SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We aimed to evaluate the effects of droxidopa (an oral synthetic precursor of norepinephrine) on the hemodynamic and renal alterations of portal hypertensive rats. Sham, portal vein-ligated (PVL), and 4-week biliary duct-ligated (BDL) rats received a single oral dose of droxidopa (25-50 mg/kg) or vehicle and hemodynamic parameters were monitored for 2 hours. Two groups of BDL and cirrhotic rats induced by carbon tetrachloride (CCl4) were treated for 5 days with droxidopa (15 mg/kg, twice daily, orally); hemodynamic parameters and blood and urinary parameters were assessed. The droxidopa effect on the Rho kinase (RhoK) / protein kinase B (AKT) / endothelial nitric oxide synthase (eNOS) pathways was analyzed by western blot in superior mesenteric artery (SMA). The acute administration of droxidopa in PVL and BDL rats caused a significant and maintained increase in arterial pressure and mesenteric arterial resistance, with a significant decrease of mesenteric arterial and portal blood flow, without changing portal pressure and renal blood flow. Two-hour diuresis greatly increased. Carbidopa (DOPA decarboxylase inhibitor) blunted all effects of droxidopa. Chronic droxidopa therapy in BDL rats produced the same beneficial hemodynamic effects observed in the acute study, did not alter liver function parameters, and caused a 50% increase in 24-hour diuresis volume (7.4 ± 0.9 mL/100g in BDL vehicle versus 11.8 ± 2.5 mL/100g in BDL droxidopa; P = 0.01). Droxidopa-treated rats also showed a decreased ratio of p-eNOS/eNOS and p-AKT/AKT and increased activity of RhoK in SMA. The same chronic treatment in CCl4 rats caused similar hemodynamic effects and produced significant increases in diuresis volume and 24-hour natriuresis (0.08 ± 0.02 mmol/100g in CCl4 vehicle versus 0.23 ± 0.03 mmol/100g in CCl4 droxidopa; P = 0.014). Conclusion: Droxidopa might be an effective therapeutic agent for hemodynamic and renal alterations of liver cirrhosis and should be tested in cirrhosis patients. (HEPATOLOGY 2012;56:1849–1860)

The development of portal hypertension in liver cirrhosis is associated with arterial vasodilation, especially in the splanchnic circulation, that is initially compensated by a hyperdynamic circulation in order to maintain a normal blood pressure and effective arterial blood volume.1-3 As the disease progresses, splanchnic arterial vasodilation increases, systemic vascular resistance is markedly reduced, and because the increased cardiac output cannot compensate, arterial pressure falls. As a consequence of the severe arterial underfilling, vasoconstrictor and antinatriuretic factors become activated to maintain blood pressure, resulting in renal sodium and fluid retention and, eventually, renal failure.

An increased production or activity of vasodilatory mediators and a decrease in vascular reactivity to vasoconstrictors are the main contributory factors of the splanchnic arterial vasodilation.4-6 In addition to these humoral factors, other mechanisms may also play a role in splanchnic vasodilation. We have demonstrated an important down-regulation, both at the transcriptional and posttranslational level, of proteins implicated in adrenergic neurotransmission in the superior mesenteric artery (SMA) from portal hypertensive rats, accompanied by a marked atrophy of the sympathetic innervation in the mesenteric vascular bed.7, 8 These data suggest that a local splanchnic adrenergic inhibition, in a context of generalized adrenergic overactivity, might contribute to arterial splanchnic vasodilation.

Vasoconstrictor drugs such as vasopressin (terlipressin) and somatostatin analogs (octeotride) or α-adrenergic agonists (norepinephrine and midodrine) are being used for the treatment of some of the clinical consequences of the hemodynamic and renal alterations of liver cirrhosis.9, 10 These vasoconstrictors are targeted to improve renal function and decrease sodium and fluid retention by increasing systemic vascular resistance and suppressing the activity of endogenous vasoconstrictors. Droxidopa is a synthetic catecholamino acid that, in the presence of L-aromatic-amino-acid decarboxylase, is converted to norepinephrine.11 Several studies have shown that the oral administration of droxidopa increases standing blood pressure and improves orthostatic tolerance in patients with neurogenic orthostatic hypotension.12, 13 Similar to other α-adrenergic drugs, droxidopa might be useful in liver cirrhosis complications.

