Angiopoietin-2 inhibition attenuates kidney fibrosis by hindering chemokine C-C motif ligand 2 expression and apoptosis of endothelial cells

Plasma levels of angiopoietin-2 are increased in patients with chronic kidney disease (CKD). Moreover, mouse models of progressive kidney disease also demonstrate increased angiopoietin-2 in both plasmas and kidneys. The role of dysregulated angiopoietins in the progression of kidney disease has not been thoroughly investigated. Here, we found in a cohort of 319 patients with CKD that plasma angiopoietin-2 and angiopoietin-2/angiopoietin-1 ratios were positively associated with the development of kidney failure. In mice with progressive kidney disease induced by either ureteral obstruction or ischemia-reperfusion injury, overexpression of human angiopoietin-1 in the kidney tubules not only reduced macrophage infiltration in the initial stage post-injury but also attenuated endothelial cell apoptosis, microvascular rarefaction, and fibrosis in the advanced disease stage. Notably, angiopoietin-1 attenuated chemokine C-C motif ligand 2 (CCL2) expression in the endothelial cells of the fibrosing kidneys, and these protective effects led to attenuation of functional impairment. Mechanistically, angiopoietin-1 reduced CCL2-activated macrophage migration and protected endothelial cells against cell apoptosis induced by angiopoietin-2 and Wnt ligands. Based on this, we applied L1-10, an angiopoietin-2 inhibitor, to the mouse models of progressive kidney disease and found inhibitory effects on macrophage infiltration, microvascular rarefaction, and fibrosis. Thus, we defined the detrimental impact of increased angiopoietin-2 on kidney survival of patients with CKD which appears highlighted by angiopoietin-2 induced endothelial CCL2-activated macrophage infiltration and endothelial cell apoptosis in their kidneys undergoing fibrosis. Plasma levels of angiopoietin-2 are increased in patients with chronic kidney disease (CKD). Moreover, mouse models of progressive kidney disease also demonstrate increased angiopoietin-2 in both plasmas and kidneys. The role of dysregulated angiopoietins in the progression of kidney disease has not been thoroughly investigated. Here, we found in a cohort of 319 patients with CKD that plasma angiopoietin-2 and angiopoietin-2/angiopoietin-1 ratios were positively associated with the development of kidney failure. In mice with progressive kidney disease induced by either ureteral obstruction or ischemia-reperfusion injury, overexpression of human angiopoietin-1 in the kidney tubules not only reduced macrophage infiltration in the initial stage post-injury but also attenuated endothelial cell apoptosis, microvascular rarefaction, and fibrosis in the advanced disease stage. Notably, angiopoietin-1 attenuated chemokine C-C motif ligand 2 (CCL2) expression in the endothelial cells of the fibrosing kidneys, and these protective effects led to attenuation of functional impairment. Mechanistically, angiopoietin-1 reduced CCL2-activated macrophage migration and protected endothelial cells against cell apoptosis induced by angiopoietin-2 and Wnt ligands. Based on this, we applied L1-10, an angiopoietin-2 inhibitor, to the mouse models of progressive kidney disease and found inhibitory effects on macrophage infiltration, microvascular rarefaction, and fibrosis. Thus, we defined the detrimental impact of increased angiopoietin-2 on kidney survival of patients with CKD which appears highlighted by angiopoietin-2 induced endothelial CCL2-activated macrophage infiltration and endothelial cell apoptosis in their kidneys undergoing fibrosis. Translational StatementUntil now, effective treatments that can halt the progression of chronic kidney disease (CKD) are still lacking. Our