Advances in immunosuppressive therapy, surgery, and medical management have made transplantation the most favorable form of renal replacement therapy. There have been progressive improvements in short-term renal graft survival. Unfortunately, long-term survival has not paralleled the short-term gains.1 The leading causes of graft loss are patient death and chronic deterioration of renal graft function. Cardiovascular disease still represents the major cause of death in kidney transplant recipients.2 Strategies are therefore needed to improve all cardiovascular and kidney graft outcomes in renal transplant recipients.
According to recent research, homoarginine exerts a variety of effects, which are potentially relevant to renal and cardiovascular health in renal transplantation. In an effort to identify potentially involved pathways, we evaluated the role of homoarginine for the occurrence of clinical outcomes in renal transplant recipients. Homoarginine is an amino acid, which is derived from lysine and mainly synthesized in the kidney.3,4 Homoarginine increases the intracellular concentration of L-arginine, which is the main substrate for nitric oxide (NO) synthase.5-8 Thus, homoarginine may increase the availability of NO5-10 and prevent or ameliorate endothelial and myocardial dysfunction.5-8 In previous studies, homoarginine was inversely correlated to concentrations of the intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 as markers of impaired endothelial function; furthermore, an inverse association with inflammation has been demonstrated.11,12 Cardiac function was markedly reduced in patients with low homoarginine, being associated with an increased prevalence of heart failure, increased levels of N-terminal pro-B-type natriuretic peptide and reduced angiographic and echocardiographic measures of left ventricular function.11,13 Importantly, low homoarginine was related to increased all-cause and cardiovascular mortality, especially due to sudden cardiac death and stroke.11-15
We reasoned that deficiency in homoarginine contributes to renal function decline, cardiovascular, and all-cause mortality in kidney transplant recipients. To that end, we analyzed data from the Assessment of Lescol in Renal Transplantation (ALERT) study, which was a randomized study to evaluate the efficacy of fluvastatin in 2102 patients after kidney transplantation.16,17
Of all 2102 patients included into the ALERT study, 829 randomized patients of the placebo group had a homoarginine measurement at baseline. The mean duration of follow-up was 5.1 years in the core trial. The patients had a mean age of 50 ± 11 years and 65% were men. The mean (standard deviation) homoarginine concentration at baseline was 1.96 (0.76) μmol/L. The baseline patient characteristics are shown in Table 1. Patients with low homoarginine concentration were younger (P = 0.04), more likely smokers (P = 0.009), and had a higher burden of diabetes mellitus (P = 0.005); furthermore, the percentage of female patients was higher (P < 0.001). Low homoarginine concentrations were associated with a lower body mass index (P < 0.001), lower estimated glomerular filtration rate (eGFR) (P < 0.001), and higher concentrations of creatinine (P = 0.004) and phosphate (P < 0.001). Lipid profile, blood pressure, the presence of hypertension, and coronary artery disease were comparable across homoarginine concentrations.
Homoarginine and Decline of Renal Function
Of all 829 studied patients, a total of 140 patients reached the composite renal endpoint of graft loss or doubling of serum creatinine (GFDSC) during follow-up. Mean homoarginine concentrations were significantly lower in the patients with progressive loss of transplant function as compared to those without (1.82 vs 1.99 μmol/L, respectively, P < 0.05). Furthermore, homoarginine concentrations were significantly correlated to eGFR (r = 0.15, P < 0.001).
