The Influence Of Epidermal Growth Factor On Glomerular Epithelia Cell

The nephrotic syndrome (NS) is the most common glomerular disease of childhood, large percentage of which is minimal change nephrotic syndrome, above 75%~90%. In spite of extensive studies in human disease and animal model being in progress, the pathogenesis of NS and proteinuria mechanisms are still unclear, thus effecting therapeutic specificity and efficiency of NS. Some patients are prone to recurrence or relapse during the course, consequently influenced children's health. Therefore to unravel the etiology and pathogenesis of NS and the cause of proteinuria is a big challenge to pediatric nephrologists all the times.More recently, insight has increased that the change of glomerular epithelial cells(GEC), also referred to as podocytes of glomerular intrinsic cells including morphological alterations and function changes, is closely and directly correlated with the development of proteinuria. The foot processes of podocyte fusion and effacement and subsequent proteinuria can be induced by abnormality of podocyte actin cytoskeleton. Many experiments in vivo and in vitro have proved that effacement of podocyte foot processes occurs associated with massive proteinuria as the diagnostic character of minimal change nephrotic syndrome(MCNS) and is accompanied by a reorganization of the actin cytoskeleton and the change of podocyte associated molecule. The molecular mechanisms regulating these structural changes and morphological alterations are poorly understood.We have found epidermal growth factor(EGF) is an up-regulation gene among gene pattern based on prophase study about the renal genes expression of MCNS children vs normal control children with Affymetrix gene chip. As we know, kidney is a main synthesis organ of EGF. EGF/EGFR mediated signal pathway can activate actin modified protein and induce F-actin depolymerization and reconstitution, affecting actin cytoskeleton reorganization, and thus play a key role in cell-cell junction and cell-matrix adhesion. Recent observations suggest that EGF-EGFR signal pathway can mediate the change of hepatocyte or intestinal cell architectureand cytoskeletal assembly. Therefore we are very interested in whether or not there is a possible link between EGF mediated signal pathway and the mechanisms regulating podocyte cytoskeleton reorganization. But to date, there are few investigations about the role of EGF induced signal pathway in onset and development of NS and the influence of EGF on glomerular epithelial cell.The present project is purposed to observe EGF and EGFR expression and the influence of EGF mediated signal pathway by constructing animal model mimicing human MCNS and culturing in vitro glomerular epithelial cell(GEC) as study objective, and try to address several questions as follow: (1) to know if EGF/EGFR signal pathway play a role in onset and development of NS or not; (2) the influence of EGF on epithelial barrier function and cytoskeleton reorganization of GEC; (3) the possible approach of EGF on glomerular epithelial cell. We hope to have a better understanding and knowledge about regulating mechanisms of GEC injury and plerosis, which can provide new thought and enlightenment for prevention and therapy of proteinuria in NS.Part I Expression of epidermal growth factor and its receptor in adriamycin nephrosis ratObjective To investigate the distribution and expression of renal epidermal growth factor (EGF) and its receptor EGFR in adriamycin nephrosis rats, and to reveal the correlation of the expression of EGF and EGFR with proteinuria level. To study EGF/EGFR play a role in pathogenesis of nephrotic syndrome(NS) and development of proteinuria.Methods (1) Adriamycin was administered as a single injection 7.0mg/kg through the tail vein of 24 Sprague-Dawley male rats. Adriamycin nephrosis rat animal model mimicing human MCNS was constructed. According to the change of proteinuria level after adriamycin injection and transformation of podocyte structure under scanning electron microscope, we select 5th day, 14th day, 28th day as three study points after adriamycin injection. There was normal control at every study points. (2) EGFmRNA was detected by real-time quantitative RT-PCR; The expression of EGF and EGFR protein was detected by immunohistochemistry and computer image analysis, 24h urine protein level was assayed; Double immunohistochemistry ofWT1 (expressed uniquely in podocyte on glomerulus) and EGFR was used to confirm the location of