Cell-Specific Roles of Angiopoietin-2 in CKD

Taipei Renal Transcriptomics and Outcomes Investigation

Taipei Renal Transcriptomics and Outcomes Investigation (TRTOI) is an ongoing prospective observation study conducted at Taipei Veterans General Hospital, Taiwan, as previously described.^24,25 Consecutive adult patients (age ≥20 years) who underwent kidney biopsies or nephrectomies have been enrolled since October 2018. Demographic data, medical history, laboratory results, and clinical outcomes were extracted from electronic medical records. Kidney function was assessed using the Modification of Diet in Renal Disease equation to estimate eGFR. All kidney biopsies were reviewed and reported by the same nephropathologist. The severity of interstitial fibrosis, expressed as percentage, was evaluated using Masson trichrome staining. The primary outcomes included KRT, a sustained ≥50% decline in baseline eGFR, and death due to cardiovascular or kidney diseases. All participants were followed until the occurrence of primary outcomes, loss to follow-up, or the end of this study (February 2, 2025), whichever came first. Patients lost to follow-up were excluded from the outcome evaluation. Data analysis and outcome evaluation were conducted between September 2024 and February 2025. Transcriptomic analysis of human kidney samples was conducted as previously reported.²⁵

Animals

Angpt2^loxP/loxP mice were generated in our laboratory (Supplemental Figure 1). Tg(UBC-CreERT2) mice (JAX stock no. 007001) were obtained from The Jackson Laboratory.²⁶ Tg(UBC-CreERT2);Angpt2^loxP/loxP mice were bred to facilitate global angiopoietin-2 deletion upon tamoxifen (T5648, Sigma-Aldrich, St. Louis, MO) administration and were hereafter referred to as Angpt2^ΔUBC. Littermates lacking the Cre transgene served as control mice. Tamoxifen was administered via oral gavage at a dose of 0.4 mg/g body weight to 8-week-old mice, including both the Tg(UBC-CreERT2); Angpt2^loxP/loxP and control groups. To minimize potential interference from tamoxifen in the induction of kidney disease, a 2-week washout period was implemented after tamoxifen administration.

Tg(Pax8-rtTA; LC1) double transgenic mice were generously provided by R. Koesters (Heidelberg, Germany).²⁷ Tg(Pax8-rtTA; LC1); Angpt2^loxP/loxP mice were bred to enable TEC-specific angiopoietin-2 deletion on doxycycline administration and were hereafter referred to as Angpt2^ΔTEC. Littermates lacking the Cre transgene served as control mice. Doxycycline (Sigma-Aldrich) was administered in drinking water at a concentration of 0.2 mg/ml, supplemented with 2% sucrose, starting at 8 weeks of age. In the kidney disease model, doxycycline administration began in 8-week-old mice, including both Tg(Pax8-rtTA; LC1); Angpt2^loxP/loxP and control groups, 1 week before kidney disease induction and continued until the end of the experiment.

Tg(Cdh5-CreERT2) mice were generously provided by R.H. Adams (Münster, Germany).²⁸ Tg(Cdh5-CreERT2); Angpt2^loxP/loxP mice were bred to achieve endothelial cell–specific angiopoietin-2 deletion on tamoxifen administration and were hereafter referred to as Angpt2^ΔEC. Littermates lacking the Cre transgene served as control mice. Tamoxifen was administered via oral gavage at a dose of 0.4 mg/g body weight to 8-week-old mice, including both the Tg(Cdh5-CreERT2); Angpt2^loxP/loxP and control groups. To minimize potential interference from tamoxifen in the induction of kidney disease, a 2-week washout period was implemented after tamoxifen administration.

