Down Expression Of Myo1e On Morphology And Functional Incoordination Of Mouse Glomerular Podocytes In Vitro

BackgroundRecent research shows that, the foot processes (FP) of renal epithelial cell and the Slit diaphragm between adjacent Foot processes are the most important physiological structure to keep glomerular filtration membrane barrier function. Maintaining the special structure of the epithelial cell mainly depends on the actin cytoskeleton system and the specific protein molecules of foot processes. Since 1998. people have found many proteins expressed in the foot processes and slit diaphragm play a very important roles in the development, maintenance, glomerular filtration membrane integrity, podocyte signal transmission, preventing proteinuria. Podocyte changes the gap between the cells by adjusting self shape to adaptive the volume change of filter glomerular filtration. This function complete dependent on podocyte powerful cytoskeleton system, and the function of myosin I closely related with the actin cytoskeleton. Research suggested that after down-regulated of Myosin I e. the zebrafish appear edema (pericardial edema), pronephric cyst, decreasing the glomerular local cells, and endosome in epithelial cell. Someone generated Myole-knockout mice and found that they exhibit proteinuria. signs of chronic renal injury, and kidney inflammation. At the ultrastructural level, renal tissue from Myole-null mice demonstrates changes characteristic of glomerular disease, including a thickened and disorganized glomerular basement membrane and flattened podocyte foot processes. These observations suggest that Myole plays an important role in podocyte function and normal glomerular filtration. But now there have no research in the cell and molecular level to clarify the effect of knock down the myosin le gene on podocyte function and relative molecular mechanism.PURPOSETo explore the effects of knockdown the myosin 1e gene on podocyte structure and function, to clarify the role of myosin 1e for maintaining foot process structure and glomerular basement membrane integrity. And to explain the mechanism of interaction between the myosin 1 e and F-actin cytoskeleton.METHODS1. Cell culture:Cultured mouse podocyte cells (MPC5) were used in present study. In 33 degrees Celsius. cells were in the period of proliferation, while in 37 degrees Celsius, cells were differentiation to mature. Through the method of immunofluorescence. to identify the podocyte-associated proteins Nephrin, CD2AP.2. To detect the distribution of myosin le and F-actin cytoskeleton in different cell state by using method of immunofluorescence.3. According to the reagent and experiment method which provided from Santa Cruz Company to knock down the gene myole of podocyte. And the experiment established control group and Scrambled negative control group.4. Use real-time quantitative fluorescence method to detect the interference effect in mRNA level. 5. Use the Western-Blot method to detect the interference effect in protein level.6. Observed morphological change of podocyte after the Myole interference by using method of immunofluorescence.7. Observed structure change of F-actin after the Myole interference by using method of immunofluorescence.8. Test cell proliferation capacity by MTT method. There were altogether six time points (24hr.48hr.96hr,72hr,120hr.144hr) to detect the OD value of MTT. respectively.9. The cells quiesced for 72 h were suspended in RPMI 1640 and added to the upper chamber of Trans-well system. These were used to determine the migration of the cells.10. Cells were cultivated for 48 hours after the treatment of PAN. then counted the adherent cells.11. Different treatment group of cells after digestion in the same density to vaccination in six orifice plate. Cultivate cells for 1 hour then discarding the medium to count the adherent cells. These were used to determine the cell adhesion ability.12. Cells to cultivate for 5 hours with culture medium which included FITC-Transferrin. Then calculated the positive rate of cells by fluorescence microscope. These were used to determine the endocytosis of the cells.Results1. MPC5 have a fusiform, polygonal shape when they were in the proliferation condition. And the cells have a shorter, thicker, less foot processes. While they have a longer, thinner, more cell's foot processes when there were in differentiation period. Nephrin and CD2AP expressed on the podocyte in specificity.2. During the undifferentiated period. Myosinle mainly distributes in nuclear and the near the nuclear place of the primary process. F-actin distributes along the axis of the primary process. After 2 weeks culturing. cells differentiate to maturation, the myole was to gather around the cell nucleus, and F-actin stretched to the newly formed foot processes of around the cell.3. Use real-time quantitative fluorescence method to detect the interference effect in mRNA level. Compared with control group the expression level of mRNA of myosin 1e was 0.857±0.154 times (P>0.05),0.250±0.0892 times (P<0.01) at the Scrambled negative control group and interference group. Use Western blot-a method to detect the differences of protein expression. Compared with control (1.638±0.061). the number of myo 1 e/β-actin reduced to 1.547±0.050 (P> 0.05). 0.851±0.0421 (P<0.01) at the Scrambled negative control group and interference group. These results indicate that interfere with success.4. Cells appeared an obvious morphological variation after successfully interference. There were fusions of foot processes of podocytes and cells were significantly smaller and irregular in size.5. After successfully interference, actin filaments in podocytes were partially disrupted, with large stress fibers spread more diffusely than in untreated cells: strongly staining, punctate concentrations of polymerized actin appeared in cells. In contrast, untreated cells and the Scrambled negative cells, parallel arrays of comparatively thin actin stress fibers that extended across the entire cell body.6. Some experiments to test cell function changes.①. The cell proliferation capacity by MTT method. Compared with control cells, the cell proliferation capacity was significantly reduced (P<0.01) at the Scrambled negative control group and interference group. But after calculating cells inhibition rate, we found that the inhibition rate of interference groups was significantly higher than scrambled negative control group (P< 0.01).②. The migration of the cultured podocytes was detected by Transwell system. Cells that had migrated below the membrane were counted in 20 high-power microscope fields. Compared with control (84.67±10.94), the number of migrated cells was reduced to 82.33±9.07 (P>0.05),26.14±4.30 (P< 0.01) at the Scrambled negative control group and interference group, per filed. Interference group podocyte migrating ability obviously constrained.③. The determination of podocyte adhesion ability showed that compared with control (132.17±19.64)the adhesion ability of interference group cells had a significantly reduced to 85.17±7.73(P< 0.01). but the Scrambled negative control group (123.17±12.98)had not a Obvious differences (P>0.05).④. Cells to cultivate for 48 hours after the treatment of PAN. then counted the adherent cells. Compared with control (104.50±6.02), the number of adherent cells was reduced to 99.00±6.13 (P >0.05),71.00±18.45 (P< 0.01) at the Scrambled negative control group and interference group, per filed.⑤.We examined uptake of fluorescent transferrin in podocyte cells. While almost all control cells and Scrambled negative control cells exhibited punctate transferring labeling, only 23.81±3.564%of cells did take up transferrin (P< 0.01).Conclusions1. The distribution of myosinl e in different periods of cells was variable, so it may be involved in the formation of foot processes.2. Myosin 1e plays an important role in maintaining cellular form and structure.3. The effects of Myole KD on podocyte functions may be mediated by the changes of actin organization in podocytes.4. Myosin 1E may contribute to receptor-mediated endocytosis. podocyte migration, cell adhesion, podocyte proliferation.