The differential activation of cardiovascular hormones across distinct stages of portal hypertension predicts clinical outcomes

Background and aims The cardiovascular hormones renin/angiotensin/aldosterone (RAA), brain-type natriuretic peptide (BNP)and arginine-vasopressin (AVP) are key regulators of systemic circulatory homeostasis in portal hypertension (PH). We assessed (i) the activation of renin, BNP and AVP across distinct stages of PH and (ii) whether activation of these hormones correlates with clinical outcomes. Methods Plasma levels of renin, proBNP and copeptin (AVP biomarker) were determined in 663 patients with advanced chronic liver disease (ACLD) undergoing hepatic venous pressure gradient (HVPG) measurement at the Vienna General Hospital between 11/2011 and 02/2019. We stratified for Child stage (A–C), HVPG (6–9 mmHg, 10–15 mmHg, ≥ 16 mmHg) and compensated vs. decompensated ACLD. Results With increasing PH, hyperdynamic state was indicated by higher heart rates (6–9 mmHg: median 71.0 [IQR 18.0] bpm, 10–15 mmHg: 76.0 [19.0] bpm, ≥ 16 mmHg: 80.0 [22.0] bpm; p < 0.001), lower mean arterial pressure (6–9 mmHg: 103.0 [13.5] mmHg, 10–15 mmHg: 101.0 [19.5] mmHg, ≥ 16 mmHg: 99.0 [21.0] mmHg; p = 0.032) and lower serum sodium (6–9 mmHg: 139.0 [3.0] mmol/L, 10–15 mmHg: 138.0 [4.0] mmol/L, ≥ 16 mmHg: 138.0 [5.0] mmol/L; p < 0.001). Across HVPG strata (6–9 mmHg vs. 10–15 mmHg vs ≥ 16 mmHg), median plasma levels of renin (21.0 [50.5] vs. 25.1 [70.9] vs. 65.4 [219.6] µIU/mL; p < 0.001), proBNP (86.1 [134.0] vs. 63.6 [118.0], vs. 132.2 [208.9] pg/mL; p = 0.002) and copeptin (7.8 [7.7] vs. 5.6 [8.0] vs. 10.7 [18.6] pmol/L; p = 0.024) increased with severity of PH. Elevated renin levels independently predicted first hepatic decompensation (adjusted hazard ratio [aHR]: 1.69; 95% confidence interval [95% CI] 1.07–2.68; p = 0.025) and mortality in compensated patients (aHR: 3.15; 95% CI 1.70–5.84; p < 0.001) and the overall cohort aHR: 1.42; 95% CI 1.01–2.01; p = 0.046). Elevated copeptin levels predicted mortality in decompensated patients (aHR: 5.77; 95% CI 1.27–26.33; p = 0.024) and in the overall cohort (aHR: 3.29; 95% CI 1.36–7.95; p = 0.008). ProBNP levels did not predict clinical outcomes. Conclusions The cardiovascular hormones renin, proBNP and AVP are activated with progression of ACLD and PH. Renin activation is a risk factor for hepatic decompensation and mortality, especially in compensated patients. Increased plasma copeptin is a risk factor for mortality, in particular in decompensated patients. Supplementary Information The online version contains supplementary material available at 10.1007/s12072-021-10203-9.

PH in ACLD is caused by both increased intrahepatic vascular resistance and hyperdynamic circulation. An abundance of vasodilators results in increased inflow into the portal venous system, aggravating PH [7]. At the same time, a decrease in peripheral vascular resistance is compensated by elevated cardiac output to maintain mean arterial pressure (MAP), which also promotes splanchnic hyperemia [8].
The renin/angiotensin/aldosterone (RAA) system, braintype natriuretic peptide (BNP) and arginine-vasopressin (AVP) not only represent key regulators of circulatory homeostasis, but also play an important role in PH and hyperdynamic circulation [9][10][11]. While AVP production is primarily regulated by central osmoreceptors sensing plasma osmolality [12], BNP is released by stretching of the myocardium [13]. The regulation of renin secretion is complex, as it is triggered by a decrease in MAP (baroreceptor stimulation), a decrease of sodium concentration in the distal tubule (macula densa stimulation), or sympathetic nervous system activity (beta-1-adrenergic stimulation) [14]. In 1980, Bosch et al. showed that plasma renin activity directly correlates with wedged hepatic venous pressure (WHVP) [15], suggesting a link between RAA activation and PH, and there is overwhelming evidence of increased levels of renin in cirrhotic patients with ascites [15][16][17]. Furthermore, previous studies have indicated that increased levels of renin and copeptin, an AVP biomarker, may indicate increased risk for mortality in ACLD patients [17][18][19][20]. However, these three systems of circulatory homeostasis are yet to be systematically investigated considering both the severity of liver dysfunction and of PH.
The objectives of this study were to assess the activation of renin, proBNP and AVP in distinct stages of (i) liver dysfunction, i.e. MELD/Child strata, (ii) of portal hypertension, i.e. HVPG strata, and (iii) to investigate whether alterations of these systems correlate with the risk for clinical events.

