Clinical And Pathological Relationship Between P-gp 170 And Glucocorticoid Resistance In Children With Primary Nephrotic Syndrome

【Backround】Nephrotic syndrome, abbreviated as NS, characterized byproteinuria, hypoproteinemia, edema, and hyperlipidemia, is a diseasefound commonly in children with a morbidity second only that of acutenephritis. Children with NS accounts for 21％of urologic disease ofinpatients in the same period, while primary nephrotic syndrome accountsfor 90％of NS according to datas from the International ScientificKidney Disease of Children of 1990. Glucocorticoid remains one of thebasic and important medicine for this kind of disease as yet, thus theresponse of NS children to GC is contributory to the prognosis to a largedegree. Clinical researches shows recently up to 80％of the childrenpresent themselves with NS at initial onset usually are sensitive to GC,while 20％shows little response even administered with GC of fulldosage, termed as glucocorticoid resistance.Besides, 50％-60％of thosesensitive to GC come to be repeated relapse during the course oftreatment, and even be dependent on GC according to findings offollow-up studys. The response of NS children to GC is a very importantfactor in judging the outcome of NS children. Clinically we found children with NS of poor response to GC often develop relapse orrepeated relapse, resulting in a high incidence of end stage renal disease,namely ESRD. In a words, mechanism of GC resistance is a focus facedby hundreds of thousands of researchers for many years. While the verymechanism of GC resistance remains poorly understood as yet. Classicresearches are focused on receptor of GC, namely GR, changes on GRmolecular structure and quantity, receptor gene polymorphism,expressing imbalance of the two subtypes of GR, GRa and GRβ, etc.Most researches are on effecting stages after GC combined with GR.Recently researchers found gene products of MDR1 P-glycoprotein170,with a molecular weight 170KD, also known as P-gp170, play animportant part in GC resistance in many kinds of disease such as tumorand immune disorder. Previously P-gp170 was believed to be a drugpump widely distributed in membrane of the peripheral bloodmononuclear cells of human or animals, combined with some drugsbefore the latter combined with the targeted receptors and consequentlylead to a decrease of drug concentration in cells and drug resistance.Some other researches showed P-gp170 may lead to GC resistancethrough various ways such as changing of inner environment,down-regulation of GR, inhibiting the cell apoptosis and interfering withrole cytokines playing in signal transduction, etc. Nowadays, researchesabout the relationship between P-gp170 and GC resistance are very limited. Only a few researchers found expression of P-gp170 onperipheral blood mononuclear cells are related to individual response toGC. Indirect testes confirmed that steroids such as GC are one of thesubstrates of P-gp170, expression of P-gp170 in children with NSsensitive to steroids, SSNS, are higher than that of SRNS, those childrenwith NS resistant to steroids. The very mechanisrn of P-gp170 in childrenwith NS of GC resistance remains poorly understood as yet. As GR mayplay roles through combing to GC to function, and GR is widelydistributed in the tissues of human, and kidney, also one of the maintargeted organs, we may make a hypothesis that abnormal P-gp170expression may exist in local kidney tissues, and relate to GC resistance.The present study try to confirm the relationship between P-gp170activity and response to GC by testing inner cell Dexamethasoneconcentration and membrane P-gp170 expression. On the other hand, wetry to further investigate what a kind of role P-gp170 may play in GCresistance in children with NS by observing the distribution, changes ofP-gp170 in renal tissues and the relationship between P-gp170 andclinical effects, and that between the former and pathological and clinicalindexes as well.【Objects】To analyze the correlation between P-gp170 expression andintracellular Dexamethasone concentration, and make regression analysis on those having correlation so as to confirm the possible role of drugpump P-gp170 may play in GC resistance, and to test and compareexpression of P-gp170 mRNA and protein in children with SSNS andSRNS, to analyze disparity on expression of P-gp170 in renal tissues,relationship between P-gp170 and repeated relapse and pathological types,to lay a gound for the researches on GC resistance.