1. Volume 85, Issue 2, Pages 393-403 (January 2014)
Suppressed soluble Fms–like tyrosine kinase-1 production aggravates atherosclerosis in chronic kidney disease 
Masaru Matsui, Yukiji Takeda, Shiro Uemura, Takaki Matsumoto, Ayako Seno, Kenji Onoue, Hideo Tsushima, Katsuhiko Morimoto, Tsunenari Soeda, Satoshi Okayama, Satoshi Somekawa, Ken-ichi Samejima, Hiroyuki Kawata, Rika Kawakami, Kimihiko Nakatani, Masayuki Iwano, Yoshihiko Saito 
Kidney International 
Volume 85, Issue 2, Pages (January 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Hypothesis representation of the balance between placental growth factor (PlGF) and soluble fms–like tyrosine kinase-1 (sFlt-1). In healthy subjects, the balance between PlGF and sFlt-1 is tightly maintained, but in chronic kidney disease (CKD) patients, this balance can be disrupted by the downregulation of sFlt-1, resulting in the progression of atherosclerosis and the promotion of plaque angiogenesis through the relative activation of atherogenic PlGF/Flt-1 signal.
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3. Figure 2
Heparin loading test. (a) Plasma soluble fms–like tyrosine kinase-1 (sFlt-1) levels were dose-dependently increased by heparin injection (n=8). (b) sFlt-1 levels were significantly lower in chronic kidney disease (CKD) patients (n=4) than in patients with normal renal function (n=4) at 5min after heparin injection. ***P<0.001.
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4. Figure 3
Plasma soluble fms–like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) levels before and after heparin injection in chronic kidney disease (CKD) patients. (a, b) Postheparin sFlt-1 levels were directly correlated with estimated glomerular filtration rate (eGFR) in CKD patients (n=273), whereas preheparin sFlt-1 levels were inversely correlated with eGFR. (c–f) PlGF levels were inversely correlated with eGFR, although low-dose heparin injection did not change PlGF or vascular endothelial growth factor (VEGF) levels. (g) Postheparin PlGF/sFlt-1 ratio was strongly correlated with eGFR (r=0.680). (h) The postheparin PlGF/sFlt-1 ratio was significantly increased according to CKD stage, including patients receiving hemodialysis (n=343). ***P< Data are mean±s.d. r, correlation coefficient.
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5. Figure 4
Placental growth factor (PlGF)/soluble fms–like tyrosine kinase-1 (sFlt-1) ratio and cardiovascular events. (a) The preheparin PlGF/sFlt-1 ratio was not relevant to the incidence of cardiovascular events (P=0.107). (b) The postheparin PlGF/sFlt-1 ratio was a strong predictor of the incidence of cardiovascular diseases (P=0.001).
Kidney International  , DOI: ( /ki )
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6. Figure 5
Soluble fms–like tyrosine kinase-1 (sFlt-1) is released from the endothelial cells by heparin administration. (a) Low-dose heparin (10IU/kg) was administered through the tail veins of C57Bl/6 mice and sFlt-1-/- mice. Blood samples (0.7–1.0ml) were drawn 10min after heparin injection and sFlt-1 protein levels were analyzed by enzyme-linked immunosorbent assay (ELISA); n=8, *P<0.05 versus wild-type (WT) mice without heparin, #P<0.05 versus sFlt-1-/- mice without heparin, respectively. (b) The thoracic aortas were excised and cut open for incubation with culture medium that included heparin (ex vivo). The medium was collected 24h after heparin treatment and sFlt-1 was measured by ELISA; n=8, *P<0.05 versus wild-type mice without heparin, #P<0.05 versus sFlt-1-/- mice without heparin, respectively. (c) Human microvascular endothelial cells (HMVECs) were cultured with heparin for 10min. The medium was collected 10min after heparin treatment and sFlt-1 was measured by ELISA; n=6, *P<0.05 versus medium without heparin. (d) sFlt-1 mRNA was analyzed simultaneously to investigate sFlt-1 production in HMVECs; n=6. Data are mean±s.e.m. