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Canadian Journal of Biochemistry

Journal canadien de biochimie

Published by

Publit par

THE NATIONAL RESEARCH COUNCIL OF CANADA

LE CONSEIL NATIONAL DE RECHERCHES D U CANADA

Volume 55 Number 12 December 1977

Volume 55 numCro 12 dCcembre 1977

Changes in renal glomerular basement membrane with age and nephritis1 N. KALANT, S. SATOMI, R . WHITE, A N D E. TEL Lady Davis Institute for Medical Research of the Jewish General Hospital, Montreal, P . Q . , Canada H3T IE2 Received March 1, 1977 Kalant, N., Satomi, S., White, R. & Tel., E. (1977) Changes in renal glomerular basement membrane with age and nephritis. Can. J. Biochem. 55,1197-1206 Glomerular basement membrane was obtained from normal, young and old adult rats and from animals with antiserum nephritis, daunomycin nephrosis, and lathyrism. With increased age there was an increase in the collagen content of whole glomeruli and of the basement membrane. About 50% of the membrane protein was solubilized in 1% sodium dodecyl sulfate, and a further 35% was solubilized by reduction and alkylation. Inhibition of formation of collagen cross-links by induction of lathyrism did not affect membrane solubility. Preparative disc gel electrophoresis permitted separation of a number of components of different composition; with decreasing molecular weight the collagen content declined from almost 100% to 0%. In antiserum nephritis, there was an increase in the noncollagen components of the membrane and marked alterations in amino acid composition, both of whole membrane and of electrophoretically separated components. In daunomycin nephrosis, the amino acid composition was similar to that of antiserum nephritis. The solubility of membrane from nephritic rats was normal. The composition of the insoluble residue was similar for all membrane preparations and resembled pure collagen. It is suggested that the presence of abnormal noncollagen proteins, associated with the insoluble collagen core by hydrophobic and disulfide bonds, as in antiserum nephritis, is associated with increased membrane permeability leading to proteinuria. Kalant, N . , Satomi, S., White, R. & Tel., E. (1977) Changes in renal glomerular basement membrane with age and nephritis. Can. J. Biochem. 55, 1197-1206 La membrane basale glomtrulaire est extraite de rats adultes normaux, jeunes et vieux et de rats atteints de ntphrite induite par un antisirum ntphrotoxique, de ntphrose induite par la daunomycine et de lathyrisme. A mesure que l'hge avance, la teneur du collagene des glomtrules entiers et de la membrane basale augmente. Environ 50% des prottines membranaires sont solubles dans le dodicyl sulfate de sodium a 1% et un autre 35% sont solubilistes par rtduction et alkylation. L'inhibition de la formation de liaisons transversales dans le collagene par induction du lathyrisme n'affecte pas la solubilitt des membranes. L'tlectrophorese prtparative sur disque de gel permet de stparer plusieurs constituants de composition difftrente; a mesure que le poids moltculaire diminue, la teneur du collagene dtcroit de presque 100% a 0%. Dans la ntphrite induite par l'antistrum, il y a augmentation des constituants non collagtniques de la membrane et des changements marquis dans la composition en acides amints et de la membrane globale et des constituants stparts par tlectrophorese. Dans la ntphrose induite par la daunomycine, la composition en acides amints est semblable a celle de la ntphrite causte par l'antistrum. La solubilitt des membranes des rats ntphritiques est normale. La composition du rtsidu insoluble est la mtme pour toutes les prtparations membranaires et elle resemble au

ABBREVIATIONS:GBM, glomerular basement membrane; OHLys, hydroxylysine; OHPro, Hydroxyproline; PAPN, P-aminopropionitrile; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; lipid-P, lipid phosphorus; glc, glucose; gal, galactose; NTS, nephrotoxic antiserum. 'Supported by a grant from the the Medical Research Council of Canada.