We performed crude cumulative hazard estimates and Cox regression analyses considering the time to reach the renal endpoint (Figure 1A and Table 2). For patients in the lowest homoarginine quartile, the unadjusted hazard to achieve the renal endpoint almost increased 3-fold as compared to patients of the highest homoarginine quartile (hazard ratios [HR], 2.95; 95% confidence interval [95% CI], 1.78-4.90). The association remained significant after adjustment for potential confounders including age, sex, diabetes mellitus, coronary artery disease, smoking status, systolic blood pressure, low-density lipoprotein cholesterol, and baseline eGFR (HR, 2.34; 95% CI, 1.36-4.02). In our extended Cox regression analyses, the multivariate model was additionally adjusted for inflammation (high sensitive C-reactive protein, interleukin-6), serum phosphate, serum calcium, time since last transplantation, treatment for cytomegalovirus, total time on renal replacement therapy, treatment for rejection, proteinuria, HLA-DR mismatches, delayed graft function and panel reactive antibodies. The HR for patients of the lowest homoarginine quartile was 2.44 (95% CI, 1.17-5.09), thus remaining virtually unchanged by the extensive adjustments.
Finally, to rule out potential confounding by diabetic nephropathy (due to differences with diabetes mellitus as a comorbidity), we performed sensitivity analyses including this factor in multivariate analyses. The results remained robust, yielding an HR of 2.45 (95% CI, 1.42-4.23) for patients of the lowest as compared to the highest homoarginine quartile.
Homoarginine and the Risk of Cerebrovascular Events
Lower homoarginine concentrations at baseline were associated with a higher incidence of cerebrovascular (CBV) events. Of all 53 CBV events, 22 occurred in patients in the lowest homoarginine quartile, 11 in patients in the second quartile, 11 in those in the third quartile, and 9 in patients in the highest homoarginine quartile. By Cox regression analyses, the crude risk of CBV events significantly increased 2.7-fold in patients of the lowest as compared to patients of the highest homoarginine quartile (HR, 2.70; 95% CI, 1.24-5.86; Table 2). The association persisted after adjustment for confounders (HR, 2.56; 95% CI, 1.13-5.82). Crude cumulative hazard estimates are shown in Figure 1B.
Homoarginine and the Risk of Cardiovascular Events, Noncardiovascular and All-Cause Mortality
In contrast to the results seen for CBV events, homoarginine was not meaningfully related to the risk of cardiac death or nonfatal myocardial infarction (CDNFMI; Table 2). Similarly, the composite endpoint of major adverse cardiovascular events was not affected. There was a significant association with noncardiovascular mortality, however. Patients in the lowest homoarginine quartile had an adjusted more than 4-fold increased risk compared to patients in the highest quartile. In further analyses investigating the pattern of the association, association with non-cardiovascular mortality, the risk seemed to increase rather steadily with lower homoarginine concentrations. Therefore, analyses using homoarginine as a continuous variable were justified, showing a clear pattern in favor of high homoarginine concentrations (adjusted HR per 1 μmol increase in homoarginine 0.50; 95% CI, 0.31-0.81).
The incidence of all-cause mortality was significantly higher in the presence of low homoarginine concentrations in crude analyses (HR 1st versus 4th quartile, 2.42; 95% CI, 1.39-4.21); this association persisted after adjustment for confounders (HR, 2.50; 95% CI, 1.38-4.55). Similar to non-cardiovascular mortality, the association between homoarginine and all-cause mortality showed a virtually linear pattern supporting analyses with homoarginine as continuous variable. These yielded an adjusted HR of 0.69 (95% CI, 0.51-0.94) per 1 μmol increase in homoarginine. Crude cumulative hazard estimates for all-cause mortality are shown in Figure 1C.
The novel finding of this study is that a low baseline homoarginine concentration was a strong risk factor for CBV events and the composite endpoint of graft loss or doubling of serum creatinine in stable renal transplant recipients. However, we found no relation of homoarginine with cardiac endpoints. Additionally, a significant association between homoarginine and noncardiovascular as well as all-cause mortality has been observed.
Competing risks may explain the lack of associations between homoarginine and myocardial infarction (MI) in this population. Patients in the lower homoarginine quartile had a significantly increased risk of death from all causes in our study. These subjects could have been predisposed to cardiovascular events compared with patients in the higher homoarginine quartiles, but died of other causes rather than MI. In this context, there is no known mechanism of action to fully explain the significant association between low homoarginine and increased risk of noncardiovascular death, the main causes in this population being infections and cancer. Further investigations into possible associations between homoarginine and noncardiovascular death in transplant recipients as well as other populations would be of interest to examine the robustness of this finding.