EGFR on glomerulus. (3) Statistical analysis of all data was performed by Independent-Samples T test , One-Way ANONVA and Person Correlate with SPSS 10.0 statistics software.Results (1) Constructing animal model of adriamycin-induced nephrosis rat: At 5th day after adriamycin injection, the urinary protein began to increase, serum albumin began to decrease ,but the podocyte structure was nomal; at 14th day, rats showed a large amount of proteinuria and partial fusion of foot process, serum albumin decreased continuously ; at 28th day, the urinary protein level increased continuously and was peak, the podocyte structure showed fusion and effacement of foot process, serum tirglyceride and cholesterol also obviously increased. (2) EGF expression: At 5th day , the expression level of EGFmRNA was higher than that of control group (P<0.01). And in adriamycin group , the expression level of EGFmRNA at 28th was much higher than that of 5th and 14th (P<0.01) . The distribution of EGF was primarily along distal convoluted tubule and Henle's loop in control rat, whereas in nephrosis rat proximal convoluted tubule and collecting duct showed the distribution of EGF as well. There were a weak distribution in endothelial cell on glomerulus at 14th and 28th, not in mesangium and epithelia cell. Accompanying with the augment of proteinuria level, the intensity and scope of EGF staining increased. (3) EGFR expression: The distribution of EGFR staining was widely in renal tubule epithelia cell, especially in proximal convoluted tubule and collecting duct. And the intensity and area of EGFR staining along renal tubule in nephrosis group was higher than that in control group (P<0.01) , however in nephrosis group there was no statistical significance among various study points 5th ,14th and 28th day (P>0.05) .The glomerular positive staining of EGFR increased accompanied following the augment of proteinuria level (P<0.01) . (4) correlation with 24h urine protein level: the expression level of EGFmRNA and the intensity of EGF staining along renal tubule was positively correlated with 24 hours proteinuria leveKr =0.608,P=0.001; r =0.887, P=0.000) . The intensity of EGFR staining along glomerulus and renal tubule was positively correlated with 24 hours proteinuria level (r =0.884,P=0.000; r =0.70, P=0.001). (5 ) Double immunohistochemistry of WT1 and EGFR showed EGFR was co-expressed with WT1 in podocyte in nephrosis rat, seldom in control rat.Conclusions The expression and distribution of EGF and EGFR in adriamycin induced nephrosis group was different at different study points, and their expression level was positively correlated with 24h urine protein level, which suggested the change of EGF and EGFR expression was accompanied by the development of proteinuria and prior to the transformation of podocyte, but these changes was apparent at the point of fusion and effacement of foot process. Because of EGFR expressed in podocyte in nephrotic rat, we can reasonably suspect that EGF can exert on podocyte to yield biological effect through EGF/EGFR signal pathway in paracrine pattern.Part II The effect of epidermal growth factor( EG F) on glomerular epithelial ce!ls(Gï¿¡Cs) cytoskeletonObjective To study adriamycin-induced glomerular epithelial cells (GECs) permeability and cytoskeleton changes and to explore the role of epidermal growth factor (EGF) on adriamycin-induced glomerular epithelial barrier function changes.Methods (1) To select proper adriamycin-induced concentration to injury rat glomerular epithelial cell(GEC) cultured in vitro, then to investigate the effect of in the absence or presence of exogenous EGF before GEC exposed to adriamycin(0.5um) 24h. (2) Paracelluar permeability of rat GEC on Millicell-PCF Inserts was evaluated by measuring bovine serum albumin(BSA) conjugated with Naphthol blue black passage through GEC monolayer. Cell viability was evaluated by detecting live cell number with Typan Blue staining method and lactate dehydrogenase (LDH) release with chemical method; cytoskeleton changes including cytoskeletal architecture n F-actin reorganization or disassemble rate and alpha-actinin distribution was analyzed by Leica laser scan confocal immunofluorescence microscopy ; RT-PCR was used to determinate alpha-actinin mRNA and CD2AP mRNA level. (3 ) Statistical analysis of all data was performed by One-Way ANONVA and LSD method or Tukey method with SPSS 10.0 statistics software.Results Compared with control group, adriamycin group evidently increased paracelluar permeability