Study population
Patients with ACLD undergoing measurement of HVPG at the Vienna General Hospital between 11/2011 and 02/2019 showing PH were included. At the time of HVPG measurement, hemodynamic parameters (heart rate, MAP) were recorded and blood samples were withdrawn in supine position after the patients rested for at least 30 min. Notably, as HVPG measurement is usually performed as an outpatient procedure, most of our patient were not hospitalized at the time of catheterization.
Patients on non-selective beta-blockers, with invalid HVPG measurements and lack of critical clinical or laboratory data were excluded. Patients after liver transplantation (LT) or transjugular intrahepatic portosystemic shunt (TIPS), with cardiac cirrhosis or hepatocellular carcinoma (HCC) out of Milan criteria were excluded.
Further information on measurement of HVPG, laboratory parameters and statistical analysis is provided in the supplementary material.

Ethics
The study was approved by the ethics committee (EC) of the Medical University of Vienna (1493/2016) and performed according to the current version of the Helsinki Declaration. Due to the retrospective design of the study the EC waived the need for informed consent. (Table 1) A total number of n = 663 patients with a median age of 56.6 [IQR 15.5] years and male predominance (n = 452; 68.2%) were included. 307 (46.2%) patients had   Association of the severity of portal hypertension with renin, proBNP and copeptin levels ( Fig. 1 [18.6] pmol/L, p = 0.024) increased with severity of PH-although the findings were not very consistent for both proBNP and copeptin, and significant increases were only observed in the HVPG ≥ 16 mmHg vs. HVPG 10-15 mmHg strata.

Biomarkers of circulatory homeostasis across Child stages and MELD strata (Fig. 1), in hyponatremia and hypotension (Fig. S2)
Hepatic dysfunction impacted on all three main parameters, as plasma levels of renin (Child-A: 17.7 [31.4]

Discussion
In our thoroughly characterized cohort of ACLD patients, we observed progressive hemodynamic alterations with increasing severity of PH. Stratifying our population not only by hepatic dysfunction (by Child stage and MELD), but also for portal pressure (by the diagnostic gold-standard HVPG), we were able to evaluate the activation of key cardiovascular hormones across the full spectrum of ACLD severity. Renin, proBNP and copeptin levels all increased with severity of PH, indicating activation of all three cardiovascular hormonal systems induced by PH-associated systemic hemodynamic alterations. Notably, the increase of these parameters between patients with HVPG 6-9 mmHg vs. 10-15 mmHg was not significant, suggesting that only pronounced stages of PH have a clinically relevant impact on the RAA, proBNP and AVP pathways. This confirms and goes beyond the findings of Bosch et al. and Arroyo et al. [15,20], who showed correlations of renin activity and WHVP. It is also in line with previous studies that showed elevated plasma levels of renin, proBNP and copeptin particularly in cirrhotic patients with ascites or other PH-related complications [18,21,22]. Considering the implications of RAA activation in the pathogenesis of ascites, these findings explain why HVPG-values ≥ 16 mmHg identify cACLD patients at a particularly high risk for hepatic decompensation [6,23]. Moreover, renin, proBNP and copeptin levels increased throughout CTP stages and MELD strata, underlining the close correlation of these parameters with the degree of hepatic dysfunction [17,18,22]. While there was no correlation of ejection fraction or IVC diameter with severity of portal hypertension, future studies should investigate how changes of the splanchnic/systemic circulation that are primarily caused by portal hypertension, may affect cardiac dysfunction in patients with cirrhosis.

Renin
Plasma renin levels were significantly increased in patients with arterial hypotension and hyponatremia and renin was independently associated with serum sodium concentration, suggesting renin release upon impaired kidney perfusion or due to reduced sodium concentration sensed by the macula densa [14]. Moreover, renin levels correlated with HVPG, which affirms the association between plasma renin levels and the severity of PH. It has been reported that RAA activation plays a major role in the development of the hemodynamic alterations observed in cirrhotic patients with ascites [21,24]. However, there is also evidence that RAA is mechanistically involved in splanchnic vasodilation and increased intrahepatic resistance already in early stages of fibrosis and PH [21].
Importantly, cACLD patients with elevated renin plasma levels experienced first hepatic decompensation earlier with plasma renin levels being an independent predictor of first hepatic decompensation, while such an association was not found for further decompensation/ death in more advanced dACLD patients. Elevated plasma renin levels were also associated with shorter transplantfree survival and predicted mortality in cACLD, as well as in the overall cohort on adjusted Cox regression analysis. Notably, patients in the cACLD cohort were strictly compensated without (previous) ascites or ascites-related intake of diuretics. These findings are of clinical relevance, as they extend beyond findings of previous studies that reported increased plasma renin levels mostly in patients with ascites [15,17,25]. While most (78.2%) of our cACLD cohort had renin levels within the physiological range, our data suggest that a pathological RAA activation may already occur in compensated patients with PH, which is also paralleled by an increased risk for hepatic decompensation and mortality. Since RAA blockade is safe in compensated Child-A patients [26] and also seems to be particularly effective at lowering portal pressure in early ACLD stages [27], the use of RAA biomarkers may identify subgroups of patients, who particularly benefit from RAA blockade.

