Interleukin-8 in chronic renal failure and dialysis patients

A total of 105 patients participated in this study, including10 with chronic glomerulonephritis with normal renal function(CGN patients), 36 uraemic patients (CRF patients), 19 continuousambulatory peritoneal dialysis patients (CAPD) without peritonitis,three CAPD patients with peritonitis, 37 patients undergoingchronic haemodialysis (HD) divided into short-term HD, 15 patients;medium-term HD, 12 patients; and long-term HD, 10 patients.IL-8 and two other proinflammatory cytokines, IL-6 and TNFweretested using a specific immunoassay. IL-8, IL-6, and TNFc serumlevels were significantly increased in patients with chronicrenal failure compared to their levels in normal individuals(P<0.000l, P<0.05 and P<0.000l respectively). The mostpronounced incre ment in IL-8, IL-6 and TNF serum levels wasobserved in CAPD patients (P<0.000l). CAPD patients withoutperitonitis showed relatively low levels of IL-8 or IL-6 inperitoneal dialysate effluents (PDE), whereas PDE-TNF were notdetectable in almost all patients tested. Patients with peritonitisshowed very high serum and PDE levels of IL-8, IL-6 and TNF.The clinical recovery from peritonitis was characterized bya rapid fall in IL-8, IL-6 and TNF in serum and dialysate. HDpatients showed a significant increase in serum levels of IL-8and also IL-6 and TNFcompared to normal individuals (P<0.05,P<0.05 and P<0.01 respectively). HD duration influencedserum levels of IL-8 and TNF since they were significantly higherin short-term HD patients than medium- or long-term HD patients(respectively P<0.05, P<0.00l for IL-8, and P<0.01,P<0.001 for TNF Pre-HD IL-6 levels were not influenced byHD duration. No major modification of IL-8 serum levels couldbe evinced after and before HD sessions in the short-term group,but concentrations of this cytokine were significantly higherafter HD in medium- and long-term HD patients (P<0.05, P<0.0lrespectively). In contrast, HD session did not influence IL-6and TNF levels. We conclude that the cytokine profile is perturbedin uraemia and during dialysis, and that this should be consideredas an inflammatory status.     

Effect of calcium-channel blocker on tumour necrosis factor alpha (TNF{alpha}) production in cyclosporin-treated renal transplant recipients

PURPOSE OF THE STUDY.: The influence of calcium-channel blocker treatment on in-vitroTNF production by peripheral blood mononuclear cells (PBMC)from renal transplant recipients treated with cyclosporin wasstudied. DESIGN.: We compared spontaneous and OKT3-induced TNF production of 12renal transplant recipients treated with calcium-channel blockertherapy with that of 18 renal transplant recipients who werenever treated with a calcium antagonist. RESULTS.: The two groups were similar with regards to age, time aftertransplantation, dosage of immunosuppressive drugs, and bloodcyclosporin levels. Spontaneous (481±161 versus 319±74pg/ml, n.s.) and OKT3-induced (745±182 versus 632±112pg/ml, n.s.) TNF production were similar in both groups. CONCLUSIONS.: The results indicate that in cyclosporintreated renal transplantrecipients calcium-channel blockers do not affect TNF production.     

Interleukin 6 production by human proximal tubular epithelial cells in vitro: analysis of the effects of interleukin-1{alpha} (IL-1{alpha}) and other cytokines

Proximal tubular epithelial cells (PTEC) from human renal tissueobtained from biopsy or nephrectomy were grown in monocultureand evaluated in vitro at passage 2–4 for interleukin6 (IL-6) production in response to medium alone or to interleukin1 alpha (IL-1), tumour necrosis factor alpha (TNF), interleukin2 (IL-2), interferon gamma (INF) or lipopolysaccharide (LPS).IL-6 bioactivity was quantitated using the IL-6-dependent murinehybridoma cell line (B9) and expressed as IL-6 units/ml/10⁵PTEC. PTEC cell lines exposed to medium alone produced intermediateamounts of IL-6 with substantial variability between cell lines.Introduction of IL-1 resulted in a dose- and time-dependentincrease in IL-6 production by PTEC that was maximal at 1 ng/mlIL-1 at 24 h. All PTEC cell lines showed an increased IL-6 productionon exposure to IL-1 varying from 1.3- to 24-fold increase overbaseline production. This response was completely blocked byanti-rIL-1. No significant IL-6 production by PTEC could beinduced by TNF, IL-2, IFN, or LPS over a broad dosage range.Cycloheximide inhibited IL-6 production without irreversiblecell toxicity, indicating de-novo synthesis. IL-6 produced byPTEC had a molecular weight of 26-29 kDa as demonstrated byWestern blot analysis. Using PCR analysis we could demonstrateupregulation by IL-1 of IL-6 mRNA in a dose-response fashion,indicating that IL-1 regulates IL-6 production at a pretranslationalvalue of protein synthesis. These results show that human culturedPTEC produce IL-6 under both normal and IL-1-stimulated conditions,and suggest that they may have a regulatory function in responseto cytokines in the setting of inflammation in the renal cortex.     

