Ramesh Saxena*, Charlott Johansson, Per Bygren and Jorgen Wieslander
Department ofNephrology, University Hospital ofLund, S-221 85 Lund, Sweden
Introduction
Ultrafiltration of blood is perhaps the most impor-tant function of kidneys and is accomplished byglomeruli. Hence, the disorders of glomerularstructure and function constitute one of the majorproblems encountered in nephrology practice. Theterm glomerulonephritis (GN) denotes an inflam-matory process involving glomeruli and forms alarge and an important group of kidney diseases inman. Glomerulonephritis can occur as a primarydisease process involving kidney or can occur inassociation with systemic disorders. Studies inexperimental models as well as widespread use ofpercutaneous renal biopsy have uncovered to agreat extent, the spectrum of glomerulopathicprocesses and have provided new insights intovarious possible pathogenic mechanisms initiatingand propagating glomerular injury. Eventually,evidence has accumulated over the past few yearswhich suggests that autoimmunity is more widelyinvolved in the pathogenesis of nephritis thanpreviously realized. Consequently, a number ofauto-antigens in various renal disorders have beenidentified. Some of them are listed in Table I.However, the pathogenic role of most of theseantigens and of the antibodies against them has notyet been elucidated. Nevertheless, some of them
have proven to be important markers of diseaseactivity as well as indices to disease prognosis.Analysis of the autoantibodies can also aid in early,as well as differential, diagnosis of severalglomerulonephritides. In the present review,autoantibodies associated with variousglomerulonephritis in humans will be discussed.
The classical anti-GBM antibody-inducedglomerulonephritis
The classical example of antibody-mediated glo-merulonephritis is Goodpasture syndrome, a termcoined by Stanton and Tange in 1958' in recogni-tion of a patient described in 1919 by E.W.Goodpasture.2 This disorder consists of a triad offindings: pulmonary haemorrhage, glomerulo-nephritis and antibodies to the glomerular base-ment membrane (GBM). It typically affects youngindividuals but may appear at any age. Pulmonaryhaemorrhage may be severe and life threatening ormay be extremely mild and easily overlooked.Rapidly progressive glomerulonephritis is the mostcommon clinical feature of the syndrome.3'4
Light microscopic studies of renal biopsy mostoften reveal an extensive extracapillary prolifera-tion (crescents) in glomeruli while immunofluor-escence studies show typical linear deposits of IgGalong the GBM.3'4
Circulating anti-GBM antibodies are found inover 90% of patients if sera are examined early inthe course of the disease by sensitive immuno-assays.35 Anti-GBM antibodies can also be elutedfrom kidneys of such cases using appropriatetechniques.346 Most often the disease is progres-sive, occasionally fulminant, leading to irreversiblerenal failure and sometimes death despite activetreatment.
Traditionally the diagnosis of Goodpasture syn-drome is confirmed by the finding of linear depositsof immunoglobulins along the GBM on directimmunofluorescence examination.7 Although fair-ly simple, it is time consuming and difficulties ininterpretation can arise in relatively damaged tis-
Table I Auto-antigens in renal auto-immune disease in man
Disease Auto-antigen
Goodpasture syndrome NC1 domain of the a3 chain of collagen IVPrimary proliferative GN Entactin/nidogenIgA nephropathy IgA-fibronectin complexWegener's granulomatosis and c-ANCA antigen (Proteinase 3)
primary small vessel vasculitis p-ANCA antigen(leucocytic myeloperoxidase (MPO))
Idiopathic pauci-immune extracapillary MPO(crescentic) GN Proteinase 3Membranoproliferative GN (MPGN) C3 convertase of alternate complement
pathway (C3bBb)Anti-tubular basement membrane disease TBM antigen/sPost-streptococcal GN Proteoglycans, collagen IV and lamininSLE and other collagen vascular diseases DNA, entactin
sue.7 Moreover, the biopsy tissue may not beadequate on some occasions. This has led todevelopment of methods to estimate circulatinganti-GBM antibodies by RIA89 and ELISA.'0However, their specificity depends upon the type ofantigen used. Recently, there has been considerableprogress in identifying and characterizing theantigen involved in Goodpasture syndrome. It islocated at the non-collagenous (NC 1) globulardomain ofthe carboxyl terminal end ofthe a3 chainof type IV collagen in the GBM""2 (Figure 1).
