Objectives: As a vasodilator, nitric oxide is considered to play a significant role in the homeostatic regulation of renal hemodynamics. To test the hypothesis that a kidney graft is capable of producing nitric oxide immediately after renal transplant surgery, we examined the possibility that it positively affects local metabolic acidosis.

Materials and Methods: In kidney transplant recipients, we analyzed renal vein and central vein blood samples, which reflect local and systemic metabolic alterations, respectively. Samples were taken immediately after kidney recirculation (that is, the first blood passing through after clamps are released) and at 5, 15, and 30 minutes thereafter. Levels of nitric oxide metabolites (nitrites, nitrates, and their sum), malondialdehyde (an indicator of oxidative damages), and parameters of acid-base balance (pH level, actual excess base, hemoglobin, actual bicarbonate, partial pressure of carbon dioxide, partial pressure of oxygen) were analyzed. Living kidney donors (the recipients’ parents) were controls.

Results: In renal vein samples, nitrates and the sum of nitrites and nitrates were significantly higher than that shown in control (P < .001) and central vein (P < .05) samples, suggesting an immediate increase in nitric oxide production in the transplanted organ. Metabolic acidosis occurred in both the renal and central vein, indicated by decreased pH and actual bicarbonate level as well as by negative actual base excess level. Only in the renal vein was an increased nitrite and nitrate associated with a reduction of negative actual excess base, thereby suggesting a decrease in anion formation.

Conclusions: Transplanted kidneys increase nitric oxide production immediately after organ transplant surgery, which positively affects local metabolic acidosis. The mechanism for this effect is likely local circulation improvement.

Key words : Acid-base balance, Endogenous nitrate vasodilator, Nitrate, Nitric oxide, Nitrite, Renal graft, Transplantation

Introduction

Nitric oxide (NO) is a potent vasodilator also known as endothelium-derived vasorelaxant factor, which is produced from L-arginine and oxygen in a reaction catalyzed by nitric oxide synthase.^1,2 As a vasodilator, NO is considered to play a significant role in the homeostatic regulation of renal hemodynamics in both normotensive and hypertensive states.3 Nitrates and nitrites represent final products of NO metabolism, whereas their sum (NOx) is used as an indicator of total NO formation in various physiologic samples.^4-6 There are accumulating data indicating disturbances of NO formation in kidney diseases, particularly in chronic renal insufficiencies.^7-11 Some findings indicate a reduction of NO formation in human kidney recipients,9 whereas other studies report no alterations in NO formation during the early period after surgery.^12,13 In recent years, increasing attention has been focused on the role and importance of the L-arginine-NO pathway, as well as the role of acid-base balance disturbances in restoring function of the transplant organ.^9,14,15 The importance of defects in the arginine-NO pathway in renal failure was suggested,16 and our recent findings have indicated that the first days after kidney transplant surgery are associated with reduced NO synthesis, mainly due to a lack of arginine.⁹ A common consequence of chronic kidney disease is metabolic acidosis, which is mainly caused by insufficient production of bicarbonate combined with endogenous acid production and intake.¹⁷ Metabolic acidosis, usually defined by a low serum pH and low serum bicarbonate levels (< 22 mmol/L), is a typical complication in kidney transplant recipients, with a prevalence of 12% to 58%.^18-22 Metabolic acidosis is also a consequence of ischemia during surgery, which represents one of the main problems encountered during an organ transplant procedure. Ischemic-reperfusion injury, occurring during a kidney transplant procedure, is also associated with increased oxygen free radical production.²³ Malondialdehyde (MDA) is usually used as an indicator of lipid peroxidation and represents a good biomarker of oxidative stress in the body.^23,24

In the present study, we hypothesized that the transplanted kidney is capable of producing NO very early after surgery, and we questioned whether this could have an effect on local metabolic acidosis. Thus, we analyzed blood samples obtained from the renal vein (RV), which reflects local metabolic alterations, and the central vein (CV), which reflects general metabolic disturbances. Analyses were performed during the first minutes after renal transplant surgery. Plasma nitrites, nitrates, and NOx were used as indicators of NO formation, whereas oxidative cell damage and metabolic acidosis were evaluated by plasma MDA levels and parameters of acid-base balance, respectively. The relationships between local NO formation and parameters of acid-base balance were also analyzed.