CAN. J . BIOCHEM. VOL. 55. 1977

collagene pur. La presence de proteines non collageniques anormales, associees au collagene insoluble par des liaisons hydrophobes et des liaisons disulfure, comme c'est le cas dans la nephrite induite par I'antiserum, pourrait etre reliee a une permeabilite membranaire accrue conduisant a la proteinurie. [Traduit par le journal]

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Introduction

The renal GBM, in all species examined, consists of collagen-like peptides2 bearing a disaccharide of glc and gal, and noncollagen peptides2 bearing an oligosaccharide of gal, mannose, fucose, sialic acid, and hexosamine. The structural relationship between these components is not established though several hypotheses have been formulated (1,2). It is clear that both glycoprotein and collagen of the native membrane are relatively insoluble, that some solubilization occurs with detergents (3), and that much of the protein is solubilized after reduction and alkylation of disulfide bonds (3, 4). While there is evidence that the collagen contains borohydridereducible cross-links, it is not known whether such bonds play a role in the stabilization of the GBM and in the determination of its permeability. There is an even greater lack of knowledge about structural changes in disease states, particularly membranous nephritis and diabetes, which may affect the morphology and function of the GBM. In experimental antiserum nephritis in the rat there is a change in the carbohydrate moiety of the glycoprotein as shown by a reduction in the sialic acid content (3,a reduction in the relative amount of collagen, as measured by the OHPro content (9,and a change in the x-ray diffraction pattern (6). Similarly, membranous nephritis in man is associated with a decrease in the sialic acid (7) and OHLys content of the GBM (7), and in the monosaccharides associated with collagen (8). Westberg and Michael (9) also noted a decrease in GBM OHPro. An alteration in the degree of glycosylation of the GBM collagen was reported to occur in human diabetes (lo), but this is now disputed (1 1, 12). The significance of these changes to the function and molecular structure of the GBM is not known. The purpose of the present work was to obtain further information on the relations between the collagen and the glycoprotein, and on the alterations which occur in experimental renal diseases associated with gross proteinuria. Materials and Methods Experimental Animals Male Sprague-Dawley rats were used throughout. NTS nephritis was induced by intravenous injections of rabbit antiserum against rat GBM obtained from adult male rats (13). For the acute stage, rats weighing 250-275 g were -

-

2For the sake of brevity collagen-like and noncollagen peptides will be referred to as collagen and glycoprotein respectively.

given antiserum and killed 2 weeks later; for the chronic stage, rats weighing 140-160 g were injected with antiserum and killed 7 weeks later. Unless specified otherwise, NTS results refer to the acute stage. Daunomycin nephrosis (14) was induced by a single intravenous injection of daunomycin, 12 mglkg body weight, into rats of 185-200 g; they were killed 6 weeks later. Lathyrism was produced by the addition of 0.1% PAPN to standard powdered laboratory diet for a period of 10 weeks beginning with rats weighing 125-150 g. All groups were thus 12-15 weeks of age at the time kidneys were obtained. Preparation of GBM Glomeruli were prepared from pools of 20-50 kidneys by the method of Gang and Kalant (13) with minor modifications: a 200 mesh rather than a 325 mesh stainless steel sieve was used in the last step to remove cellular debris, and the final centrifugation was for 25 min at 100 x g . Purity of glomerular preparations was routinely verified microscopically; all preparations were 92-96% pure glomeruli, free of Bowman's capsule. GBM was obtained as before (13); three to four periods of sonication for 30 s each were usually sufficient to disrupt all glomeruli. For the preparations of SDS, D T T , and residue fractions GBM was suspended (1 mglml) in 0.1 M sodium phosphate buffer pH 7 containing 1% SDS, incubated with shaking for 24 h at 37"C, then centrifuged at 1000 x g for 15 min. The insoluble material was washed twice with buffer solution and the washings were added to the original extract, which was then dialyzed for 48 h against distilled water, and lyophilized to constitute the ,CDS fraction. The SDSinsoluble material was suspended in 0.1 M sodium phosphate buffer pH 7 (1 mllmg of starting material), containing either 8 M urea or 1% SDS; D T T was added to provide a concentration of 0.02 M. The solution was shaken for 3 h at 37°C. A second addition of DTT, equal to the first, was made at that time (final D T T concentration, 0.04 M ) and the shaking was continued for another 3 h. The suspension was centrifuged at 100 x g for 15 min. The pellet was washed twice with phosphate buffer - urea or phosphate buffer - SDS and the washings were added to the first extract. This was dialyzed for 48 h against water and lyophilized giving the D T T fraction. The insoluble residue was washed several times with distilled water and lyophilized. Chemical Procedures Urinary protein was determined by a micromodification of a biuret method (15). OHPro measurement, lipid-P measurement, and amino acid analysis were performed as previously described (16). Specific enzymatic methods were used for the assay ofglc (17) and gal (18). Other procedures included measurement of DNA (19), of protein in GBM fractions (20), and of half-cystine as cysteic acid (21). Polyacrylamide Disc Gel Electrophoresis Polyacrylamide disc gel electrophoresis in SDS was performed by the method of Laemmli (22). Samples of GBM for gel electrophoresis were incubated for 6 h in 0.1 M