Identification of the antigen involved in Good-pasture syndrome has prompted the developmentof a highly sensitive, specific and rapid ELISA forthe diagnosis of Goodpasture syndrome.5 Theassay can be completed in 30 minutes thus permit-ting early commencement of specific therapycomprising plasma exchange'3 or protein A immu-no-adsorption,'4"15 especially in severely ill patientsand contributing to regression in morbidity andmortality from the disease.
Other anti-GBM antibody-associatedglomerulonephntides
Although several glomerular antigens have beendescribed in experimental glomerulonephritis inanimals; such as 330K and 90K glycoproteins(gp330 and gp9O) in Heymann nephritis,'6"18 lamin-in and collagen IV in mercuric chloride-inducednephritis in rats" and laminin in murine model ofgraft versus host (GvH) disease,20 the role ofpossible antigenic determinants of the GBM, otherthan the Goodpasture antigen, in the pathogenesisof anti-GBM glomerulonephritis remains unde-fined.9,10,21
Lately, we discovered a middle-aged malepatient with a mild form of glomerulonephritis.Direct immunofluorescence examination of the
kidney biopsy specimen revealed weak linearimmune deposits along the GBM. The patientpossessed circulating antibodies directed againstthe NCl domain of type IV collagen (Figure 1) butnot to the a3 (IV) antigen (Goodpasture antigen).Instead, the antibodies reacted to another subunitin the NCl region namely ml (IV). This suggeststhat anti-NC1 antibodies that are not directedagainst the Goodpasture antigen a3 (IV) can beassociated with glomerulonephritis in humans.However, they are accompanied by trivial renaldamage and possibly lead to milder variants ofanti-GBM nephritis (Johansson, C. et al., inpreparation).
Recently, we observed that almost 40% of morethan 200 patients with different types of biopsy-verified glomerulonephritis possessed antibodiesdirected against antigens present in crude 6Mguanidine-HCI extract of GBM.22 Since the crudeextract contains a variety of glycoproteins, eachone of which could be a potential auto-antigen, itwas interesting to isolate these proteins and studythe fine specificity of antibodies against them. Inthis process we isolated entactin (nidogen),23-29 a150 kD dumbbell-shaped GBM glycoprotein thatreacted, in a definite pattern, with sera from severalpatients with glomerulonephritis in a renal biopsyseries.30Two distinct groups of patients with anti-
entactin antibodies were identified. The largergroup consisted of young patients (peak incidence18-30 years) with primary mesangio-proliferativeglomerulonephritis associated with significantproteinuria which responded poorly to steroids andimmunosuppressive treatment. The other groupcomprised middle-aged patients (peak incidence51 - 60 years) with glomerulonephritis secondary toSLE or SLE-like collagen vascular disease with amilder degree of proteinuria which was well res-ponsive to therapy.30 A highly significant correla-
tion was also observed between the presence ofcirculating anti-entactin antibodies and the deposi-tion of a corresponding class of immunoglobulinsalong the GBM on immunofluorescence examina-tion. However, most of these patients had granularimmune deposits along the GBM which wouldtraditionally classify them as having immune com-plex disease. Studies in experimental models ofautoimmune nephritis such as mercuric chloride-induced nephritis in Brown Norway rats'9'3 andthe murine model oflupus nephritis20'32 have shownthe presence of anti-GBM antibodies that aredeposited initially in a linear fashion along theGBM but gradually their pattern becomes granularprobably because of the reorganization of theantigen- antibody complexes. Such a phenomenon
possibly also exists in humans but will require earlyas well as sequential renal biopsies for its elucida-tion. Nevertheless, the observations in experimentalmodels implicate that anti-GBM glomerulonephri-tis can have granular immune deposits along theGBM, which is probably the case with patientspossessing circulating anti-entactin antibodies.To summarize, our observations suggest that
entactin may be involved in the pathogenesis ofcertain forms of glomerulonephritis (non-Good-pasture anti-GBM nephritis) in humans. As onegroup of patients with anti-entactin antibodiescomprises young individuals with relatively severeand progressive disease, identification of suchpatients may have diagnostic and therapeuticimplications.
