Distribution of the α1 and α2 chains of collagen IV and of collagens V and VI in Alport syndrome

Kidney International, Vol. 42 (1992), PP. 115—126

Distribution of the al and a2 chains of collagen IV and of collagens V and VI in Alport syndrome CLIFFORD E. KASHTAN and YOUNGKI KIM Department of Pediatrics, Division of Pediatric Nephrology, University of Minnesota Medical School, Minneapolis, Minnesota, USA

and renal functional deterioration typical of Alport syndrome are unknown. Characterization of the composition of the extrawith Alport syndrome to their distribution in normal glomeruli and cellular matrix of Alport glomeruli may provide some insight into this question. In this report we describe ectopic distribuglomeruli from patients with non-Alport renal diseases. aI(IV), a2(IV), collagen V and collagen VI are normally restricted to the mesangium tion of the al and a2 chains of collagen IV, and of collagen V and the subendothelial aspect of the glomerular basement membrane and collagen VI, in Alport glomeruli. Distribution of the a! and a2 chains of collagen IV and collagens V and VI in Alport syndrome. We compared the distribution of the a! and a2 chains of collagen IV and of collagens V and VI in glomeruli of males

(GBM). In contrast, these proteins were present throughout the entire width of the GBM in Alport glomeruli. These alterations were apparent in "early" Alport glomeruli, that is, those exhibiting minimal abnor-

Methods

malities by light microscopy, and they were further accentuated in

Tissues

sclerosing Alport glomeruli. Obsolescent Alport glomeruli, in which the capillary tuft had collapsed and few remaining cell nuclei were present, exhibited nearly complete loss of al(IV) and a2(IV), like obsolescent

Kidney specimens were obtained from six unrelated males aged 12 to 22 years in whom the diagnosis of Alport syndrome glomeruli in non-Alport diseased kidneys. However, the matrix of was established on the basis of clinical and pathologic findings, obsolescent Alport glomeruli stained intensely for collagen V and as previously described [16]. The transmission of the disease in collagen VI, while these collagen types were not prominent in obsolescent glomeruli of non-Alport diseased kidneys. These observations the family of each patient was consistent with inheritance of an suggest that the process of glomerulosclerosis in Alport kidneys has X-linked dominant mutation. In each case, monoclonal antibodattributes unique to this disease. It would also appear that mutations

affecting the Alport gene product have secondary effects on the ies directed against the a3(IV) and a4(IV) chains stained no

renal basement membranes, as previously described (Fig. 1)

distribution of other GBM constituents.

[17]. The tissues represent the spectrum of the natural history of

Alport syndrome is a genetic renal disorder characterized by

the Alport renal lesion, from biopsies obtained early in the course of the disease when glomerular filtration rate was unimpaired, to nephrectomy specimens from dialysis-depen-

a progressive glomerulopathy that is often associated with dent patients undergoing preparation for renal transplantation. sensorineural deafness and ocular lesions [1]. The disease is Figure 2 depicts the range of glomerular alterations observed usually transmitted as an X-linked dominant trait [2]. Recently, mutations in an X-chromosomal gene, COL4A5, that encodes the a5 chain of collagen IV were found in several kindreds with Alport syndrome [3—8].

Males with Alport syndrome exhibit hematuria early in life, and develop increasing proteinuria and impairment of glomerular filtration with age [9]. Their glomerular basement membranes (GBM) are initially thin but undergo progressive thickening with time [10]. The homogeneous lamina densa of the normal GBM is replaced by multiple, interlacing strands of lamina densa-like material, which surround electron-dense bodies of varying size [11—131. Alport GBM does not bind antiGBM autoantibodies from patients with Goodpasture syndrome [14]. This abnormality appears to result from the absence of the a3 and a4 chains of collagen IV from Alport GBM [15]. The mechanisms that drive the progressive GBM thickening Received for publication May 22, 1991 and in revised form December 26, 1991 Accepted for publication February 6, 1992.