【Methods】24 children with NS on their first episode were divided into two groups,SSNS and SRNS, those sensitive to glucocorticoid and resistant toglucocorticoid according to follow-up studys, and 10 healthy childrenserving as control, peripheral blood mononuclear cells were chosed astargeted cells and treated with Dexamethone, cultured totally 36 hoursand harvested at different time point, namely Oh, 12h, 24h and 36h, andblank control was those without treatment at the time points. Reversehigh efficiency liquid chromatography was used to test inner cellDexamethasone concentration both in SSNS and in SRNS at differenttime point; Western blotting was used to test P-gp170 activity onperipheral blood mononuclear cells both in SSNS and in SRNS atdifferent time point; In the second part, 96 children with NS in theirdifferent course were selected as objects and divided into three groups,those without treatment, those treated with prednisone within an half yearand those with repeated relapse within one year, Excisional renal tissues from 10 patients because of surgical trauma. Renal biopsy wasperformed, renal tissues were treated with conventional dying, afterdifferent pathological types were difinited, the pathological scores werecounted, And immunehistochemistry was used to test P-gp170 proteinand hybridization in situ was adopted to test P-gp170 mRNA expressionon renal tissues both in SSNS and in SRNS【Results】1. Dex concentration in children with NS varied with differenttime of cell culture, the longer the culture time, the higher of intracellularDex concentration;2. A positive correlation was found in SSNS between intracellularDex concentration and P-gp170 expression on cell membrane, andregression analysis showed intracellular Dex concentration varied withP-gp170 activity, Dex concentration decreased with the increasingexpression of P-gp170 activity, while there is no correlation betweenintracellular Dex concentration and P-gp170 activity on cell membrane inSRNS, P＞0.05;3. There was significant differences of P-gp170 expression onperipheral blood cells between groups treated with Dex and no treatmentin children with SSNS, P＜0.05, and no difference was found betweenthem in children with SRNS;4. Expression of renal P-gp170, including mRNA and protein weremainly seen in tubular by way of immunohistochemistry and hybridization both in SSNS and in SRNS, expression of P-gp170 mRNAwere consisted with that of protein, while a significant difference wasfound between SSNS and SRNS, expression of P-gp170 in SRNS werehigher than that in SSNS according to statistics, P＜0.05;5. There was a significant correlation between pathological scoresand Expression of renal P-gp170, those having higher scores had higherexpression of P-gp170 than lower ones, and there was differentexpression of P-gp170 in children with NS presented themselves withdifferent pathological types; FSGS, MN and SGN having higherexpression of P-gp170, while MCD and MsPGN having lowerexpression level, P＜0.05;6. There was no difference in between NS children aged no morethan 6 and more than 6; those with relapse had higher expression ofP-gp170 than those with no relapse, and the expression of P-gp170 wasclosely related with Urine protein fixed quantity of 24 hours; Asignificant positive correlation was found between expression ofP-gp170 and days Urine protein turned negative, and a significantnegative correlation was also found between expression of P-gp170 andinterval days between first attack and the first relapse; Besides, asignificant positive correlation was found between expression ofP-gp170 and total dosage and the course of treatment.【Conclusions】 1. Expression of P-gp170 were found both in children with NS andhealthy control, there was increased expression of P-gp170, and P-gp170play different part in physiological and pathologic status2. The higher expression of P-gp170 in PBMCs was associated withGC resistance, increased expression of P-gp170 may result in somechildren with NS developing GC resistance;3. Dex was one of the substrates of P-gp170, the latter was involvedin transportation of GC, from outside into inside;4. Level of P-gp170 expression may be related with GC quantity, andGC resistance of some children with SRNS may be induced by thequantity of GC administrated;5. Increased expression of P-gp170 in PBMC in children with NSmay be as marker of GC resistance;6. Expression of P-gp170 were found in normal renal tissues, mainlyin tubular cell membrane;7. Expression of P-gp170 in renal tissues of children with NS wasassociated with renal lesions, thus we may predict GC sensivity bycombing pathological types, degree of injury and expression of P-gp170;8. Expression of P-gp170 in renal tissues of children with NS wasrelated to the dosage of GC, some of the children with GC resistance wasdue to increased expression of P-gp170 resulting from GCadministration; 9. Increased expression of P-gp170 was related to activity andrelapse of NS, and may be kooked as a marker to predict the prognosis ofNS...