Animals. All animal experiments were performed in accordance with the National Institutes of Health guidelines for the use and care of animals. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Mississippi Medical Center. Pregnant Sprague Dawley (SD) rats were purchased from Envigo (Indianapolis, IN) and housed in a temperature-controlled room (75˚F) with 12-hour light–dark cycles per day and with food and water provided ad libitum.
Postpartum reduced uterine perfusion pressure (RUPP) model and n7AAc infusion. Pregnant SD rats were randomly divided into 3 groups during pregnancy: Normal pregnant (NP; n = 16), reduced uterine perfusion pressure (RUPP; n = 15), and RUPP + AT1-AA inhibitory peptide (‘n7AAc’; n = 16). On gestational day (GD) 14, RUPP surgery was performed as previously described 40. This model reduces blood flow to the utero-placental unit by ~ 40% in pregnant rats 40. One group of RUPP rats randomly received a mini-osmotic pump placed intraperitoneal (IP) to deliver the capped AT1-AA inhibitory peptide (‘n7AAc’; Thermo Fisher Scientific, Waltham, MA,) at a dose of 144 µg/day at day 14 of gestation. This dose is based on previous studies performed in our laboratory 30,41. ‘n7AAc’ blocks the binding of AT1-AA chronotropic activity. 32,33,39. The capped peptide does not bind or alter the function or activity of the AT1 receptor, but specifically binds circulating AT1-AAs without affecting ANG II 33. All rats were allowed to give birth and nurse their pups. At 10 weeks postpartum, systemic, cardiac, and renal measurements were made on the dams; and kidneys and the heart was collected for molecular and mitochondrial assays.
Systemic Measurements- Blood Pressure Measurement. At 10 weeks postpartum, a PE50 catheter was implanted in the right carotid artery and tunneled out of the back of the neck to measure blood pressure. Mean arterial pressure (MAP) was measured with rats in a restrainer cage. Measurements were taken over 30 min after a 30 min equilibration period. Afterwards, animals were sacrificed and blood and tissue collected. Body weight, heart weight, and kidney weight were recorded. Blood plasma and serum were collected and tissue samples stored at -80˚C for further use 30,41.
Systemic Measurements- Plasma NOx and Antioxidant Capacity. Plasma nitrate and nitrite was measured using the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions, as previously described 39. Plasma total antioxidant capacity was measured using the Antioxidant Assay Kit (709001, Cayman Chemical, Ann Harbor, MI), according to the manufacturer's instruction as previously described 42. In summary, this assay measures the ability of all aqueous and lipid-soluble antioxidants to inhibit the oxidation of 2, 2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) to ABTS+ by metmyoglobin. The total antioxidant capacity is quantified as millimolar Trolox equivalents (mM Trolox) at the 750 nm wavelength.
Cardiac Function- Echocardiography. All measurements and assessments were performed using the Visual Sonics Echo system (Vevo 3100, VisualSonics, Inc., Toronto, Canada) and the 15–30 MHz (MX250) linear transducer (VisualSonics). Animals were placed in the echocardiography room for at least 30 min before examination. Rats were maintained unconscious using 2% isoflurane and placed in a supine position on a heating platform. The chest was shaved and ultrasonic gel applied to the thoracic area to allow maximal visibility of the heart chambers. The ultrasonic probe was placed on the chest along the long-axis of the left ventricle and adjusted to obtain clear two-dimensional B-mode and M-mode parasternal long axis images. Five minutes was allowed for each animal to stabilize in that position before acquiring any measurements. Heart rate was maintained constant throughout the procedure (350–400 beats/min).
