J Clin Pathol 1997;5O:919-922

Normal distribution of collagen IV in renalbasement membranes in Epstein's syndrome

Ichiro Naito, Shinsuke Nomura, Satoko Inoue, Megumi Kagawa, Shinichiro Kawai,Yoshimi Gunshin, Kensuke Joh, Chizuko Tsukidate, Yoshikazu Sado, Gengo Osawa

Divisions ofUltrastructuralBiology andImmunology, ShigeiMedical ResearchInstitute, Okayama,JapanI NaitoS InoueM KagawaY Sado

Nephrology Division,Department ofMedicine, KawasakiMedical School,Kurashiki, JapanS NomuraG Osawa

Division of InternalMedicine, ShigeiHospital, Kurashiki,JapanS Kawai

Department ofPediatrics, Faculty ofMedicine, UniversityofTokyo, Tokyo, JapanY Gunshin

Department ofPathology II, The JikeiUniversity School ofMedicine, Tokyo, JapanK Joh

Division of Pediatrics,Japanese Red CrossNagoya First Hospital,Nagoya, JapanC Tsukidate

Correspondence to:Dr Naito, Division ofUltrastructural Biology,Shigei Medical ResearchInstitute, 2117 Yamada,Okayama, 701-02 Japan.

Accepted for publication15 July 1997

AbstractBackground-Epstein's syndrome is de-fined as a subtype of Alport's syndrome,and is distinguished from the other sub-types by accompanying macrothrombocy-topenia. Mutations in collagen IV genesare known to be involved in the pathogen-esis of typical Alport's syndrome. How-ever, the presence ofan underlying geneticdefect has not been demonstrated inEpstein's syndrome.Aim-To clarify the involvement of colla-gen IV in Epstein's syndrome.Methods-The distribution of the a(IV)chain was studied in renal specimensobtained from three patients with Ep-stein's syndrome using chain specificmonoclonal antibodies and an antigenretrieval procedure.Results-The patients showed a normaldistribution of a(IV) chains: al(IV) anda2(IV) were expressed ubiquitously,whereas expression of a3(IV) through toa6(IV) chains was limited to the glomeru-lar basement membrane, Bowman's cap-sular basement membrane, and/or aportion of the tubular basement mem-brane.Conclusions-These results suggest thatgenes other than those encoding a(IV)chains are responsible for the pathogen-esis of Epstein's syndrome.(7 Clin Pathol 1997;50:919-922)

Keywords: Alport's syndrome; Epstein's syndrome;macrothrombocytopenia; collagen IV; deafness

Alport's syndrome is a progressive hereditaryrenal disease accompanying multiple abnor-malities including sensorineural deafness, ocu-lar abnormalities, macrothrombocytopenia,and diffuse leiomyomatosis.' These abnormali-ties vary in expression and severity amongfamilies and patients.' Epstein et al2 describedtwo unrelated families affected with hereditarynephritis, deafness, and macrothrombocyto-penia (Epstein's syndrome). This isdistinguished from other subtypes of Alport'ssyndrome by the presence of accompanyingmacrothrombocytopenia. Mutations in colla-gen IV genes are involved in the pathogenesisof X linked dominant3'5 and autosomal reces-sive Alport's syndrome,6 7and abnormalities inthe a(IV) chain distribution have been demon-strated using immunohistochemicalmethods.8'-0 Previously, we have studied all 51exons of the COL4A5 gene in two cases of

Epstein's syndrome, but no mutations werefound.4 This result does not belie the possibilitythat mutations in the COL4A5 gene mightaccount for the pathogenesis of Epstein'ssyndrome, because even in the X linkedAlport's syndrome, mutations in the COL4A5gene have been demonstrated in fewer than50% of patients.45 Epstein's syndrome isknown to be an autosomal dominant disorder,2which suggests the involvement of COL4A3 orCOL4A4, rather than COL4A5 in the patho-genesis of the disease.

Six different a(IV) chains have been identi-fied genetically and immunohistochemically.We prepared monoclonal antibodies specificfor the human a 1 (IV) through to a6(IV) chainsand used them for the immunohistochemicalanalysis of fresh frozen sections of humantissues." Antigen retrieval has extended theirapplication to paraffin embedded sections.'2 Acombination of monoclonal antibodies andantigen retrieval has enabled a retrospectivestudy of the ca(IV) chain in rare diseases. Toclarify the involvement of collagen IV in thepathogenesis of Epstein's syndrome, the distri-bution of all six a(IV) chains was determined inrenal specimens obtained from Epstein's syn-drome patients using these immunohisto-chemical procedures.

