MD Dorothee H L Bail
email: dodobail@web.de

Volker Steger

Uli Heinzelmann

Sandra Schiller

Anita I Geim

Benjamin Brüllmann

Gerhard Ziemer

Administration of brain natriuretic peptide improves cardiac function following operations using the extracorporeal circulation in an animal model

Keywords: BNP, cardiac surgery, extracorporeal circulation, cardiac output, catecholamines

The critical phase during cardiosurgical procedures is weaning the diseased heart from extracorporeal circulation. Postischemic heart failure sometimes requires the administration of inotropic and/or vasconstrictive agents. The natriuretic peptides influence pre-and afterload through their natriuretic, diuretic and vasodilating actions. To date, there are only few informations concerning the therapeutic effect of brain natriuretic peptide administration during cardiosurgical procedures. The goal of this study was to evaluate the effect of brain natriuretic peptide (BNP) administration following extracorporeal circulation in an animal model.

Surgery was performed on twenty swine using extracorporeal circulation. A 30-minute ischemic episode was simulated. Following declamping, BNP was administered to the BNP group (n=10) by an IV bolus at 0.3 µg/kg/min followed by an infusion at a rate of 0.015 µg/kg/min for 60 minutes. The animals in the control group (n=10) did not receive BNP, but instead a saline solution. Hemodynamic and clinical chemistry parameters as well as the amount of catecholamines that were required were documented.

All of the animals in the BNP group had a significantly better cardiac output and cardiac index at the end of the experiment. Seven out of 10 animals from the control group needed catecholamines, whereas only one animal from the BNP group did. Creatine phosphokinase levels were significantly lower in the BNP group. Systemic vascular resistance was markedly lower in the BNP group.

Administration of BNP is highly effective in treating post-ischemic heart failure following extracorporeal circulation. Hemodynamics are greatly improved, and there is almost no need for pharmacological support.

Introduction

The natriuretic, diuretic and vasodilating effects of the natriuretic peptides lead to a decrease in preand afterload, an improvement in myocardial ischemia by vasodilating the coronary arteries, and an increase in stroke volume, which in turn improves left ventricular function [START_REF] Baxter | The natriuretic peptides[END_REF]. Patients with heart failure or left ventricular dysfunction have elevated plasma concentrations of BNP proportional to the severity of disease.

Various studies have examined the intravenous administration of BNP/Nesiritide [START_REF] Hobbs | Hemodynamic effects of a single intravenous injection of synthetic human brain natriuretic peptide in patients with heart failure secondary to ischemic or idiopathic dilated cardiomyopathy[END_REF][START_REF] Marcus | Hemodynamic and renal excretory effects of human brain natriuretic peptide infusion in patients with congestive heart failure[END_REF][START_REF] Yoshimura | Hemodynamic, renal, and hormonal responses to brain natriuretic peptide infusion in patients with congestive heart failure[END_REF]. All of these studies demonstrated a distinct improvement in hemodynamic parameters. A dose-dependent reduction in pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP), pulmonary arterial pressure (PAP), mean arterial pressure (MAP), systemic vascular resistance (SVR) and as a result, an increase in stroke volume and cardiac index (CI) were shown. Studies of exogenously administered BNP during the stage of acute decompensated heart failure (ADHF) showed a reduction in ventricular filling pressure, which resulted in improved cardiac output (CO) and CI without increasing heart rate (HR) or significantly decreasing the MAP [START_REF] Colucci | Intravenous Nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure[END_REF].

Compared with other inotropic substances, administration of BNP has not yet been associated with severe adverse effects, especially ventricular arrhythmia, as is seen with dobutamine or phosphodiesterase inhibitors [6;7].

Weaning the patient from extracorporeal circulation (ECC) and the period immediately thereafter is usually the most unstable phase during cardiac surgery. The heart is in a particularly vulnerable situation, since it must resume perfusion of the entire organism after a period of ischemia. Myocardial insufficiency occurs regularly during this phase. The cardiac surgical trauma, induced ischemic cardiac arrest, disturbance in electrolyte and acid base balance, hemodilution and extracorporeal circulation per se can lead to myocardial edema and insufficiency. The administration of intravenous pharmacological support with various combinations of inotropes, vasoconstrictors and vasodilators are part of a well-established treatment concept for stabilizing and, when necessary, increasing cardiac output.

Two of the most important adverse effects of epinephrine and norepineprine are reduced kidney perfusion and tachyphylaxia. In addition, when ventricle function is already impaired preoperatively, catecholamine susceptibility can be partly diminished due to down-regulation of beta-adrenoreceptors. Phosphodiesterase inhibitors, which are administered for acute low-output syndrome or acute, refractory heart failure, lead to pronounced peripheral vasodilation. An -1 sympathomimeticum like norepinephrine is often needed to counteract these adverse effects. Phosphodiesterase inhibitors also have considerable arrhythmogenic effects and can lead to thrombopenia and hepatotoxicity. Overall, the phosphodiesterase inhibitors have many adverse effects.

Comparing the cardiac situation during and immediately following separation from ECC with the conventional stages of heart failure is very limited. There are only a few studies that have evaluated the influence of ECC on plasma BNP concentrations. These are, in part, conflicting [8;9]. There are only few studies to date on the therapeutic effects of the exogenous administration of BNP during cardiac surgery using ECC.

The goal of this study was to clarify whether the exogenous administration of BNP leads to improvement in hemodynamics during and after cardiac surgical procedures using ECC. Our goal was to determine whether BNP administration results in improvement in left ventricular function during the critical phase of cardiac surgery, which is weaning the heart from ECC. The effectiveness of BNP administration was measured in terms of an increase in CO and CI (of at least 10%), a reduction in RAP and a significant reduction in the need for intravenous agents for circulatory support.

Materials and Methods

Animals and the extracorporeal circulation

All animal procedures were approved by the local representatives for animal welfare at the University of Tuebingen, consistent with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 -23, revised 1996). Twenty young swine (27 ± 0.9 kg) of both sexes were randomised to two groups. Ten animals received intravenous BNP (BNP group). The ten animals who did not receive BNP comprised the control group.

On the day of surgery the swine were premedicated with ketamine (Katavet®; Pharmacia, Piscataway, NJ), diazepam, azeperone (Stresnil®; 40 mg/ml; Janssen-Cilag, Neuss, Germany) and atropine applied intramuscularly (dorsal neck muscle). Before induction of anaesthesia, indwelling catheters were placed in two ear veins.

Anaesthesia was maintained intravenously with 0.0072 mg/kg/min fentanyl, 0.06 mg/kg/min flunitrazepam and 0.104 mg/kg/min propofol. Before median sternotomy, fluid-filled catheters were inserted into the common carotid artery and the internal jugular vein to monitor arterial and central venous pressures, take arterial and venous blood samples, and to administer fluids.

All swine received 300 IU/Kg heparin for systemic anticoagulation. In addition, all animals received 250 mg of methylprednisolone (Urbason® solubile forte, Aventis Pharma Deutschland GmbH, frankgurt a.M., Germany) to prevent cross reactions with foreign swine blood which was used to prime the ECC.

ECC was initiated by cannulating the ascending aorta (14 Fr arterial perfusion cannula, Jostra, Hirrlingen, Germany) and the right atrium (32-34 Fr 2-stage venous cannula, Stöckert CAPS, Munich, Germany). After connection to the ECC, a catheter was also inserted in the left atrial appendage for measuring the left atrial pressure (LAP). Cardioplegic arrest was induced by antegrade infusion of a cold (4°C) blood cardioplegic solution (Buckberg), accomplishing external cooling with instillation of a cold saline solution into the pericardium. The heart was left in cardioplegic cardiac arrest for 30 minutes. The rewarming period to reach a normal body temperature was begun several minutes before the aortic cross clamp (AXCL) was removed. The entire duration of aortic cross clamping was 30 minutes. After the AXCL was removed and defibrillation was performed to convert the heart to a normal rhythm, a reperfusion period (with ECC maintained) of approximately 10 minutes followed, which was equivalent to 1/3 of the AXCL-time. During the subsequent 60 minutes (after weaning the heart from the ECC) pharmacological support was used when necessary (epinephrine, norepinephrine, dopamine) to sustain a minimum MAP of approximately 60 mmHg. At the end of the experiment, the animals were euthanized with an overdose of anesthesia and intracardial instillation of potassium (60 mval).

Intravenous administration of BNP

Various studies [5;10] favor a single BNP bolus application as a loading dose with subsequent continual administration in a lower dose over a defined period of time. We prepared the following protocol on the basis of information in the literature. BNP group: (n=10) 0.3 µg/kg BNP was administered by an IV bolus directly after reopening the AXCL followed by an infusion at a rate of 0.015 µg/kg/min for 60 minutes [START_REF] Colucci | Intravenous Nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure[END_REF].