In the present work we aimed to evaluate the acute and the chronic effects of the oral administration of droxidopa on the hemodynamic and renal alterations of portal hypertensive rats. For this purpose, two different approaches were designed: (1) an acute study that consists of the oral administration of a single dose of droxidopa or vehicle to sham rats, prehepatic portal hypertensive rats caused by a portal vein ligation (PVL), and cirrhotic rats induced by bile duct ligation (BDL); and (2) a chronic study where the proadrenergic drug or vehicle were orally administrated over 5 days to BDL rats and cirrhotic rats induced by carbon tetrachloride (CCl4). In addition, the acute effects of the combination of droxidopa and propranolol were also examined.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Experimental Models of Portal Hypertension.

Studies were performed in male Sprague-Dawley rats (Charles River, Barcelona, Spain) weighing 250-300 g. The acute effects of droxidopa were evaluated in prehepatic portal hypertensive and cirrhotic rats and in sham rats. Prehepatic portal hypertension was induced by PVL. Rats were anesthetized with inhaled isoflurane and the portal vein was freed from the surrounding tissue after a midline abdominal incision. A ligature (silk gut 3-0) was placed around a 20G blunt-tipped needle lying along the portal vein. Subsequent removal of the needle yielded a calibrated stenosis of the portal vein. Intrahepatic portal hypertension caused by secondary biliary cirrhosis was induced by common BDL. Animals were anesthetized with inhaled isoflurane and the common bile duct was occluded by double ligature with a 5-0 silk thread. The bile duct was then resected between the two ligatures. In sham-operated rats, the portal vein was isolated but not ligated. Also, intrahepatic portal hypertension was induced by oral administration of CCl4. Rats received 1.5 mmol/L of phenobarbital in drinking water (ad libitum) and 20 μL/kg/week of CCl4 in water by gavage.14 All animals in the different groups ate a grain-based chow (Teklad 2014, Harlan Laboratories, Indianapolis, IN) containing a fixed formula of ingredients with 0.1% of sodium (Na). The average food intake in 4-week BDL animals was 7 g/100 g of body weight representing a mean Na intake of 0.007 g/100 g/day. All models reproduced the expected hemodynamic alterations of portal hypertension (Supporting Table).

Drug Administration.

Acute model: Rats were randomly assigned to receive a single dose of droxidopa (Chelsea Therapeutics, Charlotte, NC) or vehicle by gastric gavage after 14 and 28 days of portal and common bile duct ligation, respectively. Sham animals received droxidopa at a dose of 50 mg/kg (n = 5) or vehicle (1% carboxymetylcellulose solution and 0.2% Tween 80 emulsifier) (n = 5). PVL rats received droxidopa at a dose of 50 mg/kg (n = 7), 25 mg/kg (n = 7), or vehicle (n = 9). Carbidopa (DOPA decarboxylase inhibitor) (30 mg/kg) was given intraperitoneally 30 minutes before droxidopa to four PVL rats. Likewise, droxidopa at 25 mg/kg (n = 8) or vehicle (n = 6) was administered to BDL rats. A second set of BDL rats was used to measure renal hemodynamics with the same doses of droxidopa (n = 6) or vehicle (n = 6). Finally, a third group of BDL rats were used in a combination protocol using the following scheme: 25 mg/kg (n = 7) of propranolol followed by 25 mg/kg of droxidopa 2 hours later. Propranolol was also administrated by gavage. Comparisons were made with the corresponding BDL animals treated with droxidopa alone (n = 7), propranolol alone (n = 7), or vehicle (n = 6). All rats were fasted for 16 hours before drug administration.

Chronic model: Droxidopa at a dose of 15 mg/kg or vehicle was randomly administered orally by gavage, twice a day, for 5 days. In BDL rats treatment began (droxidopa, n = 15 and vehicle, n = 15) after 28 days of the bile duct resection. Ascites was present in 20% of these animals. Eight rats of each group were used to measure hemodynamic parameters. In CCl4 cirrhotic rats treatment began (droxidopa, n = 6 and vehicle, n = 6) after 14 to 20 weeks of induction with CCl4, once the animal presented ascites.

Hemodynamic Measurements.