Endotoxins modulate chronically tumour necrosis factor {alpha} and interleukin 6 release by uraemic monocytes

We examined in vivo the release of tumour necrosis factor alpha(TNF) and interleukin 6 (IL-6) by uraemic monocytes upon stimulationwith endotoxin-contaminated bicarbonate concentrate. Twelveuraemic patients underwent 1-month-subsequent periods of standardhaemodialysis (SHD) with cuprophane (CU), a high-complement-activatingmembrane (6 patients), or haemodiafiltration (HDF) with polyacrylonitrile(PAN), a low-complement-activating membrane (6 patients), byusing a dialysate prepared with either non-sterile bicarbonateconcentrate tanks (phase 1) or sterile bicarbonate concentratebags (phase 2). TNF and IL-6 concentrations were determinedin monocyte supernatants by ELISA; endotoxin levels in bicarbonateconcentrates were measured by a chromogenic limulus amoebocytelysate (LAL) assay. A significant increase in LAL reactivity was found in bicarbonateconcentrate tanks compared to sterile bags (P<0.001). Non-steriledialysate caused a significant (P<0.001) predialytic increasein monocyte TNF release as compared to controls and nondialyseduraemic patients. One month treatment with sterile bicarbonatesignificantly decreased TNF predialytic activity in monocytesupernatants (P<0.001) to levels closer to those of non-dialyseduraemic patients. A similar decrease was observed for IL-6 production.Dialytic treatment induced a further increase in both TNF andIL-6 production, particularly in phase 1. When uraemic patientswere examined separately according to the different dialyticprocedures (SHD-CU or HDF-PAN), the use of sterile dialysate(phase 2) caused a significant decrease of predialysis TNF releasein both SHD CU patients (24.1±8.4 pg/ml versus 55.3±5.7pg/ml, P<0.001) and HDF PAN-treated patients (16.6±5.3pg/ml versus 29.1±5.4pg/ml, P<0.005), so that thedifferences between the dialytic procedures were completelyabolished. In conclusion, TNF and IL-6 release may be induced by endotoxin-contaminateddialysate during haemodialysis. The use of sterile bicarbonatecan ameliorate the bioincompatibility of CU membranes and probablyinfluences the biocompatibility of PAN membranes. Therefore,regardless of the type of dialyser used, all attempts to obtainan ultrapure dialysate are important to optimize dialytic treatment,in order to attenuate the chronic monocyte activation whichoccurs during haemodialysis.     

Expression and function of fibronectin receptors on peripheral mononuclear cells in IgA nephropathy

The ß1 integrin family, major adhesive receptors forthe extracellular matrix (ECM), have been reported to be presentin normal and diseased kidneys. Attachment of glomerular cellsto ECM is mediated by ß1 integrins. Several membersof the ß1 integrins are referred to as VLA (very lateactivation) antigens. Peripheral mononuclear cells also expressVLA antigens in both resting and activated states. We examinedthe expression and function of VLA antigens on peripheral lymphocytesand monocytes in patients with IgA nephropathy using monoclonalantibodies (mAbs) specific for VLA -chains. Peripheral lymphocytesfrom patients with IgA nephropathy expressed VLA-4 and 5, butnot VLA-1 2 or 3. Peripheral monocytes from patients with IgAnephropathy expressed VLA-2 4 and 5, but not VLA-1 or 3. Theexpression of VLA adhesive receptors was observed in healthyindividuals. Adhesion assay to fibronectin revealed augmentedadhesion of mononuclear cells in IgA nephropathy (P<0.05),and this increased adhesion was inhibited by mAbs to VLA-4 and5. The expression of ß1 integrins in IgA nephropathywas similar to that of healthy individuals, but the functionof these molecules in terms of adhesion to fibronectin thoughVLA-4 and VLA-5 is increased in these patients. These findingssuggest that the activation of fibronectin receptors on peripheralmononuclear cells plays an important role in the pathogenicprocess of IgA nephropathy.     

