Proenkephalin, an Opioid System Surrogate, as a Novel Comprehensive Renal Marker in Heart Failure.

BACKGROUND
PENK (proenkephalin) is a stable surrogate for enkephalins, endogenous opioid peptides, which exert cardiodepressive effects and improve renal function. PENK has been associated with heart failure (HF) severity and renal dysfunction. We therefore hypothesized that PENK could be associated with deterioration of kidney function and could have a role as a novel renal marker in HF.


METHODS AND RESULTS
In 2180 patients with HF of a large multicenter cohort (BIOSTAT-CHF [A Systems Biology Study to Tailored Treatment in Chronic Heart Failure]), the relationship between PENK and clinical variables, plasma and urinary biomarkers, and clinical end points was established. Data were validated in a separate cohort of 1703 patients with HF. PENK was elevated (>80 pmol/L, 99th percentile) in 1245 (57%) patients. Higher PENK was associated with more advanced HF and glomerular and tubular dysfunction. The strongest independent predictor of PENK was estimated glomerular filtration rate. Others were plasma NGAL (neutrophil gelatinase-associated lipocalin) and NT-proBNP (N-terminal pro-B-type natriuretic peptide; all P<0.001). Using correlation heatmaps and hierarchical cluster analyses, PENK clustered with estimated glomerular filtration rate, creatinine, NGAL, galectin-3, and urea. Higher PENK was independently associated with increased risk of deterioration of kidney function between baseline and 9 months (odds ratio, 1.29 [1.02-1.65] per PENK doubling; P=0.038; defined as >25% decrease in estimated glomerular filtration rate) and mortality (hazard ratio, 1.23 [1.07-1.43] per doubling; P=0.004). Analyses in the validation cohort yielded comparable findings.


CONCLUSIONS
Higher PENK levels are associated with more severe HF, with glomerular and tubular renal dysfunction, with incidence of a deterioration of kidney function, and with mortality. These findings suggest that the opioid system might be involved in deteriorating kidney function in HF.

E nkephalins are endogenous opioid peptides that exert several cardiovascular effects by reducing myocardial contractility, blood pressure, and heart rate, while also inhibiting norepinephrine release and sympathetic vasoconstriction. 1 Opioid peptides also play a role in ischemic preconditioning and cardiac hypertrophy. 1 Besides cardiovascular effects, enkephalins exert renal effects predominantly by increasing renal blood flow and urinary output. 2 By their established effects on cardiac contractility, hemodynamics, and renal function, enkephalins might play a pathophysiological role in the development and progression of cardiorenal failure.
However, endogenous enkephalins are unstable and difficult to measure in plasma. PENK (proenkephalin) is a stable and accurate surrogate marker of enkephalins that can be easily measured in plasma. 3,4 A previous study showed that in patients with an acute myocardial infarction, higher PENK levels were associated with a higher risk for the development of heart failure (HF). 5 In studies investigating PENK in HF populations, PENK levels were higher in patients with HF compared to healthy individuals, 6 and higher levels of PENK were associated with worse(ning) renal function, HF severity, and adverse clinical events. 6,7 Further validation and a deeper understanding of the role of PENK in HF is however required. Because of the profound cardiovascular and renal effects of enkephalins and previously reported associations of PENK with both disease severity and renal dysfunction in HF, we hypothesized that PENK, as a surrogate of the opioid system, could become a novel renal marker in HF, reflecting both cardiac, glomerular, and tubular dysfunc-tion. To establish this, we investigated PENK in relation to a wide variety of clinical variables, plasma and urinary biomarkers, and clinical outcome, and the findings were subsequently validated in a separate patient cohort.

