Decreased level of serum NT-proCNP associates with disease severity in COVID-19

The natriuretic peptides are potent modulators of vascular homeostasis [1]. Besides their central role in the regulation of fluid/electrolyte balance, vascular tone and cardiac remodelling [2], the amino terminals of both atrial natriuretic peptide (ANP; NT-proANP) and brain natriuretic peptide (BNP; NT-proBNP) are elevated in septic patients, most probably due to depressed myocardial function and ventricular dilatation, which represent septic heart failure (HF) signalling. Moreover, NT-proBNP can be used as a biomarker to predict survival in patients with severe sepsis [3]. In addition, the mid-region fragment of the proANP (MR-proANP) also emerged as a valuable prognostic marker of outcome in a broad range of clinical conditions, such as sepsis, heart failure or pneumonia [4‐6]. As newly discovered, the elevation of both NT-proBNP and MR-proANP predicts a worse outcome in COVID-19 [7, 8].

C-type natriuretic peptide (CNP) has been proven as a predictor of poor clinical outcome in critically ill patients [9‐11], since CNP upregulation is induced by proinflammatory cytokines including interleukin (IL) 1α, IL-1β, TNFα, by physical factors, such as shear stress or hypoxemia, or directly by bacterial endotoxins [12]. CNP however, has an important anti-inflammatory effect and also inhibits platelet reactivity [13].

As natriuretic peptides are synthetized in the form of prepropeptides, they need further proteolytic processing to be converted into their biologically active form [14]. Regarding CNP, furin has a crucial role in the cleavage of proCNP into NT-proCNP and the active CNP peptide. Without the intracellular proteolytic activation by furin, CNP cannot be secreted; therefore, it cannot fulfil its role in maintaining homeostasis [15]. Due to the short serum half-life of the CNP peptide, the measurement of the NT sequence with a longer half-life (also secreted following furin proteolysis) has found its way to clinical application [16].

Furin is a ubiquitous endoprotease with numerous substrates such as cytokines, growth factors, hormones, adhesion molecules, coagulation factors, membrane channels and albumin. In animal models, the lack of furin leads to an early death of the embryo due to severe developmental abnormalities [17].

Certain pathogens including human immunodeficiency virus (HIV), Ebola virus, measles virus, respiratory syncytial virus (RSV) also utilise a furin-mediated proteolysis, in particular for cellular entry [18‐20]. Similarly, severe acute respiratory syndrome coronavirus 2 (SARS-CoV‐2) extends the list of viruses using furin cleavage for cellular entry [18]. However, to our current knowledge, there are no clinical data available exploring the possible correlation between the serum levels of furin, CNP homeostasis and disease outcome in SARS-CoV-2 infection. Given the mutual utilisation of furin by both proCNP and SARS-CoV-2, there may be a competition for furin-mediated cleavage during SARS-CoV-2 infection. Thus, the investigation of serum NT-proCNP levels in COVID-19 is not only scientifically intriguing, but also paramount from a clinical point of view in the era of ongoing COVID-19 pandemic. Here, we aimed at characterizing the changes in NT-proCNP levels in COVID-19 patients, with special regard to disease severity and outcome.

CRP, PCT, IL-6 and NT-proBNP are well known predictors of poor clinical outcome in critically ill patients [3, 11]. Recently, multiple studies have shown that the inflammatory markers CRP, PCT, leukocytes, and IL-6 are important predictors of mortality in COVID-19, too [21‐24]. Along with inflammatory biomarkers, the predictive value of elevated NT-proCNP for poor clinical outcome in critically ill patients have also been previously described [11]. Herein, our study is the first to report discordant changes in the levels of NT-proCNP compared to cardiac and conventional inflammatory markers in a patient population with COVID-19. We identified that a decreased NT-proCNP level alone or with simultaneously elevated NT-proBNP and inflammatory parameters in COVID-19 patients on admission is significantly associated with a severe disease course (defined as the composite endpoint of need of mechanical ventilatory support/ECMO or death), thus serves as a good marker of poor outcome in COVID-19.

The detected NT-proCNP levels in the non-COVID-19 group were in line with previous observations and in the range of healthy controls [25].

To resolve the unexpected low levels of NT-proCNP in our COVID-19 positive patient population, one shall study the gene regulation and post-translational processing of CNP, which is, at present, not fully understood [12]. The syntheses of CNP and other natriuretic peptide family members are different. CNP is not stored in granules, unlike ANP or BNP, thus its serum level is always dependent on the production rate. Inflammatory signals, hypoxia and NT-proBNP induce gene transcription of CNP [12]. These parameters are significantly elevated or more common in severe COVID-19, which was also the case in our study cohort. There may be currently unknown regulators of CNP gene expression, possibly leading to a reduced serum level of the molecule. Endothelial damage occurring in deteriorating COVID-19 patients may lead to severe endothelial dysfunction with reduced excretory capacity, which hypothetically could also result in lower NT-proCNP levels. This would be in line with the findings of Dietmann et al., who described decreased NT-proCNP levels in severe malaria cases in children. Similarly to SARS-CoV-2, malaria is an intracellular pathogen and it is characterized by diffuse endothelial damage and dysfunction [26]. In contrast, the level of endothelin-1, a molecule also secreted by the endothelium was found to be elevated [26], which calls into question the strong connection between endothelial damage and reduced endothelial secretory capacity. Furthermore, endothelial damage is associated with “leaky” cell membranes and a net Ca²⁺ influx. At the same time, elevated intracellular Ca²⁺ levels would rather propagate the expression of CNP, as shown by Mendonca et al. [27]. When investigating the serum Ca²⁺ levels of our patients, we found significantly lower levels in COVID-19 than in non-COVID-19 patients and a decreased Ca²⁺ level associated non-linearly and significantly with death in COVID-19 patients. This is an expected finding, as about 50% of critically ill patients develop hypocalcaemia [28]. Nevertheless, our patients did not develop severe hypocalcaemia; the lowest ionised Ca²⁺ level in our cohort was 1.03 mmol/L. Ultimately, endothelial dysfunction resulting in a decreased CNP production seems to be an unlikely explanation for this phenomenon.

