forms of hypertension, including obesity-induced hypertension.1 17 The increase in arterial pressure in response to a high-fat diet is normally associated with significant reductions in sodium excretion and marked increases in cumulative sodium balance.3 4 16 Because the sympathetic nervous system has been reported to be elevated in obesity, and it is well documented that enhanced renal sympathetic nervous activity is an important stimulus to sodium retention, we examined the potential role of the renal nerves in mediating sodium retention and hypertension during the development of obesity in dogs.3 9 10 11 12 18 19 To do this, we determined the effects of bilateral renal denervation on the hemodynamic and renal excretory responses to a high-fat diet. Ingestion of the high-fat diet for 5 weeks in control dogs resulted in significant increases in body weight (50%), arterial pressure (15%), and heart rate (39%) and cumulative sodium retention of 455 mmol. Despite slightly higher increases in body weight (54%) and similar increases in heart rate, increases in arterial pressure and cumulative sodium retention were markedly attenuated in response to a high-fat diet in the DNX dogs. Arterial pressure did not significantly increase (from 87±3 to 90±4 mm Hg) in response to the high-fat diet, and cumulative sodium retention was only 252±47 mmol in the DNX group. These findings support the concept that the renal sympathetic nerves play an important role in mediating the sodium retention and hypertension associated with obesity.
Our data indicated that the sodium retention in response to a high-fat diet plays an important role in the development of hypertension. The sodium retention in response to the high-fat diet in both groups of dogs was due to enhanced tubular reabsorption of sodium, because GFR and the filtered load of sodium were elevated during the 5 weeks of feeding. Furthermore, differences in cumulative sodium retention between the DNX and INN dogs were also most likely due to tubular mechanisms, because comparable increases in GFR occurred in the two groups. These findings suggest that the marked increase in cumulative sodium retention in response to a high-fat diet is due, in part, to enhanced tubular reabsorption as a result of enhanced sympathetic nerve activity or possibly to increased renal tubular sensitivity to adrenergic stimulation during obesity. Further studies will be necessary to quantitate the importance of these and other possible mechanisms.
The finding of increased PRA in the INN group in our study is in agreement with findings from our previous studies indicating increased renin-angiotensin activity in obesity-induced hypertension in dogs.4 5 6 However, in the present study the increase in PRA occurred after the first week of the high-fat diet and then gradually returned toward control levels. Interestingly, the transient increase in PRA did not occur in the DNX dogs. This information suggests that the renal sympathetic nervous system plays an important role in mediating the enhanced activity of the renin-angiotensin system in dogs fed a high-fat diet. These results also suggest that initial increases in sodium retention and arterial pressure elicited by the renal nerves may, in part, be mediated by angiotensin II. Supporting this suggestion are preliminary results from our laboratories demonstrating that blockade of the renin-angiotensin system delays but does not attenuate the increase in arterial pressure in response to a high-fat diet.
Increases in plasma insulin and aldosterone concentrations have also been postulated to mediate the sodium retention and hypertension associated with obesity. In the present study, we found that plasma insulin and aldosterone levels were significantly elevated in response to the high-fat diet in the INN and DNX groups. Because the increases in plasma aldosterone and insulin were the same in both groups before and after the period of the high-fat diet, it is unlikely that the attenuated sodium retention and hypertension in the DNX group were due to differences in plasma levels of insulin or aldosterone. Further evidence that insulin per se is not involved in the hypertension observed in this dog model of obesity are studies indicating that long-term increases in plasma insulin in normal dogs do not result in chronic sodium retention and hypertension, as observed in dogs on a high-fat diet.20
Although the results of this study indicate that enhanced sympathetic nervous system activity plays an important role in mediating sodium retention and hypertension in dogs fed a high-fat diet, the mechanisms responsible for altering nervous system activity are unclear. Ingestion of a high-fat diet elicits significant changes in metabolic function in various tissues throughout the body. Enhanced activity of the sympathetic nervous system may be a direct result of altered metabolic activity of neuronal cells or an indirect result of increased circulating levels of various hormonal or humoral factors that can have an effect on peripheral or central neural regulatory sites. Because there is increasing evidence for an important link between obesity, sympathetic nervous system activity, and hypertension in animal models and humans, future studies examining the mechanisms responsible for this interaction remain an important area of investigation.
We thank John Stuart Williams for excellent technical assistance and Susie Zuller for secretarial assistance. Dr Granger is an Established Investigator of the American Heart Association. This work was supported by grants HL-51971, HL-33947, HL-23502, and HL-39399 from the National Institutes of Health.
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