For the acute model, the hemodynamic parameters were registered for 2 hours after drug or vehicle administration, whereas for the chronic model in BDL, hemodynamic measures were registered for 90 minutes after the last dose of the treatment (morning of the fifth day). In CCl4 cirrhotic rats only a single determination of hemodynamic measures was taken 60 minutes after the last dose of drug administration. Five minutes after drug or vehicle administration, animals were anesthetized with ketamine hydrochloride (100 mg/kg) plus midazolam (5 mg/kg) intraperitoneally. One polyethylene PE-catheter (PE50) was introduced into the femoral artery to determine mean arterial pressure (MAP, mmHg) and heart rate (HR), and after a midline abdominal incision another catheter was introduced into the ileocolic vein for the measurement of portal pressure (PP, mmHg) using highly sensitive pressure transducers (Harvard Apparatus, Holliston, MA). The SMA was isolated from connective tissue and a perivascular ultrasonic transit-time flowprobe (1 mm diameter, Transonic Systems, Ithaca, NY) was placed around the artery to continuously measure the SMA blood flow (SMABF, mL/min.100 g). The same flowprobe placed on the left renal artery freed of the surrounding fat tissue was used to measure renal artery blood flow (RABF, mL/min.100 g). Finally, portal blood flow (PBF, mL/min.100 g) was measured with an ultrasonic transit-time flowprobe (2 mm diameter, Transonic Systems) placed on the dissected portal vein. Hemodynamic parameters were allowed to equilibrate for 15 minutes, each value representing the average of 30 seconds. SMA resistance (SMAR, mmHg/mL.min.100 g) was calculated as (MAP-PP/SMABF), renal artery resistance (RAR) as (MAP/RABF), and intrahepatic vascular resistance (IHVR) as (PP/PBF). Animals were maintained at 37°C throughout the study by a rectal temperature probe.

Sample Collection.

Acute model: For the determination of the diuresis volume, urine was collected from the bladder at the end of the 2-hour period of hemodynamic registration. Venous blood samples were collected in the PVL groups 40 minutes after treatment in EDTA tubes for determination of norepinephrine and aldosterone plasma concentration.

Chronic model: Urine was collected the fourth day of treatment by housing the rats in individual metabolic cages for 24 hours. Venous blood samples were obtained from the cava vein at the last day of treatment, after the 90-minute period of hemodynamic registration. A group of six sham-operated rats was used as normal control for serum and urine parameters. SMA samples from BDL rats harvested from the aortic origin were placed in liquid nitrogen and kept at −80°C until processed.

Biochemical Parameters.

Serum levels of bilirubin, albumin, liver enzymes, and serum and urinary levels of sodium, potassium, creatinine, and osmolality were determined using an automatic analyzer (Olympus AV5400, Olympus Europe, Hamburg, Germany). Norepinephrine plasma concentration was determined by RIA (KatCombi RIA, IBL, Hamburg, Germany). Aldosterone plasma concentration was also detected by RIA (Aldoctk-2, DiaSorin, Saluggia, Italy). Osmolal clearance (mL/min) was calculated with the following formula: (osmolality)urine·V/(osmolality)serum, creatinine clearance (mL/min) as follows: [creatinine]urine·V/[creatinine]serum, and free water clearance (mL/min) as: V-osmolal clearance, where V is urine flow rate (mL/min). Finally, sodium, potassium and creatinine excretion (mmol/24h) were determined as [solute]·urinary volume in 24 hours. Fractional sodium excretion was calculated as ([Na+]urine·[creatinine]serum)·100/ ([Na+]serum·[creatinine]urine).

Western Blot Analysis.

SMA samples from 28-day BDL rats treated with droxidopa (n = 9) or vehicle (n = 10) were analyzed by western blot. Tissue samples were homogenized in 250 μL RIPA buffer and 15 μL of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) Protein quantification in the supernatants was performed by BCA assay (Pierce, Rockford, IL). Equal amounts of protein extracts (15-20 μg, depending on the analyzed protein) were separated on a 4%-12% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (depending on the protein size), blotted onto polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA), and incubated with antibodies against endothelial nitric oxide synthase (eNOS) (1:500) (BD Transduction Lab, Franklin Lakes, NJ,), p-eNOS (1:250), protein kinase B (AKT), p-AKT (1:1000) (Cell Signaling Technology, Boston, MA), Rho kinase (RhoK 2), moesin, and p-moesin (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were quantified by the Quantity One software (Bio-Rad Laboratories, Hercules, CA).

Statistical Analysis.

All values are expressed as the mean ± standard error of the mean and compared using Student's t test (SigmaStat 3.0). Statistical significance was established at P < 0.05.

Ethics.