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Serum from chronic kidney disease (CKD) patients induces endothelial damage and suppression of soluble fms–like tyrosine kinase-1 (sFlt-1) production. (a–c) Human umbilical artery endothelial cells (HUAECs) were cultured with serum from patients (Control, CKD5, CKD5D), and mRNA of sFlt-1, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) was measured by real-time reverse transcription–polymerase chain reaction (RT-PCR) after 48h; n=8. (d–f) The markers of endothelial damage were also analyzed by RT-PCR; n=8, *P<0.05 versus CKD1, #P<0.05 versus CKD5, respectively. Data are mean±s.e.m. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Increased oxidative stress is associated with the suppression of soluble fms–like tyrosine kinase-1 (sFlt-1) production in the endothelial cells. (a) Levels of the oxidative stress marker heme oxygenase-1 (HO-1) were increased in tissues rich in the endothelial cells in 5/6 nephrectomized (NR) apolipoprotein E (ApoE)-/- mice; n=8, *P<0.05 versus Control. (b) Intracellular reactive oxyen species (ROS) production was increased in HUAECs by cultivation with serum from chronic kidney disease (CKD) patients; n=8, *P<0.05 versus Control. (c) Suppression of sFlt-1 mRNA production in HUAECs was rescued by vitamin E administration in the CKD5D group; n=4, *P<0.05 versus CKD5D without vitamin E. (d) Placental growth factor (PlGF) production in HUAECs was increased in the CKD5D group and vitamin E suppressed this elevation; n=4, *P<0.05 versus CKD5D without vitamin E. (e–h) The expression levels of HO-1 and Nox-2 mRNA were increased in nephrectomized ApoE-/- mice compared with control ApoE-/- mice; n=6. Data are mean±s.e.m. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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9. Figure 8
Soluble fms–like tyrosine kinase-1 (sFlt-1-/-) mice demonstrate increased atherosclerotic formation and macrophage infiltration. (a, c, and e) sFlt-1+/+ ApoE-/- mice and (b, d, and f) sFlt-1-/- ApoE-/- mice were fed a high-cholesterol diet for 10 weeks, and atherosclerotic lesions were compared between the two strains. The thoracoabdominal aortas were excised and stained with Oil Red O. The aortic roots were sectioned and stained with hematoxylin–eosin (HE) (original magnification, × 5). Atherosclerotic lesions of the (a, b, and g) thoracoabdominal aorta and (c, d, and h) aortic root were exacerbated in sFlt-1-/- ApoE-/- mice compared with sFlt-1+/+ ApoE-/- mice. (e, f, i) F4/80-positive macrophages (original magnification, × 20) were increased in the plaques of the aortic roots in sFlt-1-/- ApoE-/- mice compared with sFlt-1+/+ ApoE-/- mice; n=8, *P<0.05 versus sFlt-1+/+ ApoE-/- mice. Data are mean±s.e.m.
Kidney International  , DOI: ( /ki )
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10. Figure 9
Hypothetical illustration for the localization and role of soluble fms–like tyrosine kinase-1 (sFlt-1). (a) In the baseline condition of patients with normal renal function, the majority of sFlt-1 is stored on the endothelial cell surface, with a smaller amount in the circulation. (b) In contrast, in the baseline condition of patients with renal dysfunction, a smaller amount of sFlt-1 is produced and stored on the endothelial cell surface, and a larger amount of sFlt-1 is in circulation. (c, d) Heparin injection mobilizes membrane-associated sFlt-1 into circulation. The plasma levels of sFlt-1 after heparin injection are higher in the context of normal renal function than in renal dysfunction, because smaller amounts of baseline sFlt-1 are stocked on the endothelial cell surface in renal dysfunction. PlGF, placental growth factor; VEGF, vascular endothelial growth factor.
Kidney International  , DOI: ( /ki )
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