A comparison of arteriovenous fistula failure between Malaysian and Australian and New Zealand participants enrolled in the FAVOURED trial.

AIM: To describe and compare de novo arteriovenous fistula (AVF) failure rates between Australia and New Zealand (ANZ), and Malaysia. BACKGROUND: AVFs are preferred for haemodialysis access but are limited by high rates of early failure. METHODS: A post hoc analysis of 353 participants from ANZ and Malaysia included in the FAVOURED randomised-controlled trial undergoing de novo AVF surgery was performed. Composite AVF failure (thrombosis, abandonment, cannulation failure) and its individual components were compared between ANZ (n = 209) and Malaysian (n = 144) participants using logistic regression adjusted for patient- and potentially modifiable clinical factors. RESULTS: Participants' mean age was 55 ± 14.3 years and 64% were male. Compared with ANZ participants, Malaysian participants were younger with lower body mass index, higher prevalence of diabetes mellitus and lower prevalence of cardiovascular disease. AVF failure was less frequent in the Malaysian cohort (38% vs 54%; adjusted odds ratio (OR) 0.53, 95% confidence interval (CI) 0.31-0.93). This difference was driven by lower odds of cannulation failure (29% vs 47%, OR 0.45, 95% CI 0.25-0.80), while the odds of AVF thrombosis (17% vs 20%, OR 1.24, 95% CI 0.62-2.48) and abandonment (25% vs 23%, OR 1.17, 95% CI 0.62-2.16) were similar. CONCLUSIONS: The risk of AVF failure was significantly lower in Malaysia compared to ANZ and driven by a lower risk of cannulation failure. Differences in practice patterns, including patient selection, surgical techniques, anaesthesia or cannulation techniques may account for regional outcome differences and warrant further investigation.

Center-Effect of Incident Hemodialysis Vascular Access Use: Analysis of a Bi-national Registry.

Background: Commencing hemodialysis (HD) with an arteriovenous access is associated with superior patient outcomes compared with a catheter, but the majority of patients in Australia and New Zealand initiate HD with a central venous catheter. This study examined patient and center factors associated with arteriovenous fistula/graft access use at HD commencement. Methods: We included all adult patients starting chronic HD in Australia and New Zealand between 2004 and 2015. Access type at HD initiation was analyzed using logistic regression. Patient-level factors included sex, age, race, body mass index (BMI), smoking status, primary kidney disease, late nephrologist referral, comorbidities, and prior RRT. Center-level factors included size; transplant capability; home HD proportion; incident peritoneal dialysis (average number of patients commencing RRT with peritoneal dialysis per year); mean weekly HD hours; average blood flow; and achievement of phosphate, hemoglobin, and weekly Kt/V targets. The study included 27,123 patients from 61 centers. Results: Arteriovenous access use at HD commencement varied four-fold from 15% to 62% (median 39%) across centers. Incident arteriovenous access use was more likely in patients aged 51-72 years, males, and patients with a BMI of >25 kg/m2 and polycystic kidney disease; but use was less likely in patients with a BMI of <18.5 kg/m2, late nephrologist referral, diabetes mellitus, cardiovascular disease, chronic lung disease, and prior RRT. Starting HD with an arteriovenous access was less likely in centers with the highest proportion of home HD, and no center factor was associated with higher arteriovenous access use. Adjustment for center-level characteristics resulted in a 25% reduction in observed intercenter variability of arteriovenous access use at HD initiation compared with the model adjusted for only patient-level characteristics. Conclusions: This study identified several patient and center factors associated with incident HD access use, yet these factors did not fully explain the substantial variability in arteriovenous access use across centers.

Allograft and Patient Outcomes Between Indigenous and Nonindigenous Kidney Transplant Recipients.