All animals received humane care in compliance with institutional guidelines from the European Commission and the National Institutes of Health (USA) on the protection of animals used for experimental and other scientific purposes. All animal experiments were approved by the Animal Care Committee of the Vall d'Hebron Institut de Recerca (VHIR, Barcelona, Spain) and conducted in the animal facilities of VHIR.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Acute Droxidopa Effects in Sham Rats.

As expected, droxidopa produced a significant increase in MAP and HR in sham animals that disappeared at 90 minutes (Fig. 1; Supporting Fig. 1). This effect was accompanied by an initial increase both in PP and SMABF; PP returned rapidly to normal, whereas SMABF decreased at the end of the study period. Mesenteric resistance did not change significantly.

thumbnail image

Figure 1. Hemodynamic parameters of sham rats treated with 50 mg/kg of oral droxidopa (n = 5) ( equation image) or vehicle (n = 5) ( equation image). MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric blood flow; SMAR, superior mesenteric resistance. *P < 0.05, **P < 0.005.

Download figure to PowerPoint

Acute Droxidopa Effects in PVL Rats.

The high dose of droxidopa (50 mg/kg) caused a sharp and prolonged increased in MAP, accompanied by significant increases in HR in PVL rats (Fig. 2; Supporting Fig. 1), along with an initial significant increase in PP, a nonsignificant decrease of SMABF, and a nonsignificant increase of SMAR. By reducing droxidopa dosage (25 mg/kg), the effect in MAP and HR was maintained, PP was not significantly changed, whereas SMABF was significantly decreased and SMAR increased. Carbidopa administration clearly blunted all effects of droxidopa (Fig. 2). Two-hour diuresis was significantly increased (5 times) in PVL animals treated with droxidopa as compared to vehicle-treated animals (1.47 ± 0.26 ml/2hr versus 0.28 ± 0.08 ml/2hr; P < 0.001) (Supporting Fig. 2). Norepinephrine plasma levels increased more than 10 times in droxidopa-treated animals (n = 5) (6.9 ± 0.88 μg/L) as compared to vehicle-treated animals (n = 5) (0.50 ± 0.11 μg/L; P < 0.001); carbidopa (n = 4) inhibited norepinephrine plasma level increase (0.50 ± 0.12 μg/L). Finally, aldosterone plasma levels were lower in the droxidopa-treated rats (39.9 ± 2.5 ng/dL) compared to the vehicle-treated rats (55.9 ± 8 ng/dL; P = 0.09).

thumbnail image

Figure 2. Hemodynamic parameters of PVL rats treated with 50 mg/kg of oral droxidopa (n = 7) ( equation image), 25 mg/kg (n = 7) ( equation image), a combination of droxidopa (50 mg/kg) and carbidopa (30 mg/kg) (n = 4) ( equation image) or vehicle (n = 9) ( equation image). MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric blood flow; SMAR, superior mesenteric resistance. *P < 0.05, **P < 0.005 compared to vehicle group.

Download figure to PowerPoint

Acute Droxidopa Effects in BDL Rats.

In BDL rats, droxidopa (25 mg/kg) produced similar effects as in PVL animals (Fig. 3). MAP and SMAR significantly increased during the study period, whereas SMABF decreased and PP remained unchanged; HR increased, but not significantly (Supporting Fig. 1). Portal hemodynamics demonstrated an important decrease in PBF accompanied by a significant increase in IHVR (Fig. 3). Renal hemodynamics showed a nonsignificant decrease in RABF with a slight increase in RAR (only significant 1 hour after treatment) (Supporting Fig. 3). Finally, 2-hour urine volume was significantly increased (3 times) in BDL animals treated with droxidopa as compared to vehicle-treated animals (2.42 ± 0.26 ml/2hr versus 0.85 ± 0.29 ml/2hr; P = 0.004) (Supporting Fig. 2).

thumbnail image

Figure 3. Hemodynamic parameters of BDL rats treated with 25 mg/kg of oral droxidopa (n = 8) ( equation image) or vehicle (n = 6) ( equation image). MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric blood flow; SMAR, superior mesenteric resistance; PBF, portal blood flow; IHVR, intrahepatic vascular resistance. *P < 0.05, **P < 0.005.

