Knock-down Of Cullin-5 Expression On Alteration Of Morphology And Proliferation Ability In Glomerular Podocytes

BackgroundGlomerular proteinuria is due to the causes of damage to the glomerular filtration barrier, and glomerular epithelial cells play a key role in maintaining the integrity of the glomerular filtration barrier. Many studies published in past decades revealed that, the mutation of NPHS1, NPHS2, ACTN and Synaptopodin cause congenital renal diseases. The animal models of these genes'knock-out have also presented with proteinuria and glomerular epithelial cell foot process fusion. It is clear that the expression of these molecules of glomerular epithelial cells in the foot process (such as Nephrin, Podocin, CD2AP, TRPC6) and cytoskeleton-associated protein (such as Synaptopodin,α-Actinin-4, Podocalyxin) play an important role in the maintenance of glomerular filtration membrane integrity and prevent the leakage of plasma proteins. In recent years, our research team also found that, the changes of the molecular expression and regulation of Nephrin, Podocin, and CD2AP have closely related with etiology of primary nephrotic syndrome in China. At the same time, we also confirmed, Myole (a myosin type I molecules) is cytoskeleton associated protein in glomerular epithelial cells in human and mouse. Its knock-down in zebrafish will cause proteinuria and the changes of structure in glomerular epithelial cells.Furthermore, all cells require protein degradation and metabolism mechanism to refresh its protein functions. There are mainly two ways on regulation of protein degradation in eukaryotic cells. It is Lysosomal pathway and Ubiquitin Proteasome System (UPS). UPS is composed of ubiquitin,26S proteasome, Ub-activating enzyme (Els), Ub-conjugating proteins (E2s), and Ub-protein ligase (E3s). It is an efficient and specific protein degradation system on control of the specific proteins in regular processes and elimination of misfolded proteins. The main role of Ub-protein ligase E3 is to identify the specific target protein for degradation and to transfer ubiquitin to the target protein. At last, the proteins are degradated by proteasome.Cullin-5 (CUL5) is a backbone of E3s-CRL, which takes part in the degradation of target protein (such as p53). Its encoding gene sequences (located at 11q22-23) are homologous with arginine vasopressin receptor activation of calcium transport-1(VACM-1), so these may be the same protein. Our previous study demonstrated that CUL5 in highly expressed in glomerular podocytes in human and mice, and, its knock-down in zebrafish resulted in proteinuria and change of glomerular structures.PurposeTo explore the effects of knock-down of Cullin-5 on the structure and function variation of podocytes, and to clarify the role of Cullin-5 for maintaining foot process and the glomerular filtration barrier integrity.Methods1. Culture mouse podocyte cells (MPC5). 2. Detecting the distribution of Cullin-5 and F-actin in different cell by immunofluorescence technique.3. CUL5 knockdown by Invitrogen Corporation.4. Transient transfection of Cullin-5 antisense miRNA containing plasmid to podocytes by Invitrogen's transfection reagents.5. Application of RT-PCR to test the interference effect in mRNA level. GAPDH as reference gene.6. Application of Western-blot to test the interference effect in protein level.β-actin as reference gene, semi-quantitative analysis of protein expression differences.7. Observe morphological change of podocytes after the CUL5 knock-down by immunofluorescence microscope.8. Observe structure change of F-actin after the CUL5 interference by immunofluorescence microscope.9. To test the capacity of cell proliferation by MTT. Set 4 experimental points, as 24 hours,48 hours,72 hours, and 96 hours. To test the OD value of the cells.Resultsa) Under the permissive condition the podocytes are "cobblestone" pattern in state of proliferation. Nuclei are round, located in the central cell body. Short little foot process; Under the non-permissive condition, about 14 days, podocytes are mature and extends to the surrounding cytoplasm. A large number of long thin foot processes are formed. The cells look like the bough of trees. We identify the podocytes by Nephrin expression.b) Under the permissive condition, CUL5 distribution is mainly concentrated around nuclear membrane. Under the non-permissive condition, CUL5 distributed in around the cytoplasm and the foot process.c) Under the permissive conditions, F-actin is mainly distributed at the major process; Under the non-permissive condition, F-actin radiated to the new foot processes.d) RT-PCR was used to compare the effects of CUL5 Knock down at mRNA levels among different groups. Interference group one (P=0.003), Interference group two (P=0.042), Interference group three (P=0.000), Interference group four (P=0.000). No significant difference of CUL5 expression was found between negative control group and control group (P=0.091,P>0.05).e) Westem-blot was used to compare the expression difference of CUL5 at protein-level among different groups. All interference groups are have the significant difference with the control group(P<0.05); No significant difference of CUL5 expression was found between negative control group and control group (P=0.051, P>0.05).f) Immunofluorescence assay was used to compare the difference in fluorescence intensity among different groups. Weaker signal was found in any interference group than the control group, and no significant difference of CUL5 expression was found between negative control group and control group.g) The morphological changes in podocytes: cell body became narrow, irregular shape, irregular edge and the fusion of foot processes.h) The changes in proliferation ability of podocytes:the proliferation of cell in each interference group differed with the control group (P< 0.01), and no significant difference of proliferation ability in podocytes was found between negative control group and control group (P>0.05).Conclusions1. The distribution of Cullin-5 in podocytes is different between proliferation and differentiation period.2. Cullin-5 plays an important role in maintaining normal morphology of podocytes.3. When Cullin-5 expression was knocked down, podocyte proliferation ability decreased significantly.