Targeted Disruption Of CPLA₂α Retards Cell Cycle Progression And Attenuates Renal Fibrosis In UUO Model

BackgroundRenal fibrosis is the common determinant of progression to end stage renal disease. Renal fibrosis is characterized by tubulointerstitial fibrosis, and the pathologic feature is tubulointerstitial inflammation, proliferation of fibroblast cell, excessive deposition of extracellular matrix, and tubular epithelia cells apoptosis and necrosis. The accepted mechanisms of tubulointerstitial fibrosis include chronic inflammatory cell infiltration, Epithelial or endothelia to mesenchymal transition, activation of fibroblast, but the exact cellular mechanisms of fibrosis still poorly defined.cPLA2a is a one of the phospholipase, and the activation is dependent on the intercellular calcium concentration. The active cPLA2atranslocate from cytosol to the perinuclear region including nuclear envelope and endoplasmic reticulum, and it’s will cleave the arachidonic acid from sn-2position of phospholipase. The products of cPLA2a including Arachidonic acid (AA), Lysophosphatidic acid(LPA), Thromboane A2(TxA2) and Prostaglandin E2(PGE2), and all of those have effect on the cell proliferation and DNA synthesis, but for different cell type and different conditions, they may have positive or negative effect. cPLA2a can increase some tumor cell cell cycle progression, and in vessel endothelia cell, cPLA2acould increase the expression of Cyclin A, improve S phase progression and cell cycle re-entry, that can induce endothelial cell proliferation. All of that means cPLA2a can regulate cell cycle progression. But the effects of cPLA2α on the proliferation of renal tubular epithelial cell still unknow.The potent capacity of tubular epithelial cells to proliferation and replace lost cells is crucial for repair and recover from injury. The cell proliferation is majorly controlled by cell cycle. The orderly progression through the cell cycle is regulated by the sequential synthesis, activation, compartmentalization, and degradation of proteins positive or negative controlling the cell cycle progression. Enhanced expression of cell cycle regulatory protein such as p53, p21and p16in renal proximal tubular cells has been implicated in repair in sever acute or chronic kidney injury animal model. In the condition of cell DNA damage, it might cause abnormal cell repair, that induce G2/M arrest. In our previous study, we found that epithelial cell cycle arrest in G2/M can cause kidney fibrosis after injury.There is a close connection of between fibrosis with chronic inflammation. We already know that the peritoneal macrophages from cPLA2α-/-released much less arachidonate, prostaglandin E2leukotrienes B4(LTB4) and C4(LTC4) in response to the PMA and activator or response to inflammatory stimuli than the peritoneal macrophages from cPLA2α+/+. There is a obsrvation showed that cPLA2α-/-mice can’t release AA and PAF, that reduced the inflammatory response in the pulmonary, and reduced the fibrosis in the pulmonary.Therefore, cPLA2α might promote epithelial cell proliferation, that induce more cells arrest in G2/M, and cause kidney fibrosis. Chapter I The effect of cPLA2a on the kidney epithelial cell proliferationObjective To detect the expression and activation of cPLA2a in renal epithelial cells and investigate the effect of cPLA2α on the kidney epithelial cell proliferationMethods Primary proximal epithelial cells(PTECs)were cultured in vitro, the expression of cPLA2a was detected by WB, and the activation of cPLA2α was measured by [3H]AA release assay. LLCPK cells were transfected with the plasmids of wild-type cPLA2a and mutant cPLA2αS228A.Then the expression and activation of cPLA2a were examined. The cell proliferation were test by cell counting; FACS measure the cell cycle distribution of PETCs and LLC-pCDNA LLC-cPLA2a, LLC-cPLA2αS228A;Immunofluorescence staining of Ki67on PETCs and the positive cell numbers were analyzed. The expression of cell cycle regulatory protein cyclin A, cyclin B and p27were tested after starvation synchronization. LLC-cPLA2a and LLC-cPLA2αS228A were exposed in different concentration H2O2for one hour after double thymindine synchronization for48hours, cell cycle distribution was measured by FACS, and TGF-β mRNA expression was tested through Q-PCR.Results Western blots showed that PETCs expressed cPLA2a,and there were overexpression of cPLA2a in both LLC-cPLA2a and LLC- cPLA2αS228A.AA release assay showed that the release of AA from PETCs and LLC-cPLA2α can be inhibited by the inhibitor, but the mutant cPLA2a didn’t have AA released. Cell counting assay showed that LLC-cPLA2α proliferated much faster than LLC-pCDNA and LLC-cPLA2αS228A.FACS results also showed that overexpress functional cPLA2α increased cell cycle progression and cell cycle re-entry, inhibit cPLA2αon PETCs, could reduce the cell cycle progression. Immunofluorescence staining