BACKGROUND: Kidney transplant outcomes of indigenous Australians are poorer compared with nonindigenous Australians, but it is unknown whether the type of acute rejection differs between these patient groups or whether rejection mediates the effect between ethnicity, death-censored graft failure (DCGF), and death with a functioning graft (DWFG). METHODS: Biopsy-proven acute rejection (BPAR) rates and types were compared between indigenous and nonindigenous recipients. The associations between ethnicity, BPAR, DCGF, and DWFG were examined using adjusted competing risk analyses, and mediation analysis was conducted to determine whether BPAR mediated the adverse effects between ethnicity and outcomes. RESULTS: Fifty-seven (9.3%) of 616 patients who have received kidney-only transplants between 2000 and 2010 in Western Australia were indigenous. Compared with nonindigenous recipients, BPAR rates were higher in indigenous recipients (42 versus 74 episodes/100 recipients, P < 0.01), with an excess of antibody-mediated rejections. During a median follow-up of 8 years, indigenous recipients were more likely to experience BPAR, DCGF, and DWFG compared with nonindigenous recipients, with adjusted subdistribution hazard ratio of 1.94 (1.39-2.70), 1.53 (0.85-2.76; P = 0.159), and 2.14 (1.13-4.06; P = 0.020), respectively. Although 70% of the effect between ethnicity and DCGF was mediated by BPAR, no similar association was found for DWFG. CONCLUSIONS: Indigenous recipients experienced poorer allograft and patient outcomes compared with nonindigenous recipients, with BPAR an important determinant for DCGF. Future research identifying other risk factors and mediators associated with patient survival in indigenous recipients should be considered a priority.

Fish oil and aspirin effects on arteriovenous fistula function: Secondary outcomes of the randomised omega-3 fatty acids (Fish oils) and Aspirin in Vascular access OUtcomes in REnal Disease (FAVOURED) trial.

BACKGROUND: Arteriovenous fistulas (AVF) for haemodialysis often experience early thrombosis and maturation failure requiring intervention and/or central venous catheter (CVC) placement. This secondary and exploratory analysis of the FAVOURED study determined whether omega-3 fatty acids (fish oils) or aspirin affected AVF usability, intervention rates and CVC requirements. METHODS: In 567 adult participants planned for AVF creation, all were randomised to fish oil (4g/d) or placebo, and 406 to aspirin (100mg/d) or placebo, starting one day pre-surgery and continued for three months. Outcomes evaluated within 12 months included AVF intervention rates, CVC exposure, late dialysis suitability failure, and times to primary patency loss, abandonment and successful cannulation. RESULTS: Final analyses included 536 participants randomised to fish oil or placebo (mean age 55 years, 64% male, 45% diabetic) and 388 randomised to aspirin or placebo. Compared with placebo, fish oil reduced intervention rates (0.82 vs 1.14/1000 patient-days, incidence rate ratio [IRR] 0.72, 95% confidence interval [CI] 0.54-0.97), particularly interventions for acute thrombosis (0.09 vs 0.17/1000 patient-days, IRR 0.53, 95% CI 0.34-0.84). Aspirin significantly reduced rescue intervention rates (IRR 0.45, 95% CI 0.27-0.78). Neither agent significantly affected CVC exposure, late dialysis suitability failure or time to primary patency loss, AVF abandonment or successful cannulation. CONCLUSION: Although fish oil and low-dose aspirin given for 3 months reduced intervention rates in newly created AVF, they had no significant effects on CVC exposure, AVF usability and time to primary patency loss or access abandonment. Reduction in access interventions benefits patients, reduces costs and warrants further study.

Changing survival of people with myeloma and end stage kidney disease: A cohort study using Australian and New Zealand dialysis and transplant registry 1963-2013.

BACKGROUND: It is unclear whether recent advances in myeloma therapy have improved survival for all those with myeloma and end stage kidney disease (ESKD). METHODS: Population-based registry cohort study using Australia and New Zealand Dialysis and Transplant Registry data 1963-2013. We measured survival of people with myeloma and other plasma cell dyscrasias and ESKD over time, and investigated prognostic factors for improved survival using survival analysis (results expressed as hazard ratios (HR) with 95% confidence intervals). RESULTS: We included 65 940 people (207 595 person-years); 1067 people (1.6%) with myeloma and 572 (0.9%) with other plasma cell dyscrasia. Myeloma ESKD rose from 0.8% before 1994 to 2.2% in 2004 and remained stable. People with myeloma were older, and age increased over time, from 62.5 before 1994 to 70.1 years from 2010, but the non-myeloma group age increased more steeply (52.0 before 1994; 62.2 from 2010). In myeloma patients, survival improved (P < 0.001) with recent predicted 5 year survival of 27.5% aged <55, 32.2% aged 55-64, 16.3% for 65-74 and 12.7% aged ≥75 years. Survival did not improve for plasma cell dyscrasia patients (P = 0.70). Myeloma patients on peritoneal dialysis had improved survival compared with those on haemodialysis (HR 0.7, CI 0.6-0.9), but those aged ≥65 had poorer survival (65-74 years HR 1.5, CI1.2-1.9; ≥75 HR 1.7, CI1.3-2.1), as did diabetics (HR 1.3, CI1.1-1.6). CONCLUSIONS: The proportion of people with myeloma and ESKD remains stable, but their survival has progressively improved in Australia and New Zealand. On starting ESKD treatment with myeloma, a 59 year old without diabetes on peritoneal dialysis can expect a 45% 5 year survival, where a 75-year-old diabetic on haemodialysis has 9% 5 year survival.