KALANT ET AL.

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phosphate buffer pH 7 containing 0.04 M DTT at 37°C. After removal of undissolved material by centrifugation, the solubilized protein was alkylated by the addition of recrystallized iodoacetic acid to a concentration of 0.12 M; the pH was maintained at about 9 by the addition of dilute NH,OH. The solution was dialyzed for 40 h against water and lyophilized for subsequent use. Analytical electrophoresis was performed in 7.5% gels at a constant current of 5 mAltube; gels were stained with Coomassie blue and destained by diffusion. For preparative electrophoresis an 18 x 80 mm gel was used with a current of 10 mA. At the end of the migration period the gel was removed, cut longitudinally into quarters, and stained for 16 h. It was then destained until the bands were clearly visible and sliced. The sections were lyophilized then hydrolyzed in 6 N HC1 in the presence of mercaptoethanol (23). Acrylamide was removed by centrifugation and extraction with ether (24).

Results Urine Protein Extraction Urinary protein was greatly increased in all animals given NTS or daunomycin (Table 1); PAPN did not cause proteinuria.

OHPro Content of Glomeruli It was assumed that all OHPro is contained in the collagen of the basement membrane and mesangial matrix and thus that measurement of glomerular OHPro is a reliable index of the amount of collagen in the GBM. Measurements of the OHPro were made in pools of normal glomeruli from rats of different ages and from NTS rats. In this and all subsequent results, each measurement was made on glomeruli or GBM obtained from a pool of 20-50 kidneys. It is clear (Fig. 1) that the OHPro content of the dried glomeruli increased with increasing body weight and that the NTS glomeruli contained about 25% less than weight-matched controls. The DNA content of dried, defatted glomeruli was constant at 56 + 1.5 pglmg for control rats of all weights. There was a 25% decrease in NTS rats to 42 + 2.6 pglmg ( P < 0.01); this fall was very similar in magnitude to the decrease in OHPro so that the OHPro: DNA ratio was unaltered i.e., 0.36 0.02for NTS nephritis and 0.33 + 0.01 for age-matched controls. Consequently, it may be concluded that; (a) with increasing body weight (i.e., age) there is an increase in the glomerular collagen, and (b) thickening of the GBM

I

I

I

100

200

I 300

BODY

I 400

I 500

WEIGHT ( g )

FIG. 1. Effect of age (body weight) and antiserum nephritis on OHPro content of dried, defatted, whole glomeruli. 0 ,normal; , antiserum nephritis.

in NTS nephritis (13) is not due to an increase in the amount of the collagen of the GBM. Amino Acid Composition of GBM As seen in Fig. 2 the OHPro content of GBM increased with age (body weight), though not so markedly as did that of whole glomeruli. The OHPro of GBM from NTS rats was significantly lower than that of age-matched controls; the lipid-P content of the GBM in these groups was 0.73 + 0.08 pglml and 1 .O1 + 0.07 pg/ml respectively. Amino acid analysis (Table 2) confirmed that young rats, in comparison with older rats, had a lower content of OHPro without a concomitant increase in proline; this, together with a slightly lower value for glycine, provides further evidence that the collagen content of GBM is lower in young than in old rats. The GBM of NTS rats also had a lower content of 3- and 4-OHPro (without a concomitant increase in proline), OHLys, and glycine than the controls.