Download figure to PowerPoint

When propranolol was administered to animals prior to receiving droxidopa, a decrease in PP was observed with respect to vehicle and especially to droxidopa alone (Fig. 4). This was accompanied by an impressive additive decrease in SMABF and increase in SMAR, whereas propranolol caused a partial blockade of the increase in MAP and a complete blockade of the HR increase depending of droxidopa (Fig. 4; Supporting Fig. 1). Two-hour diuresis seemed not to be affected by adding propranolol to droxidopa: 2.19 ± 0.35 ml/2hr (P = 0.02 with respect to vehicle).

thumbnail image

Figure 4. Hemodynamic parameters of BDL rats treated with 25 mg/kg of oral droxidopa (n = 7) ( equation image), 25 mg/kg of oral propranolol (n = 7) ( equation image), a combination of droxidopa and propranolol (n = 7) ( equation image), or vehicle (n = 6) ( equation image). MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric blood flow; SMAR, superior mesenteric resistance. *P < 0.05, **P < 0.005 compared to vehicle group. Combination group versus droxidopa group P < 0.05 at all points.

Download figure to PowerPoint

Chronic Droxidopa Effects in BDL Rats.

The chronic administration of droxidopa maintained the beneficial hemodynamic effects shown in the acute administration of droxidopa (Fig. 5; Supporting Fig. 1). Droxidopa treatment caused a marked and significant increase in MAP, HR, and SMAR, accompanied by a significant decrease in SMABF in BDL rats compared to the vehicle group. PP of the cirrhotic animals remained unchanged in response to the chronic droxidopa treatment.

thumbnail image

Figure 5. Hemodynamic parameters of BDL rats after chronic droxidopa (n = 8) ( equation image) or vehicle (n = 8) ( equation image) administration (15 mg/kg, twice daily orally for 5 days). MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric blood flow; SMAR, superior mesenteric resistance. *P < 0.05, **P < 0.005.

Download figure to PowerPoint

BDL animals, independently of the treatment group, showed less weight, worse liver function results, and similar serum creatinine and sodium levels than sham animals (Table 1). In addition, vehicle-treated BDL animals showed similar urinary volume and increased sodium excretion as sham rats. Chronic droxidopa treatment was associated with a significant increase (50% increase) in total diuresis volume as compared to vehicle-treated BDL rats. This was accompanied by nonsignificant increases in free water and osmolal clearance. Sodium excretion was not modified by droxidopa therapy, but creatinine excretion was increased. Droxidopa treatment in BDL rats did not change serum biochemical parameters, including liver function parameters, compared to vehicle-treated rats.

Table 1. Biochemical Parameters in Sham-Operated (Sham) and Bile Duct Ligated (BDL) Rats After Chronic Droxidopa or Vehicle Administration
 ShamBDL VehicleBDL Droxidopa
 (n=6)(n=15)(n=15)
  • *

    P < 0.05 or below compared with BDL vehicle.

  • P < 0.05 or below compared with sham.

Weight (g)395 ± 7327 ± 6 317 ± 7
Serum Na+ (mmol/L)143 ± 0.6144 ± 0.6144 ± 0.6
Serum K+ (mmol/L)4.3 ± 0.34.5 ± 0.24.6 ± 0.3
Serum creatinine (mg/dL)0.59 ± 0.10.54 ± 0.040.52 ± 0.03
Serum osmolality (mOsm/Kg)299 ± 1306 ± 2305 ± 2
Bilirubin (mg/dL)0.18 ± 0.038.05 ± 0.418.56 ± 0.19
AST (UI/L)86 ± 10557 ± 61596 ± 60
ALT (UI/L)25 ± 383 ± 1285 ± 7
Alkaline phosphatase (UI/L)125 ± 7417± 18480 ± 26
Serum albumin (g/dL)2.83 ± 0.122.58 ± 0.112.44 ± 0.13
Urinary volume (mL/24h/100g)5.3 ± 0.77.4 ± 0.911.8 ± 2.5*,
Urine flow rate (μL/min/100g)3.7 ± 0.55.1 ±0.67.9 ± 1.3*,
Urinary osmolality (mOsm/Kg)582 ± 46626 ± 39471 ± 41*
Osmolal clearance (μL/min/100g)6.9 ± 0.59.3 ± 0.310.4 ± 1.3
Free water clearance (μL/min/100g)-3.2 ± 0.3-4.2 ± 0.5-3.5 ± 0.8
Na+ excreted (mmol/24h/100g)0.17 ± 0.040.31 ± 0.040.27 ± 0.02
Fractional Na+ excreted (%)0.14 ± 0.0140.45 ± 0.070.32 ± 0.031
K+ excreted (mmol/24h/100g)0.49 ±0.020.53 ±0.020.51 ± 0.02
Creatinine excreted (mg/24h/100g)3.4 ± 0.22.9 ± 0.13.1 ± 0.1*
Creatinine clearance (mL/min/100g)0.47 ± 0.70.39 ± 0.030.43 ± 0.04

Chronic Droxidopa Effects in CCl4 Rats.

