Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy

Abstract

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.

Keywords: VEGF; VEGF-A; albuminuria; diabetes; diabetic nephropathy; endothelial glycocalyx; glomerulus; glycocalyx; permeability.

Figure 1.

VEGF-A₁₆₅b is upregulated in humans with early diabetic nephropathy and well preserved kidney function. (A) mRNA expression of exon 5/7/8 containing VEGF-A isoforms (i.e., those coding for 165 amino-acid proteins) determined by RT-qPCR relative to total RNA extracted in whole renal cortical tissue from kidneys unsuitable for transplantation from deceased donors without diabetes (“none”), donors with early diabetic nephropathy with well preserved kidney function (“early”), and kidney biopsy specimens from patients with diabetes and advanced nephropathy (“late”) (note logarithmic scale). (B) VEGF-A₁₆₅a mRNA expression (note logarithmic scale). (C) VEGF-A₁₆₅b expression (note logarithmic scale). (D) VEGF-A isoform expression ratio calculated as VEGF-A₁₆₅b mRNA per unit total mRNA divided by VEGF-A₁₆₅a mRNA per unit total mRNA. (E) Ratio of VEGF-A₁₆₅a to VEGF-A₁₆₅b mRNA in control and early diabetic samples in which it was possible to analyze mRNA from single glomeruli isolated by differential sieving. (F) Isoform-specific VEGF-A₁₆₅b protein determined by ELISA as a proportion of total VEGF-A in whole kidney cortical tissue in kidney donors with diabetes and early nephropathy (“early”) and those without diabetes (“none”). Error bars, SEM. Multiple comparisons: one-way ANOVA. Two-group comparisons: unpaired t test. *P<0.05; **P<0.01; ***P<0.005, throughout.

Figure 2.

Podocyte-specific overexpression of VEGF-A₁₆₅b ameliorates STZ-induced diabetic nephropathy. (A) Transgenic mice constitutively overexpressing human VEGF-A₁₆₅b in podocytes under control of the nephrin promoter (blue; neph^hVEGF-A₁₆₅b) received injections of STZ or vehicle at 12 weeks of age. Plasma glucose (B), urine albumin-to-creatine ratio (C), and creatinine clearance (D) measured in nondiabetic wild-type, diabetic wild-type, nondiabetic transgenic, and diabetic transgenic mice 6 weeks after diabetes induction. n=5–10 mice per group. Error bars, SEM. *P<0.05, one-way ANOVA. (E and F) Glomerular area assessed in hematoxylin and eosin–stained kidney sections from 21 to 55 glomeruli from 3 mice per group. Scale bars: 20 µm. Error bars, SEM. *P<0.05, one-way ANOVA. (G and H) Periodic acid Schiff–stained kidney sections from each group of mice. Mesangial matrix expansion (arrows) measured as a proportion of total glomerular area in 45–60 glomeruli from 3 to 4 mice per group. Scale bars: 20 µm. Error bars, SEM. *P<0.05, one-way ANOVA. (I) Low- and high-magnification electron micrographs (scale bars: 1 µm and 250 nm, respectively) of glomerular capillaries and the glomerular capillary wall (GCW) from each group of mice. CL, capillary lumen; ec, endothelial cell; gbm, glomerular basement membrane; pfp, podocyte foot process; rbc, red blood cell; US, urinary space. (J) Glomerular basement membrane width calculated from 20 to 60 measurements in 8–16 glomerular capillaries per group. Error bars, SEM. *P<0.05, one-way ANOVA.

Figure 3.