Patient Populations
The data that support the findings of this study are available from the corresponding author upon reasonable request. For the present study, we used the index and validation cohort of BIOSTAT-CHF (A Systems Biology Study to Tailored Treatment in Chronic Heart Failure), which have been described in detail before. 8 In brief, BIOSTAT-CHF was an investigator-driven multicenter clinical study consisting of 2516 patients in the index cohort with the aim to identify patients with a poor outcome despite currently recommended treatment. Patients were included after presentation with either new onset or worsening HF, which was defined as left ventricular ejection fraction (EF) ≤40% or BNP (brain natriuretic peptide) >400 pg/mL or NT-proBNP (N-terminal pro-B-type natriuretic peptide) >2000 pg/mL. Patients were expected and encouraged to be uptitrated to recommended treatment doses. 9 Data were validated in the BIOSTAT-CHF validation cohort consisting of 1738 patients with HF from 6 centers in Scotland, United Kingdom. 8 In summary, patients ≥18 years diagnosed with HF with a previous hospital admission for HF requiring diuretic treatment and current treatment with furosemide ≥20 mg/d or equivalent were included. They had previously not been treated or received ≤50% of target doses of ACE (angiotensin-converting enzyme) inhibitors/ARBs (angiotensin receptor blockers) and β-blockers as described in the 2008 European Society of Cardiology guidelines and treatment was expected to be initiated or uptitrated.
All patients enrolled in BIOSTAT-CHF provided written informed consent to participate in the study and BIOSTAT-CHF was conducted in concordance with the declaration of Helsinki, national ethics and legal requirements, as well as relevant EU legislation. The study was also approved by national and local ethics committees.

Study Design and Biomarker Measurements
PENK was measured in 2180 patients in the index cohort and in 1703 patients in the validation cohort. The study subsets used in this study did not differ greatly from the patients without PENK measurements in the BIOSTAT index and validation cohort (Tables I and II in the Data Supplement). PENK was measured using a sandwich immunoassay targeting PENK amino acids 119-159 (sphingotest penKid, Sphingotec GmbH, Hennigsdorf, Germany) as described previously. 3,5 The lower detection limit was 5.5 pmol/L and intra-and interassay coefficients of variation were 6.4% and 9.5%, respectively at 50 pmol/L and 4.0% and 6.5%, respectively at 150 pmol/L. 3 The normal range of PENK has been established in a general population cohort (the Malmö Diet and Cancer Study), where PENK was measured in 1929 healthy individuals. 10 The median (range) was 45 (9-518) pmol/L, and the 99th percentile was 80 pmol/L. The association between PENK and deterioration of kidney function was WHAT IS NEW? • The opioid system, reflected by PENK (proenkephalin), is not only independently associated with glomerular dysfunction, but also with tubular damage in heart failure (HF). • After subjection to meticulous positioning across a wide variety of clinical variables, plasma, and urinary biomarkers, PENK was repeatedly positioned in a panel of renal markers in HF. • Our study confirms the possibility of the opioid system being a common pathway affecting both the heart and kidney in HF, a cardiorenal connector. • PENK was strongly associated with increased mortality, but not with a higher risk of HF readmissions.

WHAT ARE THE CLINICAL IMPLICATIONS?
• PENK could become a novel, comprehensive renal marker in HF, reflecting both cardiac, glomerular, and tubular dysfunction. • Higher PENK levels relate to an increased risk of mortality.
analyzed with the latter defined as >25% decrease in estimated glomerular filtration rate (eGFR) between baseline and 9 months, with sensitivity analyses performed for >30% decrease and >40% decrease in eGFR. 11,12 Furthermore, associations between PENK and change of creatinine over time as a continuous variable was analyzed in additional sensitivity analyses. After blood was drawn by venipuncture, samples were stored at −80°C. If possible, analyses were performed directly based on standardized international methods, otherwise, they were performed in a central laboratory. Urinary measurements were performed using standardized methods. All urinary measurements were corrected for urinary creatinine. Urinary KIM (kidney injury molecule)-1 and NGAL (neutrophil gelatinase-associated lipocalin) were measured using an in-house developed and validated multiplex immunoassay (xMAP; Luminex, Austin, TX) as described previously. 13 IL (interleukin)-6 and endothelin-1 were measured in frozen plasma by Singulex Inc (Alameda, CA) using high-sensitive single molecule counting (SMC) technology (RUO, Erenna Immunoassay System). Bio-ADM (bioactive adrenomedullin) was measured using an immunoassay developed by Sphingotec GmbH (Hennigsdorf, Germany). 14 High-sensitivity troponin T was measured using the Roche Elecsys assay on a Cobas e411 analyzer, using standard methods (Roche Diagnostics GmbH, Mannheim, Germany). Measurement of additional biomarkers was performed as previously described. 15,16