MethodsPATIENTSRenal specimens were obtained from threepatients from different families. The diagnosisof Epstein's syndrome was based on thepresence of a combination of nephritis, deaf-ness, and macrothrombocytopenia. However,none of the patients presented ocular abnor-malities.

Patient 1At 3 years old patient 1 had been diagnosedwith idiopathic thrombocytopenic purpura(ITP). Sensorineural hearing loss was recog-nised at 6 years of age. Giant platelets werefound on her peripheral blood smear. Thepatient was treated for ITP but her plateletcount remained in the range of 1-2 x 103/ml.Subsequently, the patient had proteinuria andmicrohaematuria. At the age of 16, she under-went a renal biopsy. Light microscopy revealedan almost normal glomerular shape and mildfibrosis in the interstitium, which containedfoam cells. Electron microscopy revealed re-ticulation of the lamina densa in the glomeru-lar basement membrane (fig 1).

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Figure 1 Electron micrograph of the glomerular basement membrane ofpatient 1.Reticulation (asterisk) and lamination (arrows) of the lamina densa are observed in thethickening glomerular basement membrane.

Patient 2Since 4 years of age, patient 2 had been treatedfor ITP, but her platelet count did not recover.

Proteinuria was detected at 8 years of age andthen increased to 2 g/day. She was found tohave thrombocytopenia (4 x 1031- x 104/ml)with large platelets. She showed moderate sen-

sorineural deafness but renal function was stillin the normal range. A renal biopsy was

performed at the age of 12. Mild mesangialproliferative glomerulonephritis, interstitial fi-brosis, and tubular atrophy were found.Electron microscopy revealed irregularity ofthe glomerular basement membrane and theelectron dense granules in the partially splitlamina densa of the glomerular basementmembrane.

Patient 3Patient 3 had a history of recurrent epistaxisfrom the age of 1 year. A bone marrow biopsywas performed at 3 years of age and he was

diagnosed as suffering from ITP. He showedproteinuria of 4+ and mild sensorineural hear-ing loss when 15 years old. One year later, heunderwent a renal biopsy. Light microscopyrevealed focal segmental sclerosis of theglomerulus and the expansion of the intersti-tium, which was infiltrated with lymphocytesand foam cells. Electron microscopy revealedthinning of the glomerular basement mem-

brane. Owing to renal dysfunction, he was

started on haemodialysis at 17 years of age. Atthe time of writing his platelet count was

1.7 x 104/ml.

All three patients satisfied three or four of the10 criteria for the diagnosis of Alport'ssyndrome proposed by Gregory et al.' No his-tory of hearing abnormality, renal abnormality,or macrothrombocytopenia existed in thefamilies and relatives of these three patients. All51 exons ofthe COL4A5 gene in patients 1 and2 have been studied previously; however, nomutations have been demonstrated.4

MONOCLONAL ANTIBODIESThe monoclonal antibodies used in the presentstudy were Hi 1 (anti-al), H22 (anti-a2), H31(anti-a3), H43 (anti-a4), H52 (anti-a5), andH63 (anti-a6).` They were prepared againstsynthetic peptides of non-consensus regions ofhuman a(IV) non-collagenous domains. Spe-cificity was confirmed using western blottingand epitope mapping.

IMMUNOHISTOCHEMISTRYParaffin wax embedded renal sections wereused for the analysis, although monoclonalantibodies are not very reactive with such sec-tions; antigen retrieval was used to make themethod more sensitive. The antigen retrievalprocedure was as follows: the sections wereheated in a small autoclave (KT2322, ALPCompany Ltd, Tokyo, Japan) at 1 10-127°C forsix minutes while they were immersed in 0.2 MHCl (pH 0.9).12 Subsequently, the monoclonalantibodies were applied to the sections. Boundmonoclonal antibodies were detected using theLSAB kit (Dako, California, USA). To avoidtechnical errors, immunostaining was per-formed at least twice on sections heated atmore than two different temperatures.

CONTROLSTwo renal specimens from patients withoutAlport's syndrome or Epstein's syndrome werestained according to the same method to act aspositive controls. The recovery of antigenicitywas poor in sections stored for extendedperiods. Renal specimens from patients 1, 2,and 3 were prepared between 1989 and 1994.Controls 1 and 2 were prepared in 1994 and1989, respectively.