+_

TABLE 1. Urinary protein excretion

Normal Antiserum nephritis Acute Chronic Daunomycin BAPN

No. of rats

Protein, mg/24 h

60

28+3

50 29 67 27

745 28 502 _+ 39 682 26 33f 2

+ +

100

200

300 BODY

400

500

WEIGHT ( g )

FIG.2. OHPro content of GBM with increasing body weight (age) and in nephritis. 0, normal; , antiserum nephritis.

CAN. J. BIOCHEM. VOL. 5 5 , 1977

TABLE2. Amino acid composition of whole GBM (residues/1000 residues) Normal

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4 0g (10)

175 g (3)

NTS Acute (6)

Chronic (6)

Daunomycin (4)

Hydroxylysine Lysine Histidine Arginine 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine NOTE:Values are means

SE. Significance of difference from normal (400 g); *P < 0.05 * * P < 0.005.

FIG.3. GIC(0)and gal (A) content of basement membrane as a function of OHPro content. Closed symbols, normal; open symbols, antiserum nephritis.

There were increases in a number of other amino acids, notably threonine, serine, alanine, valine, and tyrosine. The changes tended to revert toward normal in GBM from chronic NTS rats. Measurement of cysteine as cysteic acid gave values of 181 + 10 nmol/mg for normal GBM and 191 + 12 nmol/mg for NTS GBM. The GBM in daunomycin nephrosis showed essentially the same changes as in NTS nephritis. The glc and gal contents of the GBM were each

directly related to the content of OHPro, with a relatively constant excess of gal over glc. These relationships were unchanged in NTS GBM (Fig. 3). Disc gel electrophoresis of the reduced alkylated normal GBM showed numerous bands (Fig. 4) as previously described (25) for bovine GBM. The GBM in NTS nephritis, both acute and chronic (not shown), demonstrated an altered pattern with three major bands (Fig. 4, bands A, B, and C); two of these (bands A and C) were present as minor bands in the normal GBM, while the major one (band B) appeared to be new. The realtionship of molecular weight to mobility was determined with reduced protein standards (Fig. 5). The mobility of one of these bands (Fig. 4, band B) was close to that of native rat serum albumin; however, immunodiffusion tests failed to reveal precipitin lines between the soluble, reduced, and alkylated GBM and rabbit antiserum against whole rat serum (Fig. 6). To determine the nature of the three major bands, preparative electrophoresis was done on 10-mg samples of GBM; the gels were sliced, as shown in Fig. 7, and certain segments were analyzed for amino acid content. Arginine was excluded from the analysis because it was not completely separated from the trailing edge of the ammonia peak. The results are shown in Table 3. In the normal GBM preparation, fraction I (material remaining in the stacking gel) and fraction 2 were very similar in composition to pure collagen isolated from GBM of the dog (26), the steer (27), and man

\.\

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-

FIG. 4. Disc gel electrophoresis of GBM. Left, normal (400 g); right, antiserum nephritis. Protein (150 p g ) was

applied in each case; staining of the gel carrying GBM from nephritis rats was always more intense. The molecular weight scale is extrapolated from the mobility data of Fig. 5.

(28). With increasing electrophoretic mobility there was a progressive decrease in OHLys, OHPro, and glycine and an increase in aspartic acid, glutamic acid, and alanine. Fractions 1 and 2 of the NTS preparation had much less of the collagen amino acids than the normal material; the heavy bands, particularly 8 and 13 (corresponding to bands A and B in Fig. 4), differed from the corresponding normal

Phosphorylase A

\.\

Chymotrypsinogen A

2

I

0.2

I

0.4

I

0.6

I

0.8

I

1.0

RELATIVE MOBILITY

FIG.5. Relation of relative mobility to molecular weight of protein standards in disc gel electrophoresis.

