Podocyte VEGF-A Knockdown Induces Diffuse Glomerulosclerosis in Diabetic and in eNOS Knockout Mice

Abstract

Vascular endothelial growth factor-a (VEGF-A) and nitric oxide (NO) are essential for glomerular filtration barrier homeostasis, and are dysregulated in diabetic kidney disease (DKD). While NO availability is consistently low in diabetes, both high and low VEGF-A have been reported in patients with DKD. Here we examined the effect of inducible podocyte VEGF-A knockdown (VEGF^KD ) in diabetic mice and in endothelial nitric oxide synthase knockout mice (eNOS^-/- ). Diabetes was induced with streptozotocin using the Animal Models of Diabetic Complications Consortium (AMDCC) protocol. Induction of podocyte VEGF^KD led to diffuse glomerulosclerosis, foot process effacement, and GBM thickening in both diabetic mice with intact eNOS and in non-diabetic eNOS^-/-:VEGF^KD mice. VEGF^KD diabetic mice developed mild proteinuria and maintained normal glomerular filtration rate (GFR), associated with extremely high NO and thiol urinary excretion. In eNOS^-/-:VEGF^KD (+dox) mice severe diffuse glomerulosclerosis was associated with microaneurisms, arteriolar hyalinosis, massive proteinuria, and renal failure. Collectively, data indicate that combined podocyte VEGF-A and eNOS deficiency result in diffuse glomerulosclerosis in mice; compensatory NO and thiol generation prevents severe proteinuria and GFR loss in VEGF^KD diabetic mice with intact eNOS, whereas VEGF^KD induction in eNOS^-/-:VEGF^KD mice causes massive proteinuria and renal failure mimicking DKD in the absence of diabetes. Mechanistically, we identify VEGF^KD -induced abnormal S-nitrosylation of specific proteins, including β3-integrin, laminin, and S-nitrosoglutathione reductase (GSNOR), as targetable molecular mechanisms involved in the development of advanced diffuse glomerulosclerosis and renal failure.

Keywords: GSNOR; S-nitrosylation; VEGF knockdown; diabetic kidney disease; diffuse glomerulosclerosis; laminin; β3-integrin.

FIGURE 1

Podocyte VEGF knockdown (VEGF^KD ) prevents glomerular hypertrophy in diabetic mice. (A) VEGF^KD Transgenic mouse line carries 4 transgenes: Nphs2-rtTA and tet-0-shVEGF that are activated by doxycycline to synthesize shRNA targeting Vegf-a exon 1, which inhibits expression of all Vegf-a isoforms in podocytes (Veron et al., 2012). (B) VEGF^KD mice received STZ (50 mg IP, 5 daily doses) (DM-VEGF^KD − dox), STZ + doxycycline (DM-VEGF^KD + dox), doxycycline (VEGF^KD + dox) or no treatment (VEGF^KD − dox). Dox was started a week after STZ, 2 weeks later was considered time 0 (when random blood glucose was steadily elevated) for DM-VEGF^KD mice; (C) non-diabetic control glomerulus (VEGF^KD − dox) shows normal histology; (D) diabetic control (DM-VEGF^KD − dox) glomerulus shows hypertrophy and mesangial expansion; (E) non-diabetic VEGF^KD (+ dox) glomerulus is smaller than control (C, − dox); (F) diabetic VEGF^KD (+ dox) glomerulus shows diffuse glomerulosclerosis and is smaller than control (D, − dox); Scale bars = 50 μm; (G) quantitation of glomerular size demonstrates significantly smaller glomerular volume in VEGF^KD (+ dox) vs. control (−dox) glomeruli from non-diabetic (VEGF^KD ) and diabetic (DM-VEGF^KD ) mice; unpaired t-test with Welch’s correction was used; asterisk (*) indicates p < 0.05, (***) indicates p < 0.001; control vs. VEGF^KD or non-diabetic vs. diabetic, as indicated; non-DM, non-diabetic mice, DM, diabetic mice; dox, uninduced mice; dox, doxycycline- treated mice.

FIGURE 2

Histology of eNOS ^−/−:VEGF^KD kidneys reveals diffuse glomerulosclerosis and mimics advanced DKD: PAS stain representative images: (A) eNOS^−/−:VEGF^KD − dox glomeruli are normal by light microscopy; (B) eNOS^−/−:VEGF^KD + dox glomeruli show microaneurisms (1-2, black arrowheads), mesangiolysis (3,5,6, white arrowheads), mesangial expansion (Papapetropoulos et al., 1997; Shen et al., 1999; Reidy et al., 2014; Tuttle et al., 2014), severe mesangial sclerosis (4-6, black asterisks), proteinaceous tubular casts (4-6, white asterisks) and lymphocytic infiltrates (5–6, yellow arrowheads); Scale bars = 50 μm (A, B1-3) and 100 μm (B4-6); PAS: Periodic acid-Schiff stain.