Podocyte-specific overexpression of VEGF-A₁₆₅b reduces albuminuria in the dual insult of VEGF-A₁₆₄a overexpression and STZ-induced diabetic nephropathy. (A) Heterozygous mice overexpressing human VEGF-A₁₆₅b in podocytes (blue: neph^hVEGF-A₁₆₅b) were crossed with mice in which murine VEGF-A₁₆₄a (^mVEGF-A₁₆₄a) overexpression in podocytes could be induced with doxycycline (dox): heterozygous podocin-rtTA:TetO-^mVEGF-A₁₆₄a mice (red: pod-TetO-^mVEGF-A₁₆₄a), to generate mice co-overexpressing ^mVEGF-A₁₆₄a and ^hVEGF-A₁₆₅b in podocytes. Offspring were treated with STZ at 12 weeks of age to induce diabetes in all groups. After 5 weeks of diabetes, all mice received doxycycline, inducing ^mVEGF-A₁₆₄a overexpression in the two groups carrying the podocin-rtTA:TetO-^mVEGF-A₁₆₄a transgene. After 1 week of doxycycline treatment, urinary albumin-to-creatine ratio (uACR) was measured and compared with uACR obtained before doxycycline administration. (B) Change in uACR after 7 days of doxycycline treatment in diabetic mice with no transgenes, diabetic mice overexpressing ^hVEGF-A₁₆₅b alone, diabetic mice overexpressing ^mVEGF-A₁₆₄a alone, and diabetic mice co-overexpressing ^mVEGF-A₁₆₄a and ^hVEGF-A₁₆₅b. Dashed line represents no change in albuminuria during the 1-week doxycycline treatment period. n=3–6 mice per group. Error bars, SEM. *P<0.05, one-way ANOVA.

Figure 4.

Systemic treatment with VEGF-A₁₆₅b blocks progression of albuminuria and glomerular basement membrane thickening in STZ-induced diabetic nephropathy. (A) Diabetes was induced in wild-type DBA2J mice with STZ injection (nondiabetic control mice received buffer injection alone). After 2 consecutive weeks of hyperglycemia, mice received an 11-week course of biweekly intraperitoneal injections (blue arrows) of recombinant human VEGF-A₁₆₅b (^rhVEGF-A₁₆₅b) (n=16) or vehicle (n=16). (B) Plasma glucose measured repeatedly in nondiabetic control, diabetic vehicle-treated, and 0.04 µg/g ^rhVEGF-A₁₆₅b–treated mice. *P<0.05 versus both diabetic groups; "ns" indicates P>0.05 between diabetic groups; one-way ANOVA for both. (C) Urine albumin-to-creatinine ratio (uACR) was measured repeatedly in diabetic DBA2J mice receiving injections of vehicle (n=4) or 0.04 µg/g ^rhVEGF-A₁₆₅b (n=4) for up to 11 weeks. *P<0.05, paired t test. (D) Diabetes was induced in wild-type DBA2J mice with STZ injection and vehicle or ^rhVEGF-A₁₆₅b treatment after the onset of albuminuria. (E) uACR measured repeatedly in mice receiving biweekly injections of vehicle (n=10), 0.04 µg/g ^rhVEGF-A₁₆₅b (n=10), and 0.2 µg/g ^rhVEGF-A₁₆₅b (n=6) before organ harvest. Error bars, SEM. (F) Fold change in uACR (uACR at indicated time points, divided by uACR before initiation of treatment [arrow]). Dashed line represents no change in albuminuria after start of injections. Error bars, SEM. *P<0.05, two-way ANOVA. (G) Low- and high-magnification electron micrographs (scale bars: 1 μm and 500 nm, respectively) of glomerular capillaries and the glomerular capillary wall (GCW). CL, capillary lumen; ec, endothelial cell; gbm, glomerular basement membrane; pfp, podocyte foot process; rbc, red blood cell; US, urinary space. (H) Glomerular basement membrane (GBM) width measurements in indicated groups of diabetic mice. Error bars, SEM. *P<0.05, one-way ANOVA.

Figure 5.