© 1992 by the International Society of Nephrology

by light microscopy. We defined three "stages" of glomerular

alteration for the purposes of this study: early, in which glomeruli exhibited normal cellularity and mildly increased mesangial matrix; scierosing, in which hypercellularity, increased mesangial matrix and/or hyalinosis, and periglomerular fibrosis were apparent; and obsolescent, characterized by collapsed capillary tufts with markedly decreased cellularity. The non-Alport diseased kidneys studied were obtained from

patients with diabetic nephropathy (2 patients), dense intramembranous deposit disease (DIDD; membranoproliferative glomerulonephritis, type 11—2 patients), idiopathic focal segmental glomerulosclerosis (FSGS, 1 patient), obstructive uropathy (1 patient), and refiux nephropathy (1 patient). Normal human kidney tissue consisted of uninvolved parenchyma in nephrectomy specimens from a 41-year-old male with a fibrous histiocytoma and a 29-year-old male with a germ cell tumor.

Antibodies The rat monoclonal antibody 102 is directed against the NCI domain of the al chain of collagen IV [18]. The sheep antibody anti-M24 identifies the NC 1 domain of the a2 chain of collagen 115

116

Kashtan and Kim: Collagen IV, V and VI in Alport glomeruli

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Fig. 1. lm,nunofluorescence of normal (A, C) and A/port glomeruli (B, D), stained with monoclonal antibodies against a3(IV) NCJ (A, B) and a4(IV) NCJ (C, D). These antibodies bind to GBM of normal glomeruli but not to Alport GBM. (x 500)

IV [18]. These antibodies were provided by Dr. Ralph J. South San Francisco, California, USA). All secondary antibodButkowski. ies were absorbed with normal human serum. Goat anti-collagen V was purchased from Southern Biotechnology Associates (Binningham, Alabama, USA). The antibody Immunohistologic methods

was raised against collagen V extracted from placenta, and reacts with both human and bovine collagen V. According to the manufacturer, the antibody was affinity-purified by cross-

Immunofluorescence was performed using previously described methods [161. Kidney tissues were snap-frozen in

isopentane pre-cooled in liquid nitrogen and stored at —70°C until sectioning. Cryostat sections were made using a Lipshaw cryostat maintained at constant temperature (25°C) and humidity (30%), and fixed for 10 minutes in acetone (for nondenatured gen VI [19], was obtained from the Developmental Studies sections) or ethanol (for denatured sections). Ethanol-fixed Hybridoma Bank at the University of Iowa. The Hybridoma sections were denatured by exposure to 0.1 M glycine, 6 M urea Bank is maintained by a contract from NICHD (NO1-HD-6- (pH 3.5) for one hour at 4°C, as previously described [16]. This 2915). The antibody was provided to the Hybridoma Bank by procedure was necessary for optimal visualization of epitopes Dr. Eva Engvall. reactive with the antibody against a2(IV) NC!. Sections were incubated with primary antibodies followed by Secondary antibodies consisted of fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG (Pel-Freeze, Rogers, Ar- FITC-conjugated secondary antibodies. Controls consisted of kansas, USA), FITC-conjugated rabbit anti-sheep IgG (Pci- sections incubated with secondary antibodies alone. All secFreeze), and FITC-conjugated goat anti-mouse IgG (Caltag, tions were incubated in ethidium bromide to demonstrate cell absorption against collagens I, II, III, IV and VI and by specific absorption to collagen V. Specificity for collagen V was confirmed by indirect ELISA, again by the manufacturer. The mouse monoclonal antibody 5C6, which identifies colla-

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Kashtan and Kim: Collagen IV, V and VI in A/port glomeruli

Fig. 2. Light microscopy of Alport glomeruli representing three stages of progression. (PAS, x 300) A. Early: There is segmental mesangial

hypercellularity, and the width of the mesangium is mildly increased. B. Scierosing: Cellularity is increased, with mesangial expansion, segmental hyalinosis, and periglomerular fibrosis. C. Obsolescent: The glomerular capillary tuft is collapsed and wrinkled, and there are few remaining cell nuclei.

