VEPTP inhibition with an extracellular domain targeting antibody did not restore albuminuria in a mouse model of diabetic kidney disease

Abstract

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. DKD is a heterogeneous disease with complex pathophysiology where early endothelial dysfunction is associated with disease progression. The Tie2 receptor and Angiopoietin 1 and 2 ligands are critical for maintaining endothelial cell permeability and integrity. Tie2 signaling is negatively regulated by the endothelial specific transmembrane receptor Vascular Endothelial Protein Tyrosine Phosphatase (VEPTP). Genetic deletion of VEPTP protects from hypertension and diabetes induced renal injury in a mouse model of DKD. Here, we show that VEPTP inhibition with an extracellular domain targeting VEPTP antibody induced Tie2 phosphorylation and improved VEGF-A induced vascular permeability both in vitro and in vivo. Treatment with the VEPTP blocking antibody decreased the renal expression of endothelial activation markers (Angpt2, Edn1, and Icam1) but failed to improve kidney function in db/db uninephrectomized ReninAAV DKD mice.

Keywords: diabetic kidney disease; protein tyrosine phosphatase receptor type B; renal impairment; vascular endothelial protein tyrosine phosphatase.

FIGURE 1

VEPTP inhibition with an extracellular domain targeting antibody mAb 109.1 induces Tie2 and ERK1/2 phosphorylation and protects against VEGF‐A induced vascular permeability in mouse endothelial cells. (a) Mouse endothelial cells (bEnd.3, ATCC) were treated with increasing concentrations of mAb 109.1 (purple circles) or the isotype (black circles). mAb 109.1 treatment resulted in a dose dependent increase in phospho‐Tie2:total Tie2 ratio. (b) mAb 109.1 treatment (purple circles) resulted in a dose‐dependent increase in the phospho‐ERK1/2:ERK1/2 ratio. (c) VEGF‐A (500 ng/mL) treatment significantly increased permeability of FITC‐dextran across confluent mouse endothelial monolayer on transwell membranes. VEGF‐A induced endothelial monolayer permeability was significantly blocked by mAb 109.1 treatment at both 50 and 100 nM concentrations. (d, e) qPCR analyses showed that mAb 109.1 treatment (at 50 and 100 nM) of mouse endothelial cells resulted in a significant decrease in Angpt2 and Edn1 expression. VEPTP, vascular endothelial protein tyrosine phosphatase; Tie2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2; ERK1/2, extracellular signal‐regulated kinase 1/2; VEGF‐A, vascular endothelial growth factor A; FITC‐dextran, fluorescein isothiocyanate dextran; qPCR, quantitative polymerase chain reaction; Angpt2, angiopoietin‐2; Edn1, endothelin‐1; Ppia, Peptidylprolyl isomerase A.

FIGURE 2

VEPTP inhibition with mAb 109.1 provided protection against VEGF‐A induced dermal vascular leakage in C57BL/6J female mice and induced Tie2‐phosphorylation in lung. (a) C57BL/6J female mice were injected s.c. with mAb 109.1 (100 μg) or isotype 30 min prior to Evans Blue administration. VEGF‐A induced Evans blue leakage was significantly decreased with mAb 109.1 pre‐treatment. (b) mAb 109.1 pre‐treatment resulted in a significant increase in phospho‐Tie2:total Tie2 ratio. mAb 109.1 pre‐treatment resulted in non‐significant trends in increase in (c) phospho‐AKT1/2/3 (Ser473)/AKT1/2/3 and (d) phospho‐ERK1/2 (Thr202/Tyr204)/ERK1/2 ratio. n = 3 mice per treatment group. VEPTP, vascular endothelial protein tyrosine phosphatase; VEGF‐A, vascular endothelial growth factor A; s.c., subcutaneous; Tie2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2; AKT1/2/3, akt serine threonine kinase 1/2/3; ERK1/2, extracellular signal‐regulated kinase 1/2.

