Background: The SARS-CoV-2 virus infects cells in a TMPRSS2-dependent mechanism by engaging with ACE2. ACE2 display high levels in kidneys, and risk factors for severe disease are high age and male sex. We hypothesized that in human kidneys, ACE2 and TMPRSS2 are 1) more abundant in male and with increasing age; 2) co-localize; 3) more abundant in urine and extracellular vesicles (EVs) from male patients with acute SARS-CoV-2 and 4) SARS-CoV-2 antigen is present in urine and EVs.
Methods: Kidney cortex tissue from cancer nephrectomy patients (male >75y n=6, female >75y, male <50y n=6, female <50y n=6) was analyzed for ACE2 and TMPRSS2 protein level. Urine collected from hospitalized patients with SARS-CoV-2 (male=7, female n=11) was analyzed for ACE2 and albumin and compared with healthy control (n=3) and patients with albuminuria with COVID (n=12). Urine EVs from patients with COVID-19 were used to detect ACE2, TMPRSS2 and SARS-CoV-2 antigen. Studies were approved by the Ethics Committee of the Region of Southern Denmark and the Danish Data Protection Agency and carried out in accordance with the Declaration of Helsinki. Data were tested for normal distribution, and log transformed if this yielded normal distribution. The 2-variables dataset and 1-variable dataset were analyzed by two-way-ANOVA or Student’s t-test, respectively. Correlation between parameters was assessed by linear regression with 95% confidence intervals. For all data, a p-value < 0.05 was considered significant. Normally distributed data are displayed as mean ± standard error of mean (SEM), and non-normally distributed as mean ±interquartile range. Analyses were carried out using GraphPad Prism 9.5.0
Results: Immunoblot analyses (n=6) of ACE2 and TMPRSS2 protein levels were not different with age. ACE2 was more abundant in female kidneys across ages. Immunohistochemistry (n=6) showed that ACE2 protein was associated with proximal tubule apical membranes in the cortex, while TMPRSS2 was observed predominantly in the medulla. ACE2 was elevated significantly in uEVs (immunoblot n=4) and urine from patients with severe SARS-CoV-2 with no sex difference compared with urine from controls w/wo albumin (ELISA). TMPRSS2 was elevated in EVs from male patients compared to female (immunoblot n=4). ACE2 and TMPRSS2 did not co-immunopreciptate in EVs/apical membranes (immunoblot n=6). SARS-CoV-2 antigen and mRNA were not detected in urine and EVs (PCR).
Conclusion: Higher kidney ACE2 protein abundance is unlikely to explain higher susceptibility to SARS-CoV-2 in male or elderly. Higher TMPRSS2 in male tissues could contribute. Loss of ACE2 into urine in COVID-19 could impact susceptibility and angiotensin metabolism with systemic consequences.