to BSA after 0.5uM adriamycin induced GEC 24 hours ( P <0.01) , however cell viability had no change ( -P>0.05) . F-actin reorganization rateincreased ( -P<0.01), disassemble of cortical cytoskeleton architecture was examined, stress fiber in cytoplasm disappeared, on the other hand , alpha-actinin staining was perinuclear enhancement different from normal cytosolic pattern, the expression of alpha-actinin mRNA had no change ( iJ>0.05) ; CD2AP mRNA expression level decreased ( P<0.0\) . EGF significantly reduced the effect of adriamycin- induced higher levels of BSA passage through GEC monolayer in dose-dependent manner ( P <0.01 ) , EGF prevented adriamycin-induced cytoskeletal reorganization , disassembled cortical cytoskeleton was recovered, stress fiber appeared again. Alpha-actinin staining was mainly cytosolic as control GEC. CD2AP mRNA expression level began to increase ( P<0.01) .Conclusions GEC induced by 0.5uM adriamycin (a proper pharmacological concentration to GEC) was similar to injured podocyte in adriamycin-induced nephritic rat. Adriamycin-induced increased paracelluar permeability accompanied by GEC cytoskeleton disassembly and reorganization and the change of distribution and expression of cytoskeleton associated protein. EGF can prevent adriamycin-induced GEC by restoring cytoskeleton assembly and reduce paracelluar permeability to BSA.Part HI The role of EGF-EGFR-PLCy signal pathway in glomerular epithelial cell cytoskeletonObjective To explore possible approach to EGF protection of adriamycin-induced injury GEC, and to provide a guide for confirming future intervention target against glomerular epithelial cell damage.Methods To inhibit EGF exerting on GEC by two different classes of inhibitors, EGFR tyrosine kinase inhibitor AG1478 and PLCy inhibitor U73122, then six groups according to different intervention including a) ADR group b) AG1478 + ADR group c) U73122 + ADR group d ) EGF + ADR group e) AG1478 + EGF + ADR group f) U73122 + EGF + ADR group. To observe the change of all groups GECs permeability, cell viability and distribution and expression of cytoskeleton associated protein molecule (detailed method and index see part II). Western blot was used to determinate EGFRs phosphorylated EGFR (P-EGFR) and phosphorylated PLCy (P-PLCy) protein expression level. Statistical analysis of all data was performed byOne-Way ANONVA and LSD method or Tukey method with SPSS 10.0 statistics software.Results Compared with EGF+ADR group , AG1478 or U73122 was pre-incubated before adriamycin in the presence of EGF, GEC paracelluar permeability to BSA increased (P<0.01) ; F-actin reorganization rate increased ( P<0.0\) , disassemble of cortical cytoskeleton architecture was examined again, stress fiber in cytoplasm obviously decreased; alpha-actinin staining tend to perinuclear pattern, CD2AP mRNA expression level decreased ( ^<0.01) . however compared with ADR group , AG1478 or U73122 was pre-incubated before adriamycin in the absence of EGF, there was no change of all index ( f>0.05) . The change of cell viability among all group had no statistical significance ( i^O.05) . Compared with EGF+ADR group , EGFR> P-EGFR ^P P-PLCy protein expression level of western blot in AG1478+ EGF+ADR group all decreased ( .PO.01), however in U73122+ EGF+ADR group , only P-PLCy protein expression level of western blot decreased ( -P<0.01). There was no statistical significance of EGFR, P-EGFR and P-PLCy protein expression level between ADR group and AG1478 +ADR group, U73122+ADR group as well.Conclusions The effects of EGF preventing adriamycin-induced GEC higher permeability and cytoskeleton reorganization were significantly attenuated by two inhibitors AG1478 and U73122, suggesting these protective effects of EGF was produced by the activation of some key signal molecule of EGF mediated signal pathway. The inhibiting effect of EGF-induced protection by two different inhibitors was analogical, however their P-EGFR protein expression level was different, P-PLCy protein expression level was weak and parallel, which showed EGFR was not necessarily a key mediator in the protection of GEC by EGF, PLCy may be a required downstream signal for EGF protection.conclusions1. In adriamycin induced nephrosis rat , EGFR can be expressed in visceral glomerular epithelia cell or podocyte . EGF/EGFR may play a role in the pathogenesis and development of adriamycin induced nephrosis and formation of proteinuria.2. Adriamycin increases GEC paracellular permeability to BSA as a result of...