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

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

SUBMITTER: Ku CH

PROVIDER: S-EPMC2551695 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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

Ku Ching-Hsin CH White Kathryn E KE Dei Cas Alessandra A Hayward Anthea A Webster Zoe Z Bilous Rudy R Marshall Sally S Viberti Giancarlo G Gnudi Luigi L

Diabetes 20080722 10

<h4>Objective</h4>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.<h4>Research design and methods</h4>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 ...[more]

PMID: 18647955

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Project description:BackgroundGlycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified.MethodsmRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney.ResultsmRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice.ConclusionsThese findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.

Project description:The dysfunction of mesangial cells (MCs) in high-glucose (HG) conditions plays pivotal role in inducing glomerular sclerosis by causing the imbalance between generation and degradation of extracellular matrix (ECM) proteins, which ultimately leads to diabetic nephropathy. This study was designed to determine the function of forkhead box protein O1 (FoxO1), an important transcription factors in regulating cell metabolism and oxidative stress, in MCs in HG conditions. Up-regulation of fibronectin, collagen type IV, and plasminogen activator inhibitor (PAI-1) was observed under HG conditions in vivo and in vitro, accompanied with elevation of protein kinase B (Akt) phosphorylation and reduction of FoxO1 bioactivity. After overexpression of constitutively active (CA) FoxO1 in vivo and in vitro by using lentivirus vector, in vivo and in vitro, FoxO1 expression and activity was increased, in accordance with up-regulation of antioxidative genes (catalase and superoxide dismutase, leading to alleviated oxidative stress as well as attenuated Akt activity, whereas overexpression of wild type-FoxO1 only expressed partial effect. Moreover, CA-FoxO1 decreased the expression of fibronectin, collagen type IV, and PAI-1, causing amelioration of renal pathological changes and decrease of ECM protein deposition in glomerulus. Overexpression of CA-FoxO1 in renal cortex also decreased activin type-I receptor-like kinase-5 levels and increased signaling mothers against decapentaplegic (Smad) 7 levels, and simultaneously inhibited Smad3 phosphorylation. Results from in vitro study indicated that increased combination of FoxO1 and Smad3 may interfere with the function of Smad3, including Smad3 phosphorylation and translocation, interaction with cAMP response element binding protein (CREB)-binding protein, and binding with PAI-1 promoter. Together, our findings shed light on the novel function of FoxO1 in inhibiting ECM deposition, which is beneficial to ameliorate MC dysfunction.

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

Publications

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

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