fractions in many respects with regard to amino acid composition (Table 3). Solubility of GBM Attempts were made to solubilize GBM by the use of urea, detergents, and reduction of disulfide bonds with DTT in the presence of urea or SDS. The results are shown in Table 4. SDS or urea solubilized 40-50% of the GBM protein, somewhat more than reported for bovine GBM, while prior reduction with DTT increased solubility in SDS to 85-90%, about the same as that of bovine GBM (3). There were no distinct solubility differences among the various GBM preparations: young and old normal, NTS nephritis, and daunomycin nephritis. Fractionation of GBM Since a large portion of the GBM protein was solubilized by SDS and a further fraction became soluble after reduction and alkylation of disulfide bonds, an examination of these solubilized components was made. The amino acid analysis of the SDS fraction from normal GBM (Table 5) showed a relatively low content of amino acids characteristic of collagen; the DTT fraction was closer in composition to the whole GBM. The residue was similar to electrophoretic fractions 1 and 2 (above) in its high content of collagen-associated amino acids; however, it had less lysine, OHPro, and proline. The residue of 'young' normal GBM was somewhat lower in OHPro but otherwise almost identical to the normal residue. The SDS and DTT fractions of NTS GBM were relatively low in collagen and high in the same amino acids noted in the whole GBM; the residue, however, was similar in composition to normal residue. The glc: gal ratio was 0.94-1.02 in all residues; since glc is present only in the disaccharide, and only in association with gal, the absence of 'extra' gal indicates that virtually no oligosaccharide was present. Glycosylation of the OHLys in this fraction (as

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CAN. J. BIOCHEM. VOL. 55, 1977

FIG.6. Immunodiffusion plate showing absence of precipitin reaction between reduced, alkylated basement membrane from nephritic rats (GBM), and antiserum against rat serum protein (ARS). Native rat serum albumin (RSA) showed a positive reaction.

judged by the glc: OHLys molar ratio) was 60-75% complete. Effect of Lathyrism on GBM Since the residue after reduction and alkylation was very similar in composition to collagen, it seemed possible that its insolubility was due to borohydride-reducible intermolecular cross-links, even though the typical collagen periodicity is not seen in the GBM. This possibility was tested by examining the effect on the GBM of lathyrism produced by PAPN, which prevents the formation of collagen cross-links. In evidence of lathyrism was the increased neutral salt solubility of the tail tendon collagen; 135 pg OHProIg of tendon in control rats compared with 1502 pg/g in the treated rats. Solubility of GBM was not affected; it was 45% in 1% SDS and 88% after reduction and alkylation. The insoluble residue was very similar in amino acid composition to that of the other GBM preparations (data not shown). Discussion It has previously been shown that 1% SDS or 8 M urea can solubilize a portion of bovine and canine GBM and that reduction and alkylation in the presence of SDS or urea permit solubilization of the bulk of GBM protein (3, 4). The insoluble residue was found to have a high content of glycine and OHPro (29). Similarly, rat GBM has a moiety soluble in SDS and therefore bound by hydrophobic bonds, components soluble in SDS after reduction and alkylation and therefore bound by disulfide bonds, and an insoluble residue. GBM has been shown to contain borohydride-

reducible collagen cross-links (30). Scheinman et al. (31) have reported that ingestion of PAPN for 6 weeks led to an increase in acid-soluble OHPro of whole glomeruli; however, this material constituted only about 1% of the total glomerular OHPro. They found no change in urinary protein excretion. Our data based on animals fed pAPN for a longer period are in agreement in showing no alteration in proteinuria. However, in the present work, the lathyritic state, demonstrated by the greatly increased solubility of tendon collagen, was not associated with a detectable increase in the solubility of the GBM before or after reduction of disulfide bonds. It is therefore unlikely that borohydride-reducible bonds, including intermolecular cross-links of collagen, play a significant role in maintaining the insolubility of the GBM. The degree of GBM solubility in 8 M urea, 1% SDS, and 0.5% Triton X-100 was about twice that reported for bovine GBM (3); solubility in urea or SDS after reduction and alkylation was very similar to that of bovine GBM. Thus, the fraction of the rat GBM stabilized by hydrophobic bonds is greater, and the fraction stabilized by disulfide bonds correspondingly lower than in bovine GBM. This conclusion is supported by two additional observations: ( a ) Solubilization by chaotropic agents (2 M sodium perchlorate or 2 M sodium thiocyanate) was 22-23%, i.e., two to three times greater than that reported for human GBM (32), thus confirming the greater role of hydrophobic bonding in the rat membrane, and (b) the half-cystine content of rat GBM (measured as cysteic acid) was distinctly lower than that of bovine GBM (3). The SDS fraction was primarily noncollagenous,

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K A L A N T ET AL.