Systemic treatment with VEGF-A₁₆₅b blocks early albuminuria but not later features of diabetic nephropathy in a genetic model of type 2 diabetic nephropathy. (A) After the onset of hyperglycemia at 6 or 7 weeks of age, db/db mice received biweekly intraperitoneal injections of recombinant human VEGF-A₁₆₅b (^rhVEGF-A₁₆₅b) or vehicle (blue arrows) for 8 weeks. (B) Blood glucose measurements in 6 lean and 20 db/db mice before and after receipt of twice-weekly vehicle or ^rhVEGF-A₁₆₅b (0.2 µg/g) injections. Dashed line indicates start of intraperitoneal injections. Error bars, SEM. *P<0.05 compared with both groups of diabetic mice; "ns" indicates P>0.05 between groups of diabetic mice. (C) Urinary albumin-to-creatinine ratio (uACR) determined repeatedly during the 8-week treatment period, divided by uACR in the same animal before treatment (“baseline”) to calculate fold change in uACR. Dashed line represents no fold change in albuminuria since start of treatment. Error bars, SEM. *P<0.05, paired t test. (D) Creatinine clearance measured at the end of the 8-week injection period. Error bars, SEM. *P<0.05; "ns" indicates P>0.05; one-way ANOVA for both. (E) In a separate cohort of older mice, 5 weeks of biweekly intraperitoneal injections (blue arrows) of ^rhVEGF-A₁₆₅b or vehicle were initiated in 14-week-old lean controls (n=5) and db/db mice (n=14) with long-standing hyperglycemia, established albuminuria, and decreased GFR. (F) Serial measurement of uACR in these older vehicle- and ^rhVEGF-A₁₆₅b–treated db/db mice. Dashed line indicates start of intraperitoneal injections. Error bars, SEM. *P<0.05, paired t test. (G) At the end of the 5-week injection period, GFR measurements in these older vehicle- and ^rhVEGF-A₁₆₅b–treated db/db and lean mice. Error bars, SEM. *P<0.05; "ns" indicates P>0.05; one-way ANOVA for both. (H) At the end of the 5-week injection period, mesangial matrix expansion (arrows) in periodic acid Schiff–stained kidney sections from each group of mice. Scale bar: 50 μm.

Figure 6.

VEGF-A₁₆₅b reduces apoptosis, decreases glomerular permeability, and acts via VEGFR-2. (A) Apoptosis (caspase-3 activity) was determined in human podocytes in vitro exposed to normal (10 mM) or high (30 mM) cell culture glucose concentrations with or without recombinant human VEGF-A₁₆₅b (^rhVEGF-A₁₆₅b). Experiments were done in triplicate. OD, optical density. Error bars, SEM. *P<0.05, one-way ANOVA. (B) Caspase-3 staining performed on human microvascular endothelial cells exposed to normal (5 mM) or high (30 mM) cell culture glucose concentrations in the presence or absence of ^rhVEGF-A₁₆₅b. (C) Number of caspase-3–positive cells per high-powered field (hpf) compared between conditions. Experiments were done in quadruplicate. Error bars, SEM. ***P<0.001; "ns" indicates P>0.05; one-way ANOVA for both. (D) Glomerular water permeability (volume-corrected ultrafiltration coefficient: L_PA/V_i) measured in glomeruli isolated from vehicle-injected nondiabetic (open bar), and STZ-injected diabetic rats (filled bars). Glomeruli were incubated for 1 hour in ^rhVEGF-A₁₆₅b (1 nM), VEGF-A receptor-2 antagonist ZM323881 (10 µM), pan-VEGF-A receptor blocker PTK787 (100 nM), saline, or combinations thereof before L_PA/V_i measurement. Error bars, SEM. Compared with diabetic: *P<0.05; "ns" indicates P>0.05. Compared with diabetic+^rhVEGF-A₁₆₅b: ^#P<0.05; "ns¹" indicates P>0.05. One-way ANOVA for all. (E and F) Total and phosphorylated VEGFR-2 in glomeruli of nondiabetic wild-type and neph^hVEGF-A₁₆₅b mice (E) and in glomeruli from STZ-induced diabetic DBA2J mice treated with vehicle or ^rhVEGF-A₁₆₅b (F). Controls without primary antibody are also shown. Scale bars: 20 μm. VEGFR-2 and phosporylated-VEGFR-2 by area relative to the whole glomerulus quantified for nondiabetic neph^hVEGF-A₁₆₅b versus littermates (G) and ^rhVEGF-A₁₆₅b–treated diabetic mice versus vehicle-treated diabetic mice (H). Error bars, SEM. *P<0.05, one-way ANOVA.

Figure 7.