Control group: (n = 10)

No BNP, placebo injection of 50 ml 0.9% saline.

Determination of hemodynamic parameters

CO was measured following sternotomy, pericardiotomy and preparation of the large vessels using a Transonic® perivascular ultrasonic volume flow sensor placed directly on the ascending aorta just above the aortic root (Transsonic Systems, Ithaca NY, USA). In addition, CO was measured at regular intervals until the end of the experiment. The RAP, MAP and the LAP were measured continually during the entire experiment with a catheter. The CI was calculated from CO and body surface area (BSA) (CI = CO/BSA; Table 1). The systemic vascular resistance (SVR) was calculated from the values of MAP, RAP and CO (Table 1).

Determination of metabolic and biochemical parameters

While the hemodynamic parameters were being recorded, the metabolic status (including lactate) was evaluated by analysing atrial blood gas samples. Levels of creatine phosphokinase (CPK), a sensitive and specific marker for myocardial damage, were measured once before ECC, then in regular intervals using the method of ADVIA chemistry systems from Bayer (Bayer AG, Leverkusen, Germany) together with human probes in the laboratory of the University of Tuebingen .

Determination of Plasma-BNP concentrations

Blood samples for P-BNP were collected in chilled plastic tubes containing EDTA, place immediately on ice, centrifugated within 20 min. at 4 0 C and stored at -70 0 C until assayed. Plasma levels of P-BNP were determined after extraction with a conventional available RIA-Kit for porcine BNP (Host: Rabbit, Range:1-128 pg/tube; Standard Peptide: 128 µg, Peninsula Laboratories, Inc. Division of Bachem, San Carlost, CA, USA).

The minimal detectable amount of the assay was 1pg/tube. The intraassay and interassay coefficient of variation was 8,5% and 10,5%.

Clinical

Further parameters

The amount of intravenous vasoactive agents required (dopamine, epinephrine, norepinephrine) to maintain an adequate perfusion pressure during ECC and the amount of fluids administered was documented.

Colloid and crystalloid fluids were given intravenously when MAP levels fell below 60 mmHg in combination with a low cardiac filling pressure. In the case of increased left ventricular filling pressure or high SVR, inotropic (adrenalin, dopamine) and/or vasoconstrictive agents (noradrenalin) were administered.

Statistics

All analyses were performed with SPSS 12.0 software. All results are stated as mean values SEM.

The number of test animals was calculated in collaboration with the Institute for Med. Biometrics at the University of Tuebingen (Director Prof. Dr. K. Dietz; Dr. R. Vonthein). Oneway-Anova-Analysis (with levene-Test for inhomogenous variances) was used to calculate the significance of BNP-induced hemodynamic changes between the two groups for normally distributed data (Shapiro-Wilk-Test). Non-parametric tests (Mann-Whitney) were used for non-normally distributed data. The Fisher's Exact test procedure was used to calculate an exact probablity value for the relationship between dichotomous variables. P-values < 0.05 indicated statistical significance.

Results

Cardiac output, cardiac index and cathecholamines

Prior to ECC, neither group showed a difference in CO or CI. After ending ECC, there was still no difference between the two groups. Fifteen minutes after ending ECC until the end of the experiment (60 minutes), the animals in the BNP group had a significantly higher CO (control group: 2.3 0.4 vs. BNP group: 3.3 0.2 l/min; p<0.039) and a significantly higher CI (control group: 2.47 0.4 vs. BNP group: 3.96 0.2 l/min/m²; p<0.0027) than the animals in the control group (Table 1, Figure 1 and Figure 2).

On average, the animals in the BNP group had a 41% (960 ml/min) higher CO than the animals in the control group after 60 minutes of independent cardiac activity. 2).

The CI in the BNP group was 60% higher than in the control group at the end of the experiment. The CI in the BNP group showed an increase of 23% compared with the initial values (Delta CI). CI in the BNP group was significantly higher than in the control group ( CI in the control group: -0.27 ± 0.31 vs. 0.74 ± 0.24 l/min/m 2 ; BNP group: p<0.019; Table 2).

From the beginning of the experiment, during ECC and after reopening the AXCL, a number of animals in both groups needed intravenous catecholamines (dopamine, norepinephrine, epinephrine) in order to maintain an adequate perfusion pressure during ECC. Seven out of 10 (70%) animals in the control group needed pharmacological support until the end of the experiment and during the entire phase of independent cardiac activity.

Only 3 (30%) animals from the BNP group needed catecholamines by the end of ECC. Only one animal in the BNP group needed pharmacological support in a lower dosage at the end of the experiment, which was after 60 minutes of independent cardiac activity. The animals in the BNP group needed significantly less catecholamines than those in the control group during the entire phase of independent cardiac action after ending ECC (60 minutes after ending ECC: p<0.001; Figure 3, Table 3).

Mean arterial pressure (MAP)

The mean arterial pressure (MAP) did not differ significantly throughout the entire experiment in either group (Table 1). However, 70% of the animals in the control group needed catecholamines after ending ECC to sustain a minimum MAP of 60 mmHg. MAP (preop-60 minutes) was not significantly different between groups (Table 2).

Systemic vascular resistance (SVR)

SVR did not vary between groups at the beginning of the experiment. However, clear differences were already seen 15 minutes after ending ECC. The animals in the BNP group had markedly lower SVR values until the end of the experiment, although it was not significant (Table 1; Figure 4). SVR (preop-60 minutes) was significantly lower (p<0.04; Table 2). The median SVR in the control group was not significantly higher at the end of the experiment than it was at the beginning.

Creatine phosphokinase (CPK)

There was no difference in CPK levels between the two groups at the beginning of the experiment until the start of ECC. During ECC, CPK levels increased in both groups and remained elevated in the control group.

The animals in the BNP group had significantly lower CPK values after opening the AXCL until the end of the experiment (Table 3, Figure 5). CK levels (preop-60 minutes) were significantly lower in the BNP group (p<0.0049; Copyright 2006 The Biochemical Society

Right atrial pressure (RAP)

The RAP did not vary between the groups at the beginning of the experiment. Fifteen minutes after ending ECC, the RAP in the BNP group was significantly lower (control group: 10.5 ± 0.8 vs. BNP group: 8.0 ± 0.8 mmHg; p<0.04). However, there was no other significant difference in RAP between the groups at any time throughout the experiment (Table 1 and2).

Plasma-BNP concentrations

Preoperatively the BNP concentration was equally low in the control group and in BNP-treated animals (Control group: 9.0 ± 1.2; BNP group: 9.8 1.1 pg/ml; Table 4). After connecting to the ECC, the plasma BNPconcentration increased significantly in both groups. In the control group the BNP plasma concentration declined after AXCL was removed. However, at further time points the BNP-concentrations were still higher than preoperatively. In BNP-treated animals BNP was administrated after removement of the AXCL. BNP was given as a single bolus (0,3 µg/kg) followed by continous infusion for one hour (0,015µg/kg/min). The highest BNP concentration in plasma was measured immediately after the bolus application. Further on the measured BNP values in plasma of the treated animals were constantly high until the end of the experiment (Table 4).

Other parameters (Lactate, LAP, HR)

There was no difference in mean lactate values between groups at the beginning of the experiment.

During ECC, lactate was increased in both groups. Sixty minutes after ECC, lactate values decreased slightly in the BNP group (Table 3). The LAP was low (approximately 5 mmHg) before ECC in both groups and increased up to 10 mmHg after ending ECC until the end of the experiment (Table 1 and Table 2). The mean heart rate at the end of the experiment was slightly lower in the BNP group but was not statistically significant (Table 1 and 2).

Fluid administration and urine output

The total fluid intake in the BNP group was approximately twice that of the control group (control group: 518 268 ml vs. BNP group: 1580 149 ml; p<0.003). The total urine output was markedly higher in the BNP group than in the control group (control group: 235 ± 81 vs. BNP group: 407 + 151 ml; p<0.021).

Discussion

This study shows the impressive hemodynamic effects of intravenously administered BNP during surgical procedures using extracorporeal circulation. The results demonstrate an outstanding improvement in cardiac output and cardiac index along with a decrease in systemic vascular resistance, almost without the need for inotropic support.

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136

Copyright 2006 The Biochemical Society

The present results are consistent with other studies of patients who received BNP for either chronic or acute, decompensated heart failure. There are, however, only very limited studies to date evaluating the effects of BNP after operations using ECC [11;12]. The induced myocardial ischemia and "postischemic heart failure" caused by multiple factors, can be only partially compared with other studies. Hayashida et al. [START_REF] Hayashida | Effects of intraoperative administration of atrial natriuretic peptide[END_REF] studied patients undergoing cardiac surgery who received ANP (atrial natriuretic peptide) perioperatively. Nine patients with mitral valve disease received ANP over 6 hours at a rate of 0.05 µg/kg/min after beginning ECC. The hemodynamic effect was seen in the form of a decrease in pre-and afterload, a decrease in PCWP, RAP, SVR and a significant increase in CI. At the same time, there were no adverse effects, i.e., hypotension, arrhythmias or allergic reactions to exogenous ANP.