Mouse CKD Models

In the first model, CKD was induced by feeding adult mice LabDiet 5001 (TestDiet, St. Louis, MO) containing 0.25% adenine (Sigma–Aldrich) for 5 days, followed by regular LabDiet 5001 for the remaining 2 days each week, a regimen modified from our previous study.²⁹ Mice were euthanized 1–4 weeks after initiating the adenine diet. Alternatively, an early inflammation model of adenine-induced CKD was established by administering adenine (50 mg/kg body weight) via oral gavage for 7 consecutive days. Adenine was dissolved in 5% sodium carboxymethyl cellulose (Sigma–Aldrich).

In the second model, CKD was induced through UUO surgery.¹⁸ Under anesthesia with ketamine/xylazine (100/10 mg per kg body weight), the left ureter was ligated using a 4-0 nylon surgical suture at the level of the lower pole of the kidney, with the mice in a prone position. Sham surgery, involving a left flank incision, ureter exposure, and repositioning without ligation, served as the control. Mice were euthanized 4 days after surgery.

L1-10 Administration

L1-10 was administered daily via subcutaneous injection at a dose of 5 mg/kg body weight (Amgen, Inc., Thousand Oaks, CA) to C57BL/6 mice starting from the day adenine gavage began, to block angiopoietin-2 binding to the Tie-2 receptor.¹⁸ The control mice received subcutaneous injections of sFc protein (Bethyl Laboratories Inc., Montgomery, TX).

Fluorescence-Activated Cell Sorting Analysis

Kidney samples were collected from Angpt2^ΔUBC mice and littermate controls after 3 weeks of dietary intervention. Mice were divided into three groups: (1) control mice on a regular diet, (2) control mice on an adenine diet, and (3) Angpt2^ΔUBC mice on an adenine diet. Kidney cells were dissociated using the Multi-Tissue Dissociation Kit 2 following standard protocols. Single-cell suspensions were stained with 4′,6-diamidino-2-phenylindole (to exclude dead cells), anti-Ly6C-APC (128031, BioLegend), anti-Ly6G-Buv395 (565964, BD Biosciences), anti-NK1.1-Buv395 (564144, BD Biosciences), and anti-CD11b-PE (101208, BioLegend). Data were acquired using a BD LSRFortessa flow cytometer (BD Biosciences), and analysis was performed using FlowJo V10 software (BD Biosciences). Gating involved selecting live cells (forward scatter [FSC]/side scatter), identifying single cells (FSC-Area/FSC-Height), excluding dead cells (4′,6-diamidino-2-phenylindole⁺), Ly6G⁺ and NK1.1⁺ populations, and identifying CD11b^high cells, which were further classified into Ly6C⁺ and Ly6C⁻ subpopulations.³⁰ Cell counts and percentages were compared among groups, with statistical analyses performed to assess differences.

Statistics

In the clinical study, the associations between kidney ANGPT2 mRNA levels and eGFR or interstitial fibrosis were assessed using the Pearson correlation test. Associations with clinical outcomes were evaluated using unadjusted Cox proportional hazards regression. All statistical analyses were performed using STATA SE version 16.0 (Stata Corp., College Station, TX), with a two-sided significance level of 0.05.

For in vivo and in vitro experiments, distributional assumptions were evaluated using the Shapiro–Wilk test (primary), supported by Anderson–Darling, D'Agostino–Pearson, and Kolmogorov–Smirnov tests, together with Q–Q plots. For datasets with sample size <8, group comparisons were performed using the Mann–Whitney U test or the Kruskal–Wallis test. For other datasets, when raw values were skewed, data were log-transformed and analyzed using an unpaired t test with Welch correction. A Mann–Whitney sensitivity analysis was performed to confirm the robustness of the conclusions. Reporting of statistical assumptions and methods adheres to the Animal Research: Reporting of In Vivo Experiments 2.0 guidelines. All analyses for experimental data were conducted using GraphPad Prism version 10.4.1 (GraphPad Software, San Diego, CA).

Study Approval

All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (20201053, 20201140, 20201162, 20220428, 20230126). The TRTOI cohort study was approved by the Institutional Review Board of Taipei Veterans General Hospital (2018-06-008B). All patients provided written informed consent. The clinical study protocol followed the principles outlined in the Declaration of Helsinki.