Thus, instead of the above explanation, we rather propose that a defect in one of the post-translational steps of the molecule may have caused a relative reduction of NT-proCNP in the severe COVID-19 group. There is one main post-translational step, the cleavage of NT-proCNP from proCNP, which can exclusively be performed by the specific endoprotease furin. According to Wu et al., the presence of furin is crucial in CNP processing [15]. Furin is also essential for the cellular entry of SARS-CoV-2 [18]. Recently, it was proposed that furin could hypothetically be redistributed in SARS-CoV-2 infection, especially in serious COVID-19, resulting in depleted furin activity [29]. As furin is a Ca²⁺-dependent endoprotease, relative hypocalcaemia observed in our COVID-19 group could also add to the previously proposed depleted furin activity, potentially leading to lower NT-proCNP levels in SARS-CoV-2 positive patients. However, hypocalcaemia in critically ill patients is not necessarily bound to a net Ca²⁺ loss, rather to redistribution resulting in intracellular hypercalcaemia [30, 31]. It was demonstrated in parathyreoid cell cultures that the activity of furin is not altered by the extracellular Ca²⁺ level, as no changes were found in furin-mediated proPTH-PTH conversion upon various Ca²⁺ levels [32]. Moreover, the extracellular Ca²⁺ did not alter the RNA expression of furin [33]. As furin plays a key role in the post-translational processing of numerous proteins (albumin, coagulation factors, CNP or parathyroid hormone) [34], its utilisation by SARS-CoV-2 during cellular entry may hypothetically decrease furin bioavailability for the cleavage of such proteins.

In accordance with the results of Ranta et al., who did not find any correlation between serum furin levels and the outcome of sepsis, we could not identify any difference in furin levels between any of the study groups [35]. Nonetheless, whether our measured furin levels truly represent the “whole body furin pool” or rather a relative, stable serum concentration relying on cellular redistribution, is still to be clarified. The activity of accessible furin may also influence its molecular effects [36]. Either way, it is an intriguing hypothesis that SARS-CoV-2 may channel available furin to itself in order to propagate cellular entry of the virus, which then leads to relative furin shortness and the consequent disturbance of furin-mediated post-translational processing of other proteins. If this turns out to be the case, it is of utmost importance to identify what other substrates of furin are affected and how this may influence the course of COVID-19. The novel finding of Wang et al., who demonstrated a SARS-CoV-2 infection-induced alpha-soluble NSF (N-ethylmaleimide-Sensitive Factor) attachment protein (α-SNAP)-mediated furin inhibition in HEK293 (Human Embryonic Kidney 293) and Calu-3 (non-small-cell lung cancer cell line) cells, could strengthen our furin deficiency hypothesis in SARS-CoV-2 infection [37]. Recently, Fajnzylber et al. found that viral load is associated with disease severity and mortality in COVID-19 [38]. Accordingly, the association between disease severity and serum NT-proCNP levels can be explained by a viral load-dependent furin redistribution and consequent unavailability for proteolytic cleavage. It is known that furin is a key molecule for the cellular entry of several other pathogens, too (HIV, Ebola virus, measles virus, RSV) [19]. However, in the literature, no data exist on serum NT-proCNP levels in these infections.

Our findings of a decreased serum NT-proCNP level and its association with a worse disease course in COVID-19 may highlight the clinical importance of CNP in SARS-CoV-2 infection. As CNP is a main regulator of vascular homeostasis, leukocyte activation and platelet reactivity, a decrease in NT-proCNP levels may correspond to all main complications of COVID-19 (acute respiratory distress syndrome, uncontrollable inflammation and thrombotic events) [39]. Nonetheless, it is still to be investigated whether the lack of CNP effects plays a pathophysiological role in COVID-19 deterioration. Further investigations are also needed to demonstrate a direct connection between alterations in the delicate balance of furin homeostasis and the course of infectious diseases, like COVID-19.

To the best of our knowledge, we are the first to demonstrate discordant changes of serum NT-proCNP levels compared to NT-proBNP and conventional inflammatory markers in severe COVID-19. The significant association between disease progression and reduced NT-proCNP levels was also demonstrated for the first time in COVID-19. Further investigations are required to determine if the decrease in NT-proCNP has any pathophysiological role or therapeutic potential in SARS-CoV-2 infection.