study provides evidence that the inhibition of angiopoietin-2 can reduce kidney fibrosis through the attenuation of endothelium-driven inflammation and endothelial cell apoptosis in murine models of progressive kidney disease. We are the first to demonstrate an effective therapeutic strategy directly targeting the renal endothelium. Our report supports a potential clinical trial for evaluating the inhibition of angiopoietin-2 as a treatment for CKD. Until now, effective treatments that can halt the progression of chronic kidney disease (CKD) are still lacking. Our study provides evidence that the inhibition of angiopoietin-2 can reduce kidney fibrosis through the attenuation of endothelium-driven inflammation and endothelial cell apoptosis in murine models of progressive kidney disease. We are the first to demonstrate an effective therapeutic strategy directly targeting the renal endothelium. 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Winterrowd E. et al.Angiopoietin-1 deficiency increases renal capillary rarefaction and tubulointerstitial fibrosis in mice.PLoS One. 2018; 13e0189433Crossref Scopus (24) Google Scholar the pathogenic role and causal biology of excess Angpt2 in progressive kidney disease and morbidities are not thoroughly understood. We thus hypothesized that increased Angpt2 would impose prognostic significance in CKD patients and used experimental models to study the protective effect of Angpt2 inhibition on progressive kidney disease. From December 2006 to December 2007, we enrolled 416 adult Asian patients with CKD stages 3 to 5 defined by an estimated glomerular filtration rate using the 4-variable Modification of Diet in Renal Disease Study equation.7Chang F.C. Chiang W.C. Tsai M.H. et al.Angiopoietin-2-induced arterial stiffness in CKD.J Am Soc Nephrol. 2014; 25: 1198-1209Crossref PubMed Scopus (34) Google Scholar,42Levey A.S. Bosch J.P. Lewis J.B. et al.A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group.Ann Intern Med. 1999; 130: 461-470Crossref PubMed Scopus (13286) Google Scholar After inclusion, patients were prospectively followed at the nephrology outpatient clinic until June 2020. Ancillary analysis of plasma Angpt1, Angpt2, and soluble Tie-2 receptor was performed in November 2011. Data analysis and outcome evaluation, including end-stage kidney disease (ESKD) and mortality, were performed from March 2020 to October 2020. Col1a1-GFPTg mice that synthesized enhanced green fluorescence protein (GFP) under the control of the promoter/enhancer of the gene encoding the type I collagen α1 chain were described previously.43Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar Tie2-GFP transgenic (Tie2-GFPTg) mice (stock no. 003658) were obtained from The Jackson Laboratory. Pax8-rtTA transgenic (Pax8-rtTATg) mice were able to express reverse tetracycline-dependent transactivator (rtTA) in all proximal and distal tubules and the entire collecting duct system under the control of mouse Pax8 promoter.44Traykova-Brauch M. Schonig K. Greiner O. et al.An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.Nat Med. 2008; 14: 979-984Crossref PubMed Scopus (209) Google Scholar pTRE-hAngpt1 transgenic (pTRE-hAngpt1Tg) mice were able to express human Angpt1 in cells expressing rtTA.45Ward N.L. Haninec A.L. Van Slyke P. et al.Angiopoietin-1 causes reversible degradation of the portal microcirculation in mice: implications for treatment of liver disease.Am J Pathol. 