Blood was collected from participants in the seated position to obtain plasma renin activity, aldosterone, Ang II, and Ang-(1-7). For the peptide measurements, blood samples were collected immediately in a tube containing a cocktail of inhibitors and plasma was obtained and stored at −80 °C. The plasma was thawed on ice, extracted on Sep-Pak C18 columns (Waters Corp., Milford, Massachusetts, USA), and the eluted fractions assayed by an Ang II radioimmunoassay (RIA, Alpco, Salem, New Hampshire, USA; detection limit 0.8 pmol/l; intra-assay and inter-assay coefficients of variation 12 and 22%) and an Ang-(1-7) RIA (detection limit 2.8 pmol/l; intra-assay and inter-assay coefficients of variation 8 and 20%) [11 (link)]. Aldosterone content was determined in nonextracted plasma samples by RIA (Diagnostics Products, Los Angeles, California, USA; detection limit 28 pmol/l). Renin activity was directly determined in plasma samples using an RIA (Cisbio, Codolet, France; detection limit 4 pmol Ang I/l/hour). We calculated the Ang II-to-Ang-(1-7) ratio and the aldosterone-to-renin ratio for plasma samples.
Spot urine samples were collected, immediately acidified with HCl to prevent peptide degradation, and stored at −80 °C. The urine samples were thawed on ice, extracted on SepPak columns, and the urinary levels of Ang II and Ang-(1-7) quantified by RIAs. For the ACE and ACE2 assays, separate nonacidified urine samples were collected and were concentrated 10-fold on a Millipore 5000-Da cut-off filter with the assay buffer. ACE and ACE2 assays were conducted at 37 °C in 10 mmol/l of HEPES, 125 mmol/l of NaCl, and 10 µmol/l of ZnCl2 (pH 7.4), with 0.02 ml of urine in a final volume of 0.2 ml with the indicated inhibitors and 0.02 ml of 0.1 mmol/l of either the quenched fluorescent substrate Mca-RPPGFSAFK-DNP for ACE or Mca-APK-DNP for ACE2 in a 96-well black plate. The fluorescence was read in a plate reader at an excitation λ of 328 nm and an emission λ of 393 nm. Blanks consisted of the substrate alone and the addition of the ACE inhibitor lisinopril or the ACE2 inhibitor MLN4760 for the ACE and ACE2 assays, respectively.
As the ACE and ACE2 substrates are not specific, the assays contained inhibitors against aminopeptidases (bestatin 10 µmol/l), carboxypeptidase A (benzyl succinate 10 µmol/l), serine peptidases (chymostatin 10 µmol/l), cysteine peptidases (para-chloro-mercuribenzoic acid 0.5 mmol/l), neprilysin (SCH39370, 10 µmol/l), and lisinopril (10 µmol/l) to measure ACE2 or MLN4760 (10 µmol/l) to measure ACE. ACE and ACE2 protein content (ng/mg creatinine) were based on human ACE and ACE2 standards obtained from R&D Systems (Minneapolis, Minnesota, USA). Standard enzymes were assayed under the same conditions as the urine samples. Fluorescent substrates for ACE and ACE2 were obtained from Enzo Life Sciences (VWR, Atlanta, Georgia, USA).
Creatinine levels in nonextracted urine samples were determined by a modified Jaffe assay traceable to isotope dilution mass spectrometry [11 (link)]. We calculated the urinary Ang II:Ang-(1-7) and ACE:ACE2 ratios and corrected Ang II and Ang-(1-7) concentrations and ACE and ACE2 concentrations by urine creatinine. If blood or urine sample results were below the laboratory’s lower limit of detection, the sample’s measurement was assigned a value calculated as the lower limit of detection divided by the square root of two [31 ].
Spot urine samples were collected, immediately acidified with HCl to prevent peptide degradation, and stored at −80 °C. The urine samples were thawed on ice, extracted on SepPak columns, and the urinary levels of Ang II and Ang-(1-7) quantified by RIAs. For the ACE and ACE2 assays, separate nonacidified urine samples were collected and were concentrated 10-fold on a Millipore 5000-Da cut-off filter with the assay buffer. ACE and ACE2 assays were conducted at 37 °C in 10 mmol/l of HEPES, 125 mmol/l of NaCl, and 10 µmol/l of ZnCl2 (pH 7.4), with 0.02 ml of urine in a final volume of 0.2 ml with the indicated inhibitors and 0.02 ml of 0.1 mmol/l of either the quenched fluorescent substrate Mca-RPPGFSAFK-DNP for ACE or Mca-APK-DNP for ACE2 in a 96-well black plate. The fluorescence was read in a plate reader at an excitation λ of 328 nm and an emission λ of 393 nm. Blanks consisted of the substrate alone and the addition of the ACE inhibitor lisinopril or the ACE2 inhibitor MLN4760 for the ACE and ACE2 assays, respectively.
As the ACE and ACE2 substrates are not specific, the assays contained inhibitors against aminopeptidases (bestatin 10 µmol/l), carboxypeptidase A (benzyl succinate 10 µmol/l), serine peptidases (chymostatin 10 µmol/l), cysteine peptidases (para-chloro-mercuribenzoic acid 0.5 mmol/l), neprilysin (SCH39370, 10 µmol/l), and lisinopril (10 µmol/l) to measure ACE2 or MLN4760 (10 µmol/l) to measure ACE. ACE and ACE2 protein content (ng/mg creatinine) were based on human ACE and ACE2 standards obtained from R&D Systems (Minneapolis, Minnesota, USA). Standard enzymes were assayed under the same conditions as the urine samples. Fluorescent substrates for ACE and ACE2 were obtained from Enzo Life Sciences (VWR, Atlanta, Georgia, USA).
Creatinine levels in nonextracted urine samples were determined by a modified Jaffe assay traceable to isotope dilution mass spectrometry [11 (link)]. We calculated the urinary Ang II:Ang-(1-7) and ACE:ACE2 ratios and corrected Ang II and Ang-(1-7) concentrations and ACE and ACE2 concentrations by urine creatinine. If blood or urine sample results were below the laboratory’s lower limit of detection, the sample’s measurement was assigned a value calculated as the lower limit of detection divided by the square root of two [31 ].