Dietary Empagliflozin Regulates Angiotensin Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) in Kidney Cortex of Mice

Abstract

Persons with type 2 diabetes mellitus (T2D) are at greater risk for poor prognosis, including renal pathology, when infected with Covid‐19 virus. A newer class of medications for T2D is the gliflozin class, including Empagliflozin (EMPA), which inhibits the renal proximal tubule sodium glucose cotransporter, type 2 (SGLT2) resulting in glucosuria and reduction in hyperglycemia. Whether these medications alter susceptibility and responses to Covid‐19, and similar infections, is not known. The angiotensin 2 converting enzyme (ACE2) is the receptor for Covid‐19, while transmembrane serine protease 2 (TMPRSS2) is a proteinase expressed on the surface of epithelial cells and facilitates virus uptake. In general, ACE2 is thought to be renoprotective, as it converts Ang I (Ang 1‐10) to Ang 1‐9 and Ang II (Ang 1‐8) to Ang 1‐7. Ang 1‐8, in particular, is a potent vasoconstrictor and natriferic agent. Our aim was to determine how chronic EMPA affected ACE2 and TMPRSS2 protein levels and ACE2 activity in the kidney cortex of mice. Adult male (M) and female (F) mice of three strains (n = 8/group), i.e., C57Bl6 (C57), Swiss Webster (SW), and Tally Ho/Jng (TH) were fed high‐fat control diet (C, 60% milk‐fat) or EMPA‐containing high‐fat diet (E, 0.01%) for 12 weeks. TH mice are insulin resistant and obese, with a tendency to develop T2D over time. SW are lean and the closest genetically related background strain to TH. EMPA increased glucosuria but not body weight (p = 0.38 for treatment, 3‐way ANOVA); however kidney weight (normalized to body weight) was 2‐22% increased (depending on group) by EMPA (p = 0.002 for treatment). Kidney cortex protein levels of ACE2 and TMPRSS2 were determined by conventional western blotting (to observe band size and antibody specificity) and by dot blotting (to compare all samples on the same blot). Dot blotting revealed significantly higher ACE2 protein in males (p < 0.0001) relative to females and in the TH and SW strains (p < 0.0001), relative to C57. There was a significant sex‐by‐strain interaction in that the TH females were not significantly lower than TH males. Similarly, cortical TMPRSS2 was lower in females (p < 0.0001) and in C57 mice (p < 0.0001), but also reduced by EMPA (p = 0.030). ACE2 activity was determined in 10 μg kidney cortex by calculating the slope (over 2 hours) of the difference in fluorescence generated in the presence of the ACE2 substrate (Mca‐YVADAPK(Dnp)‐OH) with the addition of the ACE2‐specific antagonist (MLN) minus fluorescence with addition of both captopril (ACE antagonist) plus MLN. Slopes were: (RFU/min, mean ± sem): MC57C‐ 19.9 ± 5.5; MC57E‐ 31.8 ± 5.2; MSWC‐ 20.2 ± 1.9; MSWE‐ 25.7 ± 3.6; MTHC‐ 18.8 ± 3.6; MTHE‐ 34.7 ± 11.3; FC57C‐ 4.7 ± 1.2; FC57E‐ 9.7 ± 3.4; FSWC‐ 4.9 ± 1.2; FSWE‐ 4.4 ± 1.8; FTHC‐ 9.0 ± 1.8; and FTHE‐ 16.2 ± 2.3. Three‐way ANOVA revealed significant effects of both sex (p < 0.0001) and treatment (p = 0.0052) in that Empagliflozin and male sex increased the slope (activity). Overall, male sex was the greatest determinant of differences in ACE2 protein and activity and TMPRSS2 protein. EMPA increased ACE2 activity, but reduced TMPRSS2 protein levels. In general, sex differences were attenuated in the TH strain. There is the potential for these relative increases to play a role in increased sensitivity of male, obese, and/or T2D subjects to pathology associated with Covid‐19; however, the beneficial effect of enhanced ACE2 to reduce Ang II may also provide countering protection.