Animal models of Alport syndrome.

Introduction The last decade of the twentieth century was a very productiveperiod for the study of Alport syndrome. Alport syndrome wasshown to result from mutations in certain members of the typeIV collagen family of proteins, the 3(IV), 4(IV), and 5(IV)chains. Several hundred different mutations in the COL4A5 gene,which encodes the 5(IV) chain, were described in patients withX-linked Alport syndrome [1]. A few dozen mutations were foundin the COL4A3 and COL4A4 genes, which respectively encode the3(IV) and 4(IV) chains, in patients with autosomal recessiveAlport syndrome [2,3]. Autosomal dominant Alport syndrome, dueto heterozygous mutations in COL4A3 or COL4A4, was establishedas an entity, and distinguished from Fechtner and Epstein syndromes,which arise from mutations in a non-collagen locus, MYH9 [4–7].Investigators established that mutations in the 3(IV), 4(IV),or      

Acute effect of oral, intraperitoneal, and intravenous 1{alpha}-hydroxycholecalciferol on markers of bone metabolism

OBJECTIVE: To study the acute effect of 1-hydoxycholecalciferol (1-OHD₃)on serum levels of alkaline phosphatase, Ca²⁺, osteocalcin,parathyroid hormone (PTH), phosphate and type I and III procollagens(PICP and PIIINP respectively) in patients undergoing peritonealdialysis. Also, 1,25-(OH)₂D₃ was measured. DESIGN: Single doses of 1-OHD₃ (80 ng/kg body wt) were given in randomizedcross-over fashion, orally, intraperitoneally (i.p.) and intravenously(i.v.) on three occasions. Blood was sampled at 0, 1, 6, 12,and 24 h after administration of 1-OHD₃. MAIN RESULTS: Following oral administration of 1-OHD₃, a decrease in serumalkaline phosphatase was seen when levels at 1 and 6 h werecompared to baseline (P<0.05). Oral and i.v. drug administrationsresulted in an increasing trend in serum Ca²⁺ throughout thestudy (P<0.05). Moreover, a difference in serum Ca²⁺ wasfound when 24-h levels after oral 1-OHD₃ dose was compared tobaseline (P<0.05). Serum osteocalcin at 12 and 24 h afteroral 1-OHD₃ compared to baseline were increased (P<0.05).Intact PTH followed a circadian rhythm after all three routesof drug delivery. After 24 h, significant decreases of intactPTH were observed in the oral and i.v. group. No changes inserum phosphate and serum PICP levels were observed over timeafter oral, i.p., and i.v. delivery of 1-OHD₃However, serumPIIINP following oral and i.p. administration of 1-OHD₃ decreasedat 1 and 6 h (P<0.05). CONCLUSION: Oral and iv. administration of 1-OHD₃ does influence serum levelsof osteocalcin, PTH, and PIINP. Noticeable is the significantincrease in serum osteocalcin after oral administration of 1-OHD₃,the remarkable increase (22.6%) in osteocalcin 24 h after i.v.1-OHD₃, though not statistically significant, the increase inserum PTH levels 12 h following oral and i.v. doses of 1-OHD₃and the moderate effect on serum Ca²⁺ levels.     

Myofibroblasts, predictors of progression of mesangial IgA nephropathy?