Figure la Type IV collagen and the organization of GBM. Type IV collagen forms the backbone of GBM. Theclassical protomer of collagen IV consists of 2 distinct a helical chains, al (IV) (185 kD) and a2 (IV) (175 kD), whichassociate to form triple helical trimers. However, other a helical chains namely a3, a4 and ac5 are also present. Eachprotomer of type IV collagen consists of 3 domains: a 7S collagenous domain at the amino terminal region; the majorcollagenous domain in the middle region and a non-collagenous globular domain, NCI, at the carboxyl-terminalregion. Four collagen molecules are joined by disulphide bonds in the 7S terminal region, while two collagen moleculesare connected to each other at the NC1 region to give rise to a 'chicken wire' network. Other components of thebasement membrane (laminin, nidogen, proteoglycans) interact among themselves and with the collagen IV to give riseto a highly organized structure of the GBM. Moreover, they assist in the attachment of collagen IV to the adjacentepithelial and endothelial cells.
Figure lb The Goodpasture antigen. NC1 domain of the a3 chain contains the antigen involved in Goodpasturesyndrome. The NC1 domain appears as a hexamer upon collagenase digestion ofGBM and dissociates into monomersand dimers under denaturing conditions, thereby releasing the free 'Goodpasture antigen'.
Circulating IgA-fibronectin complexes in IgAnephropathy
IgA nephropathy is the most common form ofglomerulonephritis in the world, usually affectingyoung males in their second and third decades oflife. It is characterized by macroscopic haematuria,often recurrent, with or without proteinuria usuallyfollowing an episode of pharyngitis or upperrespiratory tract infection.3" Light microscopytypically reveals a varying degree of segmentalmesangial proliferation and sclerosis.34 Onimmunofluorescence examination, a characteristicdeposition ofIgA and C3 in the mesangial region isobserved.3335 Circulating immune complexes con-taining IgA have been well documented in thisdisorder.33'35'36 Recently, a study showed thatpatients with IgA nephropathy have circulatingIgA antibodies that react with structures commonto collagen I, II and IV." This binding was laterfound to be mediated by a collagen-binding site offibronectin which forms circulating complexes withIgA38 (Figure 2). Fibronectin (cold insoluble globu-lin) is present both as a circulating as well as aconnective tissue protein. It is composed of twomonomeric subunits (210 kD and 230 kD) joinedby a disulphide bridge. The N-terminal ends of thetwo subunits possess collagen binding domains.39The fibronectin binding sites on collagen I havebeen identified as al-CB7 and x2-CB3,5 fragmentsafter cyanogen bromide digestion.' By using theELISA method of Cederholm et al.37'3840 a recentstudy has reported a strong association of circu-
lating IgA-fibronectin aggregates with IgA neph-ropathy, Henoch Schonlein purpura and recurrentcrescentic IgA nephropathy in transplants.4' Simi-lar results were obtained by Peter et al. using adifferent modification of this assay system.42Although the pathogenic role of circulating
IgA-fibronectin complexes in IgA nephropathyhas not been established, the results obtained bythe aforementioned studies signify that measure-ment of circulating IgA-fibronectin complexes bysimple ELISA can serve as an important serologi-cal marker for the diagnosis and follow-up ofpatients with IgA nephropathy.