Despite improvements in the care of kidney transplant recipients, life expectancy is significantly reduced after kidney transplantation and cardiovascular disease represents the major cause of death.2 Cardiovascular mortality in renal transplant recipients is partly explained by traditional risk factors including arterial hypertension, hyperlipidemia, or the development of diabetes mellitus.18 Accordingly, current treatments are focused to control these risk factors. To further reduce the high incidence of posttransplant CVD, strategies to identify new risk factors and develop novel treatments are urgently needed.
Although a variety of recent studies have investigated risk factors for cardiac events, few have addressed CBV events after kidney transplantation. One study for example particularly searched for risk factors for stroke in renal transplant recipients19 and found that diabetes mellitus, left ventricular hypertrophy, age, serum creatinine, and systolic blood pressure significantly contributed to the risk of cerebrovascular events. The present study reveals homoarginine as a novel risk factor. Low homoarginine has very recently been shown associated with adverse outcome after incident stroke.20 We show that low homoarginine is strongly associated with CBV events in renal transplant recipients, raising the question of potential mechanisms.
One mechanism may lie in the NO pathway. Nitric oxide is important for the regulation of cerebral blood flow and cell viability.21 Experimental data suggest that homoarginine may modulate the metabolism of the vasodilator NO. Homoarginine potentially contributes to improved NO availability by serving as substrate for NO synthase or as an inhibitor of arginase. However, it has to be acknowledged that homoarginine appears to be a relatively poor substrate for nitric oxide synthases22,23; the extent by which homoarginine may impact upon NO availability is not yet clear. Interestingly, it has been found that serum homoarginine concentrations were significantly higher during pregnancy; a condition with increased endothelium-dependent brachial artery flow-mediated dilation.7 Three months after delivery, the homoarginine concentrations and flow-mediated dilation were comparable to those recorded in nonpregnant females. Animal experiments have furthermore shown that homoarginine supplementation exerted antihypertensive effects and increased the excretion of nitrate, the degradation product of NO.10
In the clinical context, our results are in line with previous findings in patients from the Ludwigshafen Risk and Cardiovascular Health study, which demonstrated—in patients undergoing coronary angiography—that homoarginine was inversely related to the occurrence of stroke.14 Furthermore, we have shown that homoarginine was inversely correlated to fibrinogen (r = −0.25; P < 0.001) and d-dimer levels (r = −0.28; P < 0.001); parameters of the coagulation system involved in cardiovascular disease.12 These results support previous findings that homoarginine inhibits platelet aggregation and are consistent with the hypothesis that homoarginine may exert antithrombotic effects and thereby enhance NO availability.24,25
Another major pathway linking high homoarginine to low stroke incidence may involve the expression of the L-arginine:glycine amidinotransferase (AGAT). This is the main enzyme responsible for homoarginine formation, and is mainly expressed in the kidney, but is also found in the heart and in the brain.3,26 Experimental studies have shown that when cerebral ischemia was induced by temporary vessel occlusion in AGAT knockout (ko) and AGAT wild type (wt) mice, AGAT ko mice exhibited stroke sizes which were twice as large as those in AGAT wt mice. Interestingly, when the AGAT ko mice received homoarginine supplementation, the size of the stroke was reduced substantially. Furthermore, AGAT ko mice presented larger neurological deficits after stroke compared to the wt mice. Again, the severity of neurological deficits was reduced by homoarginine supplementation.20,27
Concerning renal endpoints, we showed in our study that low homoarginine was significantly associated with increased graft loss and doubling of serum creatinine. This finding extends our previous results from a nontransplant population with mild to moderate kidney disease. That study showed that circulating homoarginine concentrations in CKD patients were significantly lower at lower GFR, were lower in patients with kidney disease progression as compared to those without progression and were inversely associated with the risk to reach a renal endpoint.28 Our study supports the notion that homoarginine similarly may become relevant for renal allograft recipients because our findings were independent from transplantation-specific factors and characteristics differing from those in CKD patients.
