Study On The Effect Of Celecoxib On Podocytes Apoptosis Induced By Puromycin Aminonucleoside

The visceral glomerular epithelial cell,also called the podocyte,is a highly specilized and terminally differentiated cell which plays a major role in establishing the glomerular filtration barrier.Podocytes therefore have limited ability to be replaced if lost. Podocytes injury as a result of apoptosis and/or detachment of cells from the golmerular basement membrane and the inability of podocytes to replicate is a major cause of progressive glomerular damage,but underlying fators of the predisposition of podocytes to injury and the mechanisms that mediate the injury remain unclear.Cyclooxygenase-2 is the key enzyme in the synthesis of prostaglandins(PGs)from arachidonic acid.In normal adult mammalian kidney COX-2 expression is localized to the macula densa and associated cortical thick ascending limb of Henle(cTAL)in the cortex and to a subset of medullary interstitial cells near the papillary tip.COX-2 is detectable in the podocytes of human kidney.Increased podocyte COX-2 expression in rats has been reported in models of subtotal renal ablation,Thy,1 nephritis,and diabetic nephropathy,and selective COX-2 inhibitors have been shown to decrease proteinuria and retard progressive glomerular injury in models of renal ablation,diabetes,and salt- sensitive hypertension. The underlying mechanisms of which the specific cyclooxygenase-2(COX-2)inhibitors have been shown to decrease proteinuria and retard progressive glomerular injury in some animal models have not been addressed formally.Accordingly,in this study we investigated the direct effect of celecoxib,a specific COX-2 inhibitors,on podocyte apoptosis induced by puromycin aminonucleoside(PA)and the mechanisms that underlie this effect in order to further search for clinical therapeutic target on chronic progressive glomerular injury.Methods1.Experiments were performed using early-passage growth-restricted,conditionally immortalized mouse podocytes.The expression of COX-2 in apoptotic podocytes was measured by use of Western blot analysis.The effects of specific COX-2 inhibitors celecoxib on podocyte apoptosis induced by PA will be examined on the basis of podocyte culture.2.The conditionally immortalized mouse podocytes were studied and divided into eight groups.Control group(CON);puromycin aminonucleoside group(PA);losartan group (LOS);dexamethasone group(DEX);celecoxib group(CEL);losartan + puromycin aminucleoside group(LOS + PA);dexamethasone + puromycin aminucleoside group (DEX + PA);celecoxib + puromycin aminudeoside group(CEL + PA).Apoptosis was measured by staining with Hoechst 33258 at 0,8,24 and 48 h.To determine the activity of caspase-3,we used the BD ApoAlert Caspase Colormettic Assay Kit, according to the manufacturer's instructions at 0,8,24 and 48 h.Western blot analysis was performed to measured the pretein levels of p53 and theprotein levels of COX-2 at 24 h.Results1.At the permissive temperature of 33℃,the conditionally immortalized mouse podocyte grew in a cobblestone morphology.They are grown arborized under nonpermissive conditions at 37℃for 14 days.The COX-2 expression in apoptotic podocytes induced by puromycin aminonucleoside increased over time.The highest level was found at 24 hour.Afterwards it decreased gradually.2.The percentage of apoptotic cells significantly increased in the podocytes that were exposed to PA for 24 h compared with control group.No significant change was found in LOS,CEL and DEX groups.In contrast,losartan,dexamethasone and celecoxib significantly reduced PA-induced apoptosis(P＜0.05).3.The proliferetion activity significantly increased in the podocytes that were exposed to PA for 24 h compared with control group.No significant change was found in LOS, CEL and DEX groups.Losartan,dexamethasone and celecoxib significantly increased the proliferetion activity of apoptotic podocytes induced by PA(P＜0.05).4.There was no difference in the activity of caspase-3 among each group(P＞0.05).5.Western blot analysis showed that PA increased the protein levels for p53 at 24 h in arborized podocytes.The protein levels were reduced by LOS,DEX and CEL (P＜0.05).6.Western blot analysis showed that COX-2 expression was seen in control group.It was elevated profoundly after 24 hours induced by PA and reduced by use of LOS, DEX and CEL(P＜0.05).ConclusionsThe COX-2 expression in apoptotic podocytes was time-dependent.Celecoxib prevents podocyte apoptosis induced by PA,and it is p53 dependent.The preventing effect of celecoxib on podocyte apoptosis may be potential interactions with COX-2 expression.