FIGURE 3

Effect of VEGF^KD on glomerular ultrastructure of diabetic and eNOS^−/−: VEGF^KD kidneys. Representative TEM images: (A,B) diabetic control (DM-VEGF^KD (− dox) glomerular capillary loop shows GBM thickening (black arrowheads) and partial foot process effacement (FPE: black arrows, normal FP: white arrows); (C,D) diabetic VEGF^KD (+ dox) glomerulus shows mesangial sclerosis (black asterisks), extensive FPE (black arrows) and GBM thickening (black arrowheads); (E,F) eNOS^−/−:VEGF^KD (−dox) glomerulus shows preserved filtration barrier ultrastructure; (G,H) eNOS^−/−:VEGF^KD (+dox) glomerulus shows massive FPE (black arrows), GBM thickening (black arrowheads) and endotheliosis (yellow arrows). Scale bars: 1 μm in top images (A,C,E,G); 500 nm in bottom images (B,D,F,H); + dox:VEGF^KD induction with doxycycline.

FIGURE 4

Podocyte VEGF^KD downregulates nephrin in diabetic and eNOS^−/−:VEGF^KD mice: (A) WB: show nephrin downregulation in eNOS^−/−:VEGF^KD + dox and diabetic kidneys (Brown-Forsythe ANOVA, p = 0.047), no significant difference was detected between DM-VEGF^KD − dox and + dox (Welch’s t-test) ; (B) IHC: nephin IF signals are clearly decreased in glomeruli from eNOS^−/−:VEGF^KD (+dox) and DM-VEGF^KD (+dox) kidneys; (C) WB: podocin decreased in eNOS^−/−:VEGF^KD (+dox) and diabetic kidneys (Brown-Forsythe ANOVA, p = 0.0001), no significant difference was detected between DM-VEGF^KD − dox and + dox (Welch’s t-test); (D) WB: VEGFR2 decreased in eNOS ^−/−:VEGF^KD (+dox) and diabetic kidneys (Brown-Forsythe ANOVA, p = 0.015) but differences (+dox vs. - dox) were not significant; (E) WB: significant β3-integrin upregulation was detected in eNOS^−/−:VEGF^KD (+dox) kidneys (Brown-Forsythe ANOVA, p = 0.03). Scale bars = 50 μm, + dox = VEGF^KD induction with doxycycline.

FIGURE 5

Podocyte VEGF^KD causes massive proteinuria and renal failure in eNOS^−/−:VEGF^KD mice. (A) Induction of VEGF^KD in eNOS^−/−:VEGF^KD (+dox) mice (red bar) increases albuminuria ∼30 fold higher than in control eNOS^−/−:VEGF^KD (− dox) (**, p = 0.0022) but does not change albuminuria in diabetic mice (DM-VEGF^KD + dox, blue bar) (n.s., p = 0.9015); VEGF^KD causes mild albuminuria in non-diabetic mice (VEGF^KD + dox, gray bar) compared to controls (VEGF^KD − dox, white bar) (**, p = 0.0043), Mann-Whitney test. (B) SDS PAGE/Coomassie stain shows severe albuminuria in eNOS^−/−:VEGF^KD + dox and milder albuminuria in diabetic VEGF^KD + dox mice; BSA = bovine serum albumin marker, urine volume loading was normalized to creatinine. (C) Creatinine clearance decreases upon VEGF^KD induction in eNOS^−/−:VEGF^KD + dox mice (red bar) to ∼1/3 of control eNOS^−/−:VEGF^KD − dox (***, p = 0.0009), but is not significantly altered in diabetic mice (DM-VEGF^KD − dox and + dox, hatched/blue bars) (n.s., p = 0.4114) or VEGF^KD in non-diabetic mice (VEGF^KD − dox and + dox, white/gray bars) (n.s., p = 0.359) with intact eNOS; induced eNOS^−/−:VEGF^KD (+dox) mice had significantly lower Creat Cl than diabetic VEGFKD + dox and non-diabetic VEGF^KD + dox mice (*, p = 0.02 and ***, p = 0.0007, respectively).