Study End Points
In the index cohort and the validation cohort, the relation of PENK with 3 clinical outcomes was evaluated: all-cause mortality, unscheduled HF hospitalization, and a composite outcome of all-cause mortality and HF hospitalization. The end points were adjusted for the BIOSTAT risk models that were created for each specific outcome in this cohort, which included age, log blood urea nitrogen, log NT-proBNP, hemoglobin, and β-blocker use at baseline for all-cause mortality; it included age, HF hospitalization in previous year, peripheral edema, systolic blood pressure, and eGFR for HF hospitalization; and it included age, HF hospitalization in previous year, systolic blood pressure, log NT-proBNP, hemoglobin, highdensity lipoprotein, sodium, and β-blocker use at baseline for the combined end point. 17

Baseline Characteristics According to Plasma PENK Levels
In the BIOSTAT-CHF index cohort, mean left ventricular EF was 31%, 49% of patients were in New York Heart Association class III, and 7% of patients had HF with preserved EF (HFpEF). Baseline characteristics in relation to quartiles of PENK are displayed in Table 1. Median plasma PENK concentration was 86.2 (63.7-120.2) pmol/L, and 1245 (57%) patients had PENK levels >80 pmol/L (99th percentile of the normal range of PENK). Higher levels of PENK were found in older patients and in patients with more severe HF (higher New York Heart Association classification, higher natriuretic peptides) lower systolic blood pressure, poorer renal function (higher creatinine, lower eGFR, higher plasma NGAL, higher urea), a higher degree of albuminuria (urine albumin-to-creatinine ratio [UACR]), and a higher degree of tubular damage (higher urinary KIM-1, higher urinary NGAL; all P for trend ≤0.001). Table 2 shows that the strongest associations of higher levels of log plasma PENK concentrations were with lower eGFR, higher plasma NGAL, higher NT-proBNP, lower ALAT (alanine transaminase), and lower diastolic blood pressure. The adjusted R 2 for the model was 0.561 (Table 2).

PENK as a Predictor of Deterioration of Kidney Function
The relationship between PENK and a deterioration of kidney function is displayed in decrease and 40% decrease in eGFR as the definition for deterioration of kidney function provided comparable results. Doubling of baseline eGFR was univariably not predictive of deterioration of kidney function and showed a significant interaction with PENK, which led to exclusion from the adjusted model. Doubling of PENK remained a significant and strong predictor when regression analysis was repeated with serum creatinine change as a continuous variable and as an end point in receiver operating characteristic curves (Table III and Figure III in the Data Supplement). Failure; BNP, brain natriuretic peptide; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; ET-1, endothelin-1; FGF-23, fibroblast growth factor-23; gCr, gram of urinary creatinine; GDF-15, growth differentiation factor-15; HFpEF, heart failure with preserved ejection fraction; IL-6, interleukin-6; KIM-1, kidney injury molecule-1; LVEF, left ventricular ejection fraction; NGAL, neutrophil gelatinase-associated lipocalin; NT-proBNP, N-terminal pro-B-type natriuretic peptide; NYHA, New York Heart Association; PENK, proenkephalin; RAGE, receptor of advanced glycation end products; ST-2, suppression of tumorigenicity-2; TNFR-1A, tumor necrosis factor-α receptor-1a; TnT, troponin T; and UACR, urine albumin-to-creatinine ratio.

Results in the Validation Cohort
The above analyses were subsequently validated in 1703 patients of the BIOSTAT-CHF validation cohort.
In the BIOSTAT-CHF validation cohort, mean left ventricular EF was 41%, 44% of patients were in New York Heart Association class III, and 34% of patients had HFpEF. Baseline characteristics of quartiles of PENK in the BIOSTAT-CHF validation cohort are displayed in Table VII in the Data Supplement. An association between higher PENK levels and, among others, higher New York Heart Association classification, lower hemoglobin, higher NT-proBNP, higher creatinine, lower eGFR, higher urea, and higher UACR (all P for trend <0.001) was confirmed.

