| Summary: | 博士 === 高雄醫學大學 === 醫學研究所博士班 === 95 === Progressive glomerulosclerosis is characterized by abnormal expression of glomerular basement membrane (GBM) and a decrease in glomerular cell numbers. Podocyte lesions were found to correlate with glomerulosclerosis. The integrinα3β1 plays an important role in podocytes-GBM interactions. Integrins play an essential regulatory role in cell proliferation, survival, maturation and transdifferentiation, or transformation. Podocytes are found to detachment into urine in focal segmental glomerulosclerosis (FSGS). Podocyte apoptosis was found in chronic PAN (puromycin aminonucleoside)-treated rats. TGF-β1 (transforming growth factor-β1) can induce epithelial-mesenchymal transformation. The experiments described in this article were performed to test the hypothesis: (1) Detachment and loss of podocytes are associated with decrease or dysfunction of integrinα3β1; (2) Can TGF-β1 induce transdifferentiation of podocytes? Can integrins regulate this transdifferentiation? So, we performed the following studies: (1) We examined the podocyte number in patients with primary FSGS and normal controls, and the a3- and b1-integrin subunits expression of podocytes in patients with primary FSGS and chronic PAN-treated rats. We also measured their expression serially in rats received repeated PAN injection. (2) We investigated the apoptosis induced by reactive oxygen species (ROS)-inducing injury or dysfunction of integrin caused by disruption of integrin-ECM interaction cause podocyte apoptosis. Could apoptosis be caused by disruption of the actin assembly? Could activators or inhibitors of integrin-mediating downstream signalings (PKCand FAK) regulate apoptosis? (3) We investigated the apoptosis pathways (cytochrome c, Fas, Fas ligand, Bax, Bcl-2) and its related signal pathways to integrins (ERK activation) after dysfunction of integrin caused by disruption of integrin-ECM interaction. (4) The present study was designed to identify whether podocytes can transdifferentiate, with the expression of α-smooth muscle actin (α-SMA), under the effects of TGF-β1, integrins and downstream sigalings of integrins.
From the above studies, we had the following results: (1) Podocyte expression of a3- and b1-integrin subunits is significantly reduced in human with primary FSGS and chronic PAN-treated rats, before the morphological changes of FSGS are observed. The decreased podocyte expression of a3b1 integrins is closely related with podocyte depletion, glomerular sclerosis and daily protein loss in patients with primary FSGS. (2) After ROS-injury, rat podocytes exhibited increased apoptosis. Blocking integrin function led to apoptosis of potocytes. ROS-injury plus blocking integrin function increased more apoptosis. The disruption of actin cytoskeleton increased cell apoptosis. Increased PKC activity attenuated injury-induced apoptosis, whereas the inhibition of PKC activity enhanced apoptosis. The inhibition of FAK activity increased apoptosis. (3) Blocking integrin function increased the percentage of cells with apoptosis and caused cytochrome c released into cytoplasm. After blocking integrin function, the expression of Bax and Fas ligand was upregulated, while Bcl-2 expression was not changed. The expression of Fas was not found in podocytes. ERK activation was increased after blocking integrin function by disruption of integrin-ECM interaction. (4) TGF-β1 dose-dependently increased α-SMA protein and mRNA expression at 4 days. TGF-β1 also dose-dependently increased the α-SMA staining of podocytes. Blocking integrin function and inhibitors of PKC or FAK inhibited the percentage of TGF-β1-stimulated α-SMA (+) podocytes.
From the above results, we had the conclusions: Podocyte expression of a3- and b1-integrin subunits is significantly reduced in human with primary FSGS and chronic PAN-treated rats. ROS can induce apoptosis of podocytes. Blockinh integrin function can cause apoptosis of podocytes and more severe apoptosis-induced by ROS. Blocking integrin downstream-signaling pathways also cause apoptosis. The apoptosis induced by blocking integrin function is from the cytochrome c pathway, not Fas-Fas ligand pathway. TGF-β1 can induce podocytes undergoing transdifferentiation to myofibroblast-like cells. The integrin-ECM interaction can facilitate the transdifferentiation. So, decreased expression or function of integrinα3β1 after podocyte injury can facilitate podocyte loss, but lessen transformation of podocytes.
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