The hemodynamic effects of BNP in the present study coincide with studies on BNP for decompensated heart failure [START_REF] Maisel | Nesiritide: a new therapy for the treatment of heart failure[END_REF][START_REF] Hobbs | Using BNP to diagnose, manage, and treat heart failure[END_REF][START_REF] Suttner | Natriuretic peptide system: physiology and clinical utility[END_REF]. BNP led to a 28% improvement in CI [START_REF] Abraham | Sytemic hemodynamic, neurohormonal, and renal effects of a steady-state infusion of human brain natriuretic peptide in patients with hemodynamically decompensated heart failure[END_REF], and in other studies, to an increase of 400 ml/m² [5;14] in patients with severe heart failure. In our study, BNP resulted in a significant improvement in CI (60%, or 1,490 ml/m²) and CO (41%, or 960 ml/min) compared with the control group.

The administration of BNP led to a rapid vasodilative effect, which resulted in a reduction in pre-and afterload and a decrease in SVR [START_REF] Corti | Vasopeptidase inhibitors: a new therapeutic concept in cardiovascular disease?[END_REF]. At the same time, BNP led to an increase in stroke volume and cardiac output. We found a reduction in SVR in the BNP group, which coincides with findings from several other studies [5;19].

In the control group, increased cardial filling pressure and decreased CO and CI resulted in the need for catecholamines and a reduction in fluid administration. In the BNP group, the cardiac filling pressure was low.

The urine output in the BNP group was significantly higher, which resulted in a higher volume of fluid administration. The greater need for fluids in the BNP group can be explained by the low filling pressure and by the fact that the glomerular filtration rate (GFR) was higher in these animals who did not have pre-existing cardiac disease. Jensen et al. found a higher GFR for in patients with congestive heart failure after treatment with BNP. The sympatoinhibitive effect of BNP reduces renal vascoconstriction, and this reduction, combined with an overall improvement in cardiac output, results in enhanced renal perfusion [START_REF] Burger | A review of the renal and neurohormonal effects of B-type natriuretic Peptide[END_REF]. In the kidney blood flow and filtration is increased with concomitant natriuresis and diuresis after treatment with BNP [START_REF] Houben | Vascular and renal actions of brain natriuretic peptide in man: physiology and pharmacology[END_REF].

The administration of BNP did not lead to a relevant decrease in MAP (caused by vasodilation of arteries and veins), which is consistent with recent studies [14;19].

The animals in the control group required various catecholamines, sometimes in combination (i.e., epinephrine, norepinephrine and dopamine) to treat myocardial contractile dysfunction caused by post-ischemic heart failure. In contrast, the animals in the BNP group needed almost no inotropic support. Only one animal required noradrenaline at the end of the experiment, and this in a concentration approximately seven times lower than the animals in the control group. Hayashida et al. [START_REF] Maisel | Nesiritide: a new therapy for the treatment of heart failure[END_REF] found that patients in both groups (placebo and ANP) needed approximately the same amount of catecholamines postoperatively. In this study, both groups of patients had severe cardiac disease. In contrast, neither group in our study had cardiac disease. This could account for the differing in results between these studies.

BNP is produced primarily in the left ventricle, and compensatory secretion of BNP is greater than ANP in congestive heart failure. Although the effects of ANP are very similar to BNP, ANP has a lesser effect in patients with congestive heart failure [START_REF] Jensen | Renal effects of brain natriuretic peptide in patients with congestive heart failure[END_REF], and has a shorter elimination time ( Copyright 2006 The Biochemical Society BNP/nesiritide has coronary vasodilative effects on both the coronary artery conductance and resistance. When administered, the coronary artery blood flow is increased, coronary resistance is decreased, and myocardial oxygen uptake is decreased [START_REF] Zellner | Coronary vasodilator effects of BNP: mechanisms of action in coronary conductance and resistance arteries[END_REF]. BNP leads to direct vasodilation of coronary vessels [START_REF] Matsumoto | Vasorelaxing effects of atrial and brain natriuretic peptides on coronary circulation in heart failure[END_REF]. It also improves reperfusion of the induced myocardial ischemia after ECC. Creatine phosphokinase, an indicator of ischemic myocardial damage, was significantly lower in the BNP group than in the control group, which could be indicative of a positive influence of exogenous BNP on myocardial function, particularly after ischemia [23;25] A further indication of hemodynamic improvement in the BNP group is the lower lactate values after ending ECC which show that the administration of BNP leads to a significant improvement in postischemic heart failure after ECC. Further studies are needed to evaluate the effects of BNP in patients with pre-existing heart failure/impaired cardiac function.

BNP lowers pre-and afterload without reflex tachycardia [START_REF] Van Der Zander | Hemodynamic and renal effects of low-dose brain natriuretic peptide infusion in humans: a randomized, placebo-controlled crossover study[END_REF]. It also reduces the release of endogenous catecholamines by the autonomic nervous system [START_REF] Suttner | Natriuretic peptide system: physiology and clinical utility[END_REF]. We did not observe reflex tachycardia following the administration of BNP. Reflex tachycardia can cause an increase in myocardial oxygen consumption, as well as an increase in venous pooling and a decrease in vascular tone. None of the animals had a significant change in heart rate while receiving BNP. The heart rate was even slightly lower in the BNP group.

Almost all studies report a significant decrease in RAP after exogenous BNP administration [START_REF] Colucci | Intravenous Nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure[END_REF]. We, however, could not confirm this. Only at one measurement point (15 minutes after ending ECC) was RAP significant lower in the BNP treated group which was in our view due to bolus administration of the peptide. After declamping the aorta the animals in the BNP group received a bolus dosage 20 times higher than the subsequent infusion rate. The time span from the bolus until 15 minutes after termination of ECC was 25 minutes. The onset of action of BNP following intravenous bolus administration is usually observed within 15-20 Minutes.

BNP's effect is similar to nitroglycerin, although the reduction of PCWP is greater [START_REF] Elkayam | Comparison of effects on left ventricular filling pressure of intravenous nesiritide and high-dose nitroglycerin in patients with decompensated heart failure[END_REF][START_REF] Peacock | Nesiritide in congestive heart failure associated with acute coronary syndromes: a pilot study of safety and efficacy[END_REF][START_REF] De Denus | Brain natriuretic peptide in the management of heart failure.The versatile Neurohormone[END_REF]. The LAP values, which correspond to the PCWP, remained unchanged following BNP administration. However, the animals in both groups did not have heart disease and, therefore, did not have elevated PCWP/LAP values preoperatively.

The observed increase in cardiac output and cardiac index is entirely due to afterload reduction secondary to vasodilation [START_REF] Burger | A review of the renal and neurohormonal effects of B-type natriuretic Peptide[END_REF]. The positive hemodynamic effects of BNP do not cause an increase in oxygen consumption since it is not dependent on the andrenergic receptor system [START_REF] Maisel | Nesiritide: a new therapy for the treatment of heart failure[END_REF]. No severe adverse effects have been reported yet, in contrast to other drugs used to treat heart failure or low-output syndrome. The improvement in hemodynamics is not caused by a secondary increase in intracellular cyclic AMP and calcium, as is the case with positive inotropic substances (e.g. dobutamine). Therefore, arrhythmias were not seen during BNP administration. A major advantage of BNP over phosphodiesterase inhibitors is the shorter half-life, which makes it easier to control. The other adverse effects associated with phosphodiesterase inhibitors like arrhythmia, hepatotoxicity and peripheral vasodilation do not occur with BNP. Peripheral vasodilation caused by phosphodiesterase inhibitors must often be counteracted with norepinephrine, which causes decreased kidney and intestinal perfusion.

Sackner-Bernstein et al. (Circulation 2005) [START_REF] Sackner-Bernstein | Risk of worsening renal function with nesiritide in patients with acutely decompensated heart failure[END_REF] showed an impaired renal function after nesiritide treatment. However, this study should be considered carefully in respect to our data because it is meta-analysis of studies involving patients with acute decompensated heart failure (ADHF). The disease pattern of ADHF may involve many factors that could result in elevated serum creatinine levels, rather than it may be an effect by BNP therapy itself. In addition the surgical procedere involving ECC may affect renal function per se. Furthermore the analysis was based on a nonavailabilty of primary study data. We believe that in our study, 60 minutes is too One limitation of this study was that the animals did not have heart disease or heart failure preoperatively, represented by the low BNP concentration preoperatively. Nevertheless, in both groups BNP concentration rised significantly after ECC was connected. The results of the control group clearly show that procedures using ECC which cause ischemia result in a significant increase in Plasma-BNP and in a decline in myocardial function and the need for catecholamines postoperatively. In BNP-treated swines on the other side, BNP levels arised after bolus injection of BNP and stayed constantly high thereafter until the end of the experiment. Following bolus administration and inititation of a continuous infusion, 60% of the steady-state hemodynamics response is observed within 15 minutes and 95% with 1 hour [START_REF] Kayser | The use of nesiritide in the management of acute decompensated heart failure[END_REF].

