BIM deficiency differentially impacts the function of kidney endothelial and epithelial cells through modulation of their local microenvironment
ABSTRACT The extracellular matrix (ECM) acts as a scaffold for kidney cellular organization. Local secretion of the ECM allows kidney cells to readily adapt to changes occurring within the kidney. In addition to providing structural support for cells, the ECM also modulates cell survival, migration, proliferation, and differentiation. Although aberrant regulation of ECM proteins can play a causative role in many diseases, it is not known whether ECM production, cell adhesion, and migration are regulated in a similar manner in kidney epithelial and endothelial cells. Here, we demonstrate that lack of BIM expression differentially impacts kidney endothelial and epithelial cell ECM production, migration, and adhesion, further emphasizing the specialized role of these cell types in kidney function. Bim -/- kidney epithelial cells demonstrated decreased migration, increased adhesion, and sustained expression of osteopontin and thrombospondin-1 (TSP1). In contrast, bim -/- kidney endothelial cells demonstrated increased cell migration, and decreased expression of osteopontin and TSP1. We also observed a fivefold increase in VEGF expression in bim -/- kidney endothelial cells consistent with their increased migration and capillary morphogenesis. These cells also had decreased endothelial nitric oxide synthase activity and nitric oxide bioavailability. Thus kidney endothelial and epithelial cells make unique contributions to the regulation of their ECM composition, with specific impact on adhesive and migratory properties that are essential for their proper function.
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ABSTRACT: Diabetic nephropathy is the most common cause of end stage renal disease and is a major risk factor for cardiovascular disease. In the United States, microvascular complications during diabetic nephropathy contribute to high morbidity and mortality rates. However, the cell autonomous impact of diabetes on kidney endothelial cell function requires further investigation. Male Akita/+ (autosomal dominant mutation in the insulin II gene (Ins2)) mice reproducibly develop diabetes by 4 weeks of age. Here we examined the impact short duration of diabetes had on kidney endothelial cell function. Kidney endothelial cells were prepared from non-diabetic and diabetic mice (4 weeks of diabetes) to delineate the early changes in endothelial cell function. Kidney endothelial cells from Akita/+ mice following 4 weeks of diabetes demonstrated aberrant expression of extracellular matrix proteins including decreased osteopontin and increased fibronectin expression which correlated with increased α5 integrin expression. These changes were associated with the attenuation of migration and capillary morphogenesis. Kidney endothelial cells from Akita/+ mice had decreased VEGF levels but increased levels of eNOS and NO, suggesting uncoupling of VEGF mediated NO production. Knocking down eNOS expression in Akita/+ kidney endothelial cells increased VEGF expression, endothelial cell migration and capillary morphogenesis. Furthermore, attenuation of sprouting angiogenesis of aortas from Akita/+ mice with 8 weeks of diabetes, was restored in the presence of the anti-oxidant N-acetylcysteine. These studies demonstrate that aberrant endothelial cell function with short duration of diabetes may set the stage for vascular dysfunction and rarefaction at later stages of diabetes.AJP Renal Physiology 10/2012; 304(1). DOI:10.1152/ajprenal.00036.2012 · 4.42 Impact Factor
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ABSTRACT: Aim: To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway. Methods: Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H2O2 (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay. Results: Treatment of the cells with HG, H2O2, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H2O2 significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H2O2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or LY294002. Moreover, the treatment with HG or H2O2 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002. Conclusion: The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.Acta Pharmacologica Sinica 03/2013; 34(4). DOI:10.1038/aps.2012.207 · 2.50 Impact Factor
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ABSTRACT: Mutation in CYP1B1 is reported in patients with congenital glaucoma. However, the underlying mechanisms remain unknown. Here we show increased diurnal intraocular pressure (IOP) in Cyp1b1-deficient (Cyp1b1(-/-)) mice. Cyp1b1(-/-) mice presented ultrastructural irregular collagen distribution in their trabecular meshwork (TM) tissue, along with increased oxidative stress and decreased levels of Periostin (Postn). Increased levels of oxidative stress and decreased levels of Postn were also detected in human glaucomatous TM tissues. Furthermore, Postn-deficient mice exhibited similar TM tissue ultrastructural abnormalities as Cyp1b1(-/-) mice. Administration of antioxidant N-acetylcysteine (NAC) restored structural abnormality of TM tissue in Cyp1b1(-/-) mice. In addition, TM cells prepared from Cyp1b1(-/-) mice exhibited increased oxidative stress, altered adhesion and decreased levels of Postn. These aberrant cellular responses were reversed in the presence of NAC or by restoration of Cyp1b1 expression. Cyp1b1 knockdown or inhibition of CYP1B1 activity in Cyp1b1(+/+) TM cells resulted in a Cyp1b1(-/-) phenotype. Thus, metabolic activity of CYP1B1 contributes to oxidative homeostasis and ultrastructural organization and function of TM tissue through modulation of Postn expression.Molecular and Cellular Biology 08/2013; DOI:10.1128/MCB.00856-13 · 5.04 Impact Factor