nuclei, and p-phenylenediamene to retard fluorescence quenching [201. The sections were examined using an epifluorescence microscope with appropriate filters (Zeiss, Oberkochen, Germany). Results

except within the thickened Bowman's capsule (Fig. 7B), although intense GBM staining for a3(IV) was observed. Spec-

imens from patients with obstructive uropathy or reflux nephropathy did not visibly differ from the FSGS kidney in staining for al(IV) and a2(IV).

Collagen V Glomeruli from normal kidneys exhibited staining for collaconfined to the mesangium and the subendothelial region of the gen V in the mesangium and in the subendothelial region of the GBM (Fig. 3A, 4A and 5A). In Alport glomeruli showing GBM (Fig. 8A). Early Alport glomeruli also exhibited mesanminimal histologic abnormalities ("early" Alport glomeruli) gial collagen V. Rather than being restricted to the subendoal(IV) and a2(IV) were present throughout the width of the thelial region of the GBM in these glomeruli, collagen V was GBM, as well as in the mesangium (Fig. 3B, 4B and SB). A distributed throughout the width of the GBM (Fig. 8B). This similar distribution of al(IV) and a2(IV) was observed in Alport distribution pattern persisted in sclerosing Alport glomeruli glomeruli showing more advanced histologic changes ("scieros- (Fig. 8C). Obsolescent Alport glomeruli stained intensely for ing" Alport glomeruli) as shown for al(IV) in Figure 3C. GBM collagen V, with the reactivity concentrated in thick, ropy of obsolescent Alport glomeruli showed minimal staining for collections of extracellular matrix (Fig. 8D). al(IV) and a2(IV) (Fig. 3D). The expanded mesangial regions of glomeruli from patients As Kim et al have previously shown, al(IV) and a2(IV) with diabetic glomerulopathy or DIDD showed strong staining disappear from GBM during the course of diabetic nephropathy for collagen V (Fig. 9A, 9C). However, GBM in these glomeruli [211 (Fig. 6A). Likewise, the GBM of DIDD kidneys shows no did not stain for collagen V. Obsolescent glomeruli in these staining for al(IV), although there is reactivity in the expanded kidneys exhibited residual staining for collagen V in mesangial mesangium and in material within Bowman's space (Fig. 6B). regions, but no staining of GBM (Fig. 9B and D). Obsolescent glomeruli in a kidney from a patient with idioObsolescent glomeruli in a kidney from a patient with idiopathic FSGS showed virtually no staining for al(IV) or a2(IV), pathic FSGS exhibited staining for collagen V which was

al and a2 Chains of collagen IV In glomeruli from normal kidneys al(IV) and a2(IV) were

118

Kashtan and Kim: Collagen IV, V and VI in A/port glomeruli

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Fig. 3. Immunofluorescence of normal and A/port g/omeruli stained with monoclonal antibody (mAb) 102, which identifies al(IV) Nd. (X 500) A. Normal: al(IV) is present in the mesangium and the subendothelial region of the glomerular basement membrane (GBM) and is also present in Bowman's capsule, tubular basement membranes (TBM), and peritubular capillary basement membranes. B. Early Alport: al(IV) is present throughout the width of the GBM. C. Selerosing Alport: Staining of diffusely expanded mesangium and the entire width of the thickened GBM is present. Periglomerular and interstitial accumulation of al(IV) is also observed. U. Obsolescent Alport: Mesangial staining for al(IV) is virtually absent. There is residual reactivity along the collapsed capillaries; by phase-contrast microscopy this reactivity appears to localize to the epithelial aspect of the capillary wall. Substantial thickening of Bowman's capsule and TBM of atrophied tubules is observed, as in interstitial deposition of al(IV).