The limited knowledge of the cellular mediators of renal scarringhampers progress in the management of progressive chronic renalfailure (CRF). We have studied 38 patients with biopsy-provenmesangial IgA nephropathy with emphasis on attempting to definethe role of myofibroblasts(-smooth muscle actin/SMA-positivecells) in renal scarring. In 18 untreated patients, correlationswere undertaken between known histological parameters of progressionas well as the presence of myofibroblasts in tissues and theclinical outcome. -SMA staining by an avidin-biotin-peroxidasemethod was confined to a large extent to the vascular smoothmuscle cells of normal kidneys but extended to the tubulointerstitiumand periglomerular space in scarred kidneys. Mild glomerularstaining was also noted. The interstitial immunostain followeda similar distribution to that of interstitial type III collagen.Morphometric analysis showed the interstitial SMA staining tobe a reliable histological predictor of outcome as it discriminatedbetween progressors and non-progressors (²=4.923, P=0.026).The intensity of the interstitial -SMA staining correlated withrenal functional outcome; inversely with the reciprocal of serumcreatinine slopes (r=-0.466, P<0.025) and positively withthe serum creatinine value at the end of the observation period(r=0.704, P<0.00l). Other histological parameters that correlatedwith outcome included the degree of tubulointerstitial (TI)inflammatory infiltrate (r=-0.425, P<0.05 with 1/Cr slopeand r=0.760, P< with serum creatinine) and the intensityof the TI staining for collagen IV (r=-0.567 and 0.667 respectively).In 20 patients treated with prednisolone and azathioprine, asecond renal biopsy showed the persistence of interstitial myofibroblastsin the absence of progressive fibrosis. In conclusion, stainingof renal biopsies of patients with mesangial IgA for -SMA-positivecells may identify the myofibroblasts as important mediatorsof TI scarring and have useful prognostic implications.     

Sera from patients with anti-GBM nephritis including Goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen {alpha} chains

BACKGROUND.: Goodpasture (GP) syndrome is defined by the clinical associationof pulmonary haemorrhage with rapidly progressive glomerulonephritis.The disease is caused by pathogenic autoantibodies directedagainst type IV collagen, which is a major structural componentof glomerular basement membranes (GBM). METHODS.: The non-collagenous domains (NC1) of all six human type IV collagenchains was produced in E. coli as recombinant fusion proteinswith glutathione-S transferase. Sera from 10 patients with differenttypes of anti-GBM nephritis, including GP syndrome, were testedfor reactivity with the six proteins using immunoblotting ofdenatured and reduced proteins and ELISA without reduction. RESULTS.: All 10 sera reacted with the 3(IV) collagen chain by immunoblottingand ELISA. One serum also recognized the 2(IV) 4(IV), 5(IV)and 6(IV) chains by immunoblotting. ELISA measurements revealedreactivity of several other sera with 2(IV), 4(IV) or 6(IV)but not with 5(IV) collagen chains. No reactivity was observedwith the 1(IV) chain. CONCLUSION.: Autoantibodies in anti-GBM nephritis may not be directed onlyagainst the 3(IV) collagen chain and they frequently recognizeconformational epitopes.     

Serum {alpha}2-macroglobulin in haemodialysis patients: baseline and kinetic studies

The pathogenesis of dialysis related amyloidosis remains unresolveddespite the identification of ß₂-microglobulin (ß₂M)as the major protein constituent, as well as other proteinsbeing present in the deposits. Among the latter we have assessedthe serum concentrations of ₂-macroglobulin (₂M) both in thebaseline stage and during the haemodialysis (HD) procedure.We have also assessed the influence of the membrane on ₂M kinetics. Fifteen HD patients with histologically proven dialysis-relatedamyloidosis (DRA group) and 15 HD patients clinically and radiologicallyconsidered dialysis-related amyloidosis free (control group)were included in the baseline study. Blood was sampled the daybefore the second dialysis of the week and ₂M, ß₂Mand ₁, antitrypsin were determined along with the routine biologicalanalysis of these patients. Serum ₂M was greater in dialysis-relatedamyloidosis than in control patients (t = 2.35; P<0.026).Serum ß₂M was similar in both groups. The serum ₂Mand ß₂M correlated in patients with dialysis-relatedamyloidosis (r = 0.64; P<0.01), while no correlation wasfound in controls (r = 0.17; NS). Stepwise analysis taking thepresence of dialysis-related amyloidosis as the dependent variableretained the serum ₂M concentration as the first variable inthe model (F = 4.4; partial r = 0.38; P<0.046). The sameproteins were determined in another group of seven patients,before and hourly during HD as well as 2 and 8 h after the endof HD during nine consecutive dialyses (3 cycles of 3 HD eachusing AN69 and cuprophane membranes in a crossover design).Serum ₂M significantly increased from hour 3 and continued toincrease 2 hours post-HD (+11% and +9% with AN69 and cuprophanerespectively; P<0.001). Total proteins peaked at hour 4 (+4% and +3% P<0.01) and decreased after HD. Serum ß₂Msignificantly decreased with AN69 HD ( – 29% P<0.001)and remained unchanged during cuprophane HD. In conclusion, significant increases in serum ₂M are observedimmediately after and during the early post-dialysis periods,regardless of the membrane used. Further, serum ₂M correlateswith ß₂M only in patients with dialysis-related amyloidosis,and this variable was retained in the multivariate regressionanalysis to predict dialysis-related amyloidosis. Although thebaseline results require confirmation with larger studies, wepostulate that the present results are of relevance for dialysis-relatedamyloidosis pathogenesis since ₂M, previously identified indialysis related amyloid deposits, is closely related to acute-phasereactant proteins, and interacts with the main infiltratingcells of the deposits (macrophages). ₂M modifications couldrepresent a new manifestation of the inflammatory response tothe haemodialysis procedure.     