FIG.7. Preparative disc gel electrophoresis of basement membranes. Left, normal; right, antiserum nephritis. Protein (8 mg) was applied in each case. Some of the numbered segments were hydrolyzed and analyzed for amino acid composition (Table 3); others provided insufficient material. The heavily stained band near the bottom is a nonprotein artifact.

as had been reported for the comparable fraction of bovine GBM (3). The material, solubilized after reduction and alkylation, contained collagen and glycoprotein in about the same ratio as that of the whole GBM. The insolubility of this fraction may be due to disulfide bonding among the proteins within the fraction or to the residue proteins. This fraction

1203

cannot be compared directly with the reduced and alkylated fractions described by others (2, 3) since these were equivalent to a combination of the SDS and DTT fractions in the present report. The insoluble residue was largely collagen and was similar in amino acid composition to collagen extracted from GBM after proteolytic digestion (26-28). If it is assumed that the glycoprotein contains about 80 residues of glycine/1000 residues glycoprotein (34) and that collagen contains about 330 (26-28), then this fraction is almost 95% collagen. There were distinct differences in the OHLys: OHPro ratio of the various fractions derived by solubilization or electrophoresis from a single GBM preparation (Tables 3, 5), and between comparable fractions of different GBM preparations; this was least evident in the residue and most clearly seen in the DTT fraction. Such heterogeneity of collagen composition is similar to that described for lathyritic cartilage (34). Previous reports emphasized the constancy of the sum of OHLys and lysine and the reciprocal nature of changes in the amounts of these amino acids, presumably to indicate differences in the degree of hydroxylation of the lysine residues of collagen (35). Close examination of the data in those reports and in the present work (Tables 2, 5), however, reveals considerable variation in the sum of OHLys and lysine. This is not surprising, since changes in the relative amounts of collagen and noncollagen protein, which differ in their contents of both amino acids, would affect the total amount of these acids in a protein mixture even without a change in hydroxylation. The increase in the amount of collagen in the glomerulus with aging is due at least in part to an increase in the collagen content of the GBM. In young rats the bulk of the collagen is in the insoluble residue; most of the additional collagen associated with aging is found in the DTT fraction. Despite the changes in the amino acid composition, the function of the GBM did not change significantly since there was no evident increase in proteinuria. The induction of antiserum nephritis with severe proteinuria was associated with a decrease in the amount of OHProIunit weight of glomeruli; however, since the ratio of OHPro :DNA did not change, there must have been an increase in the noncollagen components of the glomeruli. The amino acid content of the insoluble residue and the normal ratio of glc or gal to OHPro (Fig. 3) suggest that the primary composition of the collagen components of the GBM was not altered. The increase in the glycoprotein components was reflected in a relative decrease in the collagen-associated amino acids of the GBM, particularly in the SDS and DTT fractions; presumably the 'extra' noncollagen material was in these two fractions.

1

*See Fig. 7. ?Measured as cysteic acid.

Hydroxylysine Lysine Histidine 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystinet Valine Isoleucine Leucine Tyrosine Phenylalanine

Component *

2

5 8

Normal 13

14

15

1

2 8

9

10

11

12

Antiserum nephritis 13

-

14

15

TABLE3. Amino acid composition of GBM components separated by polyacrylamide gel electrophoresis (residues/1000 residues)

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16

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KALANT ET AL.

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TABLE4. Solubility of rat GBM (% protein solubilized)

8 M urea in 0.1 M sodium phosphate pH 7, 37"C, 24 h 1% SDS in 0.1 M sodium phosphate pH 7, 37"C, 24 h 8 M urea in 0.1 M sodium phosphate pH 7, with 0.02 M DTT, 37"C, 24 h 1% SDS in 0.1 M sodium phosphate pH 7 with 0.02 M DTT, 37"C, 24 h

Normal

Antiserum nephrosis (acute)

Antiserum nephrosis Daunomycin (chronic) nephrosis

43 51

44 51

47 53

44 55

67

66

73

69

88

85

NOTE: Values represent the averages of three to six determinations.

TABLE5. Amino acid composition of GBM fractions (residues/1000 residues) Normal*

Young?