Similar to BDL rats, chronic droxidopa administration caused significant increases in MAP (138.47 ± 5.7 mmHg versus 102.05 ± 6.4 mmHg; P = 0.009) and HR (376.3 ± 37.4 bpm versus 296.4 ± 16.9 bpm; P = 0.065) without changes in PP (16.84 ± 1.4 mmHg versus 15.07 ± 1.3 mmHg; P = 0.39). Table 2 shows the biochemical results of blood and urine samples. Serum biochemical parameters were unchanged by the treatment, whereas diuresis volume and sodium excretion were significantly increased.

Table 2. Biochemical Parameters in Cirrhotic Rats Induced by Carbon Tetrachloride (CCl4) After Chronic Droxidopa or Vehicle Administration
 CCl4 VehicleCCl4 Droxidopa
 (n=6)(n=6)
  • *

    P < 0.05 or below compared with CCl4 vehicle.

Weight (g)438 ± 34404 ± 13
Serum Na+ (mmol/L)141 ± 1.7140 ± 0.6
Serum K+ (mmol/L)4.2 ± 0.14.6± 0.2
Serum creatinine (mg/dL)0.60 ± 0.050.45 ± 0.06
Serum osmolality (mOsm/Kg)306 ± 4304 ± 2
Bilirubin (mg/dL)1.12 ± 0.230.85 ± 0.19
AST (UI/L)465 ± 70413 ± 55
ALT (UI/L)134 ± 14113 ± 10
Alkaline phosphatase (UI/L)258 ± 36315 ± 22
Serum albumin (g/dL)2.16 ± 0.132.45 ± 0.13
Urinary volume (mL/24h/100g)3.2 ± 0.24.6 ± 0.4*
Urine flow rate (μl/min/100g)2.3 ± 0.23.2 ± 0.3*
Urinary osmolality (mOsm/Kg)787 ± 114596 ± 98
Osmolal clearance (μL/min/100g)5.5 ± 0.46.1 ± 0.7
Free water clearance (μL/min/100g)-3.3 ± 0.5-2.9 ± 0.7
Na+ excreted (mmol/24h/100g)0.08 ± 0.020.23 ± 0.03*
Fractional Na+ excreted (%)0.11 ± 0.030.25 ± 0.04*
K+ excreted (mmol/24h/100g)0.29 ± 0.030.27 ± 0.02
Creatinine excreted (mg/24h/100g)3.2 ± 0.23.1 ± 0.2
Creatinine clearance (mL/min/100g)0.38 ± 0.030.50 ± 0.06

Western Blot Analysis in SMA of BDL Rats.

In order to understand the mechanism involved in droxidopa beneficial hemodynamic effects, we quantified the level of expression of AKT, pAKT, eNOS, and p-eNOS in SMA from rats treated with droxidopa or vehicle. Droxidopa did not change the expression of AKT in SMA of BDL rats compared with those treated with vehicle. On the other hand, AKT phosphorylation was significantly lower in BDL rats treated with droxidopa, showing a significant 49% decrease in the p-AKT/AKT ratio (P = 0.03) (Fig. 6). Regarding expression of eNOS, BDL rats treated with droxidopa displayed a nonsignificant increased expression of this enzyme compared with vehicle rats. However, this increase was not associated with an increased activity of eNOS, as the p-eNOS/eNOS ratio was significantly decreased in 49% with respect to vehicle (P < 0.006) (Fig. 6). We also determined the total Rho kinase and the moesin/phospho-moesin ratio as a marker of Rho kinase activity. Our results indicate that although total Rho kinase showed no changes between BDL treated with droxidopa or vehicle, its activity must be increased in droxidopa-treated rats, because there is a significant 92% increase in the ratio of pmoesin/moesin in these animals compared to vehicle (P = 0.04) (Fig. 6).

thumbnail image

Figure 6. Western blot analysis in superior mesenteric artery of BDL rats. (A) Representative Western blots of Rho kinase (RhoK), Moesin, p-Moesin AKT, p-AKT, eNOS, and p-eNOS in vehicle (V) or droxidopa (D) treated animals, and (B) bar diagram showing protein quantitation of RhoK and ratios of p-Moesin/Moesin, p-AKT/AKT, and p-eNOS/eNOS in BDL rats treated with vehicle (black bars) or droxidopa (gray bars). GAPDH was used as an internal loading control.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The results of the oral acute and chronic droxidopa administration performed in portal-hypertensive animals indicate that this proadrenergic drug is capable of improving the systemic and splanchnic circulatory dysfunction of these animals and, at the same time, substantially increase the diuresis volume and sodium excretion.