Materials and Methods

Patients and controls
The study was conducted at the Clinic for Vascular Surgery, Clinic of Nephrology, and Institute of Medical Research of the Military Medical Academy according to the prespecified protocol, which was approved by the ethics committee of the Military Medical Academy (Belgrade, Serbia). Our study included a group of 7 patients (4 women) aged between 20 and 57 years and their living donors (kidney paired donation) and complete number of blood samples. All recipients were examined before organ transplant surgery. Patients with standard contraindications, such as malignancy, viral infections, and cardiac decompensation, were excluded. Patients were also given a multislice computed tomography scan of the aorta and iliac arteries and a color Doppler examination of the iliac veins and inferior vena cava. Blood group and human leukocyte antigen compatibility with the donor were evaluated, and the crossmatches were negative. All recipients had their own living kidney donor, mostly the parents of the recipient, except in 1 case where the living donor was the recipient’s husband.

The organ systems of all donors were evaluated and showed no pathologic findings within the coronary and peripheral arteries (confirmed by multislice computed tomography scan); no other abnormalities were detected. The kidney donors in this study experienced similar surgical trauma during open nephrectomy, a similar amount of blood loss and tissue damage, and were under general anesthesia for the same length of time as living donors of comparable studies. Furthermore, after surgery, both the transplant recipient and donor had 1 kidney that had undergone similar surgical trauma. After these evaluations, paired donors seemed to be the most suitable controls for comparison. All donors had a left kidney explanted. All explanted kidneys were perfused with 1 L of Euro-Collins solution cooled to +4°C and maintained in Ringer solution with ice (also at a temperature of +4°C). A right-sided modified Gibson incision via an extraperitoneal approach was used for all the recipients, with an anastomosis at the hypogastric artery and external iliac vein. All transplanted kidneys began to produce urine immediately after circulation was established. All operations were performed under general endo­tracheal anesthesia.25 All patients with transplanted kidneys received triple immuno­suppressive therapy consisting of corticosteroids, mycophenolate mofetil, and a calcineurin inhibitor, according to a stan­dardized protocol.26 Mean warm ischemia time was 1 minute or less, and the mean total cold ischemia time (SD) was 59 (17.1) minutes.

Blood sampling
Blood samples (2 mL), taken from controls (CV) and recipients (RV and CV), were placed into heparinized tubes kept on ice. Samples from transplant recipients were taken at the moment when circulation was established at time 0 (T0) and at 5, 15, and 30 minutes thereafter.

Quantitative analysis of biochemical parameters
Parameters of acid-base balance, nitrates, nitrites, and MDA were analyzed both in RV and CV blood samples. Parameters of acid-base balance including actual pH, partial pressure of carbon dioxide (Pco₂), partial pressure of oxygen (Po₂), actual bicarbonate (AB), actual base excess (ABE), and hemoglobin (Hb) were analyzed by a GEM premier 3000 analyzer (Instrumentation Laboratory, Bedford, MA, USA). Plasma nitrites, nitrates, and NOx were used as indicators of NO production. They were determined by capillary electrophoresis employing a P/ACE 5010 system (Beckman, Palo Alto, CA, USA) with a P/ACE system software control. This electrophoresis method does not require deproteinization or any derivatization procedure; it only requires plasma dilution with water.⁴ We analyzed MDA by the colorimetric method with thiobarbituric acid.²⁷

Statistical analyses
Data analyses were performed using the statistical package MedCalc, version 19.1 (MedCalc Software, Flanders, Belgium). Examined parameters in kidney recipients at the moment of renal transplant (T0) were compared with those of healthy subjects (living donor controls) using the nonparametric Mann-Whitney U test. Nonparametric tests (Friedman test) were used for evaluations of time-dependent alterations of examined parameters. Correlations between the parameters in RV and CV samples were determined separately by the Pearson correlation coefficient. The results were expressed as median and interquartile ranges (25%-75%). A P value < .05 was considered significant.

Results

Renal graft and alteration of biochemical parameters
The effects of renal transplant surgery on the examined parameters were performed by comparing values obtained from the moment of recirculation through the transplanted kidney (T0) to those of healthy controls (Table 1). Although in both the RV and CV, nitrites were within control limits, at the moment of recirculation in the RV, nitrates and NOx levels were significantly higher (P < .001) in kidney recipients. Molar nitrate-to-nitrite and NOx-to-Hb ratios in the RV were higher in the recipient group than in the control group (P < .01). In CV samples, nitrates, NOx, and molar nitrates-to-nitrites were within control limits. These findings suggest an immediate increase in NO formation in the transplanted organ. In kidney recipients, negative ABE and decreased AB in both the local and general circulation (Table 1) resulted in metabolic acidosis. Compared with that shown in controls, in kidney recipients, decreased pH was detected in the RV (P <.001) but not in CV samples. Moreover, ABE values were more negative (P < .03) in the RV than in the CV samples, indicating greater formation of acidic compounds in the transplanted organ. In kidney recipients, in both RV and CV samples, Hb and Pco₂ were lower (P < .001), Po₂ values were higher (P < .01), and MDA was not altered compared with the results of these parameters in healthy controls. Only in CV samples, dry fat free weight (DFFW), an indication of crude protein content, was lower compared with DFFW levels in RV and control samples.