NTS

Whole SDS DTT Residue Whole SDS DTT Residue Whole SDS DTT Residue (10) (5) (5) (4) (3) (2) (1) (3) (6) (3) (2) (4) Hydroxylysine Lysine His tidine Arginine 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

6 Trace 66 79 23 21 56 55 Trace Trace 46 13 83 89 53 57 67 67 114 105 49 52 95 135 84 78 54 58 11 16 43 43 80 83 31 23 33 32

30 15 12 37 14 97 59 28 44 87 71 301 49 29 13 28 48 13 26

13 41 20 45 4 31 81 61 70 109 68 146 65 62 15 37 78 25 31

2 56 21 45 -

2 90 75 90 117 62 104 77 66 12 41 80 32 33

14 36 20 42 10 47 80 53 70 103 62 173 61 49 13 33 69 21 29

34 15 13 37 17 99 61 31 46 90 72 274 46 32 9 29 57 11 29

*Body weight, 385425 g. ?Body weight, 165-180 g.

The nature of the 'extra' material is not established but several possibilities must be considered: ( a ) contaminating plasma membrane of renal cells; ( b ) heterlogous and autologous immunoglobulins; and ( c ) trapped serum albumin. ( a ) Westberg and Michael (36)have demonstrated an inverse relationship between the content of OHPro and that of lipid-P in normal GBM preparations; a low OHPro with a correspondingly high lipid-P in a given preparation was interpreted to indicate contamination by the cellular plasma membrane. This is not the explanation for the low OHPro in the NTS GBM however, since the lipid-P content was slightly lower than that of the normal preparations. While both of these categories of protein are demonstrable in the GBM of NTS rats (13)' indirect evidence indicates that alone they probably cannot account for the altered amino acid composition (16).( b )Furthermore,

none of the major electrophoretic components had mobilities similar to that of y-globulin H or L chains. ( c ) Westberg and Michael (36) demonstrated the presence of albumin and other serum proteins in their preparations of normal human GBM; together with the similarity in electrophoretic mobility of rat serum albumin and one of the major bands of the NTS GBM this suggested the presence of substantial amounts of albumin in this GBM preparation. However, we believe that this is not the case, since ( i ) we could not demonstrate the presence of albumin by immunodiffusion and, (ii) the amino acid composition of the GBM component with a mobility similar to that of albumin was very different from that of rat serum albumin. The difference between the present results and those of Westberg and Michael (36)may be due to the fact that we were able to use fresh kidneys perfused in situ while the latter

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1206

CAN. J. BIOCHEM. VOL. 5 5 , 1977

authors studied unperfused autopsy material. We conclude, by exclusion, that the additional glycoprotein in GBM from NTS rats in abnormal and accounts for the altered composition of the electrophoretically separable components of the GBM. Daunomycin nephrosis was also associated with severe proteinuria and the GBM was similar in composition to that of NTS membrane in showing decreases in collagen-related amino acids and increases in aspartic acid, threonine, serine, and tyrosine. Nevertheless, there were some differences from NTS nephritis in amino acid composition and the gel electrophoretic pattern was not visibly abnormal. Thus, there does not appear to be a common membrane abnormality responsible for proteinuria in these two disease states. Our data (Table 3) are in accord with the finding by Sato and Spiro (35) of a large number of components, each consisting of a collagen-like (helical) portion and a glycoprotein (polar) portion but differing in the relative amounts of the two moieties. In general, we observed that with decreasing molecular weight there was a decrease in the amount of collagen. In addition, there was an insoluble 'core' which was almost entirely collagen. It is possible that the core consists of tropocollagen molecules joined by end-to-end interchain bonds to form a high molecular weight, insoluble structure; the presence of glycoprotein-collagen 'hybrids' interspersed between such elongated aggregates, joined by hydrophobic and disulfide bonds, normally prevents the quarter-stagger array, side-to-side bonding, and thus the typical collagen banding pattern. In some pathological states molecular rearrangement may permit apposition of the aggregates in the usual collagen pattern, so that typical fibrils are seen (37). However, in experimental antiserum nephritis the composition of the 'core' collagen is essentially unchanged, while the amount of noncollagenous protein increases. We suggest that this increase, consisting of abnormal proteins as demonstrated by gel electrophoresis or amino acid composition, alters the molecular arrangement of the membrane (6) so that permeability is increased. 1. Kefalides, N. A . (1972) Connect. Tissire Res. 1, 3-13 2. Spiro, R. G. (1973) N . Engl. J. Med. 288, 1337-1342 3. Hudson, B. G. & Spiro, R. G. (1972) J. Biol. Chem. 247,4229-4238 4. Kefalides, N. A . & Winzler, R. J. (1966) Biochemistry 5,702-713 5. Lui, S. & Kalant, N . (1973) Exp. Mol. Pathol. 21, 52-62