ResultsCONTROLSA combination of chain specific monoclonalantibodies and antigen retrieval revealed welldemonstrated t 1 (IV) through to a6(IV) chainsin the renal sections of controls 1 and 2. al (IV)and a2(IV) chains were expressed ubiquitouslyin all basement membranes and mesangialmatrices, whereas the other a(IV) chainsshowed a more limited distribution. a3 (IV)and a4(IV) chains were coexpressed in theglomerular basement membrane and in a por-tion of tubular basement membrane. They alsoappeared in Bowman's capsular basementmembrane at a low intensity. The a55(IV) chainwas detected in the glomerular basementmembrane, together with the a3(IV) anda4(IV) chains. a5(IV) and a6(IV) chains weredetected in Bowman's capsular basementmembrane and a portion of the tubularbasement membrane.

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Collagen IV in Epstein 's syndrome

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Figure 2 Distribution of a(IV) chains in a renal sample from patient 1. (A) al1 (IV); (B) a2(IV; (C) a3(IV);

(D) a4(IVI); (E) a5(IV); (F) a6(IV~). All a(IV) chains are distributed in a normal pattern and show normal

antigenicity. al1 (IV) and a2(IV) chains are found in all renal basement membranes and in mesangial matrix. a3(IV) and

a4(IV) chains are detected in glomerular basement membrane, a portion of tubular basement membrane, and half of the

Bowman's capsular basement membrane. a5(IV) and a6(IV) chains are detected throughout Bowman's capsule basement

membrane and in a portion of tubular basement membrane. The a 5(IV) chain, but not the a6(IV) chain, is also detected

in the glomerular basement membrane.

PATIENTSAll three patients demonstrated significantpositive staining for all a(IV) chains in the renalbasement membranes. Figure 2 shows theresults obtained from patient 1. Antigenretrieval demonstrated positive staining ofal (IV) to a6(IV) chains, with the same

intensity and pattern of staining as seen in thecontrols. No significant abnormalities were

found in the Epstein's syndrome cases.

DiscussionAlport's syndrome is classified into severalsubtypes according to its inheritance forms andaccompanying symptoms. An X linked domi-nant form of Alport's syndrome has beenfound to result from mutations in the COL4A5gene, 5 and an autosomal recessive form hasbeen shown to result from mutations in theCOL4A3 and COL4A4 genes.67 Immunohis-tochemical studies of a(IV) chains has revealedcharacteristic abnormalities in a(IV) chain dis-tribution corresponding to the causative muta-tions in the collagen IV genes. Immunohisto-

chemical abnormalities of the a(IV) chains inX linked dominant Alport's syndrome havebeen well documented.8 9 11 12 In male patients,deletion mutations in COL4A5 result in thecomplete absence of a3(IV) to a6(IV) chains inrenal basement membranes,8 9 11 12 whereasmissense mutations (glycine substitution) re-

sult in a reduction in the antigenicity of thesechains.'2 In female patients, discontinuity of theantigenicity of a3 (IV) through to a5 (IV) chainshas been described.9 In an autosomal recessiveform of Alport's syndrome, immunohisto-chemistry has revealed a complete absence ofa3(IV) and a4(IV) chains associated with thepartial absence of a5 (IV) chains in theglomerular basement membrane."' In thepresent study, Epstein's patients demonstratednormal antigeriicity in all a(IV) chains in therenal basement membranes, raising thepossibility that genes other than those codingfor collagen IV are involved in the pathogenesisof the disease.Normal antigenicity of collagen IV has also

been reported in X linked'2 and autosomal

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recessive types'" of Alport's syndrome patients,but the incidence of such findings is very low.In contrast, normal antigenicity was recognisedin all three patients with Epstein's syndrome.Thus, the appearance of normal antigenicitysuggests that abnormalities of a glomerularbasement membrane component other thancollagen IV could be involved in the pathogen-esis of the disease.

Laminin, nidogen, and proteoglycans areexpressed in renal basement membranes.'3Laminin is a non-collagenous constituent ofbasement membranes that shows structuralheterogeneity as a result of differences in thesubunit chains. Investigation of the tissuelocalisation of these subunits has revealed thatsome are restricted to the glomerular basementmembrane. Nidogen is a glycoprotein that isdistributed abundantly in all basement mem-branes and has been found throughout theglomerular basemnt membrane. Heparan sul-phate proteoglycan is present in the glomerularbasement membrane, and has been found toplay an important role in the selective perme-ability of the glomerular basement membrane.Abnormalities in any of these glomerular base-ment membrane components can induce renaldysfunction, morphological abnormalities ofglomerular basement membrane, and possiblymacrothrombocytopenia. To identify the in-volvement of genes encoding these proteins,linkage studies must be performed using flank-ing marker genes.

