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Overexpression of VEGF165b in podocytes reduces glomerular permeability.

ABSTRACT: The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGF(xxx)b family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and homozygous transgenic mice that overexpress VEGF(165)b specifically in podocytes. We confirmed excess production of glomerular VEGF(165)b by reverse transcriptase-PCR, immunohistochemistry, and ELISA in both heterozygous and homozygous animals. Macroscopically, the mice seemed normal up to 18 months of age, unlike the phenotype of transgenic podocyte-specific VEGF(164)-overexpressing mice. Animals overexpressing VEGF(165)b, however, had a significantly reduced normalized glomerular ultrafiltration fraction with accompanying changes in ultrastructure of the glomerular filtration barrier on the vascular side of the glomerular basement membrane. These data highlight the contrasting properties of VEGF splice variants and their impact on glomerular function and phenotype.

SUBMITTER: Qiu Y

PROVIDER: S-EPMC3013528 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Overexpression of VEGF165b in podocytes reduces glomerular permeability.

Qiu Yan Y Ferguson Joanne J Oltean Sebastian S Neal Chris R CR Kaura Amit A Kaura Amit A Bevan Heather H Wood Emma E Sage Leslie M LM Lanati Silvia S Nowak Dawid G DG Salmon Andy H J AH Bates David D Harper Steve J SJ

Journal of the American Society of Nephrology : JASN 20100805 9

The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGF(xxx)b family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and h ...[more]

PMID: 20688932

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Project description:BackgroundAs a fundamental process internalizing molecules from the plasma membrane, endocytosis plays a crucial role in podocyte biology. Our previous study has identified that overexpression of Myole may enhance podocyte endocytosis. However, its potential mechanism has been not well understand. Thus, we aimed to analyze whether albumin endocytosis by mouse glomerular podocytes is dependent on Myo1e expression. Also, we aimed to elucidate whether the underlying mechanism is mediated by Dynamin.MethodsFirstly, mouse podocyte cells (MPC5) were treated with different concentrations of FITC-bovine serum albumin (BSA). The fluorescence intensity and cell viability were detected by flow cytometry and MTT assays, respectively. Afterwards, the optimal concentration of FITC-BSA was determined. Secondly, MPC5 cells were treated with Myole overexpression or knockdown. Cell morphology was observed under microscope. Immunofluorescence assay was used to determine the expression of F-actin. The protein expression of nephrin and podocin was detected by western blot. Flow cytometry was used to detect MPC5 cell apoptosis with annexin V. Finally, MPC5 cells were treated with Myole overexpression and/or Dynasore (a GTPase inhibitor of Dynamin). The fluorescence intensity was detected using flow cytometry assay.ResultsMPC5 endocytosis BSA was elevated with a concentration-dependent manner. MTT results showed that MPC5 cell viability was inhibited with a concentration-dependent manner. Myo1e overexpression promoted podocyte endocytic FITC-BSA, which was contrary to its knockdown. Under microscope, after inhibition of Myo1e, podocyte foot process fusion was observed. Myo1e overexpression promoted the expression of cytoskeleton F-actin and podocyte-specific molecules (nephrin and podocin) in podocyte endocytic FITC-BSA. Furthermore, we found that Myo1e promoted the apoptosis of podocytes. Dynasore attenuated the increase in endocytosis of FITC-BSA induced by Myo1e overexpression, suggesting that podocytes might mediate albumin endocytosis via Myo1e-Dynamin-Albumin.ConclusionOur findings revealed that overexpression of Myo1e promotes albumin endocytosis in mouse glomerular podocyte endocytic albumin mediated by Dynamin.

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Overexpression of VEGF165b in podocytes reduces glomerular permeability.

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Overexpression of VEGF165b in podocytes reduces glomerular permeability.

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