concentrated in material within Bowman's space (Fig. 7C). staining for collagen VI. Residual staining for collagen VI was Staining within the glomerular tuft itself appeared to be re- present in mesangial regions of obsolescent glomeruli in diastricted to the mesangium. The staining pattern clearly differed betic and DIDD kidneys (Fig. 11B and D). from that seen in obsolescent Alport glomeruli. Staining for Obsolescent FSGS glomeruli showed residual collagen VI collagen V in kidneys from patients with obstructive uropathy staining within the mesangium as well as in material in Bowor reflux nephropathy did not differ significantly from the FSGS man's space, but GBM was not stained (Fig. 7D). Glomeruli in kidney.

Collagen VI Glomerular staining for collagen VI was restricted to mesangium and subendothelial GBM in normal kidneys (Fig. bA). In

early Alport glomeruli, reactivity with anti-collagen VI antibody was present in mesangium and throughout the width of the

GBM (Fig. lOB). This staining pattern was preserved in sclerosing Alport glomeruli (Fig. bC). The pattern of collagen VI staining in obsolescent Alport glomeruli resembled that seen for collagen V, with staining concentrated in ropy collections of extracellular matrix (Fig. 1OD). In both diabetic and DIDD glomeruli the expanded mesangial regions reacted strongly with the anti-collagen VI antibody (Fig. 1 1A and C). However, GBM in these glomeruli showed no

kidneys of patients with obstructive uropathy or reflux nephropathy resembled the FSGS kidney in collagen VI staining.

Discussion

The extracellular matrix of the glomeruli mesangium and basement membrane (GBM) is composed of several collagenous constituents, including collagen types IV, V and VI. Five distinct collagen IV a chains have been identified. The amino acid and nucleotide sequences of al(IV) and a2(IV) have been determined, and the genes encoding these chains mapped to chromosome 13 [22—271. These chains are present in the gbmerular mesangium and in the subendothelial region of the GBM, in a completely concordant distribution [19, 28]. At this time only partial amino acid sequences for the a3(IV) and

Kashian and Kim: Collagen IV, V and VI in Alport glomeruli

119

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Fig. 4. Enlargements of panels A and B in Figure 3. The enhanced staining of Alport GBM (B) for al(IV) NC! is evident.

a4(IV) chains have been reported, and the bovine and human

the Alport antigen is present but the a3(IV) and a(IV) chains are a3(IV) genes have been partially sequenced [29—33]. In contrast not [17]. to al(IV) and a2(IV), these chains are absent from the mesanCollagen V molecules are trimeric in composition, consisting

gium and are associated with the lamina densa region of the of al(V) and a2(V) chains in most tissues, while the placenta, GBM [19, 28, 34]. The gene encoding a fifth collagen IV a chain among other tissues, appears to include an a3(V) chain as well has been cloned and mapped to the X chromosome [3—6]. [38—40]. In the kidney, collagen V has been localized to the Mutations in this gene have been found in affected members of mesangium, GBM and renal interstitium [41, 42]. three kindreds with Alport syndrome [7, 8]. Although an Collagen VI monomers are also composed of three chains, antibody generated against a synthetic a5(IV) peptide was the genes for which have been mapped to chromosomes 21 (al reported to stain GBM by indirect immunofluorescence, de- and a2 chains) and chromosome 2 (a3 chain) [43]. The collagen tailed information on the distribution of this chain in extracelVI monomer consists of a large globular domain at each end lular matrix has not yet been reported [3]. Studies in our laboratory using an anti-GBM alloantibody separated by a short triple helical region. These monomers from an Alport patient who developed anti-GBM nephritis after assemble into tetramers which associate in an end-to-end fashrenal transplantation and a monoclonal antibody have identified ion to form microfibrils [43]. Collagen VI has been demona normal basement membrane epitope, absence of which has strated in glomerular mesangium and GBM as well as in the proven to be a highly discriminating marker for Alport syn- renal interstitium [20, 44]. The structural alterations that characterize glomerulosclerodrome [16, 17, 35]. This "Alport antigen" has been mapped to a 26 kD collagenase-resistant peptide representing the carboxy- sis are accompanied by changes in the distribution of extracelterminal globular domain of a basement membrane collagen a lular matrix proteins within the glomerulus [45—49]. Studies of chain [16, 36, 37]. Whether this a chain corresponds to the a5 sclerosing and obsolescent glomeruli in kidneys of patients with chain of collagen IV, or is in fact a distinct chain, has not been diabetic nephropathy and membranous nephropathy have indiestablished. This molecule is also associated with the lamina cated that the a3 and a4 chains of collagen IV persist in GBM densa region of the GBM [17]. In general it is codistributed with during glomerulosclerosis [18, 50]. Similarly, the "Alport antithe a3(IV) and a4(IV) chains, except in certain basement gen," which has yet to be firmly identified, persists in the membranes, such as epidermal basement membrane, in which collapsed GBM of obsolescent glomeruli. On the other hand,