In conclusion, to clarify the involvement ofcollagen IV in the pathogenesis of Epstein'ssyndrome we investigated the distribution ofthe a 1 (IV) through to ct6(IV) chains in renalsections from patients suffering from thissyndrome. However, immunohistochemistryrevealed no abnormalities associated withcollagen IV molecules.

This work was supported in part by a Grant in Aid for generalScientific Research (C) from the Japanese Ministry ofEducation, Science and Culture (07671270 and 08671312) andby a Research Project Grant from Kawasaki Medical School(No.6-102, No.7-103, No.8-102).

1 Gregory MC, Terreros DA, Barker DF, Fain PN, DenisonJC, Atkin CL. Alport syndrome-clinical phenotypes, inci-dence, and pathology. In: Trvggvason K, ed. Molecu/ar,pathologgy and genetics of Alport syndronie. ContribitiOns fornephrology, Basel: Karger, 1996;117: -28.

2 Epstein's CJ, Sahud MA, Piel CF, Goodman JR, BernfieldMR, Kushner JH, et al. Hereditary macrothrombocy-topathia, nephritis and deafness. An, _7 Med 1972;52:299-310.

3 Barker DF, Hostikka SL, Zhou J, Chow LT, Oliphant AR,Gerken SC, et al. Identification of mutations in theCOL4A5 collagen gene in Alport syndrome. Scienice 1990;248:1224-7.

4 Kawai S, Nomura S, Harano T, Harano K, Fukushima 17,Osawa G, et al. The COL4A5 gene in Japanese Alport syn-drome patients: spectrum of mutations of all exons. KidneyvInt 1996;49:814-22.

5 Renieri A, Bruttini M, Galli L, Zanelli P, Neri T, Rossetti S,et al. X-linked Alport svndrome: an SSCP-based mutationsurvey over all 51 exons of the COL4A5 gene. Am 7 HiiunanGenet 1996;S8:1192-204.

6 Mochizuki T, Lemmink HH, Mariyama M, Antignac C,Gubler MC, Pirson Y, et al. Identification of mutations inthe o3(IV) and o4(IV) collagen genes in autosomalrecessive Alport syndrome. Nat Genet 1994;8:77-82.

7 Lemmink HH, Mochizuki T, van den Heuvel LPWJ, Schro-der CH, Barrientos A, Monnes LAH, et al. Mutation in thetype IV collagen o3 (COL4A3) gene in autosomal recessiveAlport syndrome. Huni Mol Geniet 1994;3:1269-73.

8 Kashtan CE, Fish AF, Kleppel M, Yoshioka K, Michael AF.Nephritogenic antigen determinants in epidermal andrenal basement membranes of kindreds with Alport-tvpefamilial nephritis. _7 Clin Invest 1986;78: 1035-44.

9 Nakanishi K, Yoshikawa N, Iijima K, Kitagawa K,Nakamura H, Ito H, et al. Immunohistochemical study ofalpha 1-5 chains of type IV collagen in hereditary nephri-tis. Kidniey IJut 1994;46:1413-21.

10 Gubler MC, Knebelmann B, Beziau A, Broyer M, Pirson Y,Haddoum F, et al. Autosomal recessive Alport syndrome:immunohistochemical study of type IV collagen chain dis-tribution. Kidney Iiit 1995;47:1 142-7.

11 Sado Y, Kagawa M, Kishiro Y, Sugihara K, Naito I, SeverJM, et al. Establishment by rat lymph node method ofepitope-defined monoclonal antibodies recognizing the sixdifferent a chains ofhuman type IV collagen. HistochJeu, CellBiol 1995;104:267-75.

12 Naito I, Kawai S, Nomura S, Sado Y, Osawa G, et al. Rela-tionship between COL4A5 gene mutation and distributionof type IV collagen in male X linked Alport syndrome. Kid-nie.y Int 1996;S0:304-11.

13 Pihlajaniemi T. Molecular properties of the glomerularbasement membrane. In: Tryggvason K, ed. Moleciular,pathology and genetics of Alport syndronie. Contribuatiois fornephrology. Basel: Karger, 1996;117:46-79.

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