BNP demonstrates excellent effects, especially in the case of severely impaired myocardial function.

Further studies are needed involving hearts with pre-existing heart disease. These patients are often difficult to wean from ECC, and need high doses of pharmacological support. Postischemic heart failure is one of the main reasons for a greater demand for catecholamines and for a decline in myocardial contractile function following cardiac operations. Therefore, a further limitation of this study is the relatively short duration of ischemia, namely 30 minutes. Even so, the animals in the control group had an increase in Plasma-BNP and a decline in contractile function and even though they received inotropic support (epinephrine).

Conclusions

The present study shows BNP to be a highly potent, readily available agent that offers significant advantages, especially in cardiac surgery. When planning this experiment, we expected an increase in cardiac output and cardiac index of at least 10% and a considerable decrease in vasoactive agents for circulatory support.

These expectations were greatly exceeded. Copyright 2006 The Biochemical Society

  Porcine BNP-32 (BNP-32 trifluoroacetate salt; Bachem, Distribution Service GmbH; Weil a. Rhein, Germany) was administered (aqua ad injectabile) intravenously through central venous cannulation immediately Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society after releasing the aortic clamp. The BNP solution was prepared by dissolving the calculated amount of porcine BNP in 5% dextrose (50 ml).

  Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society Measurement times The measurements of hemodynamic and clinical chemistry parameters took place at the following time intervals: preoperatively following induction of anesthesia and after sternotomy: preop after beginning ECC: ECC start after declamping the aorta: (iv BNP-Bolus) AXCL open immediately after ending ECC: ECC end 15 minute intervals up to 60 minutes after ending ECC: 15 min -60 min

Clinical

  Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society short to determine any change in renal function. In fact, in our study like in others diuresis was clearly improved in BNP-treated animals.

Figure 1 :

 1 Figure 1:Final data 60 minutes after the termination of extracorporeal circulation (ECC) showed that administration of BNP led to a significant increase in cardiac output in the BNP group compared to the control group.

Figure 2 :

 2 Figure 2:Final data 60 minutes after the termination of extracorporeal circulation (ECC) showed the cardiac index in the BNP group was significantly higher than in the control group, and markedly higher than the initial values within the BNP group.

Figure 3 :Figure 4 :

 34 Figure 3:The number of animals in the control group who required catecholamines to treat post-ischemic heart failure after extracorporeal circulation (ECC) was significantly higher than in the BNP group (AXCL = aortic cross clamping)

Figure 5 :

 5 Figure 5: For the time period after reopening the aortic cross clamp to the end of the procedures, mean creatine phosphokinase (CPK) was significantly lower in the BNP group than in the control group

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society

Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society approximately

  9%. Delta CO (initial values compared with values at the end of the experiment) was higher than the initial values in the BNP group, whereas CO in the control group was lower ( CO in the control group: -0.22 0.3 l/min; CO in the BNP group: 0.62 ± 0.2; p<0.035; Table

The control group's CO was slightly lower at Clinical

Table 2 ). Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136

 2

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136

1-3 min)

. BNP is used world-wide as recombinant human BNP/Nesiritide since FDA approval in 2001.

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society

Table 1 :

 1 Hemodynamic parameters before and after BNP administration: IV BNP administration started after reopening aortic cross clamping with a bolus loading dose and continuous infusion until 60 minutes after ECC end (= end of trial)

	Time	Group	MAP	CO	CI	RAP	LAP	HR	SVR
			[mmHg]	[l/min]	[l/min/m 2 ]	[mmHg]	[mmHg]	[b/min]	[dyn*sec*cm -5 ]
	Preop	BNP	70.4 ± 5.3	2.66 ± 0.15	3.21 ± 0.5	8.5 ± 1.0	5.3 ± 0.8	88.1 ± 4.1	1757.5 ± 142
		Control	58.5 ± 5.4	2.55 ± 0.28	2.74 ± 0.2	6.8 ± 0.9	4.9 ± 1.0	88.5 ± 3.1	1683.7 ± 131
			NS	NS	NS	NS	NS	NS	NS
	ECC End BNP	59 ± 3.4	2.67 ± 0.4	3.23 ± 0.42	10.2 ± 0.7	10.1 ± 0.7	121.7 ± 8.8	1744.9 ± 195
		Control	58.3 ± 4.2	2.6 ± 0.3	2.81 ± 0.15	8.0 ± 0.8	9.4 ± 0.7	113.5 ± 12.1	1636.1 ± 189
			NS	NS	NS	NS	NS	NS	NS
	60 min	BNP	65.9 ± 3.6	3.3 ± 0.2	3.96 ± 0.2	9.8 ± 0.6	9.9 ± 0.7	97.5 ± 7.3	1404.6 ± 93
	after ECC
		Control	58.5 ± 5.4	2.3 ± 0.4	2.47 ± 0.4	9.2 ± 0.9	10.2 ± 1.7	115.3 ± 13	1946.9 ± 277
			NS	P<0.039	P<0.0027	NS	NS	NS	NS

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society

Table 2 :

 2 Differences ( Delta) of hemodynamic parameters and creatine phosophokinase (CPK) at the end of the experiment compared to the beginning of the experiment. IV BNP administration started after reopening aortic cross clamping with a bolus loading dose and continuous infusion until 60 minutes after ECC end = end of trial)

	Delta	Group	CO	CI	MAP	SVR	RAP	LAP	HR	CPK
			[l/min]	[l/min]	[mmHg]	[dyn*sec*cm -5 ]	[mmHg]	[mmHg]	[b/min]	[U/L]
	Preop-60´minutes	BNP	0.62 ± 0.2	0.74 ± 0.2	-4.5 ± 6.6	-352 ± 245	1.4 ± 1.0	4.6 ± 0.9	9 ± 9	223 ± 100
		Control	-0.22 ± 0.3	-0.27 ± 0.3	0 ± 5.3	263 ± 133	2.4 ± 1.2	5.3 ± 1.8	27 ± 12	1254 ± 221
			P<0.035	P<0.019	NS	P<0.04	NS	NS	NS	P<0.0049

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society

Table 3 :

 3 Creatine phosphokinase (CPK), lactate values and frequency of catecholamines before and after BNP administration: IV BNP administration started after reopening aortic cross clamping with a bolus loading dose and continuous infusion until 60 minutes after ECC end (= end of trial)

	time	Group	CPK	Lactate	Catecholamines
			[U/L]	[mmol/L]	(n)
	Preop	BNP	1015 ± 455	2.29 ± 1.6	0
		Control	1191 ± 924	2.92 ± 1.8	0
			NS	NS	NS
	ECC End BNP	1407 ± 306	5.71 ± 2.8	3
		Control	2354 ± 1034	5.47 ± 2.8	6
			P<0.018	NS	NS
	60 min	BNP	1238 ± 287	5.27 ± 2.4	1
	after ECC
		Control	2445 ± 1361	6.47 ± 2.5	7
			P<0.013	NS	P<0.001 *
	* Fisher´s Exact Test

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136 Copyright 2006 The Biochemical Society

Table 4 :

 4 Changes in Plasma-BNP concentrations: IV BNP administration started after reopening aortic cross clamping with a bolus loading dose and continuous infusion until 60 minutes after ECC end

	(= end of trial)
	BNP	preop	ECC	AXCL	10 min Re-	ECC	15	30	45	60
	[pg/ml]		start	open	perf.	end	min	min	min	min
	BNP	9.8	268.6	363.5	340.3	267.7	332.2	374.2	315.3	367.2
		± 1.1	± 62.1	± 90.2	± 84.5	± 92.2	± 69.5	± 1.2	± 4.0	± 67.3
			P<0.36							P<0.001
	Control 9.0	270.8	98.0	63.0	131.8	63.2	42.8	36.9	39.3
		± 1.2	± 89.9	± 28.2	± 9.3	± 49.6	± 11.3	± 5.9	± 4.8	± 19.6
			P<0.004							NS

Clinical Science Immediate Publication. Published on 31 Oct 2006 as manuscript CS20060136


Pdfauthor=neil Ashman

Tatiana M Brunini

Giovanni E Mann

Claudio Mendes Ribeiro

Muhammad M Yaqoob

Dr Neil Ashman
email: neil.ashman@bartsandthelondon.nhs.uk

Claudio Mendes

Increased L-arginine transport via system b 0,+ in human proximal tubular cells exposed to albumin

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

,

INTRODUCTION

Free amino acids (AA) are re-absorbed from glomerular filtrate in the renal proximal tubule. This occurs through transmembrane AA transporters that promote AA return from the urinary space to capillary networks in the interstitium. The transport systems responsible for this flux have been extensively described over the last decade [1].