Differences between indicators of nitric oxide production, parameters of acid-base balance, and oxidative cell damage in the renal and central veins of recipients at time 0 and in the central vein of recipients and controls
The dynamics of local NO formation were evaluated by analyzing nitrite, nitrate, and NOx alterations in RV blood samples during the examined period (Table 2) using the Friedman test. At T0, there was an increase in nitrate (P < .01) and NOx (P < .0001) levels, but molar nitrate-to-nitrite ratios remained at similar levels, indicating proper nitrite-to-nitrate oxidation at the site of injury. In the local circulation of kidney recipients, nitrites were not significantly altered. Molar NOx-to-Hb levels remained stable during the entire study period. Low levels of pH persisted in the local circulation; at T0, ABE and AB levels were increased, suggesting lower production of acid compounds and improvement of local metabolic acidosis. High Po₂ levels existed throughout the examined period as a result of oxygen therapy. Some improvement in Hb levels was observed, whereas MDA and DFFW were not altered. In RV blood samples, pH positively correlated with AB (r = 0.722; P < .0001), ABE (r = 0.768; P < .0001), and Po₂ (r = 0.648; P < .0002). Positive correlations between NOx and ABE (r = 0.393; P < .05), Hb (r = 0.428; P < .05), Pco₂ (r = 0.612; P < .0005), and nitrate levels (r = 0.838; P < .001) were observed.

Time course alterations of nitric oxide production, parameters of acid-base balance, and oxidative cell damage in the renal vein during the first 30 minutes after kidney transplant
The time course alterations of nitrite, nitrate, and NOx (Table 3) in the CV were used as indicators of systemic NO formation. Nitrate and NOx in the CV were at similar levels during the examined 30 minutes after renal transplant surgery. However, nitrite levels significantly decreased (P < .05) during that time, even below control limits, but molar nitrate-to-nitrite and NOx-to-Hb ratios remained at stable levels. In kidney recipients, low pH, negative ABE, decreased AB, and high Po2 in the CV persisted. Levels of Hb, DFFW, and MDA in the general circulation were not altered during that period. In CV blood samples, pH positively correlated with ABE (r = 0.624; P < .001) and Po2 (r = 0.493; P < .05). Positive correlations between NOx and nitrate (r = 0.997; P < .001) and NOx and nitrite (r = 0.580; P < .05), as well as a negative correlation with Po2 (r = –0.441; P < .05), were observed.

Time course alterations of nitric oxide production, parameters of acid-base balance, and oxidative cell damage in the central vein during the first 30 minutes after kidney transplant Levels of NOx in the RV were higher (P < .05) than corresponding values in the CV (Figure 1). A reduction in negative ABE values was only detected in the RV. No significant time-dependent alterations of NO_x and ABE were observed in CV blood samples.

There was a minimal decrease in Pco2 as a result of intense ventilation, after which the level of Pco₂ increased. Hemoglobin levels decreased over a 5-minute period after blood dilution with Ringer lactate solution. This procedure is performed by an anesthesiologist to prepare the patient for excess urine production.

Discussion

The main finding of this study suggests that transplanted kidneys produce increased NO levels immediately after transplant surgery. This is probably the result of increased endothelial nitric oxide synthase (eNOS) activity, as plasma nitrite and nitrate levels reflect activity of this enzyme. In normal kidneys, this enzyme is located on vascular endothelial cells28 and is responsible for physiologic vasodilatation and blood flow adjustments producing small quantities of NO. In addition, the gradient increase of produced nitrate and NO_x, observed in the present study, confirms an increase in eNOS activity. It was reported that a loss of NO production by glomerular eNOS is involved in the pathogenesis of chronic renal transplant failure.² Other studies found that nitrate was not released from the reperfused kidney graft during the first minutes after reperfusion.^12,13 However, it must be considered that we used capillary electrophoresis for nitrite and nitrate measurements, permitting sensitive and precise determinations of NO metabolites without any derivatization procedure.⁴