One of the interesting findings of the present study is that the vasoconstrictive effect of droxidopa is especially important in the splanchnic circulation, causing an increase in systemic arterial pressure that is relatively more intense and long-lasting than the observed effect in sham rats. Treated PVL and cirrhotic animals reduce their mesenteric blood flows to levels similar to normal animals and show relative increases of mesenteric resistance (more than a 50% increase with respect to basal values) that double the effect detected in sham animals. The lack of cardiac output data does not allow exploring the global systemic effects of droxidopa, but mesenteric blood flow represents in part the regional cardiac output in this area. By contrast, the vasoconstrictive effect of droxidopa in other areas explored, such as the renal arteries, is much less pronounced, suggesting that the improvement in systemic hemodynamics caused by droxidopa is mainly due to the correction of the splanchnic vasodilation and, consequently, the amelioration of the severe arterial underfilling. This observation is in accordance with our prior findings of a local splanchnic adrenergic inhibition with sympathetic atrophy in portal hypertensive rats, in a context of generalized adrenergic overactivity.7, 8 Droxidopa might be in part correcting this acquired adrenergic mesenteric down-regulation. In addition, the intense splanchnic effect of droxidopa indicates that somehow this drug is capable of overcoming the largely known low reactivity to vasoconstrictors of mesenteric vessels of PVL and cirrhotic animals.5, 15 Single mesenteric vascular bed experiments would be very helpful to better characterize the molecular mechanisms involved in the droxidopa effects.

The pressor and diuretic effects of droxidopa administration have been reported in different animal species.11, 16-18 In human studies, especially in patients with neurogenic orthostatic hypotension, droxidopa shows a potent and lasting effect in arterial pressure with significant amelioration of symptoms.11-13 Some of the pharmacological properties of droxidopa might be a consequence of the fact that this drug does not have direct effects on arterial pressure and diuresis; instead, it needs to be converted to norepinephrine by a single enzymatic step with L-aromatic-amino-acid decarboxylase (DOPA decarboxylase). This is supported by our findings of carbidopa blocking the droxidopa effects. In consequence, droxidopa is to be converted to norepinephrine in cells expressing DOPA decarboxylase that are mainly postganglionic sympathetic cells, but also nonneuronal cells.19 In patients with pure autonomic failure, a disease that entails loss of postganglionic sympathetic neurons, droxidopa shows pressor effects similar to other neurogenic diseases with intact sympathetic neurons. In portal hypertension droxidopa would increase the local norepinephrine levels in the adrenergic presynaptic terminals, especially in areas with a low adrenergic tone as the splanchnic area. This mechanism would represent a more physiological approach to increase the adrenergic tone than using indirect adrenergic drugs such as midodrine. In addition, the synthesis of norepinephrine by nonneuronal cells might also have an important role in increasing the adrenergic tone in these adrenergic-deficient areas. As shown in the western blot analysis of SMA, part of the droxidopa effect would be mediated by a direct norepinephrine dependent activation of the RhoA/Rho kinase signaling pathway inducing vasoconstriction, but also indirectly by inhibiting the activation of the AKT/eNOS vasodilation mechanism.20, 21 RhoA/Rho kinase control the AKT/eNOS pathway by posttranscriptionally down-regulating eNOS messenger RNA (mRNA) and by inhibiting the protein kinase B/AKT that in turn it is necessary to phosphorylate eNOS.