Effect of Fish Oil Supplementation and Aspirin Use on Arteriovenous Fistula Failure in Patients Requiring Hemodialysis: A Randomized Clinical Trial.

Importance: Vascular access dysfunction is a leading cause of morbidity and mortality in patients requiring hemodialysis. Arteriovenous fistulae are preferred over synthetic grafts and central venous catheters due to superior long-term outcomes and lower health care costs, but increasing their use is limited by early thrombosis and maturation failure. ω-3 Polyunsaturated fatty acids (fish oils) have pleiotropic effects on vascular biology and inflammation and aspirin impairs platelet aggregation, which may reduce access failure. Objective: To determine whether fish oil supplementation (primary objective) or aspirin use (secondary objective) is effective in reducing arteriovenous fistula failure. Design, Setting, and Participants: The Omega-3 Fatty Acids (Fish Oils) and Aspirin in Vascular Access Outcomes in Renal Disease (FAVOURED) study was a randomized, double-blind, controlled clinical trial that recruited participants with stage 4 or 5 chronic kidney disease from 2008 to 2014 at 35 dialysis centers in Australia, Malaysia, New Zealand, and the United Kingdom. Participants were observed for 12 months after arteriovenous fistula creation. Interventions: Participants were randomly allocated to receive fish oil (4 g/d) or matching placebo. A subset (n = 406) was also randomized to receive aspirin (100 mg/d) or matching placebo. Treatment started 1 day prior to surgery and continued for 12 weeks. Main Outcomes and Measures: The primary outcome was fistula failure, a composite of fistula thrombosis and/or abandonment and/or cannulation failure, at 12 months. Secondary outcomes included the individual components of the primary outcome. Results: Of 1415 eligible participants, 567 were randomized (359 [63%] male, 298 [53%] white, 264 [47%] with diabetes; mean [SD] age, 54.8 [14.3] y). The same proportion of fistula failures occurred in the fish oil and placebo arms (128 of 270 [47%] vs 125 of 266 [47%]; relative risk [RR] adjusted for aspirin use, 1.03; 95% CI, 0.86-1.23; P = .78). Fish oil did not reduce fistula thrombosis (60 [22%] vs 61 [23%]; RR, 0.98; 95% CI, 0.72-1.34; P = .90), abandonment (51 [19%] vs 58 [22%]; RR, 0.87; 95% CI, 0.62-1.22; P = .43), or cannulation failure (108 [40%] vs 104 [39%]; RR, 1.03; 95% CI, 0.83-1.26; P = .81). The risk of fistula failure was similar between the aspirin and placebo arms (87 of 194 [45%] vs 83 of 194 [43%]; RR, 1.05; 95% CI, 0.84-1.31; P = .68). Conclusions and Relevance: Neither fish oil supplementation nor aspirin use reduced failure of new arteriovenous fistulae within 12 months of surgery. Trial Registration: anzctr.org.au Identifier: CTRN12607000569404.

Asymptomatic CMV infections in long-term renal transplant recipients are associated with the loss of FcRγ from LIR-1+ NK cells.