Cationic amino acids (AA + ) such as L-arginine are transported by four potential transport systems, identified through characteristic affinity for substrate, and transport capacity: systems y + (first described and ubiquitously expressed), y + L, b 0,+ and B 0,+ [1,2]. The latter three systems mediate both AA + and neutral amino acid (AA 0 ) transport but differ in their interactions with inorganic monovalent ions. System b 0,+ is Na + -independent whilst system B 0,+ is Na + -dependent. System y + L transports cationic amino acids independently of sodium, and neutral amino acids only in the presence of Na + [1].

AA + transport by epithelial cells in the proximal tubule (and intestine) occurs through grouped transporters acting in concert to effect net absorption: the so-called amino acid hetero-exchanger (Figure 1) [2,3]. Apical system b 0,+ transport provides preferential AA + (against a negative intracellular potential) and cystine transport with exchange of AA 0 into the urinary space. Prompt unidirectional re-absorption of urinary AA 0 via system B 0 carriage prevents AA 0 loss [4,5]. Baso-lateral transport system y + L supports AA + efflux into the interstitium [6,7] with parallel AA 0 recycling (through system L-like and other transport) and flux into the interstitium [8]. For AA + , the net effect is transcellular reabsorption mediated by apical system b 0,+ and baso-lateral sytem y + L. Both b 0,+ and y + L transport systems' activity is a consequence of the expression of a dimer consisting of a light subunit linked by a disulfide bridge to an associated glycoprotein (the heavy subunit) forming a functional heterodimer. b 0,+ -associated transport protein forms a complex with rBAT, a type II membrane glycoprotein, orienting the system toward the apical side of PTEC, to provide system b 0,+ . Similarly, transport system y + L activity is result of the expression of a heterodimer consisting of one of two y + L-associated transport proteins (y + LAT1 and y + LAT2) and a basolaterally directed membrane protein, 4F2hc (CD98 heavy chain) [9].

The expression of transporter proteins and function of transport systems as a whole may be influenced by changing cell requirements, or cell injury. There is clear evidence of disease-related changes in the transport of L-arginine, the precursor for a number of important signalling pathways [10][11][12][13]. L-arginine is the substrate for the synthesis of nitric oxide (NO), polyamines, agmatine, proline, glutamate and proteins. Of these, the uptake of L-arginine and its conversion to NO is best studied in cells or tissues exposed to injury, stress, growth factors, or other cytokines, principally in cells capable of high nitric oxide output (macrophages or endothelial cells) but also in platelets and red blood cells [14][15][16][17]).

Changes in L-arginine transport in cells not associated with high NO output are increasingly being recognised in the context of cell growth [18,19], cell activation [20] and cell injury [21]. From these studies (and our own work [22]), circumstantial evidence links L-arginine transport with increased arginase activity in proliferating cells (arginase converts L-arginine to urea and L-ornithine, the precursor of mitogenic polyamines). Specifically in the kidney, the kinetic characteristics of proximal tubular cell AA + transport are described in unstressed cell culture [7,23,24]. Transport (principally system y + L) has been shown to be modulated by NO production (in rat renal medullary cells [25] and animals in vivo [26]), and modified in animal models of renal injury (ischaemia-reperfusion injury [27], lipopolysaccharide-induced septic shock [28] streptozotocin-induced diabetes [29] and uraemia [30]). This data suggests renal cells may exhibit altered AA + transport in response to a changing cellular environment, be this stress, injury or growth.

Proteinuria has been strongly implicated in the tubular atrophy and interstitial fibrosis characteristic of progressive proteinuric nephropathies [31]. In vitro, albumin directly affects proximal tubular cell turnover, activation and phenotype through signalling networks dependent on the endocytosis and degradation of albumin (and albumin-bound lipid). As such cultured PTEC exposed to albumin offer an interesting model in which to examine AA + transport: we have recently shown that recombinant human albumin induces human proximal tubular cell proliferation as a result of increased L-arginine metabolism to polyamines, key regulators of the cell cycle [22]. Increased flux along the arginine-ornithine-polyamine pathway occurs in conjunction with increased L-arginine transport capacity, as has been described in proliferating non-renal cells [18,19].

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

There are no reports to our knowledge of AA + transport kinetics in human PTEC, nor any data quantifying changes in AA + (more specifically L-arginine) transport in models of proximal tubular disease. As a result of our previous work, and recognizing that growth factors influence L-arginine transport and utilization in other tissues and cell lines, we have examined albumin's effect on the apparent kinetics of L-arginine transport in human proximal tubular epithelial cells in culture, and sought to describe the role of L-arginine transport in albumin-induced cellular proliferation.

METHODS

Reagents and cell culture

All amino acids, inhibitors and other chemicals, including [2,3 3 H]-L-arginine, and [ 3 H]-L-lysine were obtained from Sigma-Aldrich Co Ltd (Poole, Dorset, UK). Recombumin  , an ultra-pure recombinant human albumin derived from Saccharomyces cerevisiae was obtained from Delta Biotechnology (Nottingham, UK). Recombinant human albumin is entirely fatty acid free, allowing study of the potential effects of albumin itself, without the confounding effect of albumin-bound factors. Immortalized HK-2 human PTEC [32] were maintained in Dulbecco's modified Eagle's Medium (DMEM)/Ham's F-12, supplemented with 20 mmol/L HEPES buffer, 2 µmol/L glutamine, 5% fetal calf serum (FCS, Labtech International, Ringmer, East Sussex, UK) and penicillin G 100 U/mL, amphotericin B 0.25 µg/mL and streptomycin 100 µg/mL. MEM Select-amine kit (GIBCO, UK) was used for experiments involving L-arginine deprivation. LLC-PK1 cells were cultured in DMEM, 10% FCS and antimicrobials as above.

Fresh growth medium was added to cells every 2 to 3 days until confluent in 16 mm diameter wells [6]. Cells were cultured in serum-free conditions for 24 hours prior to experimental manipulation. In all experiments cells were stimulated with recombinant human albumin (20 mg/mL) in serum-free medium, or serum-free medium alone. All media had comparable pH, and diluent alone in medium did not reproduce the experimental effect of albumin.

Amino acid uptake assays

Experimental medium was aspirated off prior to the assay being performed [33], and monolayers washed in warmed Krebs' buffer prior to incubation at 37 0 C for 30 minutes in Krebs' buffer containing 100 µmol/L L-arginine (to exclude any immediate osmolar effect between experimental media). In studies of sodium dependence, sodium was replaced with potassium. Uptake media were prepared by adding L-[ 3 H]-arginine or L-[ 3 H]-lysine to Krebs' buffer or sodium-free medium at a final concentration of 0.5-1 µCi/mL. Uptake was started by the addition of 0.2 ml of uptake medium, and terminated by rapid removal, before washing three times with an ice cold stop solution containing 10 mmol/L L-arginine. A time point at 4 minutes was chosen for all experiments (uptake being linear at this point, data not shown) [27,34]. Cells were lysed in 0. buffer (1% NP-40, 0.5% w/v sodium deoxycholic acid, 0.1% SDS in AnalaR ™ grade water), and removed for liquid scintillation counting in 2 mL Ecoscint (National Diagnostics, Hull, UK). Counting was performed in a LKB Wallac Rackbeta scintillation counter against appropriately quenched blanks. Background values were subtracted.

Kinetic parameters calculated by non-linear regression analysis for a saturable system plus diffusion. The maximal velocity of transport (V max , in pmol/min/10 6 cells) and affinity constant (K m , that concentration of substrate that leads to half-maximal transport velocity, in µM) were then calculated.

Strategy for identifying cationic amino acid transport systems

Cationic amino acids (like L-arginine) are transported across membranes by specific transporter proteins, themselves broadly grouped as 4 transport systems (systems y + , y + L, b 0,+ and B 0,+ ) with distinct characteristics [1,35]. Strategies for identifying specific systems utilized the following characteristics: (1) system y + transport accounts for low affinity high capacity AA + transport, insensitive to inhibition with competing AA 0 ; (2) system B 0,+ accepts a broad range of subtrates, but is wholly Na + dependent; (3) systems y + L and b 0,+ are broad-scope higher affinity systems transporting both AA + and AA 0 .

