The coronavirus disease 2019 (COVID-19) pandemic has caused millions of deaths worldwide, prompting intense research into the pathogenic mechanism behind this disease.

A new study published on the medRxiv* preprint reports find very low levels of aldosterone in a subset of COVID-19 patients, thus indicating the involvement of renal hormones in the immunothrombotic and inflammatory phenomena typically seen in severe cases of COVID-19.

Study: Unquantifiably low aldosterone levels are common in hospitalized patients with Covid-19, but may not be revealed by chemiluminescent immunoassay. Image Credit: Kateryna Kon/Shutterstock.com

introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, binds to angiotensin-converting enzyme receptor 2 (ACE2) to enter the host cell . The ACE2 receptor is also a key component of the renin-angiotensin-aldosterone system (RAAS), which regulates kidney and cardiovascular function in several ways.

ACE2 converts angiotensin II (AngII) into the anti-inflammatory and vasodilator antioxidant molecule Angiotensin 1-7 (Ang1-7), which allows RAAS inhibition. Conversely, AngII is a potent vasoconstrictor, pro-oxidant and pro-inflammatory molecule that can also trigger fibrosis, aberrant coagulation and immunothrombosis through its effect on the endothelium. This activity is mediated by endothelial production of tissue factor and plasminogen activator inhibitor 1.

AngII also binds to the adrenal angiotensin I receptor, which leads to the production of aldosterone, the main mineralocorticoid in the body. By binding to ACE2 and thereby blocking AngII metabolism, SARS-CoV-2 can activate RAAS and promote elevated aldosterone secretion. Excessive AngII activity may contribute to hypertension, thrombosis, inflammation, and pulmonary fibrosis in COVID-19.

Previous studies have failed to demonstrate RAAS activation or low aldosterone levels; however, these studies have often used non-specific methods that are very vulnerable to interference. For this reason, the researchers in the present study used mass spectrometry because it is free of interference and provides more accurate estimates.

Previously, scientists had several patients with low aldosterone levels, although this was not reported as typical. To further examine this observation, the researchers used liquid chromatography-tandem mass spectrometry (LC-MS) to estimate aldosterone levels in hospitalized COVID-19 patients.

Study results

All patients had tested positive for SARS-CoV-2, all had a median age of 64. Of 134 patients with baseline aldosterone, cortisol, or renin levels within 72 hours of the first positive test for SARS-CoV-2, approximately 60% were male. About 11% of patients were administered in the intensive care unit (ICU), with an in-hospital mortality rate of 13.4%.

Aldosterone concentrations were very low, with nearly 60% having undetectable aldosterone levels below 70 pmol/L. Cortisol was above 744 nmol/L in 20.5% of patients, which was the discriminating factor between survival and non-survival curves in previous studies.

Renin levels were also generally low, although aldosterone levels were reduced, which would normally have increased renin levels. Aldosterone levels were correlated with cortisol and renin concentrations; however, the absence of detectable aldosterone was not a marker of a clinical characteristic or outcome, survival or renal function.

Histograms of the first available result (A) Aldosterone, measured by LCMSMS (B) Cortisol (VS) Renin.

High cortisol levels exceeding 744 nmol/L were linked to lower chances of survival compared to those with lower levels. That is, 44% of people in the first group died after 28 days, compared to 11% in the low cortisol group. Additionally, the high cortisol cohort had an average hospital stay of 19 days versus 11 days in the low cortisol group.

A marked difference in survival was observed when cortisol levels exceeded 800 nmol/L in the high cortisol group.

Previous studies that used immunoassay methods to measure aldosterone levels found no significant changes in COVID-19 patients compared to controls. The very different results of this study are attributed to the use of LC-MS. When the measurements were repeated by chemiluminescent immunoassay (CLIA), they found that CLIA (pmol/L) = 14.1 + 3.16 × LC-MS (pmol/L).

Thus, there is a wide variation in the results obtained by these methods, which only increases as the average measurements increase. These differences are not explained by impaired renal or hepatic function.

However, if the serum was subjected to solvent extraction, the results were much more consistent, where extracted CLIA = -14.9 + 1.0 × LC-MS. This is explained by the presence of a water-soluble metabolite which interferes with the substance to be detected in the immunoassay.

Consequences

The lack of clinical features of hypoaldosteronism has given rise to many hypotheses, including adequate mineralocorticoid activity to maintain water and electrolyte homeostasis. Elevated levels of cortisol can act at the mineralocorticoid (MR) receptor, since the inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is saturated at these levels, thereby allowing glucocorticoid-MR crosstalk.

This may explain the absence of hypoaldosteronism clinically, but only partly because of the reduced correlation between cortisol and aldosterone levels in these patients. Aldosterone metabolites also do not appear to bind very strongly to MR, thus excluding their compensatory action.

Alternatively, activity of distal nephron epithelial sodium channels (EnaC) by AngII results in increased sodium reabsorption and increased blood volume, although potassium and hydrogen ions are excreted at a higher rate. This could trigger a vicious circle of negative feedback for RAAS, where renin and aldosterone levels fall further, even though AngII activity continues to be excessive due to the lack of inhibition of AngII. ‘ACE2.

The poor agreement between CLIA and LC-MS corroborates previous studies while showing that COVID-19 exacerbates this problem. Solvent extraction likely removes a water-soluble metabolite that cross-reacts in the immunoassay. The lead candidate is the predominant metabolite of aldosterone, aldosterone-18-glucuronide.

These data demonstrate that aldosterone cannot be accurately estimated in the serum of SARS-CoV-2 infected patients using a direct competitive immunoassay. When measured using the reference LCMSMS, serum aldosterone is found to be remarkably low in most patients with COVID-19.”

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.