Interleukin-8 during peritonitis in patients treated with CAPD; an in-vivo model of acute inflammation

CAPD-related peritonitis was used as an in-vivo model to studyI1-8 during peritoneal inflammation. Eleven episodes were studiedin nine patients, who were followed on 8 consecutive days fromthe start of peritonitis and once after recovery (control).I1-8 was measured in dialysate (night dwells) and serum. TheI1-8 time course was compared to I1-6 and TNF. In addition,an in-vivo relationship between dialysate I1-8 and intraperitonealaccumulation of neutrophils was studied. A highly increased peritoneal appearance rate of I1-8 was foundin the acute phase that decreased to control values during recovery.A remarkable parallelism was observed for dialysate I1-8 andI1-6 with respect to the time course and the peritoneal appearancerate. In contrast, the appearance rate of TNF was much lessand had a different time course. In three of four cases wherethe dialysate I1-8 peak occurred on day 2, the dialysate I1-6peak still coincided with I1-8, in contrast to TNF (always day1). Dialysate I1-8 generally exceeded serum concentrations duringthe entire follow-up, indicating intraperitoneal I1-8 synthesis.A positive correlation was present between the dialysate I1-8peak and the maximal number of neutrophils in dialysate. Thisrelationship was absent for I1-6 and TNF. In five of six episodeswhere neutrophils were quantified on both day 1 and 2, the I1-8peak occurred simultaneously with the neutrophil peak. Thesefindings suggest that I1-8 is involved in the recruitment ofneutrophils towards the dialysate during peritonitis.     

Imbalance between intraperitoneal coagulation and fibrinolysis during peritonitis of CAPD patients: the role of mesothelial cells

We compared peritoneal dialysis effluents from 18 CAPD patientswho had not suffered from peritonitis during the last 6 months(group 1) with the effluents from five patients with acute peritonitis(group 2), measuring activation markers of coagulation and fibrinolysis.These markers included prothrombin fragment F1+2 (F1+2), thrombin-antithrombinIII complex (TAT), fibrin monomer (FM), and fibrin degradationproducts (FbDP). In the dialysate of group 1 we found remarkablyhigh levels of F1+2, TAT and FM concomitant with a high concentrationof FbDP, indicating a high rate of intraperitoneal fibrin turnover.The balance between peritoneal generation and degradation offibrin was disturbed in untreated patients of group 2, who hadsignificantly higher levels of coagulation markers and a higherratio between FM and FbDP. Seven days after treatment with intraperitonealadministration of antibiotics and heparin, F1+2, TAT, FM andFbDP decreased significantly. To evaluate the role of mesothelial cells (MC) in the high peritonealfibrin turnover we investigated the expression of tissue-typeplasminogen activator (t-PA), urokinase-type plasminogen activator(u-PA), plasminogen activator inhibitor type-1 (PAI-1), andtissue factor in cultured human peritoneal MC under basal conditionsand after exposure to tumour necrosis factor (TNF) interleukin-1(IL-1), or bacterial lipopolysaccharide (LPS). The exposureof MC to TNF or to a lesser extent IL-1 or LPS reduced theirfibrinolytic activity by decreasing t-PA production and increasingPAI-1 synthesis. Furthermore the addition of TNF resulted inactivation of the coagulation cascade by the expression of tissuefactor. These in-vitro findings explain the imbalance betweenintraperitoneal coagulation and fibrinolysis during peritonitisof CAPD patients.     