Kidney ANGPT2 mRNA Levels Were Higher in CKD and Associated with Adverse Kidney Outcomes

In the TRTOI cohort, an ongoing prospective observational study at Taipei Veterans General Hospital (Figure 1A),²⁵ kidney ANGPT2 mRNA levels were higher in patients with CKD and were negatively correlated with eGFR (Figure 1B, N=127). By contrast, angiopoietin-1 (ANGPT1) mRNA levels were not correlated with either eGFR or kidney fibrosis (Figure 1, C and E, N=127), whereas kidney ANGPT2 mRNA levels were positively correlated with the degree of kidney fibrosis (Figure 1D, N=127). In Cox proportional hazards analysis, higher ANGPT2 expression was associated with a greater risk of the composite kidney outcome (hazard ratio [HR], 1.79; 95% confidence interval [CI], 1.15 to 2.79 for ANGPT2; HR, 1.01; 95% CI, 0.91 to 1.13 for ANGPT1), initiation of KRT (HR, 1.96; 95% CI, 1.24 to 3.11 for ANGPT2; HR, 1.01; 95% CI, 0.91 to 1.13 for ANGPT1), and death (HR, 1.68; 95% CI, 1.08 to 2.59 for ANGPT2; HR, 0.99; 95% CI, 0.87 to 1.11 for ANGPT1; Figure 1F, n=101). These results indicate that increased kidney ANGPT2 mRNA levels were associated with lower kidney function, greater fibrosis, and a higher risk of adverse clinical outcomes in CKD.

Global Deletion of Angiopoietin-2 Ameliorated Kidney Damage in Advanced Adenine-Induced CKD

Because deletion of angiopoietin-2 in all epiblast cells by embryonic day 6.5 resulted in a higher risk of intrauterine fetal death (Supplemental Table 1), we used Angpt2^ΔUBC mice to achieve global deletion of angiopoietin-2 before the induction of CKD with an adenine-containing diet (Figure 2, A and B). No significant differences were observed between littermate controls lacking Cre recombinase (Control) and Angpt2^ΔUBC mice 2 weeks after tamoxifen administration (Supplemental Table 2). Compared with Control mice, Angpt2^ΔUBC mice exhibited improved kidney function, as reflected by lower plasma levels of BUN and creatinine starting from the third week after CKD induction (Figure 2C). Histologic analysis revealed reduced tubulointerstitial injury (Figure 2, D and E), vascular rarefaction (Figure 2, F and G, and Supplemental Figure 2), macrophage infiltration (Figure 2, H–J), and interstitial fibrosis (Figure 2, K–M) in Angpt2^ΔUBC mice. These findings indicate that upregulation of angiopoietin-2 contributed to kidney dysfunction and structural injury in adenine-induced CKD.

Global Deletion of Angiopoietin-2 Mitigated Metabolic Derangement in Advanced Adenine-Induced CKD