While it is established that cytomegalovirus (CMV) disease affects NK-cell profiles, the functional consequences of asymptomatic CMV replication are unclear. Here, we characterize NK cells in clinically stable renal transplant recipients (RTRs; n = 48) >2 years after transplantation. RTRs and age-matched controls (n = 32) were stratified by their CMV serostatus and the presence of measurable CMV DNA. CMV antibody or CMV DNA influenced expression of NKG2C, LIR-1, NKp30, NKp46, and FcRγ, a signaling adaptor molecule, on CD56dim NK cells. Phenotypic changes ascribed to CMV were clearer in RTRs than in control subjects and affected NK-cell function as assessed by TNF-α and CD107a expression. The most active NK cells were FcRγ- LIR-1+ NKG2C- and displayed high antibody-dependent cell cytotoxicity responses in the presence of immobilized CMV glycoprotein B reactive antibody. However, perforin levels in supernatants from RTRs with active CMV replication were low. Overall we demonstrate that CMV can be reactivated in symptom-free renal transplant recipients, affecting the phenotypic, and functional profiles of NK cells. Continuous exposure to CMV may maintain and expand NK cells that lack FcRγ but express LIR-1.

The Omega-3 fatty acids (Fish Oils) and Aspirin in Vascular access OUtcomes in REnal Disease (FAVOURED) study: the updated final trial protocol and rationale of post-initiation trial modifications.

BACKGROUND: The FAVOURED study is an international multicentre, double-blind, placebo-controlled trial which commenced recruitment in 2008 and examines whether omega-3 polyunsaturated fatty acids (omega-3 PUFAs) either alone or in combination with aspirin will effectively reduce primary access failure of de novo arteriovenous fistulae (AVF) in patients with stage 4 and 5 chronic kidney disease. Publication of new evidence derived from additional studies of clopidogrel and a high screen failure rate due to prevalent aspirin usage prompted an updated trial design. METHODS/DESIGN: The original trial protocol published in 2009 has undergone two major amendments, which were implemented in 2011. Firstly, the primary outcome 'early thrombosis' at 3 months following AVF creation was broadened to a more clinically relevant outcome of 'AVF access failure'; a composite of thrombosis, AVF abandonment and cannulation failure at 12 months. Secondly, participants unable to cease using aspirin were allowed to be enrolled and randomised to omega-3 PUFAs or placebo. The revised primary aim of the FAVOURED study is to test the hypothesis that omega-3 PUFAs will reduce rates of AVF access failure within 12 months following AVF surgery. The secondary aims are to examine the effect of omega-3 PUFAs and aspirin on the individual components of the primary end-point, to examine the safety of study interventions and assess central venous catheter requirement as a result of access failure. DISCUSSION: This multicentre international clinical trial was amended to address the clinically relevant question of whether the usability of de novo AVF at 12 months can be improved by the early use of omega-3 PUFAs and to a lesser extent aspirin. This study protocol amendment was made in response to a large trial demonstrating that clopidogrel is effective in safely preventing primary AVF thrombosis, but ineffective at increasing functional patency. Secondly, including patients taking aspirin will enroll a more representative cohort of haemodialysis patients, who are significantly older with a higher prevalence of cardiovascular disease and diabetes which may increase event rates and the power of the study. TRIAL REGISTRATION: Australia & New Zealand Clinical Trial Register (ACTRN12607000569404).

Peri-operative third party red blood cell transfusion in renal transplantation and the risk of antibody-mediated rejection and graft loss.

Historic red blood cell transfusion (RBCT) may induce anti-HLA antibody which, if donor specific (DSA), is associated with increased antibody-mediated rejection (AMR). Whether post-operative RBCT influences this risk is unknown. We examined the RBCT history in 258 renal transplant recipients stratified according to prevalent recipient HLA antibody (DSA, Non-DSA or No Antibody). AMR occurred more frequently in patients who received RBCT both pre and post transplant compared with all other groups (Pre+Post-RBCT 21%, Pre-RBCT 4%, Post-RBCT 6%, No-RBCT 6%, HR 4.1 p=0.004). In the 63 patients who received Pre+Post-RBCT, 65% (13/20) with DSA developed AMR compared with 0/6 in the Non-DSA group and 2/37 (5%) in the No-Antibody group (HR 13.9 p<0.001). In patients who received No-RBCT, Pre-RBCT or Post-RBCT there was no difference in AMR between patients with DSA, Non-DSA or No-Antibody. Graft loss was independently associated with Pre+Post-RBCT (HR 6.5, p=0.001) AMR (HR 23.9 p<0.001) and Non-AMR (6.0 p=0.003) after adjusting for DSA and delayed graft function. Re-exposure to RBCT at the time of transplant is associated with increased AMR only in patients with preformed DSA, suggesting that RBCT provides additional allostimulation. Patients receiving Pre+Post-RBCT also had an increased risk of graft loss independently of AMR or DSA. Both pre and post procedural RBCT in renal transplantation is associated with modification of immunological risk and warrants additional study.