IgA
Fibronectin
Collagen
Figure 2 IgA-fibronectin complex. See text for explana-tion.
The association of ANCA with vasculitis andglomerulonephritis has opened new vistas to studypathogenic mechanisms involved in glomerulo-nephritis. These auto-antibodies which are directedagainst the constituents of primary (a or azuro-philic) granules of human neutrophils and mono-cyte lysosomes (ANCA) were first described inpatients with necrotizing glomerulonephritis andclinical evidence of primary small vessel vasculitisin 1982.43 The auto-antibodies were detected byindirect immunofluorescence staining of ethanol-fixed normal human neutrophils. Since then, twomain fluorescence patterns have been characteriz-ed. One is a distinct cytoplasmic staining withaccentuation at the centre (c-ANCA) while theother is a peripheral perinuclear or nuclear staining(p-ANCA).4 c-ANCA and p-ANCA react withneutrophils and monocytes but not with maturemacrophages, lymphocytes or eosinophils.The c-ANCA antigen is a 29 kD protein present
in a granules and lysosomes of neutrophils andmonocytes respectively.45 N-terminal sequencingof this 29 kD antigen has revealed that this targetantigen is proteinase 3 (PR 3), a recently describedhuman leukocytic serine protease.' It is identicalwith myeloblastin, a serine protease that is presentin promyelocyte-like leukemia cell line HL-60.47The p-ANCA antigen is, in 80-90% of cases,
found to be the enzyme myeloperoxidase (MPO)."8'49However, p-ANCA is not synonymous with MPO-ANCA. It has been observed that a few patientswith p-ANCA have antibodies to other consti-tuents of a granules, mainly human leukocyteelastase (HLE).48'49 It should, however, be notedthat perinuclear fluorescence of ethanol-fixed neu-trophils observed with p-ANCA is an artefact anddisappears when neutrophils are fixed with for-malin. Sera from certain patients with collagenvascular diseases, like Felty's syndrome, have per-sistent perinuclear fluorescent pattern with forma-lin-fixed neutrophils. This pattern is referred to asGS-ANA (granulocyte specific anti-nuclear anti-bodies).ANCA are mainly associated with idiopathic
small vessel vasculitis with or without granuloma(Wegener's granulomatosis and microscopic poly-arteritis respectively) and primary pauci-immune,non-linear extra-capillary GN.49`52 It has beenobserved that c-ANCA is mainly associated withmicrovasculitides with widespread systemicinvolvement whereas most patients with renalrestricted idiopathic extracapillary GN havep-ANCA.49 52Although pathogenicity ofANCA has not been
proven so far, several experimental models suggesta pathophysiological role of these antibodies.53 It
has been shown that activated neutrophils areinvolved in the pathophysiology of necrotizingglomerulonephritis by releasing proteolytic enzymesand free oxygen radicals.54 However, under normalcircumstances, these proteolytic enzymes are in-activated rapidly by protease inhibitors present inblood. It is assumed that ANCA form complexeswith lysosomal enzymes and protect them fromproteolytic degradation. The complexes might thenbe transported to other areas such as kidney andtrapped there. If proteinases in these complexesretain their activity, they might then cause localdamage.53'54
Analysis ofANCA has proven to be of immensevalue in early diagnosis of patients with rapidlyprogressive GN and GN associated with systemicvasculitis. As in the case with Goodpasture synd-rome, early diagnosis of ANCA-associated dis-orders can vastly improve the outcome of theseotherwise rapidly progressive medical emergentconditions.