VEGF-A₁₆₅b stimulates VEGFR-2 in glomerular endothelial cells in vivo. (A) Sections of kidney tissue from wild-type and neph^hVEGF-A₁₆₅b mice showing glomeruli costained for VEGFR-2 (green), and the endothelial cell marker PECAM-1 (red), and overlaid images showing colocalization (yellow). (B) High magnification of a single glomerular capillary loop showing VEGFR-2 expression and endothelial cell PECAM-1 colocalize. (C) Sections of kidney tissue from wild-type and neph^hVEGF-A₁₆₅b mice showing glomeruli costained for VEGFR-2 (green) and the podocyte marker nephrin (red). (D) High magnification of a single glomerular capillary loop showing that VEGFR-2 expression and podocyte nephrin do not colocalize.

Figure 8.

VEGF-A₁₆₅b restores the glomerular endothelial glycocalyx in early STZ-induced diabetic nephropathy. Six weeks after STZ (or buffer) injections to induce diabetes (or normoglycemia) in DBA2J mice, mice received biweekly intraperitoneal injections of vehicle or ^rhVEGF-A₁₆₅b for 4 weeks. Animals were cardiac perfusion fixed and renal cortices were processed for transmission electron microscopy. (A) Glomeruli were imaged at low magnification, the glomerular capillary wall at higher magnification, and endothelial cell surface at high magnification using transmission electron microscopy. e-glx, endothelial glycocalyx; ec, endothelial cell; fen, endothelial fenestra; gbm, glomerular basement membrane; gc, glomerular capillary; pfp, podocyte foot process; us, urinary space. (B) Endothelial fenestral density and (C) glycocalyx depth covering the glomerular endothelial cell surface quantified in electron microscopic images from vehicle-treated nondiabetic, vehicle-treated diabetic, and ^rhVEGF-A₁₆₅b–treated diabetic animals. Error bars, SEM. n=3 mice per group. *P<0.05; "ns" indicates P>0.05; one-way ANOVA for both.

Figure 9.

VEGF-A₁₆₅b restores glomerular endothelial glycocalyx acutely in diabetic rat glomeruli. Kidneys were perfused in vivo with cell membrane label (R18; red) and glycocalyx label (Alexa Fluor-488–wheat germ agglutinin lectin; green), and then glomeruli were isolated and treated with vehicle or VEGF-A₁₆₅b for 1 hour (exactly as per physiology experiments) before imaging with confocal microscopy. (A) whole glomerulus; scale bar: 20 μm. (B) Glomerular capillaries (gc) with endothelial cell (ec) bodies; scale bar: 5 μm. (C) High-magnification image (scale bar: 1 μm) of a vehicle-treated glomerular capillary from a healthy animal, demonstrating glomerular endothelial glycocalyx (GLX) lining luminal surface of endothelial cell body. gcw, glomerular capillary wall. (D) High-magnification image (scale bar: 1 μm) of a vehicle-treated glomerular capillary from a diabetic animal. Note absent glomerular endothelial glycocalyx (absent GLX) lining the luminal surface of endothelial cell body. (E) High-magnification image (scale bar: 1μm) of a VEGF-A₁₆₅b–treated glomerular capillary from a diabetic animal. Note restoration of glomerular endothelial glycocalyx. (F) Mean±SEM glomerular endothelial glycocalyx depth in five glomeruli in each of three animals per group. *P<0.05, one-way ANOVA.

Figure 10.

VEGF-A₁₆₅b normalizes permeability of diabetic mouse, rat, and human glomeruli. (A) Glomeruli were harvested from 6 diabetic (STZ-injected) wild-type, 4 nondiabetic wild-type, and 8 diabetic (STZ) neph^hVEGF-A₁₆₅b mice (102 glomeruli); from 3 diabetic (STZ) and 3 nondiabetic rats (74 glomeruli); and from untransplantable kidneys from 3 nondiabetic and 3 diabetic human kidney donors (54 glomeruli). Glomerular water permeability (volume-corrected ultrafiltration coefficient: L_PA/Vi) was measured in individual glomeruli following exposure to constitutively overexpressed ^hVEGF-A₁₆₅b (mice) or 1-hour incubation in vehicle or ^rhVEGF-A₁₆₅b (rat and human glomeruli). Error bars, SEM. *P<0.05; "ns" indicates P>0.05; one-way ANOVA for both. (B) Glomerular L_PA/Vi was measured twice in 3 diabetic human glomeruli, before (black) and after (blue) 1-hour incubation in ^rhVEGF-A₁₆₅b. *P<0.05, paired t test.