The kidney seems to play an important role in homoarginine formation. Homoarginine is mainly synthesized in the kidney by transaminidation of lysine,4 a reaction that is catalyzed by AGAT. There is an organ-specific pattern of AGAT expression, which is highest in renal tissue and supports a crucial role of the kidney in homoarginine metabolism.26 In our present study, we were able to confirm the results from the mild to moderate kidney disease study of a virtually linear association between homoarginine concentrations and GFR. In this context, it is of interest that genome-wide association studies showed polymorphisms of AGAT to be significantly associated with GFR.29,30
A number of different causes may account for graft loss and decline in kidney function in renal transplant recipients. These include alloantigen-dependent factors, such as episodes of acute rejection, HLA mismatching and sensitization, and alloantigen-independent factors, such as tissue injury, posttransplant hypertension, hyperlipidemia, recurrent glomerular disease or donor age, and illnesses. Our present study adds low homoarginine concentrations to the list, representing a potentially modifiable risk factor as target for novel future interventions. Taken together, low homoarginine concentration may therefore be an early indicator of kidney failure likely reflecting a decreasing synthesis capacity of the kidney.3
Potential limitations of the study need to be acknowledged. This was a post hoc analysis within a selected cohort of European and North American renal transplant recipients, and the results may not be generalizable to other patient populations. Despite our careful adjustments in the multivariate analysis, residual confounding cannot be completely ruled out, for example, by impaired renal function or transplantation-related conditions. However, known important confounders were considered and did not materially impact in the observed association of homoarginine and outcomes in our analyses. Thus, the effect of potential residual confounding is likely to be small. Furthermore, due to the observational nature of our study, a significant statistical association between homoarginine and the renal endpoint does not prove a causal relationship, as reduced homoarginine production may follow from previous graft damage and reduced renal function. Randomized trials are needed to prove causality. Furthermore, we could not perform homoarginine measurements right after transplantation and serial measurements during follow-up. There are also strengths to the study design. The prospective long-term follow-up, large sample size, and the prespecified and centrally adjudicated endpoints contribute to the reliability of the analyses.
In conclusion, in this first study of homoarginine in kidney transplant recipients, we have shown that low homoarginine concentrations are a strong risk factor for mortality, CBV events, and the combined endpoint of doubling of serum creatinine and graft loss in renal transplant recipients. Whether homoarginine supplementation improves posttransplant clinical outcome in these patients deserves further study including randomized controlled trials. For now, the association between low homoarginine and the higher occurence of the composite renal endpoint suggests that low homoarginine may be a marker of renal disease progression to be used for risk monitoring in renal transplant recipients.
MATERIALS AND METHODS
Study Design and Participants
The methodology of the ALERT study has previously been reported in detail.31 Briefly, this was a prospective randomized controlled trial investigating the effect of fluvastatin, 40 to 80 mg daily, on cardiac and renal outcomes in renal transplant recipients over a follow-up period of 5 to 6 years. The study included 2102 renal transplant recipients, aged 30 to 75 years, who had received a renal transplant more than 6 months before and had a serum cholesterol concentration between 4.0 and 9.0 mmol/L (155–348 mg/dL). Within the 3 months before randomization, patients should not have been taking statins and not have experienced an acute rejection episode. Further exclusion criteria were familial hypercholesterolemia and a life expectancy of less than 1 year. Study visits took place at randomization, at 6 weeks after randomization and every 6 months thereafter until the date of death, censoring, or end of the study. At each follow-up, blood samples were taken and clinical information including any adverse events was recorded. The study conformed to the principles outlined in the Declaration of Helsinki and adhered to the International Conference on Harmonisation guidelines for Good Clinical Practice. It was approved by the medical ethical committee of each participating center, and all patients gave their written informed consent before inclusion. We studied the 829 of 1052 patients in the placebo group who had available homoarginine measurements; the rest were missing at random.