120

Kashtan and Kim: Collagen IV, V and VI in Alport glorneruli

Fig. 5. Immunofluorescence of normal (A) and Alport (B) glomeruli stained for ce2(IV) NC]. Mesangium and subendothelial GBM in the normal glomerulus are stained, while the entire width of Alport GBM (arrows) is stained. (x 460)

the al and a2 chains of collagen IV disappear from the GBM of persist as the glomerular tufts collapse into balls of matrix. Gubler et al found a normal distribution of collagen IV, or Because of previous observations by ourselves and others nonspecific degenerative changes, in glomeruli of children with that Alport GBM lacks a3(IV), a4(IV) and the Alport antigen Alport syndrome [52]. Thorner and colleagues described thick[17, 51, 52], we wondered how the biochemical aspects of ening of GBM staining for collagen IV in affected male dogs glomerulosclerosis in Alport kidneys and non-Alport diseased with Samoyed hereditary nephropathy, a disease with histokidneys might differ. The present study demonstrates unique pathologic and genetic similarities to Alport syndrome [53]. Alport syndrome is the only renal disorder in which we have alterations in the localization of al(IV), a2(IV), collagen V and collagen VI in GBM of Alport glomeruli. These collagens, observed such changes in al(IV), a2(IV), collagen V and collagen which are normally confined to the subendothelial region of VI. Since mesangial proliferation, mesangial matrix expansion and GBM, are found throughout the width of Alport GBM. It would GBM thickening are characteristic histologic features of Alport appear that with advancing sclerosis, al(IV) and a2(IV) disap- syndrome [54], we compared Alport glomeruli to glomeruli from pear from Alport GBM. However, the diffusely thickened GBM patients with diabetic nephropathy or dense intramenibranous of sclerosing and obsolescent Alport glomeruli appears to deposit disease (DIDD, membranoproliferative glomerulonephriprogressively accumulate collagen V and collagen VI, which tis, type II), diseases in which mesangial expansion and GBM diseased glomeruli.

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Kashtan and Kim: Collagen IV, V and VI in A/port glomeruli

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thickening also occur. Although the expanded mesangial regions

in diabetic and DIDD kidneys exhibited intense staining for

and dense intramembranous deposit disease (B) glomeruli, stained with antibody against cil(IV) Nd. Expanded mesangial regions of these glomeruli stain positively for al(IV) Nd, but GBMs (arrows) do not. Staining for a2(IV) NC I was not distinguishable from

staining for al(IV) Nd. (X

460)

mechanism(s) by which mutations in COL4A5 result in glomerulosclerosis and renal failure have yet to be established. Mod-

collagen V and collagen VI, the thickened GBMs in these kidneys ulation of gene transcription by extracellular matrix has been did not stain positively for these collagen types. We also compared demonstrated in vitro [55]. It is possible that changes in the Alport glomeruli to glomeruli from patients with focal segmental composition of Alport GBM initiated by defects in the a5(IV)

