High Angiotensin-Converting Enzyme and Low Carboxypeptidase N Serum Activity Correlate with Disease Severity in COVID-19 Patients

Abstract

(1) Background: Angiotensin-converting enzyme 2 (ACE2) is a functional receptor of SARS-CoV-2 and counter-balances ACE in the renin-angiotensin system (RAS). An imbalance of the RAS could be associated with severe COVID-19 progression. (2) Methods: Activities of serum proteases angiotensin-converting enzyme (ACE) and carboxypeptidase N (CPN) for 45 hospitalized and 26 convalescent COVID-19 patients were investigated vs. healthy controls using labeled bradykinin (DBK) degradation with and without inhibition by captopril as a read-out. Data were correlated to clinical parameters. (3) Results: DBK degradation and CPN activity were significantly reduced gender-independently in COVID-19 and returned to normal during convalescence. ACE activity was over-active in severe disease progression; product DBK1-5 was significantly increased in critically ill patients and strongly correlated with clinical heart and liver parameters. ACE inhibitors seemed to be protective, as DBK1-5 levels were normal in moderately ill patients in contrast to critically ill patients. (4) Conclusions: CPN and ACE serum activity correlated with disease severity. The RAS is affected in COVID-19, and ACE could be a therapeutic target. Further proof from dedicated studies is needed.

Keywords: ACE; ACE inhibitor; COVID-19; CPN; TLC; bradykinin; corona virus; hypertension.

Figure 1

Influence of SARS-CoV2-infection on the renin–angiotensin system (RAS) and the kinin–kallikrein system, which are linked by angiotensin-converting enzyme (ACE). ACE2 counter-balances the RAS by inactivating angiotensin II. Being a functional receptor of SARS-CoV2, ACE2 availability decreases upon infection. ACE activity may subsequently increase with an impact on the cleavage of bradykinin (BK). Carboxypeptidase N (CPN) also degrades BK, changing receptor specificity from the B2 to the B1 receptor. ACE and CPN activity were measured in this study with a specially designed neuropeptide reporter assay [11].

Figure 2

Principle of the neuropeptide reporter assay (NRA) for the study of serum protease activity. (A) Bradykinin (BK, sequence: RPPGFSPFR) dabsylated (see star) at the N-terminus (DBK) is degraded in serum by angiotensin-converting enzyme (ACE) and carboxypeptidase N (CPN); fragments DBK1-5 and DBK1-8 serve as readout. (B) Exemplary assay data show an increase in the formation of DBK1-5 and a decrease in the formation of DBK1-8 in a 66-year-old, fatally ill ARDS (acute respiratory distress syndrome) COVID-19 patient vs. data from a healthy control (female, 25 years old). The yellow circle indicates the presence of another DBK fragment, which was observed infrequently in the patient cohort and is subject to further study.

Figure 3

Boxplots of relative values for labeled bradykinin DBK1-9 (A) and its cleavage products, DBK1-8 (B) and DBK1-5 (C), for samples of the hospitalized patients (HoP) in comparison to convalescent (CoP) and healthy (HCtr) probands. Subgroups: HoPc: critical disease, HoPs: severe disease, HoPm: moderate disease, CoP-NV: CoP subgroup, where NRA results fell within the range detected in healthy probands, CoP-DV: CoP subgroup where NRA results deviated from the values measured in healthy people. The vertical dashed lines separate the groups HoP, CoP, and HCtr, the horizontal dotted lines indicate the median in the CoP-NV reference cohort for improved visualization. DBK1-9 values were higher in HoP than in HCtr and CoP-NV, indicating reduced DBK degradation and thus lower protease activity in COVID-19. DBK1-8 values were lower in HoP due to reduced CPN activity. DBK1-5 deviated to higher values in HoP and two convalescent patients (CoP-DV). DBK1-5 correlated with disease severity; values shot up in critical disease.

Figure 4

Experimental values (dabsylated bradykinin DBK1-9 and its fragments DBK1-8 and DBK1-5) in percent (y-axis; x-axis, random numbers) with inhibition by captopril (DBK1-9/8/5C, orange squares) and without inhibition (blue dots) for hospitalized patients (HoP). (A) Sorted by DBK1-5. High values of DBK1-5 and a lower effect of captopril were associated with other diseases in addition to COVID-19 (diabetes, hypertonia, co-infection) and poor outcome (acute respiratory distress syndrome (ARDS), death) with one outlier (female (F)). (B) Hypertonic male patients, data ordered by ACEI/ARB (angiotensin-converting enzyme inhibitor/angiotensin receptor blocker) medication and fatality. High DBK1-5 was observed in critical patients, while patients on ACEI/ARBs showed balanced DBK1-5 values. No effect was seen for DBK1-9 and DBK1-8.

Figure 5

Results and hypothesis: Fragment DBK1-5 of labeled bradykinin (DBK) increased in severe COVID-19 cases due to enhanced angiotensin-converting enzyme (ACE) activity resulting from decreased ACE2 counterbalance caused by the virus. The infection led to organ damage, and clinical parameters to that effect correlated with the neuropeptide reporter assay (NRA) results. The decrease in both the serum DBK degradation and DBK1-8 formation capacity likely resulted from impaired CPN synthesis in the liver. RAS: renin–angiotensin system, LDH: lactate dehydrogenase, ɣGT: ɣ-glutamyltransferase, CK: creatine kinase, GOT: glutamate-oxalacetate transaminase, GPT: glutamate-pyruvate transaminase, PCHE: pseudocholinesterase.