Limits...
Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice.

Ku CH, White KE, Dei Cas A, Hayward A, Webster Z, Bilous R, Marshall S, Viberti G, Gnudi L - Diabetes (2008)

Bottom Line: Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04).Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05).Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, King's College London School of Medicine, Guy's Hospital, King's College London, London, UK.

ABSTRACT

Objective: Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.

Research design and methods: We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.

Results: Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an approximately 70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69-199) vs. 43 (26.8-69) mug/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.

Conclusions: Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

Show MeSH

Related in: MedlinePlus

Podocyte-inducible overexpression of LacZ/sFlt-1. A: Nuclear LacZ staining of glomerular podocytes was detected in DOX-treated but not in VEH-administered double transgenic mice (10 weeks of treatment). No staining was observed in Pod/+ administered VEH or DOX (not shown). B: Densitometry quantitative analysis of sFlt-1 expression (as a ratio with β-actin) in total kidney cortex lysate after 10 weeks of treatment with VEH or DOX. Data are expressed as mean ± SEM (*P < 0.01 for Pod/sFlt-1 DOX vs. all other groups, P = NS for Pod/+ VEH vs. Pod/+ DOX; n = 4–8/group). (Please see http://dx.doi.org/10.2337/db08-0647 for a high-quality digital representation of this figure.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2551695&req=5

f1: Podocyte-inducible overexpression of LacZ/sFlt-1. A: Nuclear LacZ staining of glomerular podocytes was detected in DOX-treated but not in VEH-administered double transgenic mice (10 weeks of treatment). No staining was observed in Pod/+ administered VEH or DOX (not shown). B: Densitometry quantitative analysis of sFlt-1 expression (as a ratio with β-actin) in total kidney cortex lysate after 10 weeks of treatment with VEH or DOX. Data are expressed as mean ± SEM (*P < 0.01 for Pod/sFlt-1 DOX vs. all other groups, P = NS for Pod/+ VEH vs. Pod/+ DOX; n = 4–8/group). (Please see http://dx.doi.org/10.2337/db08-0647 for a high-quality digital representation of this figure.)

Mentions: Eight-week-old adult double transgenic mice (Pod/sFlt-1) or single transgenic mice (Pod/+) were treated with DOX or administered VEH for up to 10 weeks (both sexes showed a similar phenotype and were analyzed together). X-gal staining revealed no signal in Pod/+ DOX and VEH kidneys, confirming that the staining protocol did not detect endogenous galactosidase activity (not shown). Furthermore, Pod/sFlt-1 mice administered with VEH also failed to give a positive signal (Fig. 1A), excluding “leakage” of LacZ expression in noninduced mice. In contrast, kidneys from adult Pod/sFlt-1 mice exposed to DOX showed positive X-gal staining (Fig. 1A), consistent with podocyte expression of the transgene (13,14). X-gal staining was analyzed at different time points after DOX administration (10 days, 5–10 weeks), and the results were superimposable (not shown). After 10 weeks of treatment with DOX, Pod/sFlt-1 animals showed a significant inducible sFlt-1 overexpression (100% increase) when assessed in kidney cortex lysate (Fig. 1B). The upregulation of sFlt-1 was associated with an increase in 24-h urine sFlt-1 levels, which was four- to fivefold higher in DOX-treated Pod/sFlt-1 compared with Pod/sFlt-1 mice given VEH or with Pod/+ control mice (P < 0.001) (Fig. 2A). This was paralleled by a reduction in urine free VEGF-A in Pod/sFlt-1 DOX-treated mice, suggesting an sFlt-1–mediated “sequestration/binding” of VEGF-A (P < 0.04) (Fig. 2B). Renal cortex VEGF-A was upregulated in Pod/sFlt-1 DOX mice in comparison with the other groups (P < 0.03) (Fig. 2C). No difference was observed in the renal cortex expression levels of VEGFR2 among the groups of mice studied (Pod/+ VEH, 1.5 ± 0.3; Pod/+ DOX, 1.7 ± 0.3; Pod/sFlt-1 VEH, 1.7 ± 0.3; Pod/sFlt-1 DOX, 1.5 ± 0.2 VEGF-R2/β-actin arbitrary units, n = 5–6/group). VEGFR2 activation, determined by phosphorylation of VEGFR2 on Tyr (951) (20,21) was reduced by ∼50%, but not abolished, in Pod/sFlt-1 mice treated with DOX (Fig. 2D).


Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice.

Ku CH, White KE, Dei Cas A, Hayward A, Webster Z, Bilous R, Marshall S, Viberti G, Gnudi L - Diabetes (2008)

Podocyte-inducible overexpression of LacZ/sFlt-1. A: Nuclear LacZ staining of glomerular podocytes was detected in DOX-treated but not in VEH-administered double transgenic mice (10 weeks of treatment). No staining was observed in Pod/+ administered VEH or DOX (not shown). B: Densitometry quantitative analysis of sFlt-1 expression (as a ratio with β-actin) in total kidney cortex lysate after 10 weeks of treatment with VEH or DOX. Data are expressed as mean ± SEM (*P < 0.01 for Pod/sFlt-1 DOX vs. all other groups, P = NS for Pod/+ VEH vs. Pod/+ DOX; n = 4–8/group). (Please see http://dx.doi.org/10.2337/db08-0647 for a high-quality digital representation of this figure.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551695&req=5

f1: Podocyte-inducible overexpression of LacZ/sFlt-1. A: Nuclear LacZ staining of glomerular podocytes was detected in DOX-treated but not in VEH-administered double transgenic mice (10 weeks of treatment). No staining was observed in Pod/+ administered VEH or DOX (not shown). B: Densitometry quantitative analysis of sFlt-1 expression (as a ratio with β-actin) in total kidney cortex lysate after 10 weeks of treatment with VEH or DOX. Data are expressed as mean ± SEM (*P < 0.01 for Pod/sFlt-1 DOX vs. all other groups, P = NS for Pod/+ VEH vs. Pod/+ DOX; n = 4–8/group). (Please see http://dx.doi.org/10.2337/db08-0647 for a high-quality digital representation of this figure.)
Mentions: Eight-week-old adult double transgenic mice (Pod/sFlt-1) or single transgenic mice (Pod/+) were treated with DOX or administered VEH for up to 10 weeks (both sexes showed a similar phenotype and were analyzed together). X-gal staining revealed no signal in Pod/+ DOX and VEH kidneys, confirming that the staining protocol did not detect endogenous galactosidase activity (not shown). Furthermore, Pod/sFlt-1 mice administered with VEH also failed to give a positive signal (Fig. 1A), excluding “leakage” of LacZ expression in noninduced mice. In contrast, kidneys from adult Pod/sFlt-1 mice exposed to DOX showed positive X-gal staining (Fig. 1A), consistent with podocyte expression of the transgene (13,14). X-gal staining was analyzed at different time points after DOX administration (10 days, 5–10 weeks), and the results were superimposable (not shown). After 10 weeks of treatment with DOX, Pod/sFlt-1 animals showed a significant inducible sFlt-1 overexpression (100% increase) when assessed in kidney cortex lysate (Fig. 1B). The upregulation of sFlt-1 was associated with an increase in 24-h urine sFlt-1 levels, which was four- to fivefold higher in DOX-treated Pod/sFlt-1 compared with Pod/sFlt-1 mice given VEH or with Pod/+ control mice (P < 0.001) (Fig. 2A). This was paralleled by a reduction in urine free VEGF-A in Pod/sFlt-1 DOX-treated mice, suggesting an sFlt-1–mediated “sequestration/binding” of VEGF-A (P < 0.04) (Fig. 2B). Renal cortex VEGF-A was upregulated in Pod/sFlt-1 DOX mice in comparison with the other groups (P < 0.03) (Fig. 2C). No difference was observed in the renal cortex expression levels of VEGFR2 among the groups of mice studied (Pod/+ VEH, 1.5 ± 0.3; Pod/+ DOX, 1.7 ± 0.3; Pod/sFlt-1 VEH, 1.7 ± 0.3; Pod/sFlt-1 DOX, 1.5 ± 0.2 VEGF-R2/β-actin arbitrary units, n = 5–6/group). VEGFR2 activation, determined by phosphorylation of VEGFR2 on Tyr (951) (20,21) was reduced by ∼50%, but not abolished, in Pod/sFlt-1 mice treated with DOX (Fig. 2D).

Bottom Line: Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04).Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05).Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, King's College London School of Medicine, Guy's Hospital, King's College London, London, UK.

ABSTRACT

Objective: Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.

Research design and methods: We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.

Results: Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an approximately 70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69-199) vs. 43 (26.8-69) mug/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.

Conclusions: Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

Show MeSH
Related in: MedlinePlus