showed that the Ki67positive cells were reduced in inhibitor groups. Overexpress cPLA2α can increased cyclin A and cyclin B expression, but not p27. LLC-cPLA2aand LLC-cPLA2αS228A were exposed in different concentration of H2O2could cause G2/M arrest, and overexpress cPLA2α can increase arrested cell population. Q-PCR showed that exposed in H2O2result in the increasing expression of TGF-β.Conclusion Mouse primary kidney epithelial cell can express cPLA2α. Overexpression of cPLA2α improves cell cycle progression, enhance cell proliferation, and increase G2/M arrest and TGF-β expression after DNA damage. Chapter Ⅱ The effect of cpla2a in kidney fibrosis in Unilateral ureteral obstruction modelObjective To observe the effect of cPLA2a in kidney fibrosis in Unilateral ureteral obstruction(UUO) model and investigate the mechanism.Methods cPLA2α-/-and cPLA2α+/+were used to build up the UUO model, and scarified in10days and14days. Renal pathological changes were observed by Masson and PAS staining. The expression of a-SMA was measured by western blot and immunofluroscence staining. The expression of Fibronectin and Collagen I were measured by Real-time PCR and immunofluroscence staining. TGF-β,CTGF and TNF-a mRNA expression were tested by Real-time PCR. The expression of F4/80(macrophage) was tested by immunofluroscence. Ki67, BrdU,p-H3positive cells were tested by immunofluroscence and the cell cycle distribution were analyzed. The expression of cell cycle regulatory protein cyclin A,cyclin B,cyclin D,cyclin E were detected by western blot.Results Masson and PAS staining showed that there were many lesions in the kidneys both cPLA2α-/-and cPLA2α+/+mice at10days UUO kidney and14days UUO kidney, such as lose of brush border, necrosis of epithelial cells and exposure of basement membrane, tubular atrophy and compensatory expansion, interstitial inflammatory cells infiltration, and interstitial fibrosis. But the pathologic changes of cPLA2α-/-UUO kidney were much less severe than cPLA2α+/+UUO kidney.Western blot, Real-time PCR and immunofluroscence staining showed that the expression of a-SMA was significant less in cPLA2α-/-UUO kidney in both10days and14days UUO mice. Real-time PCR and immunofluroscence staining showed that the expression of FN and Col I were less in cPLA2α-/-UUO kidney in10days UUO kidney, but without significant difference. But in14days UUO kidney, the expression of FN and Col I were significant less in cPLA2α-/-mice. The expression of TGF-(3and CTGF were significant less in both10days and14days cPLA2α-/-UUO kidney.There was no significant difference of F4/80positive cells measured by immunofluroscence staining quantification in both10days and14days UUO kidney. Real-time PCR showed that the expression of TNF-a were significant less in cPLA2α-/-UUO kidney in10days UUO kidney, but in14days UUO kidney, there was no significant difference in cPLA2α-/-and cPLA2α+/+mice.Immunofluroscence staining quantification of Ki67, BrdU, p-H3positive cells showed that, there were significant less proliferative cell in cPLA2α-/-UUO kidney in both10days and14days mice, but the cell cycle distribution were similar. In14days UUO kidney, there were lots of cells arrest in G2/M phase, but the ratio of G2/M arrest cells was similar. The expression of cyclin A, cyclin B, cyclin D, cyclin E were much less in cPLA2α-/-UUO kidney in14days mice.Conclusion Targeted disruption of cPLA2a inhibit kidney epithelial cell proliferation and G2/M arrest, that attenuate fibrosis induced by UUO. Chapter III The effect of cPLA2a on the interaction between macrophage and epithelial cells in cell cycle and differentiationObjective To investigate the effect of cPLA2α on the interaction between macrophage and epithelial cells in cell cycle progression and cell differentiation.Methods Isolated bone marrow macrophage (BMM) from cPLA2a-/-and cPLA2α+/+mice. Co-cultured cPLA2a-/-and cPLA2a+/+BMM with PETCs for48hours, measured PETCs cell cycle distribution by FACS, and TGF-β expression by Real-time PCR. Induced cPLA2α-/-and cPLA2α+/+BMM differentiate to Ml macrophage, ELISA tested the PGE2concentration in M1conditional medium. Then co-cultured the M1conditional medium with PETCs for48hours, measured PETCs cell cycle distribution by FACS, and TGF-β expression by Real-time PCR.Results The concentration of PGE2in conditional medium from cPLA2α+/+BMM was significantly higher than cPLA2α-/-BMM. There were no significantly difference of cell cycle distribution and TGF-β expression in between cPLA2α-/-and cPLA2α+/+BMM co-cultured PETCs.There were no significantly difference of cell cycle distribution and TGF-β expression in between cPLA2α-/-and cPLA2α+/+BMM conditional medium co-cultured PETCs. Conclusion LPS and IFN-y can active cPLA2α from cPLA2α+/+BMM. There were no affect of cPLA2α on the interaction between macrophage and epithelial cells in cell cycle progression and cell differentiation.