Plasma Concentrations of Atrial Natriuretic Peptide in Relation to Body Fluid Status in Chronic Uraemia

Basal plasma -human natriuretic peptide (-hANP) values werefound to be significantly higher in 7 fluid-overloaded chronicdialysis patients than in 13 non-dialysed renal patients withoutextracellular fluid (ECF) expansion. Iso-osmotic reduction ofthe body weight by a single 3-h ultrafiltration caused -hANPto decrease significantly in all anuric patients to values comparableto those of non-dialysed subjects. In this latter group, however,there was a significant inverse relationship between -hANP andglomerular filtration rate but not between -hANP and total bloodvolume. These findings suggest that both ECF expansion and impairedrenal removal of -hANP might be responsible for the high -hANPin chronic uraemia.     

Evaluation by histology, immunohistology and PCR of protocollized renal biopsies 1 week post-transplant in relation to subsequent rejection episodes

Renal biopsies were performed 1 week following renal transplantationat a time without clinical evidence of rejection in 43 patients(13 females, mean age 48 years range 18–60 and 30 males,mean age 43 years range 17–59 years). Thirty-six biopsieswere available for histological or immunohistochemical analysis.Immunohistochemical analyses were performed with monoclonalantibodies against leukocytes (CD45), monocytes (WT14), complementfactor 3 (C3), T-cells (Leu4), T-cell receptor ß and, tumour necrosis factor (TNF) IL-2 receptor (IL2-R, TAC), intercellularadhesion molecule-l (ICAMI) and HLA-DR. The slides were scoredsemiquantitatively with the observers having no knowledge ofclinical or patient data. TNF and IL-2R were also measured byquantative PCR. None of the studied parameters correlated todelayed graft function or graft loss. Histological analysisshowed that both focal interstitial infiltrate (18/35) and tubularbasement membrane disruption (11/35) were followed by a higherincidence of subsequent rejection (P = 0.03 and 0.02 respectively).Also positivity for WT14 around tubuli (P = 0.02) was associatedwith subsequent occurrence of rejection. The intensity of stainingof ICAM-I on PTC as well as TAC on proximal tubular cells wasassociated with the number of subsequent rejection episodes.The association between the IL-2 receptor and subsequent rejectionwas also found applying PCR to the tissue specimens. We conclude that the presence of focal interstitial infiltratesand tubulitis in 1-week biopsies from well-functioning graftscarries an increased risk of subsequent rejection. The observedinfiltrate outside the tubuli may consist of either monocytesor lymphocytes. Further studies, both in vitro and in vivo,applying immunohistochemical and molecular biological techniqueswill be necessary to further elucidate the role of adhesionmolecules and interleukins in early and ongoing rejection.     

Once-weekly erythropoietic therapy: is there a difference between the available preparations?

Sir, Iain Macdougall provided, in general, a comprehensive reviewof once-weekly administrations of epoetin , epoetin ßand darbepoetin [1]. However, additional mention could havebeen made of the differences between epoetin and epoetin ß,as well as further analysis of the data from studies of subcutaneous(s.c.) administration. With regard to the differences between epoetin and epoetin     

Tissue-specific distribution of the Goodpasture antigen demonstrated by 2-D electrophoresis and Western blotting

The target of autoantibodies in Goodpasture's disease, the Goodpastureantigen has recently been characterized as the NC1 domain ofthe 3 chain of type IV collagen. In order to study the Goodpastureantigen in different organs, NC1 domains were isolated frombasement membranes (BM) of human glomeruli (GBM), tubules (TBM),alveoli (ABM), placenta (PBM) and aorta (VBM). NC1 preparationswere separated by 2-D electrophoresis, and silver stained orimmunoblotted to determine the subunit structure and antigenicityof different basement membranes. All basement membranes containedmonomeric components of MW 26 kDa and 24 kDa, and associateddimers, corresponding to the 2-D location of 1(IV) and cc2(IV)chains respectively. However, GBM, ABM, and to a lesser extentTBM possessed an extra set of monomeric components of MW 28kDa and associated dimers corresponding to the proposed locationof 3(IV) and 4(IV) chains. 2-D-separated polypeptides were Westernblotted with autoantibodies from patients with Goodpasture'sdisease, a monoclonal antibody to the Goodpasture antigen (P1)and a monoclonal antibody to the bovine 3(IV) chain. The predominantbinding of all these reagents was to cationic 28 kDa monomersof GBM, ABM and TBM, corresponding to the 3(IV) chain, althoughautoantibodies and P1 also bound to neutral 28 kDa monomers,corresponding to the 4(IV) chain. Autoantibodies bound weaklyto more neutral components of PBM and VBM, but neither monoclonalantibody bound to these basement membranes. This study suggeststhat there are two separate type IV collagen networks: one containingthe l(IV) and 2(IV) chains appears to be present in all basementmembranes, and the other containing the 3(IV) and 4(IV) chainshas a tissue specific distribution. The latter network bearingthe Goodpasture antigen is expressed in the basement membranesof both kidney and lung, the organs involved in Goodpasture'sdisease.     