2004; 165: 889-899Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Double transgenic (DT) mice were those carrying both transgenes of Pax8-rtTA and pTRE-hAngpt1. Littermates that inherited single Pax8-rtTA or no transgene served as experimental controls. Doxycycline (Sigma) at a concentration of 0.2 mg/ml in drinking water was given to both DT and littermate control mice to induce human Angpt1 expression from the day after surgery for renal fibrosis induction to the time the mice were euthanized. L1-10 (4 mg/kg body weight; Amgen, Inc.), a Fc-fusion peptide able to block Angpt2 binding to receptor Tie-2, was injected intraperitoneally every other day from the day after surgery for renal fibrosis induction to the time the mice were euthanized according to previous reports.7Chang F.C. Chiang W.C. Tsai M.H. et al.Angiopoietin-2-induced arterial stiffness in CKD.J Am Soc Nephrol. 2014; 25: 1198-1209Crossref PubMed Scopus (34) Google Scholar,46Tressel S.L. Kim H. Ni C.W. et al.Angiopoietin-2 stimulates blood flow recovery after femoral artery occlusion by inducing inflammation and arteriogenesis.Arterioscler Thromb Vasc Biol. 2008; 28: 1989-1995Crossref PubMed Scopus (57) Google Scholar Soluble Fc fragment (4 mg/kg body weight; Bethyl laboratories Inc.) was used as a control. Unilateral ureteral obstruction (UUO) or unilateral ischemia reperfusion injury (UIRI) was performed in adult (8–12 weeks) mice as previously described.43Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar To further evaluate residual function of the fibrotic kidney, nephrectomy of the contralateral kidney was applied on day 14 after UIRI, and the mice were euthanized on day 16. Sham surgery was performed by manipulation of the renal pedicle in age-matched, background-matched mice used as controls. In the clinical study, differences in categorical variables were compared using χ2 and Fisher’s exact tests. Differences between continuous baseline variables, including angiotrophic growth factors, were analyzed using the Kruskall-Wallis test. Natural log transformation was performed for angiotrophic growth factors to assess the association with outcomes by the Cox regression analysis. The proportional-hazard assumption was confirmed by log-minus-log plots showing parallel survival curves and the Schoenfeld residuals test (globe test P = 0.33). All statistical analyses were carried out using SAS version 9.4 (SAS Institute Inc.) and STATA SE version 15.1 (Stata Corp.) under the 0.05 significance level. In animal and cell experiments, data are expressed as mean ± SEM. Statistical significance was evaluated by unpaired Student’s t test or 1-way analysis of variance with a post hoc test. Two-sided P < 0.05 was considered statistically significant. The analyses were performed using GraphPad Prism (version 9.0.0; GraphPad Software). Human and animal studies were executed under protocols approved by the National Taiwan University Hospital Institutional Review Board (200611010R, 201105023RC) and the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (20120006, 20160104, 20180336, 20201140). All patients provided written informed consent. The clinical study followed the reporting requirements of the Strengthening the Reporting of Observational Studies in Epidemiology statement. Full methods including study design and setting, participants, variables and endpoint evaluation, blood and urine measurement, animals, mouse models of progressive renal fibrosis, enzyme-linked immunosorbent assay, ex vivo micro-computed tomography, isolation and culture of endothelial cells from the kidney, transfection, transwell migration assay, immunoprecipitation and Western blot analysis, fluorescence-activated cell sorting (FACS) analysis, tissue preparation for histology and enzyme-linked immunosorbent assay, tubular injury score, whole-mount X-gal staining of mouse kidneys, polymerase chain reaction (PCR), and statistical analysis are available in the Supplementary Methods. From December 2006 to December 2007, we enrolled 416 adult Asian patients with CKD stages 3 to 5 and reported the positive association between plasma Angpt2 and pulse wave velocity in CKD in a cross-sectional analysis.7Chang F.C. Chiang W.C. Tsai M.H. et al.Angiopoietin-2-induced arterial stiffness in CKD.J Am Soc Nephrol. 