FIGURE 6

Effect of VEGF^KD on circulating and urine VEGF-A and NO in diabetic and eNOS^−/−:VEGF^KD mice. (A) plasma VEGF-A is similarly elevated in eNOS^−/−:VEGF^KD mice (red bars) irrespectively of VEGF^KD , as compared to non-diabetic eNOS intact mice (VEGF^KD − dox, white bar) (****, p =<0.0001) or VEGF^KD + dox mice (gray bar) (*, p < 0.005); in diabetic mice VEGF^KD (DM-VEGF^KD + dox, blue bar) significantly decreases circulating VEGF-A (**, p = 0.0013), but all diabetic mice have plasma VEGF-A >2-fold higher than non-diabetic mice with intact eNOS (VEGF^KD , white/gray bars) (****, p < 0.0001 and ***, p = 0.0006). (B) Urine VEGF-A: podocyte VEGF^KD does not alter VEGF-A excretion in eNOS^−/−:VEGF^KD (red bars) or diabetic mice (blue bars); VEGF^KD significantly inhibits VEGF-A excretion in non-diabetic mice (VEGF^KD + dox, gray bar) (**, p = 0.0043). (C) Plasma NO: podocyte VEGF^KD (+ dox) does not significantly alter plasma NO in any experimental group; plasma NO is lower in eNOS^−/−:VEGF^KD (red bars) than diabetic (blue bars) (**, p = 0.001 and ***, p = 0.0009) and non-diabetic mice with intact eNOS (white bar) (*, p = 0.047); plasma NO is higher in diabetic (DM-VEGF^KD − dox, hatched blue bar) than in non-diabetic mice (VEGF^KD − dox, white bar) (*, p = 0.015) and VEGF^KD abrogates this change (DM-VEGF^KD + dox, blue bar). (D) Urine NO: VEGF^KD increases NO excretion in eNOS^−/−:VEGF^KD + dox mice (*, p = 0.0272, red bar); all diabetic mice (blue bars) have several fold higher NO excretion than non-diabetic mice (white/gray bars), irrespectively of VEGF^KD .

FIGURE 7

Podocyte VEGF^KD induces thiol-mediated mechanisms in diabetic and eNOS^−/−:VEGF^KD mice. (A) WB: Kidney GSNOR expression is not altered by diabetes or VEGF^KD , tubulin is shown as loading control. (B) GSNOR S-nitrosylation (SNO-GSNOR) detected by BST: VEGF^KD (+dox) decreases SNO-GSNOR in eNOS^−/−:VEGF^KD and diabetic kidneys; podocyte and kidney lysates are used as SNO positive and negative BST controls, respectively, input shows equal loading for BST. (C) Urine Cys thiol excretion (normalized to creatinine): podocyte VEGF^KD increases ∼2.5 fold Cys thiol excretion in eNOS^−/−:VEGF^KD + dox mice (red bar) (*, p = 0.013); diabetic mice (blue bars), irrespective of VEGF^KD , have ∼6-fold higher Cys thiol excretion than uninduced non-diabetic mice with intact eNOS (VEGF^KD − dox, white bar) (**, p = 0.004) or eNOS^−/−:VEGF^KD − dox (hatched red bar) (**, p = 0.004). (D) IHC: podocyte VEGF^KD (+ dox) increases S-nitrosylation of glomerular proteins in eNOS^−/−:VEGF^KD kidneys, SNO-Cys quantification is shown in (E), (****, p = 0.0003). (F) PLA: shows that podocyte VEGF^KD (+ dox) increases laminin S-nitrosylation (SNO-laminin) in eNOS^−/−:VEGF^KD kidneys, SNO-laminin PLA quantification is shown in (G) (*, p = 0.025). (H) PLA: shows β3-integrin S-nitrosylation (SNO-β3-integrin) in eNOS^−/−:VEGF^KD glomeruli, which is increased by podocyte VEGF^KD (+dox); SNO-β3-integrin quantification is shown in (I) (*, p = 0.036).

FIGURE 8

Proposed model of podocyte VEGF^KD driven diffuse glomerulosclerosis in DM-VEGF^KD and eNOS:VEGF^KD mice. (A) Strong compensatory NO and thiol generation prevents GFR loss, attenuates proteinuria and diffuse glomerulosclerosis in diabetic VEGF^KD mice, while limitation of this compensatory mechanism in eNOS:VEGF^KD mice worsens the renal phenotype, leading to renal failure. (B) Reduced GSNOR S-nitrosylation increases GSNO and promotes increased S-nitrosylation of proteins, altering their signaling pathways: (C) decreased nephrin and VEGFR2 signaling and high SNO-β3-integrin inhibit β3-integrin activity leading to podocyte and endothelial cell injury; high SNO-laminin and low VEGFR2 signaling may contribute to the severe diffuse glomerulosclerosis described herein in eNOS:VEGF^KD + dox mice.