Pre-transplant donor specific anti-HLA antibody is associated with antibody-mediated rejection, progressive graft dysfunction and patient death.

BACKGROUND: The long term effect of donor specific antibodies (DSA) detected by Luminex Single Antigen Bead (SAB) assay in the absence of a positive complement-dependant cytotoxicity (CDC) crossmatch is unclear. DSA at the time of transplant were determined retrospectively in 258 renal transplant recipients from 2003 to 2007 and their relationship with rejection and graft function prospectively evaluated. After a median of 5.6 years follow-up 9% of patients had antibody mediated rejection (AMR) (DSA 11/37 (30%), DSA-Neg 13/221 (6%), HR 6.6, p<0.001). Patients with anti-HLA class II (HR 6.1) or both class I+II (HR 10.1) DSA had the greatest risk for AMR. The Mean Fluorescent Intensity (MFI) of the DSA was significantly higher in patients with AMR than those with no rejection (p=0.006). Moreover, the strength of the antibody was shown to be important, with the risk of AMR significantly greater in those with DSA >8000 MFI than those with DSA <8000 MFI (HR 23, p<0.001). eGFR progressively declined in patients with DSA but was stable in those without DSA (35.7 ± 20.4 mls/min vs 48.5 ± 22.7) and composite patient and graft survival was significantly worse in those with class II (HR 2.9) or both class I+II (HR 3.7) but not class I DSA. Class II DSA alone, or in combination with class I DSA had the strongest association with graft loss and patient death. Patients with DSA not only have increased rates of acute AMR, but also chronic graft dysfunction, graft loss and death. Antibody burden quantified by SAB assay may identify patients at highest immunological risk and therefore influence patient management and improve long-term patient outcome.

Rosiglitazone does not improve vascular function in subjects with chronic kidney disease.

BACKGROUND: Thiazolidinediones such as rosiglitazone (RSG) are insulin-sensitizing agents, which may improve inflammation and vascular function, and thus potentially lower cardiovascular risk in patients with chronic kidney disease (CKD). However, there is growing concern about the adverse cardiovascular effects of RSG in diabetic patients without CKD, and the data in patients with CKD remain conflicting. This study examines the effect of RSG on vascular function in patients with CKD. METHODS: A randomized, double-blind placebo-controlled study comparing RSG 4 mg daily (n = 35) with placebo (n = 35) for 8 weeks was performed in CKD subjects. Primary outcome measures were flow-mediated dilatation (FMD), systemic arterial compliance (SAC) and augmentation index (AIx). Secondary outcomes included glyceryl trinitrate-mediated dilatation (GTN-MD), pulse-wave velocity (PWV), lipids, blood pressure, homoeostasis model assessment (HOMA), adiponectin, high-sensitivity C-reactive protein (hs-CRP) and high-sensitivity interleukin 6 (hs-IL-6) and in vivo marker of endothelial function [von Willebrand Factor (vWF)]. RESULTS: RSG lowered HOMA score [RSG geometric mean 1.7 (95% confidence interval 1.3-2.3); placebo 1.9 (1.4-2.5), P = 0.04], hs-CRP [RSG 1.2 (0.9-1.7) mg/L; placebo 1.6 (1.2-2.3), P = 0.04] and vWF [RSG mean 126.1 ± SD 45.7%; placebo 132.7 ± 41.7, P = 0.01] but not hs-IL-6. RSG did not significantly change arterial function (FMD, GTN-MD, SAC), arterial stiffness (AIx, PWV) or blood pressure. RSG increased triglyceride concentration [RSG 1.8 (1.3-1.9) mmol/L; placebo 1.5 (1.3-1.9), P = 0.01] without affecting other lipid and lipoprotein concentrations. CONCLUSION: Short-term RSG therapy reduced insulin resistance, in vivo markers of inflammation and abnormal endothelial function but had no effect on arterial function and stiffness in patients with CKD.

Maintenance of elevated versus physiological iron indices in non-anaemic patients with chronic kidney disease: a randomized controlled trial.

