Experimental animals
All studies using mice were approved by an Institutional Animal Care and Use Committee at the University of Kentucky and were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Female mice with loxP sites flanking exon 4 of the Ace2 gene on a C57BL/6 background (Ace2fl/fl) were bred to male Ace2fl/y hemizygous transgenic mice expressing Cre recombinase under the control of the adipocyte-specific promoter, adiponectin. The resulting offspring were either experimental animals with adipocyte-ACE2 deletion (Ace2Adipo) or littermate controls (females Ace2fl/fl; males Ace2fl/y). Mice were maintained on a standard murine diet (Harlan Laboratories, Indianapolis, IN) until 8 weeks of age.
Initial studies characterized the efficiency and specificity of adipocyte ACE2 deficiency using 8-week-old male and female mice (n = 7–8 mice per genotype). Kidney, heart, liver, subcutaneous (SubQ), and retroperitoneal fat (RPF) were dissected, frozen in liquid nitrogen, and stored at − 80 °C until use. For Cre expression studies, female mice carrying the transgene with the ROSA26-stop-lacZ reporter (Jackson Laboratory, Bar Harbor, ME, stock # 0003474) were bred to male Ace2Adipo mice.
For blood pressure studies, 8-week-old male and female mice of each genotype were randomly assigned to receive ad libitum either a low fat (LF, 10% kcal from fat; D12450B, Research Diets Inc, New Brunswick, NJ) or a high fat diet (HF, 60% kcal from fat; D12492, Research Diets, New Brunswick, NJ) for 4 months (n = 6–13 mice/genotype/diet group). Bodyweight was quantified weekly. Fat and lean mass were measured at week 14 of diet feeding by EchoMRI (EchoMRI-100TM, Echo Medical Systems, Houston, TX). Blood pressure was measured by radiotelemetry in a subset of mice (n = 5 mice per genotype/diet group) at week 16 of diet feeding for 5 consecutive days, and again following acute administration of AngII (subcutaneous, 20 μg/kg). The method for blood pressure measurement is described previously [13]. Briefly, anesthetized (isoflurane, to effect) mice were implanted with carotid artery catheters advanced to the aortic arch and radiotelemeter implants (model PA-C10) inserted in a subcutaneous pocket on the right flank. After 1 week of recovery, blood pressure was monitored continuously, with values reported every 5 s. Inclusion criteria for blood pressure measurements were (1) pulse pressures > 20 mmHg and (2) pulse pressures > 1 standard deviation of the mean. At study endpoint, mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.) for exsanguination and tissue harvest.
Acute administration of AngII
HF-fed female Ace2fl/fl and Ace2Adipo and male Ace2fl/y and Ace2Adipo mice (n = 4 mice per group) with radiotelemetry implants were subcutaneously (interscapular) administered 20 μg/kg of AngII (Sigma-Aldrich) in 0.9% sterile saline. Blood pressure was recorded via telemetry continuously for 60 min after administration of AngII. Baseline (time = 0 min) blood pressure reported is the average blood pressure over 15 min prior to administration of AngII. Blood pressure at time = 2, 5, 10, 15, 20, 30, 40, 50, and 60 min following AngII administration is the average value per minute. Data are reported as time course and as integrated area under the curve (AUC).
Detection of β-galactosidase activity in tissues
Whole organs were fixed in formalin at 4 °C for 1 h, then rinsed three times with buffer (100 mM sodium phosphate, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40). Organs were incubated overnight in X-gal staining buffer (rinse buffer with 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/mL X-gal) and then visualized, where blue staining indicates expression of Cre recombinase.
Tissue DNA and RNA extraction and PCR
Adipose tissue genotyping was performed using DNA extracted from RPF (DNeasy, Qiagen, Alameda, CA). cDNA was generated using the forward primer: 5′–AGCTCATAGAGAAAGAGGGAGCACG and either the reverse primer: 5′–ACAGCCAGGGTGATACAGAGAAACC (generates products demonstrating the presence [912 bp] or the absence [723 bp] of the floxed ACE2 gene) or the reverse primer 5′–AAGGGTAATGTGTGAGCTGGAACCC (generates a 912 bp product demonstrating the deletion of exon 4 of the ACE2 gene).
