Sublytic Effects Of Complement Membrane Attack Complex Assembled In Vitro On Human Renal Proximal Tubular Cells

Background and ObjectiveTubulointerstitial fibrosis is the common pathway through which kidney diseases develop into terminal renal failure.Its characteristic is excessive accumulation of extracellular martix(ECM) in renal interstitial tissue.Human kidney proximal tubular cells(HKCs),which tightly connected with renal interstitial,are the core cells of renal tubular interstitial lesion,and their lesion or post-lesion effects are the mostly factors resulting in the renal tubulointerstitial fibrosis.sMAC deposit can be found in glomerulus and renal tubule in the most patients with glomerulonephritis,and is tightly related to the renal lesion.So it is important and interesting to study the sublytic stimulating effects of sMAC generated by the activated complement in kidney.In present,studies have demonstrated the stimulating effects of sMAC on many types of karyocytes such as endothelial cells,lymphocytes and glomerular cells.However,the sublytic stimulating effects of sMAC on HKCs remain poor clear.It is well known that HKCs is the most obviously location of the expression of various complement components,but express poor complement regulatory proteins comparing with other renal intrinsic cells,which make the HKCs more frequently damaged by immune lesion of actived complement complex.Collagenâ…£(Colâ…£),partly derived from HKCs,is an important ingredient of ECM. Many studies have showed that some impaired factors can up-regulate Colâ…£expression in HKCs.Furthermore,C₃ and MAC can deposite at the injured regions of the tubulointerstitium with the Colâ…£accumulation,but the relative research is limited. Based on the above analysis,it is presumed that sMAC can exert stimulating effects on HKCs and regulate the expression of collagenâ…£in HKCs. In the present investigation,HKCs were chosed as target cells to assembl sublytic complement membrane attack complex in vitro,and establish HKCs sublytic model.By extracting human serum complement euglobulin C₅₆,and detect sublytic effect of sMAC on HKCs.This study furthered our understanding of the roles of sMAC on HKCs in pathogenesis of tubulointerstitial fibrosis.Method1.The complement euglobulin C₅₆ was extracted from acute stage patient serum with a high ESR of more than 30mm/h by zymosan.The activity of C₅₆ was detected by microplate methods;purified C₅₆ was titrated by microassay of reactive hemolytic.2.sMAC was assembled by C₅₆ and C_7-9 drived from normal human serum(NHS) on cultured HKCs in vitro,sMAC deposition was identified by luorescent microscope and laser confocal microscopy.The sublytic dose of sMAC was determined by CCK-8 assay.3.Morphologic changes of HKCs were observed by inverted phase contrast microscope on different time points after induction of sMAC.Cell activity and the change of cell adhesion were determined by cast-off counting and trypan blue staining assay.The dose-dependent and time-dependent manner of Colâ…£expression induced by sMAC was determined by fluorescence activated cell sorting(FACS).Results:1.The 20%-30%of serum activated by Zymosan from patients with a high ESR of more than 30mm/h occured hemolytic effects.The hemolytic effect was dose-dependent.2.sMAC was assembled accurately on the cellular surface of HK-2 by purified C₅₆ with C₇-C₉ came from NHS.Sublytic dose of sMAC was determined as 1:480 C₅₆ and 1:40 EDTA-NHS.sMAC could significantly deposit on the surface of HKCs with the integrited cellular membrane and normal cellular shape.These data suggested that the close of sMAC applied in this study was sublytic effect,and HKCs could tolerate the lytic effect caused by sMAC.3.After incubation of sMAC,activity of HKCs but not cell adhesion was not significantly impaired comparing with the control group and the expression of collagenâ…£significantly increased with a dose-dependent and time-dependent manner. Conclusion:1.Complement euglobulin C₅₆ was extracted successfully by yeast extract method.2.sMAC model on HKCs was established successfully in vitro,and the optimal sublytic dose of sMAC were determined.3.The established sublytic dose of sMAC could activate HKCs and up-regulate the expression of collagenâ…£,which revealed that sMACwas an important factor in pathogenesis of tubulointerstitial fibrosis.