Cardiac mitochondria isolation, respiration, and membrane potential. Intact mitochondria were isolated from excised rat hearts by differential centrifugation 10,43. In brief, rat hearts were isolated and quickly washed in ice-cold MSM buffer (220 mM mannitol, 70 mM sucrose, 5 mM Mops, pH 7.4). Hearts were covered in ice-cold MSM buffer supplemented with 1 mg/ml bacterial proteinase type XXIV (Sigma) and rapidly minced with a razor blade on a cold and clean plastic surface. The minced tissue was added to ice-cold MSM buffer, supplemented with 2 mM EDTA and 0.2% fatty acid-free BSA, phenylmethylsulfonyl fluoride (PMSF) was added to 0.1 mM, and the tissue was homogenized on ice with a glass homogenizer and a loosely-fitting Teflon pestle using three to four hand driven strokes. The homogenate was centrifuged at 300×g for 10 min at 4º C. The supernatant was centrifuged at 3000×g for 10 min at 4º C, the supernatant was discarded and the pellet containing the mitochondria was resuspended by pipetting with cold MSM buffer with EGTA/BSA, and centrifuged again at 3000 x g. The final mitochondrial pellet was resuspended in a minimal volume of MSM buffer with EGTA/BSA and the protein concentration was determined using the DCA protein assay (Bio-Rad).
Immediately after isolation, mitochondria were used to measure respiration and membrane potential simultaneously with an Oroboros FluoRespirometer (Oroboros Instruments). The reaction mixture includes 2.1 mL of respiration buffer (100 mM KCl, 5 mM KPi, 1 mM EGTA, 1 mg/ml BSA, 50mM MOPS, pH 7.4) and 2 µM safranin O (described below). Mitochondria (100–200 µg in 30 µL) were added immediately after oxygen signal stabilization to record respiration (O2 consumption) driven by endogenous substrates in the isolated mitochondria. State 2 respiration was initiated by injecting glutamate (10 mM) and malate (2 mM) into the chamber. More rapid State 3 respiration was initiated by adding ADP (5 mM), which allows proton flow back across the inner mitochondrial membrane through ATP synthase. Then oligomycin (2.5 µM) was added, to inhibit proton flow through ATP synthesis, yielding the slower rate of State 4 respiration. Rotenone (0.5 µM) and antimycin A (2.5 µM) were injected to inhibit electron transfer to O2 that is specific to oxidative phosphorylation. The resulting slow rates of O2 consumption, due to processes other than oxidative phosphorylation, were subtracted from other rates of O2 consumption. Rates of respiration are expressed as nmol e-/min/mg mitochondrial protein.
Mitochondrial membrane potential was measured by using the membrane-permeable cationic, fluorescent dye safranin O (Ex/Em = 485 nm/586 nm) as previously described (16, 21). Safranin is taken into the matrix in proportion to the density of negative charge on the matrix surface of the inner mitochondrial membrane. Since the crowding of safranin in the matrix causes quenching of its fluorescence, safranin fluorescence is inversely proportional to the magnitude of the membrane potential (24). The fluorescence signal of the Oroboros FluoRespirometer is calibrated using known concentrations of safranin up to 2 µM maximum. Hence, the fluorescence readout of the Oroboros software is in units of 0–2 µM safranin, where the concentration of safranin is that of the fluorescent population on the outside of the mitochondria. This is easily converted to percent uptake of the total amount of safranin in the reaction, therefore our relative measure of the magnitude of mitochondrial membrane potential is “percent safranin uptake”. The maximum percent safranin uptake value, reported here, is taken at the point where safranin fluorescence quenching is greatest. The magnitude of the membrane potential is maximum during State 2 and State 4 respiration, it declines slightly during State 3 respiration, and the membrane potential is completely lost (safranin fluorescence returns to maximum) upon the addition of an uncoupler, such as FCCP.
Uncontrolled complex IV activity was measured as the rate of oxygen consumption catalyzed by complex IV in broken, KCl-washed rat heart mitochondria as described previously 44 with some modifications. Electrons were provided to complex IV in 10 µg broken mitochondria by horse heart cytochrome c (20 µM), which was kept reduced by ascorbate (3 mM) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD; 0.3 mM). The reaction mixture also contained 50 mM Tris (pH 7.4), 8 mM KCl, 1 mM EDTA, 2 µg/ml catalase, 5µM antimycin A, at 25°C. Inhibition of complex IV activity upon the addition of 25 mM ZnSO4 and 5 mM MgCl2 allowed measurement of the slow, non-enzymatic consumption of O2 by ascorbate/TMPD, which was subtracted. Complex IV activity is reported as nmol e-/min/mg protein.