Linear Regression Analysis for PENK
The strongest predictors for log PENK were eGFR, log glucose, log urea, log NT-proBNP, and log ALAT, adjusted R 2 for the model=0.639 (Table 2).

Biomarker Position of PENK
As a sensitivity analysis, we repeated these analyses in the validation cohort, including only variables available in the BIOSTAT-CHF validation cohort. This resulted in a comparable correlation heatmap ( Figure

DISCUSSION
In 2 large independent cohorts of patients with HF, we demonstrated that plasma PENK levels were elevated and related to more severe HF, and worse renal function, both reflected by glomerular and tubular renal markers. These findings were validated in an independent cohort of patients with HF. Remarkably, higher PENK levels were a predictor of deterioration of kidney function independent of UACR and tubular markers. Finally, higher PENK levels were associated with increased mortality, but not with a higher risk of HF readmissions. The role of PENK in HF, and thereby the possible role of the opioid system, might be defined by (1) its prominent association with renal dysfunction, which might lead to cardiac dysfunction, (2) cardiac depression, which might lead to renal dysfunction, or (3) a factor that both reflects cardiac and renal dysfunction. We suggest that the opioid system can be such a common pathway affecting both the heart and kidney in HF, a cardiorenal connector, which can be defined as "factors that are modulated by either heart or kidney failure, affect both organs, interact, and are associated with functional or structural, renal or cardiac consequences", 18 in addition to previously described common denominators. 19,20 Based on our results, the way the opioid system affects both the heart and kidney could be either of a beneficial Negative correlations are expressed in green, neutral associations in yellow, and positive associations in red. Correlations are based on Spearman ρ as a correlation coefficient. ALAT indicates alanine transaminase; ASAT, aspartate aminotransferase; BNP, brain natriuretic peptide; CRP, C-reactive protein; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; FGF-23, fibroblast growth factor-23; HR, heart rate; KIM-1, kidney injury molecule-1; LVEF, left ventricular ejection fraction; NGAL, neutrophil gelatinase-associated lipocalin; NT-proBNP, N-terminal pro-B-type natriuretic peptide; SBP, systolic blood pressure; and UACR, urine albumin-to-creatinine ratio. nature, or of a disadvantageous, damaging nature, as explained further on.