Another important finding of the present study is the marked effect in diuresis volume and natriuresis observed after the acute and chronic droxidopa treatment in the different animal models. In normal rats, droxidopa has no direct renal effects by itself when directly infused in renal arteries, and when renal perfusion pressure is protected from the systemic pressor effect of droxidopa by partial clamping, the drug-induced diuresis is abolished.18 From our results it is quite clear that at least in our BDL model the increase in diuresis volume is not a consequence of an increase in blood flow in the main renal arteries; possibly an intrarenal redistribution of blood flow is responsible for the improved water clearance. The increase in urine production in portal-hypertensive animals induced by droxidopa is most probably secondary to its systemic hemodynamic effects. As a consequence of the amelioration of the systemic hemodynamics and the subsequent improvement of the arterial vascular underfilling, the pressure diuresis would increase and at the same time the release of systemic and endogenous renal vasoconstrictors (antidiuretic hormone, renin-angiotensin-aldosterone system, endothelin) would be inhibited, leading to an improvement of the intrarenal perfusion and diuresis. The decrease in plasma aldosterone levels in PVL animals treated with droxidopa would point to this direction. Other possible mechanisms contributing to the diuretic effect of droxidopa via norepinephrine might be inhibition of renin release22 and renal vasopressin action23 through activation of renal alpha-2 adrenergic receptors. Another question that arises from our results is whether droxidopa is able to substantially increase diuresis volume in portal-hypertensive animals despite sharply increasing the already high norepinephrine blood levels. This apparent paradox could be explained by assuming that the indirect effects on kidney function of the improved systemic hemodynamics caused by droxidopa predominate over the renal vasoconstrictive effects of norepinephrine, or by contrast, that the role of the activation of the adrenergic system in causing renal dysfunction in cirrhosis might have been overestimated.24 Finally, despite the significant diuretic effect of droxidopa in BDL rats, increased sodium urinary excretion was not observed. This is probably explained by the fact that our 4-week BDL model did not reproduce many of the typical features of renal dysfunction in human cirrhosis. Serum sodium and creatinine levels were equal in sham and BDL rats, and natriuresis was actually increased in cirrhotic rats, whereas oligoanuria was absent. In fact, it is already known that the BDL model is characterized by polyuria rather than oliguria.25, 26 By contrast, in the CCl4 model natriuresis that was low in the vehicle-treated animals was sensibly increased with droxidopa treatment, accompanied by a mild nonsignificant improvement in creatinine clearance. Regardless of some discrepancies in the literature with respect to renal dysfunction in BDL rats,25, 26 the best way to analyze the renal effects of droxidopa is probably to test it directly in cirrhosis patients. Renal alterations in patients are well defined and droxidopa is already used in humans.

The important reduction in portal blood flow observed in our cirrhotic rats treated with droxidopa should be accompanied by a substantial reduction in portal pressure. However, portal pressure did not change significantly because the decrease in portal blood flow was compensated by an increase in intrahepatic vascular resistance secondary to a direct intrahepatic effect of norepinephrine.27 This intrahepatic vasoconstriction caused by droxidopa does not seem to significantly alter liver function in BDL rats, but it is a consequence that should be taken into consideration for long-term therapies with droxidopa. For similar reasons, the evidence demonstrating that hepatic alpha-adrenergic stimulation activates hepatic stellate cells and promotes liver fibrosis28 should also be kept in mind. Strategies aimed at minimizing these intrahepatic effects of chronic adrenergic stimulation and, consequently, achieving a net reduction in portal pressure should probably contemplate the use of combination therapies with proven beneficial intrahepatic effects such as statins, nitrates, and others. In this sense, our preliminary observations with the use of combined therapy with droxidopa and propranolol indicate that some of the effects of droxidopa could be improved without substantially affecting its beneficial systemic effects.

Vasoconstrictors are currently being used for the treatment of hemodynamic and renal alterations of cirrhosis.9, 10 Among them alpha-adrenergic drugs have been shown to be useful to improve these complications. The acute and chronic administration of droxidopa, a norepinephrine precursor, is able to improve the systemic and hemodynamic alterations of cirrhotic rats by increasing mean arterial pressure, reducing mesenteric and portal flow, and increasing mesenteric resistance. In addition, droxidopa produces a marked diuretic and natriuretic effect. Droxidopa seems to have a very potent pressor effect in these animals and has the advantage of oral administration. In conclusion, our data suggest that droxidopa might be an effective therapeutic agent for the hemodynamic and renal alterations of liver cirrhosis and should be tested in cirrhotic patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Chelsea Therapeutics Inc. for providing droxidopa and Dr. Robert Catalán for performing norepinephrine and aldosterone tests.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25845_sm_SuppFig1.tif8506KSupporting Information Figure 1.
HEP_25845_sm_SuppFig2.tif8502KSupporting Information Figure 2.
HEP_25845_sm_SuppFig3.tif8503KSupporting Information Figure 3.
HEP_25845_sm_SuppTable.doc21KSupporting Information Table 1.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.