BACKGROUND: An optimal haemoglobin (Hb) response to erythropoietin requires elevated iron indices in dialysis patients; however, it is unknown if the same applies in chronic kidney disease (CKD). METHODS: One hundred patients [CKD Stages 3-5, Hb >or= 110 g/L, iron replete, erythropoietin-stimulating agent (ESA)-naive, 47% diabetic, median age 69.5 years] were block-randomized in an open-label study to receive up to 200 mg intravenous iron sucrose (Group A, n = 52) bimonthly or oral iron sulphate (Group B) to maintain raised and normal iron indices (respectively) over 12 months. The primary endpoint was the change in Hb concentration at 12 months or at termination after at least 6 months of treatment. RESULTS: Eighty-five patients reached the primary endpoint (43, Group A; 42, Group B). Initial Hb was 119 +/- 7 vs 116 +/- 12 g/L (mean +/- standard deviation); ferritin 122 (71-176), median (inter-quartile range), vs 90 microg/L (58-150); transferrin saturation (TSat) 22 (18-26) vs 21% (15-24); and creatinine 240 (195-313) vs 230 micromol/L (184-352). Ferritin and TSat differed by month 2 [157 (103-220) vs 96 microg/L (73-162), P = 0.003] and month 6 [25 (20-31) vs 21% (17-27), P = 0.02], respectively. At study end, Hb did not differ between groups (121 +/- 10 vs 117 +/- 13 g/L). Ferritin was 362 (310-458) vs 125 microg/L (84-190), P < 0.001; TSat 30 (23-34) vs 21% (18-24), P < 0.001; and creatinine 229 (188-326) vs 272 micromol/L (195-413), P = NS. For patients (Groups A and B, n = 27 in each group) whose creatinine regression slope increased (indicating worsening function), the fall in Hb over 12 months also did not differ between groups despite adequate separation in iron indices. Serious adverse events overall did not differ between groups. CONCLUSIONS: Elevated iron indices did not increase Hb synthesis in ESA-naive, iron replete, pre-dialysis patients with Hb >110 g/L.

Insulin resistance, dyslipidaemia, inflammation and endothelial function in nephrotic syndrome.

BACKGROUND: Nephrotic syndrome (NS) is associated with an increased risk of cardiovascular disease (CVD). We have shown previously that endothelial function, measured by post-ischaemic flow-mediated dilatation (FMD) of the brachial artery, is impaired in NS. In this study our aim was to assess the potential roles of insulin resistance, plasma non-esterified fatty acids (NEFAs) and inflammation in endothelial dysfunction in NS patients. METHODS: FMD was compared between NS patients (n=19) and controls (CS, n=19). Plasma glucose, insulin and NEFAs were measured. Insulin resistance was calculated using the Homeostasis Model Assessment (HOMA) score. C-reactive protein (CRP), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFalpha) and fibrinogen were measured as markers of inflammation. RESULTS: FMD was significantly lower in the NS group (mean+/-standard error, NS 5.1+/-0.7%, CS 7.3+/-0.7%, P=0.02). Fasting insulin (NS 12.5+/-1.5 mU/l, CS 6.8+/-0.7 mU/l, P<0.01), fasting glucose (NS 5.3+/-0.2, CS 4.8+/-0.1, P=0.02) and the HOMA score (NS 3.0+/-0.4, CS 1.5+/-0.2, P=0.001) were significantly higher in NS. These differences persisted after adjusting for waist circumference. Of the inflammatory markers, only fibrinogen (P<0.01) and IL-6 (P=0.01) were significantly increased in NS. Despite significantly lower plasma NEFAs in NS, the NEFA:albumin ratio showed a non-significant trend to higher levels in NS (NS 10.7+/-0.1 micro mol/g, CS 8.7+/-0.1 micro mol/g, P=0.06). Within the NS group, multivariate backward regression analysis showed that NEFAs (P<0.01) and low-density lipoprotein (LDL) cholesterol (P=0.05) were significant negative independent predictors of FMD. CONCLUSION: Endothelial function in NS is inversely correlated with plasma concentrations of NEFAs and LDL cholesterol. Dyslipoproteinaemia and NEFAs probably contribute to the increased risk of CVD seen in NS. We also postulate that in NS, hypoalbuminaemia increases the delivery of NEFAs to endothelial cells thereby impairing the synthesis and release of nitric oxide.