2014; 25: 1198-1209Crossref PubMed Scopus (34) Google Scholar It is poorly understood whether the elevated baseline Angpt2 is associated with poor renal outcomes. Therefore, we prospectively followed the enrolled patients at the nephrology outpatient clinic until June 2020. Ninety-seven patients were excluded from outcome evaluation because of loss of follow-up for more than 1 year during the study period. Therefore, 319 patients are reported in this study (Table 1).Table 1Baseline patient characteristics of patientsCKD stageCharacteristicsAll (n = 319)3 (n = 134)4 (n = 90)5 (n = 95)PaThe χ2 test or Fisher’s exact test was used for in categorical variables and the Kruskall-Wallis test for continuous variables.Clinical characteristicsAge, yr62 (52–71)60 (49–69)64 (53–72)62 (54–71)0.198Male sex206 (64.6)108 (80.6)51 (56.7)47 (49.5)<0.0001Diabetes115 (36.1)46 (34.3)32 (35.6)37 (38.9)0.768Hypertension274 (85.9)115 (85.8)78 (86.7)81 (85.3)0.963Dyslipidemia93 (29.2)43 (32.1)23 (25.6)27 (28.4)0.563Smoker38 (11.9)22 (16.4)11 (12.2)5 (5.3)0.037BMI, kg/m224.0 (21.8–26.7)24.8 (22.0–27.1)23.4 (21.8–27.1)23.4 (21.6–25.9)0.134MedicationACEI/ARB205 (64.3)96 (71.6)63 (70.0)46 (48.4)0.0006CCB146 (45.8)44 (32.8)44 (48.9)58 (61.1)<0.0001β-Blocker128 (40.1)44 (32.8)40 (44.4)44 (46.3)0.075α-Blocker68 (21.3)27 (20.1)21 (23.3)20 (21.1)0.847Diuretic108 (33.9)41 (30.6)35 (38.9)32 (33.7)0.437Statin80 (25.1)36 (26.9)20 (22.2)24 (25.3)0.733Acetylsalicylic acid54 (16.9)24 (17.9)11 (12.2)19 (20.0)0.342Laboratory characteristicseGFR, ml/min per 1.73 m225.1 (13.2–40.1)42.8 (36.9–49.3)23.2 (19.3–25.6)9.5 (7.4–12.0)<0.0001Creatinine, mg/dl2.4 (1.7–4.4)1.6 (1.5–1.9)2.7 (2.3–3.1)5.5 (4.6–7.2)<0.0001Albumin, g/dl4.5 (4.2–4.7)4.6 (4.3–4.8)4.5 (4.1–4.6)4.4 (4.1–4.6)<0.0001Urine albumin-to-creatinine ratio, mg/g361.5 (71.0–886.5)117.5 (23.5–525.5)451.0 (105.0–934.0)658.0 (311.0–1304.0)<0.0001Calcium, mmol/l2.3 (2.3–2.4)2.4 (2.3–2.5)2.3 (2.3–2.4)2.3 (2.2–2.3)<0.0001Phosphate, mg/dl3.8 (3.4–3.6)3.4 (3.1–3.7)3.8 (3.5–4.3)4.9 (4.2–5.4)<0.0001Hemoglobin, g/dl11.4 (9.7–13.5)13.5 (11.9–14.5)11.2 (9.6–12.2)10.1 (8.9–11.0)<0.0001Total cholesterol, mg/dl194 (167–217)196 (169–214)189 (168–219)192 (163–218)0.990HDL cholesterol, mg/dl44 (39–51)43 (39–50)46 (38–52)44 (37–52)0.545Triglyceride, mg/dl137 (98–195)140 (101–192)145 (104–214)125 (91–174)0.141Uric acid, mg/dl8.2 (7.1–9.5)7.8 (6.8–8.9)8.3 (7.2–9.5)8.7 (7.9–10.1)0.0001Ferritin, ng/ml170.0 (105.0–283.0)183.5 (115.0–284.0)157.0 (100.5–274.0)167.0 (103.0–270.0)0.684iPTH, pg/ml91.3 (53.8–181.5)60.3 (40.7–84.3)103.0 (66.2–152.0)269.0 (166.0–372.0)<0.0001hsCRP, mg/dl0.11 (0.06–0.21)0.11 (0.07–0.23)0.13 (0.07–0.28)0.11 (0.06–0.18)0.304Angiogenic growth factorsAngpt1, pg/ml6859.0 (2850.0–12,679.9)6933.1 (2530.7–13,045.9)7340.9 (3505.4–11,800.5)6487.9 (2223.3–13,923.3)0.669Angpt2, pg/ml2294.5 (1674.0–3182.1)1886.0 (1492.9–2570.7)2488.5 (1783.2–3308.5)2831.3 (1975.5–3872.3)<0.0001Angpt2/Angpt10.3 (0.2–0.9)0.3 (0.1–0.8)0.3 (0.2–0.8)0.5 (0.2–1.4)0.137sTie-2, pg/ml12.3 (10.1–14.5)12.5 (10.7–14.7)12.3 (10.1–14.7)11.4 (9.2–13.8)0.015ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin Ⅱ receptor blocker; BMI, body mass index; CCB, calcium channel blocker; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; HDL cholesterol, high-density lipoprotein cholesterol; iPTH, Intact parathyroid hormone; hsCRP, high-sensitivity C-reactive protein; Angpt1, Angiopoietin-1; Angpt2, Angiopoietin-2; sTie-2, soluble Tie-2 receptor.Values are median (interquartile range) or n (%).a The χ2 test or Fisher’s exact test was used for in categorical variabl