To uncover the molecular mechanisms underlying the protective effects of angiopoietin-2 deletion, we performed RNA sequencing (RNA-Seq) on kidney tissues from Control and Angpt2^ΔUBC mice 3 weeks after adenine-induced CKD. Principal component analysis revealed distinct transcriptomic profiles in Angpt2^ΔUBC kidneys compared with Control kidneys (Figure 3A). Ingenuity pathway analysis showed significant upregulation of metabolic pathways in Angpt2^ΔUBC kidneys, particularly peroxisomal lipid metabolism, fatty acid β-oxidation, triglyceride metabolism, and peroxisome proliferator–activated receptor signaling (Figure 3B). Gene Ontology biologic process analysis indicated enrichment of categories related to energy substrate metabolism, amino acid and organic acid metabolism, mitochondrial energy metabolism, cofactor and redox balance, and transport (Figure 3C), suggesting higher bioenergetic activity in Angpt2^ΔUBC kidneys. Notably, peroxisomal, rather than mitochondrial, β-oxidation was preferentially upregulated (Figure 3D). Key peroxisomal molecules, including Abcd3 (encoding 70-kDa peroxisomal membrane protein), Acox1 encoding peroxisomal acyl-coenzyme A oxidase 1, and Cat (encoding catalase), were significantly increased at both the transcript and protein levels (Figure 3, E–G, and Supplemental Figure 3). Immunofluorescence confirmed increased expression of peroxisomal acyl-coenzyme A oxidase 1, catalase, and 70-kDa peroxisomal membrane protein in TECs, as demonstrated by colocalization with the basement membrane marker nidogen (Figure 3H). By contrast, both in vivo and in vitro studies showed that TECs lacked Tie-2 expression, and treatment with exogenous angiopoietin-2 did not alter metabolic gene expression in primary TECs (Supplemental Figure 4). These results indicate that angiopoietin-2 deletion promoted metabolic reprogramming in TECs, characterized by enhanced peroxisomal lipid catabolism and a shift toward a protective bioenergetic state, through an indirect mechanism that remains to be fully defined.

Global Deletion of Angiopoietin-2 Attenuated Inflammatory Signaling and Reduced Macrophage Infiltration in Progressive Adenine-Induced CKD

Inflammatory pathways, including neutrophil degranulation and triggering receptor expressed on myeloid cells signaling, and fibrosis-related pathways, such as extracellular matrix organization and cell surface interactions at the vascular wall, were downregulated in Angpt2^ΔUBC kidneys compared with Control kidneys (Figure 3B). Gene Ontology biologic process analysis indicated that inflammation- and adhesion-related categories were significantly downregulated in Angpt2^ΔUBC kidneys (Figure 4A). Consistently, expression of key proinflammatory chemokines, cytokines, and adhesion molecules—including Ccl2, Cx3cl1, Ccl17, Csf1, Ccr2, Cx3cr1, Icam1, and Vcam1—was significantly reduced in Angpt2^ΔUBC kidneys (Figure 4, B and D, and Supplemental Figure 5). Flow cytometry analysis revealed elevated CD11b^high macrophage populations in Control kidneys following adenine-induced CKD, which were significantly attenuated in Angpt2^ΔUBC kidneys (Figure 4, E and F, and Supplemental Figure 6). Moreover, CD11b^high macrophages sorted from Angpt2^ΔUBC kidneys exhibited reduced expression of proinflammatory (Il1b) and profibrotic (Ccl17, Ccl22) markers, suggesting suppression of both proinflammatory and profibrotic macrophage subsets (Figure 4G). Stratification by Ly6C expression revealed that both CD11b^highLy6C⁺ and CD11b^highLy6C⁻ populations were significantly reduced in Angpt2^ΔUBC kidneys (Figure 4, H and I). These findings indicate that angiopoietin-2 deficiency limited macrophage recruitment and activation, thereby attenuating inflammation and fibrosis in CKD.

Angiopoietin-2 Enhanced Proinflammatory and Profibrotic Effects of Endothelial Cells

CD31⁺ endothelial cells, the primary kidney cell type expressing the Tie-2 receptor, were isolated from Control and Angpt2^ΔUBC kidneys 3 weeks after adenine-induced CKD. Consistent with whole-kidney findings, endothelial cells from Angpt2^ΔUBC kidneys showed significantly reduced expression of Angpt2 compared with Control cells (Supplemental Figure 7A). Expression of proinflammatory (Vcam1) and profibrotic (Tgfb1) genes was also reduced (Supplemental Figure 7A). Western blot analysis confirmed lower expression of C–C motif chemokine ligand 2 and VCAM1 proteins in endothelial cells from Angpt2^ΔUBC kidneys (Supplemental Figure 7, B and C). These findings suggest that angiopoietin-2 promoted endothelial activation and enhanced proinflammatory and profibrotic signaling in endothelial cells during adenine-induced CKD.