They may be discriminated in that system y + L AA 0 transport is Na + dependent, whilst system b 0,+ AA 0 transport is Na + -independent. Removing Na + from experimental medium limits system y + L AA 0 transport (allowing unrestricted [ 3 H]-L-arginine influx). Also, Lcystine is transported through system b 0,+ but not system y + L. N-ethylmaleimide (NEM, as 0.2 mM in Krebs solution pre-incubated at 37 0 C for 10 minutes prior to assays) is a non-competitive sulfhydryl reagent inhibitor of system y + . p-Chloromercuribenzoic acid (pCMB, as 1 mM in 0.01% DMSO choline chloride 10 mM with 10 µM EDTA pre-incubated for 5 minutes prior to assays) is an organic mercurycontaining compound that modifies thiol bridges, thus preventing heterodimer formation crucial for systems y + L and b 0,+ . Inhibition was measured as the transport rate (velocity) of a fixed concentration of substrate (1 µM L- L-arginine transport velocity by 50% (IC50) is used with the known K m for transport to derive the inhibition constant (K i ):

K i = IC50 (1 + [substrate])/K m
An increasing K i implies less efficacious competitive inhibition: higher concentrations of inhibitor are required to limit substrate influx.

Efflux studies [6] were performed by loading monolayers for 60 min with 100 µM [ 3 H]-L-arginine, withdrawing this and washing three times with L-arginine-free Krebs solution at 37 0 C. 300 µL warmed Krebs solution alone or Krebs containing 1 mM L-arginine was then added, and efflux into this medium measured at 4 minutes (on the linear part of the efflux curve as measured over 30 s to 10 min).

Reverse transcriptase polymerase chain reaction

Total RNA was prepared from HK-2 cells from independent experiments as previously Cycling temperatures and times were as follows: b 0,+ -AT was initiated for 5 min at 94 0 C, then run for 39 cycles of 95 0 C (60s), 55 0 C (90s) and 72 0 C (90s) with a final extension at 72 0 C for 10 min. y + LAT-1 and -2 were initiated for 3 min at 94 0 C, then run for 35 cycles of 94 0 C (45s), 59 0 C (45s) and 72 0 C (120s) before a final extension at 72 0 C for 5 min.

CAT-1 and -2B were initiated for 3 minutes at 94 0 C, then run for 34 cycles at 94 0 C (30s), 59 0 C (45s) and 72 0 C (120s). After amplification, RT-PCR products were separated on a 1% (wt/vol) agarose gel containing 0.2 µg/mL ethidium bromide. Densitometry was performed using Gel Blot Pro ® software (UVP Systems, Ultraviolet Products Ltd, UK).

Cell proliferation

Cell proliferation was measured using the [ 3 H]-thymidine incorporation assay.

Monolayers at 70% confluence in experimental media had 2 µCi [ 3 H]-thymidine added for the final 2 of 24 hours. Each well was then washed five times in ice-cold DMEM-Hams F12, after which 1 mL of ice-cold 5% v/v trichloracetic acid (TCA) was added for 1 hour at 4 0 C. TCA was then aspirated off, and a further TCA wash performed.

Thereafter 2 mL ice-cold ethanol with 200 µM potassium acetate was added for 5 minutes and removed. Two further 15 minute incubations in 2 mL ethanol/ether (3:

1 v/v)
were performed, before the monolayers were dried in air and solubilized in 1 mL 0.1M

NaOH. This was withdrawn into scintillation vials to which 3mL Ecoscint A (National Diagnostics, UK) was added, and counted in a beta-counter.

Statistics

All data are expressed as mean ± SD of measurements in n different cell cultures. Curve fitting was performed by non-linear regression using curve fitting software (GraphPad Prism ™ , San Diego, CA, USA). Shapiro-Wilk testing was done to determine the distribution of data. Statistical analysis was generally performed using one-way ANOVA with post-hoc Bonferroni testing for multiple comparisons. If non-parametric, Kruskal-Wallis ANOVA with appropriate post-hoc testing was used. A P value of <0.05 was accepted as significant. In a second proximal tubular cell line (LLC-PK1), total L-[ 3 H]-arginine influx was also significantly increased after albumin incubation (n=6 independent experiments, table 1), excluding a cell-specific effect. These data confirm that albumin, a mitogen to which the proximal tubule is exposed in both health and disease, leads to increased cationic amino acid transport at 20 mg/mL in cultured PTEC.

Identification of AA + transport systems in albumin-exposed PTEC

The identity of potential AA + transport systems (systems y + , B 0,+ , b 0,+ and y + L) was established as described in the Methods. Firstly, the influx of 1 µM L-[ 3 H]-arginine was examined in the presence of increasing concentrations of competing AA + (0 -1000 µM), and the degree of inhibition expressed as an inhibition constant (K i , see Methods).

Unlabeled L-arginine, L-ornithine and L-lysine (all AA + ) potently inhibited L-[ 3 H]arginine influx (n = 6 independent experiments for each AA + , table 2), confirming universal transport of cationic AA + . Although the neutral amino acid L-leucine is not significantly transported by the system y + , increasing concentrations of L-leucine were equally potent in inhibiting influx of 1 µM L-[ 3 H]-arginine (n = 6 independent experiments). This suggests that L-arginine is transported into PTEC via cationic and neutral amino acid transport systems such as systems B 0,+ , b 0,+ and y + L, but not y + .

AA + transport through system B 0,+ is sodium-dependent, however, no statistically significant change in inhibition was observed in the absence of Na + in experimental medium, excluding system B 0,+ transport. To distinguish between the remaining two AA + transport systems, two strategies were used: firstly, system b 0,+ , but not system y + L, mediates the uptake of L-cystine. In the concentration range of 0 -1000 µM, L-cystine significantly inhibited 1 µM L-[ 3 H]-arginine, though to a lesser extent than seen with Lleucine (n = 4 independent experiments, table 2). Secondly, system y + L requires the presence of sodium to mediate AA 0 (but not AA + ) transport, unlike b 0,+ which is sodiumindependent. In the absence of sodium, L-leucine was a significantly less potent inhibitor of 1 µM L-[ 3 H]-arginine transport (a rise in K i in the absence of Na + strongly suggests system y + L activity, n = 5 independent experiments, table 2). These findings are consistent with AA + transport activity via systems b 0,+ and y + L as reported previously in healthy opossum kidney cells [7].

We could not find evidence for system y + transport. N-ethylmaleimide (NEM), a noncompetitive and selective sulfhydryl inhibitor of system y + transport, did not affect L-[ 3 H]-arginine transport in our cells (n = 4 independent experiments, Figure 3). Pchloromercuribenzoic acid (pCMB) is an organic mercury compound that disrupts thiol bridges (and thus heterodimer formation) inhibiting broad-scope transport via systems b 0,+ and y + L. In our cells L-[ 3 H]-arginine transport capacity was significantly reduced after pre-treatment with pCMB (n = 4 independent experiments, Figure 3), confirming our kinetic data using competitive AA.

Systems b 0,+ and y + L appear to be functioning in tandem. Cells pre-loaded with L-[ 3 H]arginine (see Methods) were then washed and incubated with an excess of unlabeled Larginine. This apical (unlabelled) trans-stimulation was associated with increased L-[ 3 H]arginine efflux from pre-loaded cells. Increased activity of inward transport is thus coupled with increased outward transport, characteristic of a functioning heteroexchanger system as described by Chillaron et al (Figure 1). A five-fold increase in L-[ 3 H]-arginine efflux was demonstrated in both control and albumin-incubated cells (n=4 independent experiments, Figure 4).

Albumin is associated with increased transporter mRNA in PTEC

We examined expression of potential transporter proteins after albumin incubation. b 0,+associated transporter protein (b 0,+ AT) is a 12-domain transmembrane protein encoded on SLC7A9 [3,38]. b 0,+ AT forms a disulfide-bridge heterodimer with the rBAT heavy chain, (encoded on SLC3A) to orientate the transporter complex apically and mediate b 0,+ transport activity [3]. b 0,+ AT mRNA was significantly increased at 8 hours by 20 mg/mL albumin exposure (n=5 independent experiments, Figure 5). System y + L transport is mediated by a heterodimer composed of an immunoglobulin-family protein 4F2hc (CD98, thought to orientate its' coupled transporter basolaterally) and a light subunit, y + L associated transporter (y + LAT). The light subunit may one of two proteins, y + LAT-1 or -2, encoded on SLC7A7 and SLC7A6 respectively [38]. Appreciable y + LAT-1 mRNA was detectable in HK-2 PTEC, but no significant change was seen with albumin incubation (n=5 independent experiments, Figure 6). Only very small amounts of y + LAT-2 mRNA were detected. System y + provides pure cationic amino acid transport through at least 4 transporters the cationic amino acid transporters (CATs). CAT-1, encoded on SLC7A1, is thought to be ubiquitously expressed in mammalian cells, and has been described in embryonal kidney cells [39]. CAT-2B, encoded on SLC7A2, is frequently linked to NOS-2 activity, and has been described in tubular preparations [27]. CAT-1 mRNA was found in small quantities in HK-2 PTEC, and was not influenced by albumin incubation (n=5 independent experiments, Figure 7). CAT-2B mRNA was not detected.

