Online Supplement. Title: Angiotensin II Type 1 Receptor Autoantibody as a Novel Regulator of. Aldosterone independent of Preeclampsia

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1 Online Supplement Title: Angiotensin II Type 1 Receptor Autoantibody as a Novel Regulator of Aldosterone independent of Preeclampsia Authors: Jie Yang, MS 1, Li Li, MS 2, Jian-Yu Shang, BS 1, Lin Cai, MD 3, Li Song, MS 1, Su-Li Zhang, PhD 1, Hao Li, MS 1, Xiao Li, MS 1, Wayne Bond Lau, MD 4, Xin- Liang Ma, PhD 1,4,, Hui-Rong Liu, PhD 1,5,6,*. Affiliation: 1 Department of Physiology & Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, China; 2 School of Heath Management and Education, Capital Medical University, Beijing, China; 3 Department of Gynaecology & Obstetrics, The Third People s Hospital of Chengdu, Chengdu, China; 4 Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, United States; 5 Beijing Key Laboratory of Cardiovascular Diseases and Related Metabolic Dysfunction, Capital Medical University, Beijing, China; 6 Department of Cardiology, Capital Medical University, Beijing, China. This PDF includes all of the supplemental material related to this manuscript. It is intended for publication as an online data and method supplement. 1

2 Supplementary Method Patients All patients and 50 normotensive pregnant women were informed of the study s purpose and protocol. Written consent was obtained from all 152 enrolled subjects, thirty-five of who were diagnosed with sever PE on the basis of ISSHP (International Society for the Study of Hypertension in Pregnancy) [1] and US National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy [2]. The criteria included high blood pressure of 160/110 mmhg and urinary protein exceeding 3.0 g/24hours. Other criteria included the presence of visual disturbance, persistent headache, hemolysis, elevated liver enzymes or low platelet count syndrome with hypertension ( 140/90 mmhg) and placental abruption. Mild PE was defined as blood pressure reading of 140/90 mmhg and urinary protein exceeding 0.3 g/24hours. For patients with gestational hypertension (GH), the criteria were 140/90 mmhg and urinary protein less than 0.3 g/24hours. Protocol: introduction of AT 1 -AA from PE patients into pregnant rats All studies were performed upon female Wistar rats housed in a temperature controlled room (25 C) with a 12: 12 light. All experimental procedures were in accordance with the National Institute of Health Guidelines for experimental use and care for animals. All protocols were approved by the Institute of Animal Care and Use center in Capital Medical University. Purified human AT 1 -AA from PE patients including early- and late-onset type (or nsigg) (8 μg/g, matched to clinically-relevant levels of PE patients based upon our preliminary data, Supplementary Fig. S1) was concentrated by lyophilization, resuspended in saline, and injected by sublingual vein into rats anesthetized with chloral hydrate (0.3 ml/g) on days 13 and 15 of pregnancy ( g, n=8 per group), respectively. Rat serum was collected on gestation days 13, 15, 18 when pregnant rat systolic blood pressure (BP) was measured both by tailcuff (AD Instruments). The arterial pressure of pregnant rats on day 18 was measured as previously described [3, 4]. Briefly, pregnant rats were catheterized under anesthesia (isoflurane) using a catheter of V-3 tubing (SCI) which was inserted into the carotid artery for BP monitoring. Arterial pressure was monitored with a pressure transducer and was recorded continuously for a 1.5-hour period after 1-hour stabilization. Five days after the first injection, V7 dimension ultrasound device with 10.0-MHz convex transducer was used to determine the fetal intrauterine growth on gestational day 18. Pregnant rats were euthanized on day 18 of pregnancy at the time kidneys and adrenal glands were harvested, and the fetus was carefully removed, sucked and weighted. Blood creatinine was measured by the Roche enzymatic creatinine method [5] and serum uric acid was quantified using enzymatic-colorimetric methods. Histopathology Rats were anesthetized and the adrenal gland and kidneys were collected, fixed in 10% (v/v) formalin, embedded in paraffin, and applied to hematoxylin & eosin staining (H&E) or Masson staining where red stain represented cytoplasm and green stain represented collagen fibers [6]. Immunofluorescence 2

3 NCI-H295R cells were grown on glass cover slips for 24 hours. For labeling AT 1 R, cover slips were rinsed with PBS buffer (137 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4, 2 mm KH 2 PO 4, PH=7.4) three times and fixed in 4% formaldehyde in PBS for 20 minutes at room temperature (RT). To quench autofluorescence and reduce unspecific binding to aldehyde groups, cells were treated with freshly prepared, icecode 0.13 M NaBH 4 for 2 X 10 min on ice, followed by 10% Bovine serum albumin incubation at RT for 30 min. Cells were washed with PBS for 3 X 5 min and then incubated for 1 h at 37in a humidified chamber with primary antibodies as indicated (Goat anti human angiotensin type 1receptor antibody, 1:50, abcam). Cover slips were rinsed with PBS for 3 X 10 min and then incubated with corresponding secondary antibodies for 1 h at 37 (Alexa-Fluor-488 donkey anti human IgG, 1:500, Cell Signaling Technology; Rhodamin chicken anti goat IgG, 1: 1000, Rockland. Inc). Finally, cover lips were washed with PBS for 3 X 10 min in the dark and mounted in Mowiol. Confocal images were required by a Leica TCS SPE confocal microscope. Determination of in vitro cellular injury Cellular injury was determined by LDH release. 0.1 ml of culture medium from NCI- H295R cells post AT 1 -AA treatment was mixed up with 0.1 ml freshly prepared working buffer (0.03 M INT, 0.02 M PMS, 0.05M NAD, 0.2 M Tris, 0.06M lactic acid), and then incubated at 37 for 2 hours. The changes of OD values of samples were monitored via ELISA reader at 490 nm (Spectra Max Plus, Molecular Devices, Sunnyvale, California, USA) every 20 min. LDH release was expressed as change in absorption per min. Reference 1. Brown, M.A., Lindheimer, M.D., de Swiet, M., Van Assche, A., Moutquin, J.M. The Classification and Diagnosis of the Hypertensive Disorders of Pregnancy: Statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy. 2001; 20(1): p. ix-xiv. 2. Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am J Obstet Gynecol. 2000; LaMarca B, Wallukat G, Llinas M, Herse F, Dechend R, Granger JP. Autoantibodies to the angiotensin type I receptor in response to placental ischemia and tumor necrosis factor alpha in pregnant rats. Hypertension Dec; 52(6): LaMarca B, Parrish M, Ray LF, Murphy SR, Roberts L, Glover P, Wallukat G, Wenzel K, Cockrell K, Martin JN Jr, Ryan MJ, Dechend R. Hypertension in response to autoantibodies to the angiotensin II type I receptor (AT1-AA) in pregnant rats: role of endothelin-1. Hypertension Oct; 54(4): Korpi-Steiner, N.L., Williamson, E.E., Karon, B.S. Comparison of Three Who l e Blood Creatinine Methods for Estimation of Glomerular Filtration Rate Before Radiographic Contrast Administration. Am J Clin Pathol. 2009; 132(6): p Zhou, C.C. et al. Angiotensin receptor agonistic autoantibodies induce preeclampsia in pregnant mice. Nat Med. 2008; 14(8): p