TEC-Specific Deletion of Angiopoietin-2 Reduced CKD Severity

Given increased angiopoietin-2 expression in injured and proliferating TECs in murine CKD models (Supplemental Figure 8),^17,31 we generated Angpt2^ΔTEC mice to achieve TEC-specific deletion of angiopoietin-2 before adenine-induced CKD (Figure 5, A and B). No significant differences were observed between littermate controls lacking Cre recombinase (Control) and Angpt2^ΔTEC mice 2 weeks after doxycycline administration (Supplemental Table 3). Compared with control mice, Angpt2^ΔTEC mice displayed reduced kidney Angpt2 expression (Figure 5B); improved kidney function (Figure 5C); reduced tubulointerstitial injury (Supplemental Figure 9), vascular rarefaction (Figure 5, D and E), interstitial inflammation (Figure 5, F–H), and fibrosis (Figure 5, I–K); and decreased expression of endothelial adhesion molecules (Figure 5, L–N). These findings indicate that TEC-specific angiopoietin-2 deletion protected against functional and structural deterioration in progressive adenine-induced CKD.

Endothelial Cell–Specific Deletion of Angiopoietin-2 Did Not Impact Progressive Adenine-Induced CKD

Given the expression of angiopoietin-2 in endothelial cells,^17,21,22,31 we used Angpt2^ΔEC mice to assess the role of endothelial cell–derived angiopoietin-2 in progressive adenine-induced CKD (Figure 6A). No significant differences were observed between littermate controls lacking Cre recombinase (Control) and Angpt2^ΔEC mice 2 weeks after tamoxifen administration (Supplemental Table 4). Unlike global or TEC-specific deletion, endothelial cell–specific deletion of angiopoietin-2 did not markedly reduce kidney Angpt2 mRNA or angiopoietin-2 protein expression 3 weeks after CKD induction, although a significant reduction was observed before and 1 week after CKD induction (Figure 6B). These findings suggest that endothelial cells are not the predominant source of angiopoietin-2 at this advanced stage of disease. Moreover, endothelial cell–specific deletion did not significantly improve kidney function or histologic features (Figure 6, C and D, and Supplemental Figure 10).

Endothelial Cell–Derived Angiopoietin-2 Drove Early Inflammation in CKD

To investigate the role of endothelial cell–expressed angiopoietin-2 in early inflammation, Angpt2^ΔEC mice were subjected to adenine gavage for 7 consecutive days (Figure 6E). CD31⁺ endothelial cells isolated from Angpt2^ΔEC kidneys showed reduced expression of Angpt2, Ccl2, Cx3cl1, Icam1, and Vcam1 genes (Figure 6, F and G), as well as their corresponding proteins (Supplemental Figure 11, A and B). Similar anti-inflammatory effects were observed after treatment with L1-10, an antiangiopoietin-2 peptide (Figure 6, H–J, and Supplemental Figure 11, C and D).

We previously demonstrated that macrophage accumulation significantly increased in the murine kidney by day 4 after UUO surgery.¹⁵ Here, we demonstrated that both Angpt2^ΔUBC and Angpt2^ΔEC mice exhibited reduced macrophage accumulation and lower expression of proinflammatory markers in the kidneys on day 4 postsurgery (Supplemental Figures 12 and 13), whereas Angpt2^ΔTEC did not affect early inflammation (Supplemental Figure 14). CD31⁺ endothelial cells isolated from both Angpt2^ΔUBC and Angpt2^ΔEC kidneys also showed reduced expression of Angpt2 and Icam1 (Supplemental Figure 15). These findings highlight the role of endothelial cell–derived, rather than TEC-derived, angiopoietin-2 in initiating the early inflammatory response in CKD. As capillary rarefaction progressed and the disease advanced, TEC-derived angiopoietin-2 sustained a cycle of inflammation, fibrosis, and metabolic dysregulation (Figure 6K).