Analysis Of Nuclear Factor-Kappa B Activity In The Kidney And Plasma Levels Of Tissue Factor And Tissue Factor Pathway Inhibitor In Children With Idiopathic Nephrotic Syndrome

Background: Idiopathic nephrotic syndrome(INS) is one of the commonest renal diseases in children,with the clinical charasteristics of intensive proteinuria, hypoalbuminemia, edema to some degree and hypercholesterolemia. Glucocortoid is the most commonly used drug at the moment and Steroid responsiveness is the major determinant of prognosis in INS. Approximately 85% to 90% of patients with INS respond to glucorticoid therapy and have complete remission of proteinuria;The rest 10% to 15% experience partial or no response to corticosteroid treatment. Children with steroid-sensitive nephrotic syndrome(SSNS)have a favorable long-term outcome, whereas half of steroid-resistant nephrotic syndrome(SRNS)patients progress to end-stage renal failure within 1 to 4 years. There are no definitely clinical predictors of the response to steroids in INS. The mechanism of INS has not yet been identified. It is generally accepted that cellular immune disturbances, particularly T-lymphocyte abnormalities, have been implicated in the pathologenesis of the disease.The absence of renal histologic alterations on lightmicroscopy, and no significant immune deposits in glomeruli have suggested that INS is a T-cell disorder associated with a functional renal compromise.Many inflammatory mediators including cytokines are thought to render glomerular basement membrane more permeable to proteins.The activation of nuclear factor Kappa B(NF-k B) plays a vital part in regulating the expression of these inflammatory mediators. NF-k B,composed of two subunits P50 and P65,exists extensively in mesangial, epithelial cells of glomeruli and epithelial cells of renal tubules. In resting cells, it remains in the cytoplasm in an inactive form bound to the inhibitory subunit I k B.Upon stimulation by certain pathological factors, NF-k B is translocated into the nucleus .bound to the DNA consensus sequcnce(k B site) and activates the transcription of target genes of several cytokines, chemotactic and matrix proteins that are involved in inflammatory and immunologic responses.As a result, NF- k B takes part in regulating the expression of these fundermental mediators, and plays a vital role in injury and renovation of immune glomerulonephritis. Tissue factor (TF),with another name of coagulative factor III, is a single-chain transmembrane glycoprotein composed of 263 amino acid residues generated by such cells as endothelial eel Is,platelets and macrophages/monocytes,and exists abundantly in endothelial cells of vessels,renal fibrocyst and glomerular epithelium.lt is one of the most potent initiators of the blood coagulation system which acts as a cofactor for factor VII.When stimulated by certain pathological factors,TF is produced and released into the circulation to trigger extrinsic coagulation.NF-k B regulates the transcription and expression of TF as well as immediate early genes.Besidesprocoagulative function,TF is also conceived to be involved in the pathogenesis of glomerulonephritis and prethromblic state of INS, through mediating the transduction of many cellular signals. In addition, tissue factor pathway inhibitor(TFPI),as an endogenous anticoagulant, regulates the coagulatory function of tissue factor by forming two complex: FXa-TFPI-FVIIa-TF and FXa-TFPI.To our knowledge,quite a few experimental researches have been conducted to investigate the effects of activated NF-k B and TF pathway on renal diseases and some achievements of experimental models of renal injury acquired.But few combined studies were carried out on the activity of NF- k B and plasma levels of TF-TFPI system in children with idiopathic nephrotic syndrome,and their relationship with different histopathological and clinical categories of it.This study is aimed to explore the significance of abnormal activation of NF-k B in the kidney,plasma antigen of tissue factor and tissue factor pathway inhibitor in the pathophysiology and advance of childhood INS ,thus provides a theoretical basis for clinical diagnosis and administration.Materials and methods:patients and control subjects: From August 2002 to January 2005,69 children with INS,diagnosed according to the criteria, were adopted in our study. 41 of them were boys and 28 girls with mean age of 6.5 years old(range from 2.3 to 13). All the patients were absent of such secondary glomerulonephritis as purpural nephritis and pericutaneous renal biopsy under the localization of B-ultrasound was conducted on each of them with parental informed consent, with the following results of histopathological changes: 27 children with minimal changenephrotic syndrome(MCNS)and 42 with non-minimal change nephrotic syndrome(NMCNS, including 26 mesangial proliferative glomerulonephritis, 2 focal and segmental glomerulosclerosis, 5 membranonephrology, 7 membranoproliferativeglomerulonephritis, and 2 IgM nephrology).None of the patients who experienced renal biopsy had initiated steroid therapy or been on therapy for less than 4 weeks before puncture, including all patients with nephritic nephrotic syndrome,5 with non-selective proteinuria and the rest unfavorable response to steroid. In addition,29 children with simple nephrotic syndrome and 40 nephritic nephrotic syndrome were classified based on the clinical manifestation. Follow-up of these children revealed that 36 of them showed steroid resistant(including partial response ? no response and steroid dependent),11 complete response but frequent relapse and 19 infrequent relapse.13 age and sex-matched children receiving renal operation served as the control subjects.Samples collecting:2.7ml empty venous blood was collected into plastic tubes containing 0.3ml 3.8% sodium citrate (9 volumes blood:1 volume anticoagulant) from each