Total RNA was extracted from tissues using the Maxwell RSC (Promega, Madison, WI). RNA concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE); 400 ng of RNA was used for reverse transcription to make cDNA using qScript cDNA Supermix (Quanta, Gaithersburg, MD). The following mouse primers were used to probe gene products from cDNA amplified using SYBR Green PCR Master Mix (Quanta, Gaithersburg, MD): ACE2, forward 5′–TCCAGACTCCGATCATCAAGC, reverse 5′–GCTCATGGTGTTCAGAATTGTGT; 18S, forward 5′–CGGCTACCACATCCAAGGAA, reverse 5′–GCTGGAATTACCGCGGCT. Data are expressed as ΔΔCt relative to 18S rRNA.
Studies in humans
This study was approved and work was completed in compliance with approval from the Institutional Review Board of the University of Kentucky. Study participants were transgender women (biological males) seeking gender-affirming hormone therapy recruited from the endocrine clinic at the University of Kentucky (n = 4 subjects). Inclusion criteria were biologic male subjects between the age of 21 and 60 with a body mass index (BMI) between 30 and 45 kg/m2 seeking initiation of estrogen therapy for the first time. Exclusion criteria were fasting blood sugar > 126 mg/dL, or use of diabetes medications, current use of angiotensin-converting enzyme (ACE) inhibitors or angiotensin I receptor blockers (ARBs), anti-inflammatory medications (e.g., steroids), prior estrogens, or any other medication or condition that may affect the RAS pathway. Note that subjects participating in this study delayed the use of spironolactone until after at least 12 weeks of estradiol therapy. Subjects were in overall good health and had no significant hepatic, cardiac, or renal impairment. Subjects were seen at baseline (prior to initiation of estrogen therapy) and 12 weeks after estradiol treatment (estradiol, 1–2 mg/day, orally, dose determined by the endocrinologist). Blood pressure and anthropometric measurements took place during office visits at the endocrine clinic. Blood pressure was measured by arm cuff in the seated and resting position. Blood collection took place at the outpatient Clinical Service Core (CSC) of the institutional Center for Clinical and Translational Science (CCTS). For blood collection, subjects were fasted overnight and arrived at the outpatient CSC at 8am. Plasma was collected after centrifugation and stored at − 80 °C until analysis.
Quantification of plasma parameters in humans
Estradiol concentrations were quantified using a commercial ELISA kit (Calbiotech, ES180S, Spring Valley, CA; analytical sensitivity of 3 pg/mL). Angiotensinogen concentrations were quantified using a commercial kit (IBL, 27412, Minneapolis, MN; analytical sensitivity of 0.03 ng/mL). Ang-(1-7) peptide concentrations were quantified using a commercial kit (Peninsula Labs, San Carlos, CA, S-1330; analytical sensitivity of 0.01 ng/mL). Plasma renin activity (IBL, IB59131; analytical sensitivity of 0.14 ng/mL) and AngII peptide concentrations were quantified by enzyme- and radioimmunoassay, respectively, as described previously [6, 13, 14].
Statistical analyses
Data are presented as mean ± SEM. Statistical analyses were performed using SigmaPlot version 12.3. All data passed normality or equal variance tests or logarithmic transformation was used to achieve normality. Two-tailed Student’s t tests were used for the analysis of data between two groups. For two-factor analysis, a two-way ANOVA was used to analyze end-point measurements with between-group factors of genotype and diet, followed by Holm-Sidak for post hoc analyses. Response to acute AngII administration was analyzed as a time course using repeated measures (RM) two-way ANOVA, and as the integrated area under the curve (AUC). Correlation analyses were performed for plasma parameters and blood pressures of humans. Values of p < 0.05 were considered to be statistically significant.