Higher PENK Levels Are Associated With More Advanced HF
Higher PENK levels were associated with more advanced HF, lower blood pressure, and lower heart rate, which can be explained by the direct cardiodepressive effects of PENK.
Alternatively, higher PENK levels with more advanced HF could have beneficial effects by influencing neurohormonal activity, which is a key mechanism in HF pathophysiology 21 with sympathetic nervous system activation being one of the responses. 22 The opioid system acts as an inhibitor of sympathetic activation; it inhibits norepinephrine release and sympathetic vascular constriction. Enkephalins are even coreleased with catecholamines in the heart. 23 The opioid system could therefore be an important mediator in opposing neurohormonal activity in HF, 1,2 which is supported by our finding of a strong correlation between higher levels of PENK and higher levels of renin. Similarly, natriuretic peptides are activated, and a relationship between the two has been demonstrated before. 24 With sympathetic stimulationhaving deleterious effects-being in overdrive in HF, it is possible that increased PENK levels are, in the end, a beneficial, adaptive mechanism limiting this overdrive, somewhat in similar vein as natriuretic peptides oppose activation of the renin-angiotensin Hierarchical cluster analysis of PENK in the index cohort based on the Spearman ρ correlation coefficient. The further down the scale a variable is positioned, the higher the correlation coefficient between a cluster of variables. ALAT indicates alanine transaminase; ANP, atrial natriuretic propeptide; ASAT, aspartate aminotransferase; Bio-ADM, bioactive adrenomedullin; BNP, brain natriuretic peptide; CRP, C-reactive protein; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ET-1, endothelin-1; FGF-23, fibroblast growth factor-23; GAL-3, galectin-3; GDF-15, growth differentiation factor-15; HR, heart rate; IL-6, interleukin-6; KIM-1, kidney injury molecule-1; LVEF, left ventricular ejection fraction; NGAL, neutrophil gelatinase-associated lipocalin; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PENK, proenkephalin; RAGE, receptor of advanced glycation end-products; SBP, systolic blood pressure; ST-2, suppression of tumorigenicity-2; TNFR-1A, tumor necrosis factor-α receptor-1a; and UACR, urine albumin-to-creatinine ratio. system. 1,24 The presence of a relationship between higher PENK levels and more advanced HF is furthermore supported by the fact that the highest proportion of enkephalins in the heart originate from cardiomyocytes themselves, 25,26 and the strong association between higher PENK levels and poor outcome in our study, especially the highest quartile of PENK. This was with the exception of HF hospitalization and the combined end point after adjustment, from which one could hypothesize that the association with allcause mortality may be largely through renal dysfunction. Interestingly, the association with all-cause mortality remained after extensive adjustment including multiple renal and other disease severity markers and the BIOSTAT risk model, which also includes several markers of HF severity and renal function, suggesting that PENK provides additional prognostic information. In baseline analyses, higher PENK also associated with a higher degree of cardiomyocyte stretch (ANP), remodeling (eg, galectin-3 and ST-2 [suppression of tumorigenicity-2]), and inflammation (eg, CRP [C-reactive protein] and IL-6). Because these are all related to more severe disease, this further strengthens the relationship between higher PENK and more advanced HF. A higher degree of inflammation, another key component in HF pathophysiology, 21 could furthermore be explained by the presence of endogenous opioid peptides at inflammatory sites through immune cells to provide analgesia. 27
*Base 2 log-transformed. †Adjusted for BIOSTAT risk score, log NT-proBNP, eGFR, and log UACR. Variables in BIOSTAT risk score: all-cause mortality: age, log blood urea nitrogen, log NT-proBNP, hemoglobin, and β-blocker use at baseline HF hospitalization: age, HF hospitalization in the previous year, peripheral edema, systolic blood pressure, and eGFR. Combined end point: age, HF hospitalization in the previous year, systolic blood pressure, log NT-proBNP, hemoglobin, high-density lipoprotein, sodium, and β-blocker use at baseline. ‡In the validation cohort, the variables included in adjusted model 2 as used in Table 4 were not available. §P value <0.05. ciated with higher albuminuria and higher levels of tubular damage markers, suggesting not only an association with glomerular dysfunction but with tubular damage as well. Finally, PENK was a predictor of deterioration of kidney function independent of UACR and tubular markers. The relationship between PENK and renal function emerges in existing literature. In renal transplant recipients, PENK associated with poorer renal function and an increased risk of graft failure. 29 Both after cardiac surgery and in septic patients PENK predicted acute kidney injury and strongly associated with creatinine and eGFR. 30,31 In a prospective cohort of patients without chronic kidney disease, PENK predicted deterioration of kidney function and chronic kidney disease onset. 32 PENK has also previously demonstrated a strong relation with renal function in HF. 6,7,33 Our study adds to these existing data by providing a more detailed position of PENK in a large number of HF patients including an extensive range of relevant renal function parameters, including several glomerular and tubular renal markers. PENK appears to be freely filtrated in the glomerulus, and it is indeed the case that, with declining eGFR, PENK levels rise. The association between PENK and renal function could thus be explained by PENK being a filtration signal, for which interest has already previously been shown to use PENK as an easy and reliable determinant of current GFR and possibly a better early indicator of kidney function decline and kidney injury, 34 but PENK might also serve a functional purpose.
Similar to the relationship between PENK and more advanced HF, the relationship between PENK and renal dysfunction could be either of a beneficial nature or of a disadvantageous, damaging nature. PENK could be secreted as a counterregulatory response to renal dysfunction through the effects of enkephalins on renal blood flow and urinary output 1,2 ; activation of δ receptors of enkephalins produces significant diuretic and natriuretic responses without changes in heart rate or blood pressure. 2,35 Alternatively, through the cardiodepressive response to PENK, 1,2 this could, in turn, result in reduced kidney perfusion.
With regards to the association between PENK and tubular damage specifically, this could be explained by increased chronic renal hypoxia and a higher degree of congestion (because of worse HF) with increased tubular damage as a consequence. Furthermore, both renal and HF are associated with a prooxidant status 18 including in tubular cells, 36 and PENK expression is upregulated in response to oxidative stress conditions. 37 In a previous study including both chronic and acute HF, 6 PENK was not associated with tubular damage markers. The chronic HF cohort that focused on tubular damage was conducted in stable, ambulatory patients, whereas our study included patients with either new onset or worsening HF with suboptimal treatment, hence possibly representing more severely ill patients, including presence of tubular damage. Furthermore, the previous data on tubular damage was limited to only 95 chronic HF patients, compared with 2180 in the present study.