FIGURE 3

Systemic pharmacokinetics, kidney Tie2 phosphorylation and gene expression following mAb 109.1 administration in C57BL/6J female mice. (a) Plasma concentration‐time profile of mAb 109.1 after single dose s.c. administration of 5, 10 and 20 mg/kg antibody measured on Days 1, 2, 4, 7 and 14 using ELISA‐based methods. The mAb 109.1 was detected in plasma for 1–4 days at all three‐doses. After Day 4, decline in plasma concentrations of the antibody were noted and at Day 14, mAb 109.1 concentrations were below lower limit of quantification (LLOQ) for the two lower doses (5 and 10 mg/kg). (b) Plasma concentration‐time profile of mAb 109.1 after multiple dose administration of the antibody. C57BL/6J female mice were injected s.c. twice per week for 4‐weeks with two doses (5 and 20 mg/kg) of the mAb 109.1. Plasma mAb 109.1 concentrations were measured on Days 2, 15 and 29 following antibody administration. Systemic concentrations of the antibody were maintained for 4‐weeks following repeated dose s.c. administration of the antibody in mice. (c) Kidneys were collected from the mice injected with the single dose of the mAb 109.1 and analyzed for phospho‐Tie2 (Tyr992) and Tie2. mAb 109.1 treatment at 20 mg/kg resulted in a significant increase in phospho‐Tie2:total Tie2 ratio on Day 4. (d–g) Kidneys were collected from the mice injected with single dose of mAb 109.1 and analyzed for angiopoietin‐2 (Angpt2), endothelin 1 (Edn1), intercellular adhesion molecule 1 (Icam1) and vascular cell adhesion molecule 1 (Vcam1) mRNA expression by qPCR. A dose‐dependent decrease in Angpt2, Edn1, Icam1 and Vcam1 expression were noted with mAb 109.1 treatment. n = 3–5 mice per treatment group. Tie2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2; s.c., subcutaneous; AKT1/2/3, akt serine threonine kinase 1/2/3; ERK1/2, extracellular signal‐regulated kinase 1/2; Ppia, Peptidylprolyl isomerase A.

FIGURE 4

Effects of VEPTP inhibition with mAb 109.1 in the db/db uninephrectomized ReninAAV mice model for DKD. (a) Experimental scheme depicting the timeline and age of mice corresponding to Renin‐AAV injection and dosing with mAb 109.1 or lisinopril. Dosing with mAb 109.1, vehicle or lisinopril was initiated 4 weeks after Renin‐AAV injection and continued for 4‐weeks. (b) Body weight monitoring of the db/db uninephrectomized ReninAAV mice over the course of dosing with mAb 109.1 or lisinopril. (c) db/db Unx ReninAAV female mice were treated with two doses (5 and 20 mg/kg) of mAb 109.1, administered s.c., twice weekly for 4‐weeks. At the end of the study, plasma concentrations of mAb 109.1 were measured using ELISA‐based methods. (d) After treatment completion, kidneys were analyzed for phospho‐Tie2 (Tyr992) and total Tie2. Lisinopril and mAb 109.1 treatment at both 5 and 20 mg/kg doses resulted in a significant increase in phospho‐Tie2:total Tie2 ratio. (e) Urinary albumin to creatinine ratio (uACR) was measured before treatment initiation (baseline) and after 2 and 4‐weeks of treatment with lisinopril or mAb 109.1. Lisinopril treatment resulted in a significant decrease in uACR after 2 and 4‐weeks of treatment compared to vehicle. mAb 109.1 treatment at both doses resulted in no significant change in uACR after 2 and 4 weeks of treatment. (f) Plasma creatinine (g) Blood Urea Nitrogen (BUN) and (h) Blood glucose was measured in the DKD mice after 4‐weeks of treatment with lisinopril or mAb 109.1. Treatment with lisinopril or mAb 109.1 at both doses exhibited no significant changes in plasma creatinine, BUN and blood glucose compared to vehicle treatment. n = 14–16 mice per treatment group. VEPTP, vascular endothelial protein tyrosine phosphatase; DKD, diabetic kidney disease; AAV, adeno‐associated virus; s.c., subcutaneous; Tie2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2.

FIGURE 5

Effects of VEPTP inhibition with mAb 109.1 on kidney mRNA expression of endothelial activation, fibrotic, inflammatory and injury markers in the db/db uninephrectomized ReninAAV mice model for DKD. Female db/db uninephrectomized ReninAAV mice were injected with either vehicle, lisinopril or two doses (5 and 20 mg/kg) of mAb 109.1 for 4‐weeks. After treatment completion kidneys were collected and analyzed for mRNA expression of (a–d) endothelial activation (e–h) fibrosis (i, j) inflammatory and (k) kidney injury marker by qPCR. n = 14–16 mice per treatment group. Angpt2, angiopoietin 2; Edn1, endothelin 1; Icam1, intercellular adhesion molecule 1; Igfb7, insulin like growth factor binding protein 7; Fn1, fibronectin 1; Ctgf, connective tissue growth factor; Acta2, actin alpha 2 smooth muscle; Col1a1, collagen type I alpha 1 chain; Ccl2, chemokine (C‐C motif) ligand 2; Lcn2, lipocalin 2; Havcr1, hepatitis A virus cellular receptor 1; Ppia, peptidylprolyl isomerase A.