Analysis of ANCA also facilitates differentialdiagnosis and sub-classification of patients withextracapillary GN. This is exemplified by 16 caseswho presented with haemoptysis and rapidly pro-gressive glomerulonephritis (clinically as Goodpas-ture syndrome). In fact, a majority of them (9patients) did not have anti-GBM (anti-NC1) anti-bodies but had ANCA. These findings have clinicalimplications since anti-NC1 antibodies are assoc-iated with more severe disease and require moreintensive management than those with ANCA.5'
Auto-antibodies against C3b convertase inglomerulonephritis
IgG auto-antibodies to C3 convertase of the alter-native complement pathway (C3bBb) have beendiscovered recently in several patients with hypo-complementemic membranoproliferative GN(MPGN). These autoantibodies are called C3nephritic factor (C3NeF).5557 C3NeF stabilizes thelabile C3bBb from inactivation, thus resulting in anincreased degradation of C3.55-57 This in turnresults in depression of haemolytic complementactivity of the serum accompanied by low C3 levelsin patients with MPGN. However, C3 levels can benormal when C3NeF is present in a high concentra-tion and vice versa.55-57Not all patients with MPGN possess C3NeF. It
is more common in patients with type II MPGNthan in patients with type 1.55,57.58 It has also beenfound in some cases of secondary MPGN particu-larly in patients with SLE, shunt nephritis andidiopathic cryoglobulinemia.55 There is no directrelationship between the levels ofC3NeF and renaldamage nor is there any correlation between thepresence of C3NeF and disease activity.55,57,58
The role of C3NeF in the pathogenesis ofMPGN is questionable and it should only beconsidered as a marker of a subset ofMPGN. 55'57'58
Other auto-antibodies in human nephritis
Auto-antibodies in post-streptococcalglomerulonephritis
Recently, Fillit et al.59 demonstrated that sera frompatients with acute post-streptococcal GN (APSGN)contain antibodies directed against heparan sulphateproteoglycans oftheGBM. Kefalides et al.%extendedthese observations by showing that these sera notonly react with heparan sulphate proteoglycans butalso with laminin and collagenase-resistant frag-ment of type IV collagen corresponding to the 7Sregion (Figure 1). No reaction was observedagainst fibronectin. They suggested that tissueinjury occurring in post-streptococcal GN canrelease antigen fragments from critical tissueregions into circulation, leading to development ofspecific auto-antibodies. However, another possi-bility remains that auto-antibodies in post-strep-tococcal glomerulonephritis are developed againstcross-reactive determinants shared by streptoco-ccus and basement membrane antigens. Thesecross-reactive determinants need necessarily not beproteins but may also represent cross-reactivecarbohydrate epitopes.5960 However, the patho-genic or diagnostic significance of these autoanti-bodies in APSGN remains unexplored.
Streptococcal neuraminidase activity has alsobeen held responsible for development of auto-immune reactivity in APSGN.6' Neuraminidasereacts with sialic acid-rich sites found on immuno-globulins and glomerular capillary epithelial andendothelial cells.61~" Sialic acid depletion fromthese sites may evoke autoimmune response result-ing in formation of anti-immunoglobulins (rheu-matoid factors) and possibly, antibodies againstyet unidentified glomerular antigens.6'65
Auto-antibodies in tubulointerstitial nephritis
Tubulointerstitial nephritis (TIN) in humansusually occurs as a secondary process followingglomerular or vascular disorders but can also occuras a primary process in a minority ofcases. Most ofthe TIN probably have an immunological basisregardless of the inciting event.' Three differentpatterns of TIN have been identified on renalbiopsy.' One is characterized by cellular infiltra-tion with a negative immunofluorescence pattern.The second is similar to the previous one exceptthat immunofluorescence shows granular immunedeposits along the tubular basement membrane(TBM). The third pattern also resembles the first
except that a linear immunofluorescence along theTBM is observed. The third form is called anti-TBM disease and is presumed to be associated withantibodies directed against TBM antigens. Althoughseveral TBM antigens have been isolated by differ-ent workers, like 70 kD antigen by Graindorge andMahieu,67 58 kD antigen by Fliger et al.,68'69 48 kDantigen by Clayman et al.70 and 30 kD antigen byWakashin et al.,7' the immunopathogenesis ofanti-TBM nephritis remains incompletely under-stood.