Cycloheximide inhibits RNA-protein translation. Pre-incubation with cycloheximide (Cx) 5 µg/mL abolished increases in 50 µM L-[ 3 H]-arginine influx observed after 24 hours incubation with rHSA to control levels in HK-2 PTEC, suggesting new transporter protein synthesis (n = 4 independent experiments, Figure 8).

Albumin's effects are not mediated through PI 3-kinase

Receptor effect not significantly altered by the addition of L-lysine 1 mM to albumin-containing medium for 24 -48 hours (Figure 9). HK-2 PTEC cultured in medium containing all essential amino acids but excluding L-arginine (and L-citrulline) were exposed to albumin 20 mg/mL. At 48 hours (but not 24 hours) L-arginine deprivation significantly reduced albumin-induced [ 3 H]-thymidine incorporation to control levels (Figure 10).

DISCUSSION

AA + transport capacity in human PTEC

Albumin is a growth factor in vitro for proximal tubular epithelial cells. As L-arginine has important metabolites regulating cell growth and function, we examined the transport of L-arginine in PTEC exposed to albumin, and the role of transport in subsequent proliferation. AA + transport has been described in the renal proximal tubule in vivo, in isolated tubular perfusion [42], cell culture [6,7] and membrane vesicle preparations [24,43]. AA + are transported through broad-scope, high affinity systems consistent with system b 0,+ , and to a lesser extent system y + L in cultured human PTEC acting as a functioning hetero-exchanger. Transport activity and b 0,+ -associated transporter expression was in association with albumin, through a pathway independent of PI 3kinase. Inhibition of L-arginine transport with L-lysine did not restrict PTEC proliferation, although L-arginine deprivation did.

Albumin's effect on AA + transport capacity

Albumin induces proliferation and survival of PTEC in culture [22,41,44]. After 24 h incubation with 20 mg/mL albumin, a significant increase in AA + transport was observed.

Prior work has shown this increase to be dose-and time-dependent, and albumin-specific rapidly available for metabolism, unlike L-lysine (principally available for protein synthesis). That albumin increases the transport capacity of both AA + excludes transport artefact and effects of L-arginine metabolism [46].

The increased transport capacity observed after albumin incubation was again largely through the broad-scope high affinity system b 0,+ , with increased expression of b 0,+associated transporter mRNA. Pre-treatment with cycloheximide abolished any albumininduced increase in L-arginine influx, confirming that new (b 0,+ AT) transporter protein synthesis is important in the observed effect.

Consistent with our kinetic findings, only small quantities of both CAT-1 and -2B mRNA was found in control or albumin-exposed PTEC. CAT-1 and CAT-2 mRNA has been described in the rat proximal tubule exposed to hypoxia (ischaemia-reperfusion) or bacterial lipopolysaccharide: specific AA + transporters appear to be co-induced in parallel with inducible NOS in association with proximal tubular injury [27,28]. Our data in healthy or proliferating proximal tubular cells (where only very small quantities of NO should be produced by constitutively expressed NOS) does not support system y + activity. Interestingly, L-arginine transport in embryonal HEK 293 cells transfected with iNOS is described via system y + transport activity induced by CAT-1 and -2B protein expression [39]. It is possible that cellular injury may induce a reversal of phenotype to a more primitive state in which increased AA + transport capacity is required to support the metabolic needs of such cells, with increased CAT-1 expression and activity.

Albumin and L-arginine: transport-or arginine-dependent proliferation?

Why should albumin, avidly re-absorbed by the proximal tubule in health, modulate AA + transport? Albumin is an important signalling and regulatory molecule in PTEC, activating STAT proteins [47] and MAP kinase pathways with downstream effects on the NF-κB-dependent production of chemo-attractants, pro-fibrotic agents, matrix proteins and vasoconstrictors [31]. Albumin also modifies cell turnover, acting as a mitogen in cultured cells (at the dose range used in this study) [41]. This is due in part to increased arginase activity, with shunting of L-arginine toward the synthesis of pro-proliferative polyamines [22].

In arginine-utilising cells such as macrophages (and others capable of high NO output), increased system y + AA + transport, increased CAT synthesis [15] and increased NO production occurs in tandem. This occurs despite a high intracellular L-arginine concentration (well above the K m for nitric oxide synthase): the 'arginine paradox'. In cells not capable of high output of NO (and in the absence of increased NO production), Other AA + competitively inhibit of L-arginine transport: in albumin exposed PTEC, coincubation with 1 mM L-lysine did not restrict albumin-induced proliferation. Although this suggests L-arginine transport to be an epiphenomenon, there is an important caveat to this data: PTEC are the principal site of L-arginine synthesis from L-citrulline, at a rate dependent on L-citrulline availability [50]. L-arginine may be available to arginineutilizing enzymes such as arginase through de novo synthesis, or even through the degradation albumin or other proteins to L-arginine (and L-citrulline). This is also consistent with the concept of arginine compartmentalization: transported L-arginine may be required to replenish cytosolic L-arginine stores, but not directly necessary for arginase activity. Equally, over 24 hours, sufficient L-arginine may be transported into cells to maintain arginase activity despite L-lysine. It has been shown that in models of cytokine-induced NO production in both endothelial cells [15] and vascular smooth muscle cells [51], excess L-lysine was not able to reverse increased NO generation, despite inhibiting transport, ascribed to compartmentalization of L-arginine.

In contrast, L-arginine deprivation significantly inhibited albumin-induced PTEC proliferation at 48 but not 24 hours: no L-citrulline was present in the arginine-free medium, and it is tempting to speculate that once de novo L-arginine synthesis was exhausted for lack of substrate, arginase-dependent proliferation through the polyamines was reversed. Tumour cells share with PTEC the ability to recycle L-citrulline to Larginine. Tumours are notably polyamine-dependent for proliferation: arginine deprivation prevents growth in such cells through an arginase-dependent pathway (rather than impaired protein synthesis) [52,53]. A similar mechanism appears to be at work here.

The proximal tubular endocytosis of albumin, and albumin's mitogenic effects are in part mediated via the cytosolic enzyme phosphatidylinositide 3-kinase (PI 3-kinase) (and a subsequent downstream MAP kinase pathways) [40]. PI 3-kinase inhibition had no effect on albumin-induced [ 3 H]-L-arginine transport, but was a potent inhibitor of proliferation in our model [22,40]. This result, together with our data demonstrating L-lysine to have no effect on proliferation, suggests the inward transport of L-arginine is not directly influenced by albumin endocytosis or critical to proliferation, but rather associated with it. Similar changes may be seen with the synthesis, surface expression and activity of the Na + /H + exchanger isoform 3 (NHE3) in response to albumin in kidney cells [44].

We believe that human PTEC proliferate in response to recombinant human albumin in an L-arginine (but not L-arginine transport) dependent manner, with increased arginase activity converting L-arginine into L-ornithine, and subsequently into polyamines required for cell cycling and growth. This study demonstrates that L-arginine influx is increased in albumin-exposed PTEC through increased system b 0,+ transport activity and increased b 0,+ -associated transporter mRNA by a mechanism not related to PI 3-kinase.

Such transport is not critical to proliferation, and may be required to replenish specifically compartmentalized L-arginine stores. However, L-arginine deprivation reverses albumin's actions as a mitogen, re-inforcing the importance of the arginineornithine-polyamine axis. phosphate dehydrogenase (GAPDH) cDNA from PTEC incubated in control medium (lane 1) and rHSA 20mg/ml (lane 2) at 8 hours. B: Relative amount of mRNA quantitated by densitometry and expressed as y + LAT-1 mRNA/GAPDH mRNA ratio (*P<0.05).

Figure and table legends

Similar findings were found at 24 hours.

  [ 3 H]-arginine) in the presence of increasing concentrations of unlabelled inhibiting amino acids (0-1000 µM). L-cystine was prepared as cysteine 0 -1000 µM in the presence 10 mM diamide [35]. The inhibitor concentration that reduces Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

  described [22]. Primers were obtained from MWG Biotech (Ebersberg, Germany) as follows. b 0,+ -AT, sense 5'-GTTCATCTCGACAGTGAACT-3', antisense 5'-CACGTTGATGAGGATGTAGCA-3' (330 bp) [36]. y+LAT-1, sense 5332bp) [37]. CAT-1, sense 5'-CGTCCCTCTTGATCTGCTTC-3', antisense 5'-GGCTGGTACCGTAAGACCAA-3' (427bp) [37]. CAT-2B, sense 5'-CCCAATGCCTCGTGTAATCT -3', antisense 5'-ACATTTGGCTGGTCGTAAG -3' (262bp) [37].