glomeruloscierosis of various etiologies, since the histological chain affect transcription of genes encoding matrix proteins, lesion of focal segmental glomerulosclerosis is a characteristic leading to enhanced synthesis and deposition of al(IV), ci2(IV), feature of the advanced Alport nephropathy [54]. While the Alport collagen V and collagen VI and perhaps other GBM constituGBM stained intensely for collagen V and collagen VI even in ents by glomerular endothelial cells and/or visceral epithelial advanced stages of sclerosis, histologically similar glomeruli from cells. GBM thickening and glomeruloscierosis in Alport kidneys patients with focal segmental glomerulosclerosis showed no GBM may reflect the accumulation of these products over time. staining for collagen V or collagen VI.

Acknowledgments The primary event in the pathogenesis of the Alport glomerulopathy is mutation in COL4A5, the gene encoding the aS This work was supported by the Variety Club and the Viking chain of collagen IV, as demonstrated by Barker et at [7}. The Children's Fund. The rat monoclonal antibody 102 and sheep antibody

122

Kashtan and Kim: Collagen IV, V and VI in A/port glomeruli

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FIg. 7. Jmmunofluorescence of obsolescent glomeruli in a nephrectomy specimen from a child with focal segmental glomerulosclerosis (X 500).

A. Anti-a3(IV) NC!: Note preservation of reactivity with collapsed, wrinkled GBM. B. Anti-a!(!V) NC!: There is minima! reactivity with the glomerular tuft. There is intense staining of the thickened Bowman's capsule and tubular basement membranes. Antibody against the a2(IV) Nd gave identical results. C. Anti-collagen V: There is persistent staining within the collapsed giomerular capillary tuft which appears to be localized to the mesangium and to Bowman's space (arrow). D. Anti-collagen VI: There is persistent glomeru!ar staining which appears confined to the mesangium.

anti-M24 used in this study were a gift from Ralph J. Butkowski, M.D. Dr. Jurgen Wieslander provided monoclonal antibody 17 against a3(!V) Nd, Monoclonal antibody 85 against a4(IV) NC! was provided by Dr.

Mary Kieppel. The authors wish to thank Crystal Blocher, Kathy Divine, Kim Pinkham and David Hurd for their assistance, and Alfred F. Michael for valuable advice. Reprint requests to Cltfford E. Kashtan, M.D., University of Minnesota Medical School, Department of Pediatrics, Box 491 UMHC, 515 Delaware Street, S.E., Minneapolis, Minnesota 55455, USA.

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Kashian and Kim: Collagen IV, V and VI in A/port glomeruli

123

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r

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Fig. 8. Immunofluorescence of normal and A/port glomeruli stained with anti-collagen V (X 500). A. Normal: Staining is predominantly

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Kashtan and Kim: Collagen IV, V and VI in Alport glomeruli

¼

t'

j

Fig. 9. Immunofluorescence of glomerulifrom patients with diabetic nephropathy (A and B) or dense intramembranous deposit disease (C and D), stained with anti-collagen V. Glomeruli in panels A and C exhibit mesangial expansion. Glomeruli in panels B and D are obsolescent (x 500), A and C. Staining for collagen V in the expanded mesangium is apparent. The thickened GBMs (arrows) are not stained. B and D. There is residual mesangial staining for collagen V, but no staining of GBM (arrows).

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Fig. 11. Immunofluorescence of glomeruli from patients with diabetic nephropathy (A and B) or dense intramembranous deposit disease (C and D), stained for collagen VI. Glomeruli in panels A and C exhibit mesangial expansion. Glomeruli in panels B and D are obsolescent (x 500). A and C. Expanded mesangial areas exhibit intense staining for collagen VI. The thickened GBMs show no reactivity (arrows). B and D. Obsolescent glomeruli show some residual staining for collagen VI. Compare with Figure 3D.

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