Bone marrow transplantation rescues Alport mice.

In the 9 May 2006 issue of Proc Natl Acad Sci USA, Sugimotoand colleagues [1] described fascinating data on a potentialapproach to treat Alport's syndrome, a rare genetic diseaseleading to renal failure, which so far could not be cured. Thework was highly publicized and discussed in both scientificjournals and the lay press. Alport's syndrome derives from a mutation of either the 3, 4or 5 chain of type IV collagen, i.e. collagen types that constitutebasement membranes in the renal glomerulus, the ear and theeye. Mice     

Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy

Background. We have previously documented that human mesangialcell (HMC)-derived tumour necrosis factor- (TNF-) is an importantmediator involved in the glomerulo-tubular communication inthe development of interstitial damage in IgA nephropathy (IgAN).With the strategic position of podocytes, we further examinedthe function of podocytes in IgAN. Methods. Podocyte markers were examined in renal tissues byimmunofluorescence. In vitro experiments were conducted withpodocytes cultured with polymeric IgA (pIgA) or conditionedmedium prepared from HMC incubated with pIgA (IgA–HMCconditioned medium). Results. Glomerular immunostaining for nephrin or ezrin wassignificantly weaker in patients with IgAN. The immunostainingof IgA and nephrin was distinctly separate with no co-localization.In vitro experiments revealed no effect of pIgA on the expressionof these podocyte proteins as IgA from IgAN patients did notbind to podocytes. In contrast, IgA conditioned medium preparedfrom IgAN patients down-regulated the expression of these podocyteproteins as well as other podocyte markers (podocin and synaptopodin)in cultured podocytes. The mRNA expression of nephrin, erzin,podocin but not synaptopodin correlated with the degree of proteinuriaand creatinine clearance. The down-regulation was reproduciblein podocytes cultured with TNF- or transforming growth factor-β(TGF-β) at concentration comparable to that in the IgA–HMCconditioned medium. The expression of these podocyte proteinswas restored partially with a neutralizing antibody againstTNF- or TGF-β and fully with combination of both antibodies. Conclusion. Our finding suggests podocyte markers are reducedin IgAN. An in vitro study implicates that humoral factors (predominantlyTNF- and TGF-β) released from mesangial cells are likelyto alter the glomerular permeability in the event of proteinuriaand tubulointerstitial injury in IgAN.     

Identification of post-transplant anti-{alpha}5(IV) collagen alloantibodies in X-linked Alport syndrome

X-linked Alport syndrome (AS) is a heritable disorder whichis associated with mutations in the type IV collagen 5(IV) chaingene (COL4AS) located on chromosome X. Following renal transplantation,an average of 6% of male AS patients develop anti-GBM nephritis.We studied the specificity of the antibodies against type IVcollagen in the serum of a patient with COL4A5 partial deletion.The specificity of these alloantibodies was determined againstcollagenasedigested GBM, as well as against recombinant noncollagenous(NCl) domains of the type IV collagen 1(IV)—6(IV) chainsexpressed in Escherichia coli. Immunoblotting and ELISA demonstratedthat these antibodies bound specifically to the NCl domain of5(IV) collagen. There was no binding to the NCl domain of theother chains, including the Goodpasture antigen. CompetitiveELISA confirmed the results obtained by ELISA and immunoblotting.This patient developed alloantibodies directed against antigenspresent in the grafted kidney, but absent from his Alport kidney.The pathogenesis of post-transplantation glomerulonephritisin the Alport patient studied is thus similar to that of Goodpasturesyndrome, with the exception that the pathogenic antibodiesare targeted to another chain of type IV collagen.