Activated Glomerular Parietal Epithelial Cells Lose The Ablility Of Defferentiating Into Podocyte

BACKGROUNDThe glomerular parietal epithelial cells(PECs)are the potential stem cells in the glomerulus,characterizing the ability of self-renewal and differentiation into other cell types.PECs can differentiate into podocytes under specific conditions in certain diseases,which is one of the important mechanisms for recruiting the lost podocytes in glomerulonephropathy.However,the mechanism of regulating the differentiation of PECs into podocytes remains unknown,and the role of PECs in glomerular diseases is still not clear.PECs is engaged in the occurrence and development of some glomerular diseases.They are the predominant constituent cells in crescent nephritis,playing an important role in the pathophysiological process of crescent formation and transferring to fibrous crescent.Abnormal activation of PECs in crescent glomerulonephritis is often accompanied by high expression of CD44(activation).In other glomerular diseases,especially in diabetic nephropathy,high expression of TGF-β1 in PECs has been observed,nevertheless,whether it is involved in the occurrence and development of glomerular diseases still needs further studies.Whether these activated or highly expressed TGF-β1 PECs still have the ability to differentiate into podocytes,and the role of recruiting lost podocytes has not been studied.OBJECTIVEPrimary culture of PECs and maintaining the biological features;To investigate the number of mitochondria,the changes of oxidative phosphorylation key enzymes and mitochondrial dynamics during PECs’ differentiation into podocytes in vitro;Activate PECs by administration of ANCA-associated glomerulonephropathy(AAVN)serum or TGF-β1 in vitro,and observe if PECs will lose its ability of differentiating into pocytes,and the underlying mechanisms.METHODSWe obtained mouse glomerulus using the method of cell sieving combined with magnetic separation,and obtained PECs by primary culturing separated glomerulus.Real-time PCR,western blot and immunofluorescence staining were used to identify the expressions of claudin-1,a PECs specific marker,CD133 and CD24,stem cell markers.Differentiation of PECs into podocytes was induced by VRADM in vitro,and podocyte-specific protein expression was identified by western bloting and immunofluorescence staining.PECs were induced to differentiate into pocytes in vitro.Realtime PCR was used to detect mitochondrial DNA(mtDNA)copies,and western blot was used to detect the expressions of key regulatory factors of mitochondrial biosynthesis,mitochondrial proteins and dynamic related proteins.Immunohistochemistry staining was used to detect the expression and localization of claudin-1,CD44 and TGF-β1 on consecutive sections of renal biopsy specimens from ANCA-associated crescentic glomerulonephropathy(AAVN)patients.In vitro,PECs were stimulated by AAVN serum and TGF-β1,ERK1/2 signaling pathway was inhibited by U0126,and differentiation of PECs was induced by VRADM.The experiment grouping was described as follows:(1)control group and AAVN serum group;(2)control group and TGF-β1 group;(3)DMSO group,TGF-β1 group,TGF-β1 + U0126 group;(4)control group,VRADM group,AAVN serum group,AAVN serum + VRADM group;(5)control group,VRADM group,TGF-β1 group,TGF-β1 + VRADM group;(6)control group,VRADM group,TGF-β1 group,TGF-β1 + VRADM group,U0126 + TGF-β1 + VRADM group.CD44 and fibronectin mRNA expression were determined by real-time PCR,and the expression of PECs activation markers,podcyte-related protein,intracellular signaling MAPK and mitochondrial related protein were detected by western blot.RESULTSHigh purity of glomerulus were obtained by cell sieving combined with magnetic separation.Primary culture of PECs expressed claudin-1,a PECs-specific marker,and CD24 and CD133,stem cell markers,while E-cadherin and aquaporin 1,tubule epithelium protein,and mesangial specific protein α-SMA were not detected.Claudin-1,CD24 and CD133 were still expressed by PECs after 6 passages in vitro.PECs can be induced to differentiate into PECs and express podocyte-specific proteins in vitro.During differentiation of PECs into podocytes,the number of mitochondria increased,including increased of mtDNA copy number,and PGC-1α was up-regulated.Also,the expression of mitochondrial respiratory chain COX IV and the key enzyme of tricarboxylic acid cycle pyruvate dehydrogenase(PDH)and its active form p-PDH(S293)were increased.In the process of PECs differentiating into podocytes,the expression of mitochondrial dynamic-related proteins,Mfn-1 and p-DRP1(S637),were increased,and mitochondrial dynamics favored fusion rather than division.Claudin-1 and CD44 were highly expressed in crescent of renal biopsies from AAVN patients,and the consecutive sections staining showed that location of claudin-1 and CD44 was the same as TGF-β1 in AAVN patients,showing positive expression.In vitro,both AAVN serum and TGF-β1 can induce PECs activation(expression of CD44)and enable PECs synthesizing extracellular matrix protein(fibronectin),which is activated by ERK1/2 pathway.The expression of CD44 and the synthesis of extracellular matrix were inhibited by ERK1/2 specific inhibitors,U0126.After the activation of PECs by AAVN serum or TGF-β1,VRADM could no longer induce PECs to express podocyte-specific markers,and mitochondrial biogenesis was blocked.Inhibition of ERK signaling can partially improve the differentiation.CONCLUSIONClaudin-1,CD24 and CD133 were expressed in primary to P6 PECs cultured in vitro.Primary to P6 culture of PECs could be induced to differentiate into pocytes.The number of mitochondria increased during the differentiation,and mitochondria tended to fusion.The ERK1/2 signaling pathway is involved in the abnormal activation of PECs induced by AAVN serum and TGF-β1.Abnormally activated PECs lost its ability to differentiate into podiocytes,accompanied by the blocked mitochondria biogenesis and alteration in mitochondrial dynamics.