Auto-antibodies in lupus nephritis
Nephritis associated with SLE and other relatedcollagen vascular diseases is a well-known entityand is perceived to arise from autoimmune mecha-nisms.72'" SLE has been associated with variousauto-antibodies, especially anti-nuclear antibodies(ANA) and particularly anti-DNA antibodies.72'73However, the pathogenic role ofANA, anti-DNAantibodies or DNA-anti-DNA immune complexesin lupus nephritis is widely disputed. Several recentexperiments have suggested that anti-DNA anti-bodies probably do not have pathogenic signifi-cance in lupus nephritis.74-" Furthermore, inmurine models of GvH disease, which resemblesSLE, it was observed that antibodies were deposit-ed along theGBM in a linear pattern which later onchanged to a granular arrangement. 20'32'78 Thelinear phase corresponded to the presence ofanti-GBM (anti-laminin) antibodies whereas thegranular phase with anti-RTE (renal tubularepithelial antigen) antibodies in the kidneyeluates.32'78 This, along with our observations ofthepresence of anti-entactin antibodies in SLE,30 sug-gests the possible pathogenic role of anti-GBMantibodies in lupus nephritis.
Other probable auto-immune glomerulonephritis inhumans
Membranous glomerulonephritis
The autoimmune nature of human membranousglomerulonephritis (MN) has been debated strong-ly over the past few years. Most discussions arebased upon the findings in Heymann nephritiswhich is regarded as a unique experimental modelfor human MN. This model was first introduced byHeymann and Lund in 1951 by immunization ofrats with homologous kidney cortex homogenate.79The disease is non-inflammatory, associated withnephrotic syndrome and characterized by glome-rular aggregates localized exclusively to theepithelial side of the GBM, similar to the MN inman.80 The antigen involved is a glycoprotein witha molecular weight of 330 kD (gp 330) and is
Figure 3 An algorithm showing distribution of various auto-antibodies in primary and secondary glomerulo-nephritides.
present on the cell membrane of glomerularvisceral epithelial cells.'6"'7 In the early seventies, apassive form ofHeymann nephritis was introducedin rats by a single injection of heterologousantibodies directed against gp 330.81 Bagchus etal.'8 showed that eluates from rat kidneys withpassive Heymann nephritis contained antibodiesreacting with a glycoprotein with a molecularweight of 90 kD (gp 90), in addition to the anti-bodies directed against gp 330.Although similar auto-antigens or auto-anti-
bodies have not yet been identified, there is a strongsuspicion for the existence of such an antigen-antibody system in human MN.8283
Summary
Autoimmunity is now unequivocally regarded asthe predominant pathogenic process underlyingmost forms of primary and secondary glomerulo-nephritis in humans. Most of the investigations sofar have been focused upon humoral mechanisms.Consequently, the role of cell-mediated immunityin nephritis is still incompletely understood. None-theless, as a result of contemporary studies, anumber of previously unidentified auto-antibodies
in association with glomerulonephritis have beendiscovered. However, apart from anti-NC1 anti-bodies in the classical Goodpasture syndrome, theexact role of these auto-antibodies in the patho-genesis of glomerulonephritis yet remains un-defined. This fact, however, does not underminethe relevance of exploring these auto-antibodies.They have been of immense help in sub-classifyingglomerulonephritis previously thought homo-geneous (Figure 3). Besides, analysis of auto-antibodies has assisted tremendously in the earlydiagnosis of rapidly progressive glomerulone-phritis. This, in turn, has aided in early commence-ment of therapy thus contributing to regression inmorbidity and mortality resulting from these dis-orders. Moreover, investigation of these auto-antibodies is of enormous value for future studiesaimed at understanding the pathogenic mechan-isms involved in glomerulonephritis.
Acknowledgements
A part of this study was supported by grants fromSwedish Institute, Swedish Medical Research Council(MFR 16X-09487) and Medicinska Fakultetens Forsk-ningsanslag.
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