The present study also suggests that gradual increases in nitrate and NOx levels in the RV may indicate a continuous local increase of NO formation during the very early posttransplant period. Nitrate and NOx levels in the general circulation were within control limits, and similar levels existed throughout the examined period. The early local increase in NO formation after renal transplant surgery could be physiologically important due to its cytoprotective effects through regulation of vascular resistance and blood flow through tissues.²⁹ In recent years, increasing attention has been focused on the role and importance of the L-arginine-NO pathway in restoring organ transplant function in the early period.^9,14,15 During the first week after organ transplant, due to a lack of arginine, decreased NO formation occurs.⁹ Increased NO formation is a physiologic consequence of ischemia, which is intensified after organ reperfusion.14 Nitrite is the lowest oxidation product of NO, which is further oxidized to nitrate in the presence of sufficient oxygen.^4,30 The molar nitrate-to-nitrite ratios were at similar levels in both the renal and general circulations throughout the examined period, suggesting proper nitrite oxidation and oxygen delivery to the tissues. Interestingly, the observed increase in local NO production was associated with an improvement in local metabolic acidosis (ie, reduction of negative ABE, normalizing trends of AB and pH alterations), which deserves further examination. The increased local NO production very early after renal transplant surgery has to be considered as a positive action of NO, known as the “Janus-faced” molecule.

Kidney recipients had higher levels of blood Po₂ than controls as a result of the higher percentage of oxygen they were inhaling while under general anesthesia during the transplant procedure. In the RV, the levels of Po₂ positively correlated with pH, ABE, and AB, whereas in the general circulation, Po₂ levels correlated only with pH values.

There was a positive effect on metabolic acidosis at the site of injury in conjunction with an increase in NO formation. Metabolic acidosis was documented during the transplant procedure; acidosis occurs as a result of lactic acid accumulation from anaerobic metabolism due to renal ischemia.^19,22 To compensate for this metabolic acidosis, the respiratory rate of the kidney recipients increased, leading to an increase in expiratory carbon dioxide within the first 3 minutes after release of clamps and start of kidney functioning, reaching a maximum after 15 minutes.²² An increase in Pco₂ in exhaled air correlates with a high incidence of early graft loss.¹⁹ Our study findings suggest that almost at the moment of kidney transplant, pH, ABE, and AB levels decreased in the local circulation compared with those of healthy controls. This is to be expected considering that the transplanted organ is out of circulation for approximately 1 hour. Acidosis in the general circulation is delayed for some time but, after 5 minutes, pH and AB were decreased in the CV as well. Other studies have also reported a high prevalence of compensated metabolic acidosis after renal transplant manifested by low serum bicarbonate levels and impaired renal acid excretion.³¹ Our patients began producing urine almost immediately after kidney transplant surgery. Considering that kidney transplant recipients are characterized by a higher prevalence of metabolic acidosis and that alkali treatment has a renoprotective effect in patients with chronic kidney disease, it seems reasonable to conduct further studies to clarify whether metabolic acidosis correction affects renal function and the severity of anemia in kidney transplant recipients.²¹ The relationships between local NO production and reduction of metabolic acidosis needs further elucidation.

When RV and CV samples from the kidney recipients were compared, a high level of local acidosis in the transplanted kidney (RV) was noted, which gradually became stable and equal to that of the CV. Samples from CV in controls compared with CV in recipients in T0 shows that there are no differences in the general circulations of both groups. The level of MDA concentration, a good indicator of oxidative damage in various conditions,³² was not significantly altered at the site of injury or in the general circulation. These results suggest proper reperfusion of the transplanted organ and surgical and therapeutic success.

Conclusions

Our study results suggest that a transplanted kidney is capable of producing NO immediately after organ transplant surgery, which is followed by a gradual increase in nitrate and NOx levels in the local circulation during the very early posttransplant period. A local increase in NOx formation is associated with a positive effect on local metabolic acidosis, probably due to an improvement in local circulation. Increased NOx formation results in a decrease in negative ABE levels, thereby indicating a reduction of acid compounds at the site of injury. Both pH and AB levels showed positive trends in the local circulation as well. In the general circulation, no alterations in NO formation or acid-base balance were documented. Metabolic acidosis is a systemic effect of kidney transplant surgery that can persist up to 6 months posttransplant. Pulmonary ventilation increases to compensate for decreasing pH but with insufficient effect because of renal acidosis. Future studies should evaluate whether arginine supplementation before organ transplant surgery can improve these results.

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