Renal Function. Renal function was determined by glomerular filtration rate (GFR) using FITC-sinistrin as previously described 39,45. Briefly, 1 to 2 days before rats were 10 weeks postpartum a catheter was inserted into the jugular vein to infuse FITC-sinistrin under isoflurane anesthesia. At 10 weeks, postpartum rats were anesthetized and hair on the upper back below the ears was removed to reduce interference. For determining GFR, a miniaturized device (NIC-Kidney, Mannheim Pharma & Diagnostics, Mannheim, Germany) composed of 2-light-emmiting diodes that transcutaneously excite and measure FITC-sinistrin was used and baseline fluorescence collected for 10–15 minutes, followed by a bolus injection of FITC-sinistrin (3 mg/100 g body weight in 0.2 mL 0.9% irrigation saline). Continuous fluorescence was measured for 2 hours and clearance curves analyzed using the MPD Lab Ver 1.0RC3 software. The half-life (t1/2) for the clearance of FITC-sinistrin was determined 45 min post-injection using a one-compartment model. The t1/2 value was converted to GFR (mL/min/100 g body weight) using the following semi-empirical equation developed and validated by the manufacturer: GFR = 31.26 [mL/100g body weight]/t1/2 [min] 45.
Plasma Creatinine. Plasma creatinine was measured using the LabAsay Creatinine kit (Jaffe method; Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s instructions and as previously published 39. The standards ranged from 1.25–10 mg/dl. The amount of plasma creatinine was measured calorimetrically and expressed as mg/dL.
Proteinuria. Proteinuria was measured by a BCA protein assay from Bio-Rad using bovine albumin as standard. Briefly, rat urine was collected for 24 hours in metabolic cages at 10 weeks postpartum and diluted 1:10 with 1x phosphate buffered saline (PBS). Diluted samples were pipetted into duplicates wells and measured by the spectrophotometer at 540 nm. Proteinuria was expressed as mg/day.
Urine Nephrin Excretion. Urine nephrin excretion was measured by the Exocell rat nephrin ELISA kit (kit #:1019; Exocell Inc.) according to the manufacturer’s instructions. Urine nephrin excretion was expressed as µg/mL. The urine used in this assay collected for 24 hours in metabolic cages at 10 weeks postpartum.
Western blot analysis. Cardiac tissue was homogenized in a RIPA buffer with protease and phosphatase inhibitors using the Fisher Scientific PowerGen 125 electric homogenizer at a low speed. Lysates were separated into a membrane and soluble fraction by centrifugation (10,000 g) for 30 min. Samples containing 100 µg of protein from the soluble fraction were loaded on 4–20% precast Criterion gels (Bio-Rad, cat # 5671093). Separated proteins were transferred to nitrocellulose membranes using a trans-blot turbo apparatus (Bio-Rad), which were blocked for one hour at room temperature in blocking buffer (LI-COR Biosciences, Lincoln, NE) diluted 1:1 with PBS. Membranes were incubated overnight at 4°C with electron transport chain primary antibody total OXPHOS (1:250; Abcam, Cambridge. MA; cat # ab110413). This cocktail of antibodies recognizes complex I-V proteins of the electron transport chain. Membranes were washed 3x in PBS with 0.1% Tween-20) and incubated with IRDeye700-conjugated anti-mouse IgG (1:5000, LI-COR Biosciences, cat # 928-68070) for one hour and scanned using the LI-COR Odyssey Infrared Imaging System.
Statistical analysis. Mean values ± SEM for n rats or independent observations are presented. The significance of differences among mean values was analyzed by a one-way ANOVA with Bonferoni’s post hoc test. Two means were compared using an unpaired t-test. All statistical analysis was performed with Prism 8 (GraphPad Software, La Jolla, CA). P < 0.05 was considered statistically significant.