Future Perspectives
Our data confirm and extend the relationship between the heart and kidneys in HF. Dysfunction of both organs often coexists, and cardiac factors can lead to renal damage or vice versa, or there are common pathways that influence both cardiac and renal function. 18,19 The opioid system, represented by PENK, has mechanistic grounds to be such a common pathway affecting both the heart and kidney (Figure 4). A causal relationship can however only be established by prospective intervention studies. It therefore needs to be established whether the opioid system plays a causal role in the onset and progression of cardiorenal failure. Apart from being used as an analgesic agent in experimental animal models, therapeutic administration of PENK with respect to cardiovascular effects in HF has, to our knowledge, not been investigated.
Furthermore, it remains to be elucidated what the effects of the use of HF therapy, and specifically sacubitril/valsartan will be on circulating PENK levels, because this study was conducted before the incorporation of this The relationship of higher PENK levels with (worsening) glomerular dysfunction might be through chronic reduced kidney perfusion, but the response of higher levels of PENK to renal dysfunction might also be of a counterregulatory nature. Higher PENK levels could be connected to tubular dysfunction/damage through chronic renal hypoxia, a higher degree of congestion, and oxidative stress. A wide variety of (renal) biomarkers illustrates this relationship. This association of PENK with renal dysfunction could, in the end, largely contribute to its relationship with increased risk of mortality. eGFR indicates estimated glomerular filtration rate; FGF-23, fibroblast growth factor-23; GAL-3, galectin-3; Hb, hemoglobin; and UACR, urine albumin-to-creatinine ratio. Illustrations were adapted from Servier Medical Art (https://smart. servier.com/). Copyright © 2019, Servier. These are Open Access images distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. drug in HF management. Because enkephalins are a substrate of neprilysin, this will be a relevant issue to address.

Strengths and Limitations of the Study
The in-depth analysis of PENK across a wide variety of clinical variables and biomarkers is a strength of our study, since it allowed for meticulous positioning of PENK. Furthermore, results were based on a large number of patients that were included in this multicenter, multinational cohort. The likelihood that results are applicable to the general chronic HF population are increased due to this cohort being a very heterogeneous HF population and validation of results in a separate validation cohort that yielded very comparable results.
Shortcomings of this study are the inability to extend results beyond the European population and to show causality instead of associations. The precise stability of the centrally determined biomarkers after frozen storage time is unknown, but we feel it is unlikely that this significantly impacted their concentration, especially for PENK due to extensive testing in this regard. 38 Some analyses included less patients because of missing values. PENK was measured in a subset of patients, and even though baseline characteristics did not differ between in-and excluded patients, informed censoring cannot be fully ruled out. The study populations mainly included HF patients with reduced EF, with different proportions between the study populations, which can be traced back to their study designs. We were also not able to show changes of PENK levels over time which could have contributed to a better understanding of its dynamics.

Conclusions
This study shows that higher levels of PENK are associated with worse HF, deterioration of kidney function beyond creatinine, and notably both glomerular dysfunction and tubular damage in a large chronic HF study population. PENK was independently related to poor outcome in HF. This study therefore provides clues for potential pathophysiological mechanisms of PENK and the opioid system and positioned PENK as a potential novel comprehensive renal marker in patients with HF.