6. Kalant, N . , Misra, R. P.. Manley, R. St. J. & Wilson, J. (1966) Nephron 3. 167-172 7. Mahieu, P., Winand. R. J. & Nusgens, B. (1972) Ad\!. Nephrol. 2,25-34 8. Mahieu, P. (1972) Kidney Int. 1, 115- 123 9. Westberg, N. G. & Michael, A. F. (1973) Acta Med. Scand. 194,49-57 10. Beisswenger. P. J. & Spiro, R. G. (1970) Science 168, 596-598 11. Westberg, N. G. & Michael, A. F. (1973) Acta Med. Scand. 194, 39-47 12. Kefalides. N. A . (1974) J. Clin. Invest. 53,403-407 13. Gang, N. G . & Kalant, N. (1970) Lab. In~3est.22, 53 1-540 14. Sternberg, S. S . (1970) Lab. Invest. 23,39-51 15. Gornall, A . G., Bardawill, C. J . & David. M. M. (1949) J . Biol. Chem. 177,75 1-766 16. Fung, K . K . & Kalant, N . (1972) Biochem. J. 129, 1-9 17. Klotzsch, H. & Bergmeyer, H. B. (1965) in Methods yf Enzymatic Analysis (Bergmeyer, H. V., ed.), pp. 156- 159, Academic Press, London. 18. Hu. A . S. L. & Grant, S . ( 1968) Anal. Biochem. 25, 22 1-227 19. Croft, D. N. & Lubran, N . (1965) Biochem. J. 95, 6 12-620 20. Lowry, 0. H., Rosebrough, N . J . , Farr, A. L . & Randall, R. J . (1951) J. Biol. Chem. 193,265-275 21. Shram, E., Moore, S . & Bigwood, E. J. (1954) Biochem. J . 57,33-37 22. Laemmli, U. K . (1970) Nature (London)227,680-685 23. Houston, L. L . (197 1) Anal. Biochem. 44,81-88 24. Schwartz, M. L., Pizzo, S. V., Hill, R. L . & McKee, P. A , (1971) J. Biol. Chem. 246,5851-5854 25. Myers, C. & Bartlett, P. (1972) Bioc-him. Biophys. Acta 290. 150-157 26. Kefalides, N. A . (1968) Biochemistry 7, 3103-31 12 27. Daniels, J. R. & Chu, G. H. (1975) J. Biol. Chem. 250, 353 1-3537 28. Kefalides, N. A . (1971) Biochem. Biophys. Res. Cornmun. 45,226-234 29. Hudson, B. G . & Spiro, R. G. (1972) J. Biol. Chem. 247,4239-4247 30. Tanzer, M. L . & Kefalides, N . A. (1973) Biochem. Biophys. Res. Commirn 51,775-780 31. Scheinman, J. I., Brown, D. M., Vernier, R. L. & Michael, A. F . (1973) Proc. Soc. Exp. Biol. Med. 144, 753-758 32. Marquardt. H . , Wilson, C. B. & Dixon, F. J. (1973) Biochemistry 12, 3260-3266 33. Huang, F. & Kalant, N . (1968) Can. J. Biochem. 46. 1523- 1532 34. Trelstad, R. L., Kang, A. H . , Toole, B. P. & Fross, J. (1972) J. Biol. Chem. 247,6469-6473 35. Sato, T . & Spiro, R. G. (1976) J. Biol. Chem. 251, 4062-4070 36. Westberg, N. G. & Michael, A. F. (1970) Biochemistry 9, 3837-3864 37. Kuriyama, T. (1973) Lab. In~vest.28,224-235

Changes in renal glomerular basement membrane with age and nephritis.

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