Natriuretic peptide proBNP
ProBNP levels increased with advanced liver dysfunction and in pronounced PH (HVPG ≥ 16 mmHg). Increased proBNP levels may indicate a hyperdynamic state in ACLD patients, as they are associated with arterial hypotension and creatinine, potentially linking proBNP to kidney dysfunction. This finding may explain, why elevated proBNP was associated with further decompensation and/or death in already decompensated ACLD patients, although its natriuretic and diuretic functions [13] should actually be beneficial in hyperdynamic ACLD patients by counteracting volume overload. Kidney dysfunction may trigger proBNP secretion through associated volume overload [28], with BNP being insufficient to overcome this volume overload in this particular setting of PH-associated renal dysfunction. Future studies should specifically address the value of proBNP levels in assessing cardiac dysfunction and subsequent renal hypoperfusion and/or acute kidney injury in patients with dACLD. Finally, proBNP levels could also be indicative of cirrhotic cardiomyopathy [29], which is supported by an association Fig. 2 Incidence of first decompensation in a-c cACLD patients binary for elevated/non-elevated renin, proBNP and copeptin plasma levels. d-f Incidence of further decompensation in dACLD patients. Time to any first/further decompensation event (development/worsening of ascites or HE, or development of variceal bleeding), death or end of follow-up. Log-rank test is used to determine differences between the groups. proBNP probrain-type natriuretic peptide, ULN upper limit of normal ◂  of proBNP levels and heart failure in our cohort, as well as a recent study reporting increased risk for post-TIPS heart failure in patients with elevated BNP levels [30].

Copeptin
Copeptin was negatively associated with serum sodium concentration, indicating an inadequate activation of AVP in ACLD similar to the syndrome of inappropriate antidiuretic hormone secretion (SIADH) [31], causing additional water retention and worsening of dilutional hyponatremia. This hypothesis is in line with our finding of increased copeptin levels in hyponatremic ACLD patients. The activation of AVP in hyperdynamic portal hypertensive patients likely indicates progressive worsening of the hemodynamic state, as previous studies have reported that plasma copeptin may be a surrogate marker for hemodynamic derangement and worse prognosis in patients with ACLD [18,32]. In our cohort, elevated copeptin levels were linked to shorter time to first hepatic decompensation in cACLD patients, but failed to reach statistical significance on multivariate analysis, potentially due to the small sample size of this subgroup. Moreover, elevated plasma copeptin levels were independently associated with increased mortality in dACLD patients and in the overall ACLD cohort.

Limitations
A limitation of this study is that proBNP and copeptin were not evaluated in all patients and thus, duration of follow-up   Fig. 3 a-c Transplant-free mortality in ACLD binary for elevated/ non-elevated renin, proBNP and copeptin plasma levels. Transplantfree mortality in d-f cACLD patients and in g-i dACLD patients.
Time to death, liver transplantation or end of follow-up. Log-rank test is used to determine differences between the groups. proBNP probrain-type natriuretic peptide, ULN upper limit of normal  was not similar for the different outcome analyses. Additionally, a single assessment of cardiovascular hormones has limitations and is to be seen as additional tool for risk stratification, rather than replacement of HVPG measurement. Moreover, for copeptin, sample size was limited. Intake of diuretics and angiotensin-converting-enzyme inhibitors/ angiotensin-1-receptor blockers (ACEi/ARB) was unequally distributed throughout the strata of PH and hepatic dysfunction, as patients with ascites are usually treated with diuretics, while ACEi/ARB are discontinued due to the risk of acute kidney injury. Importantly, proBNP and copeptin levels were not influenced by the intake of diuretics or ACEi/ ARB. However, renin levels correlated with diuretic treatment, which was not routinely discontinued before HVPG measurement and blood withdrawal. Of note, no patient was treated with AVP analogues.

Conclusions
Our results demonstrate a differential activation of renin, natriuretic proBNP and AVP as critical hormones of circulatory homeostasis across distinct stages of PH in patients with ACLD. Increased renin levels predict first decompensation and mortality in ACLD and especially cACLD patients, while elevated copeptin levels were independently associated with mortality, in particular in dACLD patients. Future studies should investigate if these biomarkers may identify ACLD patients who benefit from specific therapeutic interventions that target the molecular signaling axis of these cardiovascular hormones.
Author contributions All authors contributed either to research design (LH, MM and TR) and/or the acquisition (LH, MJ, DS, CD, BS, RP, EE, TR), analysis (LH, MJ, MM, TR) or interpretation (all authors) of data. LH, MM and TR drafted the manuscript, which was critically revised by all other authors.
Funding Open access funding provided by Medical University of Vienna. This study was supported by a Gilead International Liver Disease Research Scholarship (2018-FA716E0930) to T.R.