  -mediated endocytosis of albumin in PTEC occurs through the specific transmembrane protein megalin, a process in part controlled by.Inhibition of this enzyme restricts tubular albumin uptake and albumin-induced cellular proliferation[40,41]. Pre-treatment and co-incubation with PI 3-kinase inhibitors (wortmannin 100 nM or LY294002 20 µM, used in tandem to confirm specific PI 3kinase-mediated effects[41]) had no effect on the observed albumin-induced increase in AA + transport. 50 µmol/L L-[ 3 H]-arginine influx in HK-2 PTEC treated with 20 mg/mL albumin was 223 ± 6.6, with 100 nM wortmannin was 201.3 ± 39.5 and with LY294002 20 µM was 201.9 ± 25.9 (all results pmol/min/10 6 cells, n = 5 experiments, P = NS). In studies of cell proliferation, we confirm that wortmannin 100 nM and LY294002 20 µM Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158 as used above reversed the significantly increased [ 3 H]-thymidine incorporation observed with 24 hours incubation with 20 mg/mL albumin (data not shown). It appears that an unrecognized complementary pathway is responsible for increased L-arginine transport capacity in albumin-exposed PTEC. L-arginine deprivation restricts albumin-induced PTEC proliferation Its is well-established that recombinant human albumin induces PTEC proliferation [40], depending in part on the arginine-ornithine-polyamine axis [22]. Non-competitive inhibitors of short-term AA + transport such as pCMB are too toxic for use in 24 hour culture. L-lysine 1mM is an efficient competitive inhibitor of L-[ 3 H]-arginine transport (table 2) in short-term experiments (< 60 min). HK-2 PTEC co-incubated with 20 mg/mL albumin showed increased proliferation as measured by [ 3 H]-thymidine incorporation, an

[

  22]. Albumin-induced increases in AA + transport capacity were confirmed by using a second labelled AA + ([ 3 H]-L-lysine influx). L-lysine and L-arginine have marginally different transport characteristics [45], but very different intracellular fates. L-arginine is

  growth factors may lead to increased AA + transport[20, 48], with L-arginine made available to arginase for subsequent synthesis of the mitogenic polyamines required for cell proliferation[19]. Equally, nutrients themselves induce signalling cascades and promote anabolism[49]. Amino acid transporters themselves may initiate signalling through protein-protein interactions, through cell volume and pH changes, or through direct intracellular sensing[38]. A number of pathways have been implicated in nutrient signalling, including mammalian target of rapamycin (mTOR) and other regulators of transcription[38]. Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Figure 1 .

 1 Figure 1. Co-ordinated amino acid transport in proximal tubular epithelial cells (after Chillaron et al [3]) Apical b 0,+ transport accounts for AA + (and cystine) uptake toward the electronegative intracellular milieu, with baso-lateral y + L transport exporting AA + into the interstitium (intracellular Na + concentrations favouring AA + transport). Parallel AA 0 shuttling ensures high intracellular concentrations of AA 0 to maintain inward AA + flux.

Figure 2 .

 2 Figure 2. L-[ 3 H]-arginine transport capacity is significantly increased by exposure to albumin for 24 hour in PTEC. (control as open squares and rHSA as closed squares [22]).

Figure 3 .

 3 Figure 3. System y + -like transport is not responsible for [ 3 H]-L-arginine influx in HK-2 PTEC. N-ethylmaleimide (NEM) is a non-competitive inhibitor of system y + transport. p-chloromercuribenzoic acid (pCMB) is a non-competitive inhibitor of heterodimeric transporters responsible for b 0,+ and y + L transport. (*P < 0.01 for control compared to pCMB).

Figure 4 .Figure 5 .

 45 Figure 4. Trans-stimulation is observed in HK-2 PTEC in both control (open bars) and albumin-incubated (closed bars) cells. (*P < 0.01 compared to L-arginine-free medium)

Figure 6 .

 6 Figure 6. y + LAT-1 mRNA is not increased with apical albumin. A: Ethidium-stained agarose electrophoresis gel showing RT-PCR amplified y + LAT-1 and glyceraldehyde-3-

Figure 7 .

 7 Figure 7. CAT-1 mRNA is not increased with apical albumin. A: Ethidium-stained agarose electrophoresis gel showing RT-PCR amplified CAT-1 and glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA from PTEC incubated in control medium (lane 1) and rHSA 20mg/ml (lane 2) at 8 hours. B: Relative amount of mRNA quantitated by densitometry and expressed as CAT-1 mRNA/GAPDH mRNA ratio (*P<0.05).Similar findings were found at 24 h.

Figure 8 .Figure 9 .

 89 Figure 8. Increased L-arginine influx in albumin-exposed PTEC is limited by cycloheximide pre-treatment (Cx). (*P < 0.05 for rHSA compared to control, Cx)

Figure 10 .

 10 Figure 10. Arginine deprivation reverses albumin-induced proliferation at 48 (but not 24 hours). ( * P < 0.05 for rHSA compared to arginine deprived rHSA. Significantly increased proliferation is observed with rHSA compared to control).

Figure 2 L

 2 Figure 2

  Figure 5

  Figure 8

  Figure 10

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

3 mL RIPA Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158 Copyright 2006 The Biochemical Society 10 RESULTS Cationic amino acid (AA + ) transport in albumin-exposed PTEC

	Total L-[ 3 H]-arginine influx in HK-2 PTEC was carrier-mediated, exhibiting an apparent
	K m of 64 ± 13 µM and an apparent maximal velocity (V max ) of 401 ± 17 pmol/min/10 6
	cells. Incubation with 20 mg/mL recombinant human albumin for 24 h significantly
	increased total L-[ 3 H]-arginine transport capacity with an apparent V max of 628 ± 32
	pmol/min/10 6 cells (and similar K m of 74 ± 13 µM) (n= 12 independent experiments,
	Figure 2). We have previously observed this increase in total L-[ 3 H]-arginine transport to
	be dose-and time-dependent [22]. To exclude any effects of metabolism on L-arginine
	transport, experiments were also performed with a different AA

+ , L-[ 3 H]-lysine, using the same experimental protocol. L-[ 3 H]-lysine uptake in HK-2 PTEC was similarly increased by albumin (n=3-7 independent experiments, table

1

).

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158 TablesTable 1

 1

			Control	Albumin-incubated
		Km	vmax	Km	vmax
	L-arginine in HK-2 PTEC	64 ± 13	401 ± 17	74 ± 13	628 ± 32 *
	L-lysine in HK-2 PTEC	62 ± 16	283 ± 20	66 ± 18	382 ± 28 *
	L-arginine in LLC-PK1 PTEC	94 ± 21	235 ± 16	77 ± 17	348 ± 21 *
	Table 2
	Inhibition of L-arginine transport in HK-2 PTEC
	Condition		Ki (inhibition constant)
	Cationic amino acids
	L-arginine		17.3 ± 5.6
	L-lysine		21.3 ± 4.4
	L-ornithine		49.3 ± 13.8
	L-arginine (in the absence of sodium)		29.6 ± 8.8
	Neutral amino acids
	L-leucine (in the presence of sodium)		23.2 ± 6.1
	L-leucine (in the absence of sodium)		121.5 ± 34.1*
	L-cystine		48 ± 11

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

Table 1 . Cationic amino acid transport is significantly increased in albumin-exposed PTEC after 24 hours

 1 . ( * P<0.01 for control compared to albumin-incubated, all K m in µM, and all V max in pmol/min/10 6 cells).

Table 2 . Competitive amino acids inhibit L-arginine influx consistent with broad- scope transport.

 2 An increasing K i (inhibition constant) implies less efficacious

Clinical Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158 competitive

  inhibition. (*P < 0.05 for L-leucine inhibition of [ 3 H]-L-arginine influx in the presence of sodium compared to its' absence).

Table 3 . Relative amount of transporter mRNA after albumin incubation at 8 and 24 hours

 3 . mRNA quantitated by densitometry and expressed as a ratio compared to GAPDH. (*P < 0.05 compared to control).

	Clinical

Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

	Figures
	Figure 1
			AA + , cystine
					Urinary space
	Electrical		AA 0
	gradient	+	b 0,+	B 0
		-			Apical
					membrane
			AA +
				AA 0
					AA 0 systems,
	Baso-lateral			including system L
	membrane		Low Na +
			y + L	High Na +	Interstitium
			AA +	AA 0

Science Immediate Publication. Published on 23 Aug 2006 as manuscript CS20060158

		24 hours		48 hours
	[ 3 H]-thymidine incorporation as % control	1.4 1.6 1.8
		Control rHSA 20	rHSA 20	Control rHSA 20	rHSA 20
		mg/mL	mg/mL	mg/mL	mg/mL
			(Arg-		(Arg-
			deprived)		deprived)

*

Clinical

Supported by a grant from the Joint Research Board of St Bartholomew's and the Royal London Hospitals, UK Running head: L-arginine transport in albumin-exposed PTEC