Homoarginine was measured in blood samples taken at baseline and stored at −80 °C, using a reverse phase HPLC method.32,33 Within-day coefficients of variation were 4.7% (1.21 μM) and 2.2% (3.53 μM), and between-day coefficients of variation were 7.9% (1.25 μM) and 6.8% (3.66 μM), respectively. All blood samples were taken in the morning before the administration of medication. The measurements of homoarginine were performed at the Department of Clinical Chemistry at the Medical University of Graz, Austria. Furthermore, measurements of fasting lipids, serum creatinine, creatine kinase, and hepatic enzymes were performed centrally at Medinet Breda, the Netherlands.
The primary endpoint of the ALERT study was defined as a composite of death from cardiac causes, nonfatal MI, or coronary revascularisation procedure, whichever occurred first (major adverse cardiovascular event [MACE]). Coronary revascularization procedures included coronary artery bypass grafting or percutaneous coronary interventions. An MI was classified as definite if a new Q-wave, or pathological ST elevations and T-wave changes developed in the presence of abnormal cardiac markers plus symptoms. An MI was classified as probable if pathological ST elevations and T-wave changes developed in the presence of abnormal cardiac markers or symptoms. Predefined secondary endpoints were the individual cardiac events, combined CDNFMI, combined CBV events, noncardiovascular death, all-cause mortality, and the composite renal endpoint of GFDSC. The ALERT study endpoints were centrally adjudicated by 4 members of the endpoint committee blinded to study treatment and according to predefined criteria.17,31
For the present analysis, the primary endpoint of MACE, combined CDNFMI, combined CBV events, noncardiovascular death, all-cause mortality, and the composite renal endpoint of GFDSC were all chosen as separate outcome measures. The categorization of these events was based on the primary judgement of the endpoint committee during the ALERT study.
The study population was divided into 4 groups, according to quartiles of homoarginine levels at baseline: 1.40 μmol/L or lower, higher than 1.40 to 1.86 μmol/L or lower, higher than 1.86 to 2.34 μmol/L or lower, higher than 2.34 μmol/L. Demographic and clinical baseline characteristics were compared using analysis of variance for continuous and χ2 tests for categorical variables respectively. Continuous variables were expressed as mean with standard deviation or median with interquartile range as appropriate, and categorical variables were expressed as percentages. Variables with obvious non-normal distribution were logarithmically transformed before running tests for linear trend. We assessed the association of baseline homoarginine with the specific cardiovascular and renal events: primary endpoint of MACE, combined CDNFMI, combined CBV events, noncardiovascular death, all-cause mortality, and the composite renal endpoint of GFDSC. For continuous analyses, risk is estimated per unit increase in homoarginine, whereas in the categorical analyses, patients of the highest homoarginine quartile were used as the reference group. Nelson-Aalen cumulative hazard estimates were performed in each group, and the log-rank test was computed to compare the curves. Relative risks were determined by Cox regression analyses, that is, HRs and corresponding 95% CIs. The Cox regression analyses were adjusted for potential confounders including age, sex, diabetes mellitus, coronary artery disease, smoking status, systolic blood pressure, low-density lipoprotein cholesterol, and eGFR. The confounders were chosen based on previous knowledge from the literature. In additional analyses, we also adjusted the multivariate model for inflammation (high sensitive C-reactive protein, interleukin-6), calcium and phosphate, time since last transplantation, treatment for cytomegalovirus, total time on renal replacement therapy, treatment for rejection, proteinuria, HLA-DR mismatches, delayed graft function and panel reactive antibodies. All P values are reported two-sided. Analyses were performed using SPSS version 19.0 and STATA version 11 (StataCorp, College Station, TX).
The authors thank all patients who participated in the ALERT study. The authors are grateful to all investigators, study nurses, and collaborators involved in patient recruitment, sample and data handling, and laboratory staffs.
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