child at 7 ~ 8 am in the morning, and citrated plasma was separated by centrifugation at 3000r/min for 15 minutes with a high-speed, low-temperature centrifuge, removed and frozen for use.Percutaneous renal puncture was performed on the patients and renal tissues were sent for regular pathological diagnosis and the rest embedded with paraffin for use, together with sections from normal areas of nephrectomy specimens of the controls.Detection of NF- k. B activity: Two-step immuno-histochemical staining system(Beijing Zhongshan Golden bridgeBiotechnology Co. Ltd)provides a method for measuring the activity of NF- k Bin kidney. Formalin-fixed,paraffin-embedded kidney tissue sections, each with the thickness of 4 u m , were deparaffinized and hydrated, incubated at room temperature for 10 minutes in 3% hydrogen peroxide to quench endogenous peroxidase activity,washed in PBS once for five minutes.The slides were placed in a container and covered with 0.Olmol/L sodium citrate buffer,PH 6.0,heated at middle-fire of microwave stoven for 10 minutes,al lowed to cool in the buffer for half an hour, then washed in PBS three times for 2 minutes each.The slides were incubated with mouse anti-human NF-k B P65(Santa Cruz , USA) diluted at 1:100 in PBS for 2 hours at 37 °C ,washed in PBS three times for 2 minutes. The slides were incubated for 30 minutes at room temperature with Goat anti-mouse IgG horseradish peroxidase-conjugated (Santa Cruz , USA) washed with 3 changes of PBS for 2 minutes each. Each slide was incubated in diaminobenzidine (DAB) solution until desired stain intensity developed, immediately washed with deionized H20, dehydrated through alcohol and xylenes,added with 1 drop of hismoumt, covered for microscopic observation. Measurement of plasma TF and TFPIrPlasma antigen were detected with a sandwich enzyme-linked immunosorbent assay according to the notification.ELISA plates were coated overnight at 4°C with 50uL/well of mouse antihuman monoclonal antibody of TF or TFPI ( Diagnostica Inc,USA) diluted in PBS.All further incubation were at a temperature of 37°C.Each well was blocked for 30 min with 1% of bovine serum albumin(BSA). 100u L of sample dilution was added to each well and incubated for 90 minutes.Horseradish peroxidase-conjugated mouse antihuman IgG (DiagnosticaInc, USA) dilution was added (100uL)to each well and incubated for 150min.The plates were washed three times with 100 u L/well of PBS(pH7.40 0. 01mol/l) after coating, blocking samples and conjugate incubations.The color was developed by adding 100uL of o-phenylenediamine (8mg) in phosphate-citrate buffer containing hydrogen peroxide for 30 min.Then the reaction was teminated by adding 50u L/well of 0. 5mol/l sulphuric acid. The plate were read at 450 nm using a roboticized Elx800 microplate reader (Bio-Tek instruments INC, USA ) within 30 min.All sera samples were run in duplicate, and for each serum the optical density(0D)measured on blank wells was subtracted from the OD obtained in antigen-coated wells.Linear regression equation was deduced for the calculation of plasma levels of TF(pg/ml) and TFPI(ng/ml). The inter- and intra-assay coefficients of variations for IgG assay were <6% and <10%,respectively. Data analysisAll data are expressed as mean ± standard deviat ion(SD) . Significant differences between two groups were estimated by student's t-test, multiple comparisons by Student-Newman-Keuls(SNK,namely q test) test with the basis of one-way analysis of variance (ANOVA),and x2 test(Chi-square test) for categorical variable at specific time points. Simple linear regression was used to examine the relationship between NF-k B activity in kidney and plasma TF, and that of plasma TF and TFPI as well. A P value of less than 0.05 indicated statistical significance. The SPSS Version 11.0 program was used for all statistical calculations. Results NF-k B activity in glomeruli and renal tubules inchildren with INS and the control was [ (0. 85 + 0. 10) % vs (0.21 ±0.06)%, in glomeruli] and [(38. 74 + 3.80)% vs (13. 12 + 2. 35) %, in tubules], respectively (t=22. 26 and 23. 40, respectively, y°<0. 01) ; There were statistical differences of NF- k B activity in glomeruli between MCNS, NMCNS and controls(q=l1.42, 43.26 and 40. 05, respectively, P<0.01),and so were the activation of NF-k B in renal tubules between the above three groups(P<0.01);In addition, NF-k B activity in glomeruli and renal tubules of children with nephritic nephrotic syndrome was statistically different from that of simple nephrotic syndrome(P<0.01).As a result,TF and TFPI antigen in plasma of children with INS was drastically increased compared with that of the control(P<0. 01),and the ratio of TF:TFPI as well (P<0.01) ;Children with NMCNS had higher levels of plasma TF and TFPI than those with MCNS (P<0.01 and 0. 05, respect ively. ), just the same as the ratio of TFPI:TF (P<0.01).But there was no difference of plasma TFPI between simple INS and nephritic INS(q=2.01, P>0.05), and that between MCNS and NMCNS (q = 2.65, P>0. 05) . NF-k B activity of glomeruli and renal tubules in children with INS had positive correlation with plasma TF (r=0.3681 and 0. 6055, respectively,P<0.01)and 24-hour urinary protein excretion (r=0.3870 and 0. 7031, respectively, P<0.01),while no significant correlation was observed between elevated level of plasma TFPI and TF(r=0. 1612,P>0.05).Conclusions:Up-regulated activation of NF- k B in glomeruli and renal tubules of children with INS was closely related to pathological and clinical types,and aggravated proteinuria; NF-k B induced expression of plasma TF contributed to immunedamage of kidney and prethrombotic state of INS. Elevated level of TFPI specifically inhibited TF activity incompletely.Both NF- k. B and tissue factor pathway played a crucial role in the pathogenesis of INS,and were related to clinopathological manifestations and prethrombotic state.