4 Supplementary data Supplementary data 1. Selection of clinically-relative dose for animal model Supplementary Fig. S1. The selection of clinically-relative dose for animal model was performed. 8 μg/g was chosen as an optimal dose for animal models since its AT 1 -AA level was in the range of clinical levels of preeclamptic women (AT 1 -AA OD value, to ), compared with 4 μg/g and 16 μg/g. * P < 0.05, versus rats free from AT 1 -AA injection. AT 1 -AA = angiotensin II type 1 receptor agonistic autoantibody. Supplementary data 2. The half life of AT 1 -AA in rats Supplementary Fig. S2. The half life of AT 1 -AA in rats was determined. 1μg/g AT 1 - AA was injected into rats to monitor the serum levels of this antibody to find an optimal time interval for passive immunization. The half life of AT 1 -AA was around 14 days. AT 1 -AA = angiotensin II type 1 receptor agonistic autoantibody. Supplementary data 3. The bioactivity test of AT 1 -AA either from the serum of patients or rats 4

5 Supplementary Fig. S3. AT 1 -AA bioactivity was measured via in-vitro vasoconstriction of thoracic aorta. Like ANG II, AT 1 -AA either from human or rats serum constricted rat thoracic aorta obviously. * P < 0.001, versus nsigg. AT 1 -AA = angiotensin II type 1 receptor agonistic autoantibody, ANG II = angiotensin II. Supplementary data 4. The blood pressure in response to elevated AT 1 -AA levels in pregnant rats Supplementary Fig. S4. Systolic blood pressure increased 5 days after injection of AT 1 -AA. The autoantibody increased the blood pressure from ± 5.2 to ± 8.6 mm/hg, * P < In contrast, the abnormality was absent in the nsigg-injected pregnant rats. AT 1 -AA = angiotensin II type 1 receptor agonistic autoantibody. Supplementary data 5. Renal dysfunction caused by AT 1 -AA 5

6 Supplementary Fig. S5. Plasma creatinine and serum uric acid were increased dramatically 5 days after injection of human AT1-AA from PE patients. (A) human AT1-AA dramatically increased plasma creatinine. *P < 0.05, versus nonpregnant group, #P < 0.05, versus pregnant group. (B) human AT1-AA dramatically increased serum uric acid. *P < 0.05, versus nonpregnant group, #P < 0.05, versus pregnant group, &P < 0.05, versus pregnant group injected nsigg. AT1-AA = angiotensin II type 1 receptor agonistic autoantibody. Supplementary data 6. Marked pathological alterations in renal histology of AT1AA-positive pregnant rats Supplementary Fig. S6. HE (1-4) and Masson (5-8) staining of rat kidney tissues. The red stain represents cytoplasm and the green stain represents collagen fibers. The majority of kidneys treated with patient AT1-AA had obvious congestion of the glomeruli with glomerular atrophy, presented as a characteristic bloodless consolidated appearance; Masson staining also suggested an evident renal fibrosis in pregnant rats injected by AT1-AA from women with PE. Magnifications: 1, 3, 5, 7:40x; 2, 4, 6, 8:100x. n = 8/group. *P < 0.05 vs. nonpregnant rats; #P < 0.05 vs. pregnant rats. Supplementary data 7. Changes in fetal growth caused by AT1-AA 6

7 Supplementary Fig. S7. Intrauterine growth retardation caused by AT1-AA injection on gestation day 13 to pregnant rats. (A) On gestation day 18, the fetal body weight in AT1-AA group was obviously less than that of both nsigg-injected and normal pregnant rats. *P < 0.05, versus pregnant rats. (B) AT1-AA increased fetal heart beats 5 days later. *P < 0.05, versus pregnant rats. AT1-AA = angiotensin II type 1 receptor agonistic autoantibody. Supplementary data 8. The immunofluorescence imaging of AT1R on NCIH295R cells Supplementary Fig. S8. NCI-H295R cells were fixed with 4% formaldehyde and immunolabeled with an antibody specific against AT1R (Rhodamine: red, middle column), which is mainly localized on the cellular plasma membrane and co-stained with DAPI for nuclei (Blue, left column). Merged images are presented in the right column. Magnification: upper panels, 64 X; lower panels, 256 X. 7