European Journal of Clinical Investigation (1 977) 7, 177- 180
Composition of the glomerular basement membrane in the congenital nephrotic syndrome of the Finnish type* KARL TRYGGVASON, Department of Medical Biochemistry and Department of Pediatrics, University of Oulu, Oulu, Finland Received 23 August 1976
Abstract. The composition of the glomerular basement membrane (GBM) was studied in three patients with the congenital nephrotic syndrome of the Finnish type (CNF). A decrease was found in the relative amounts of amino acids characteristic of collagen, i.e. 3-hydroxyproline, 4-hydroxyproline, hydroxylysine and glycine. The decrease in hydroxyproline was also apparent in the ratio of 4-hydroxyproline to proline and in direct assays of 4-hydroxyproline in whole glomeruli. The ratio of 3-hydroxyproline to 4-hydroxyproline was decreased by about 40%. Slight increases were found in the amounts of some amino acids. No significant change was found in the glucose content of the GBM whereas the galactose content was slightly decreased. The results suggest a decrease in the relative amount of the collagen component in the GBM of CNF patients, but a decrease in the relative amount of 3-hydroxyproline indicates that other changes also exist.
logically normal and improved clinically during 9 months of haemodialysis, but died after an unsuccessful transplantation [4]. This suggests that clinically the CNF gene affects the kidneys alone. The number of glomeruli is increased in kidneys of CNF patients by about 70% [ 7 ] , but the role of this finding in the pathogenesis is uncertain. A fault in the chemical structure of the GBM might be important in the pathogenesis of the disease [3] and studies of the GBM in two non-Finnish patients with the congenital nephrotic syndrome revealed an increase in the content of hydroxylysine and 3- and 4-hydroxyproline as well as glucosylgalactosylhydroxylysine [8] . It is possible that the clinical symptoms of the CNF are due to a faulty structure in the GBM, and it is the purpose of this study to examine and compare the chemical properties of the GBM in CNF and normal kidneys. Materials and Methods
Key words. Basement membrane, collagen, kidney,
nephrotic syndrome. Introduction
The congenital nephrotic syndrome of the Finnish type (CNF) seems to form a distinct entity among the rare congenital nephrotic syndromes (CNS). The incidence of the disease in Finland is exceptionally high or 12.4: 100 000 births [2] . The disease is inherited as an autosomal recessive disease [3] and it is characterized by a large placenta, early onset of massive selective proteinuria and oedema, steroid resistance and a fatal outcome after 1-3.5 years. A great many of the glomeruli are fibrotic or hyalinized but by electron microscopy the glomerular basement membrane (GBM) looks mostly normal; in some places i t may be thickened but does not contain any dense aggregations and there is a general fusion of the pedicels [4-6]. The pathogenesis of the disease is unknown but an apparently important finding is that a patient who was nephrectomized at the age of 4 months became sero*Part of these results were presented as a preliminary communication [ 1 ] . Correspondence: Dr Karl Tryggvason, Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, SF-90220 Oulu 22, Finland.
Six kidneys were obtained from healthy individuals (4, 28 and 32 years of age) who had died in accidents and six kidneys from three children with CNF up to 3 years of age. The glomeruli were isolated by graded sieving as described elsewhere [ 7 , 9 ] and the GBM was prepared after sonication of the glomeruli for 8 min in a MSE sonicator with 1.5 amperes [ 101 . Amino acids were determined after hydrolysis in 6 mol/l HCl at 110°C for 2 0 h on a Jeol JLC-SAH automatic amino acid analyser. The 3-hydroxyproline was determined separately [ 111 using equal colour values for 3 - and 4-hydroxyproline. The cysteine was determined as cysteic acid and methionine as methionine sulfone [I21 The neutral sugars were isolated [13] and assayed for glucose and galactose with glucose- and galactoseoxidase reagents (Glox, Galax, AB Kabi, Sweden). The 4-hydroxyproline content in whole glomeruli was assayed by a specific procedure [14]. The GBM was examined for purity by determining DNA content ~ 5 1 . Results
Preparation of glomeruli The glomeruli were examined for purity in a phase 177
178
KARL TRYGGVASON Table 1. Amino acid composition of GBM from normal and diseased kidneys*
Normal kidneys (n = 6) Hydroxylysine Lysine Histidine Arginine 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonine Serine Glutamatic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
CNF 2 (n = 2)
CNF 1 (n = 2)
16.5 i 1.9 33.6 f 1.8 17.1 f 1.5 51.4 f 3.3 4.6 f 0.1 50.4 f 1.1 74.4 f 2.4 41.5 f 0.9 52.8 f 0.6 99.3 f 4.1 71.3 f 0.4 194.7 f 1.6 67.8 f 1.7 22.1 f 1.1 43.2 f 0.1 18.0 f 0.6 32.4 f 0.2 66.2 i 2.8 13.0 f 0.7 29.7 f 0.1
18.3 i 0.1 40.7 f 0.8 20.5 f 0.7 57.3 f 2.4 5.4 i 0.5 55.4 f 5.8 86.4 f 0.8 44.1 f 0.8 54.6 f 1.3 94.4 t 5.0 66.9 f 4.0 163.1 f 5.2 67.9 f 1.1 25.6 f 0.3 41.8 i 0.1 18.1 f 0.2 32.0 f 0.1 68.2 f 0.7 10.8 f 0.8 28.5 f 0.2
23.4 f 1.7 25.5 ?I 2.2 15.5 f 1.0 42.2 f 1.5 12.4 i 2.3 77.2 i 2.4 72.5 f 2.5 37.9 f 2.5 49.0 f 1.5 89.1 f 1.5 70.6 i 1.6 227.6 f 3.1 59.8 f 3.7 19.8 f 1.0 31.2 f 1.3 15.0 i 0.7 28.1 k 0.4 61.9 f 1.0 12.9 i 3.4 28.4 f 1.2
CNF 3 t (n = 2) 19.9 f 2.1 25.8 f 1.3 13.8 f 0.4 47.8 i 4.1 7.0 f 0.9 66.9 f 3.3 78.3 f 3.3 40.1 f 4.0 48.0 f 0.2 91.0 f 1.5 75.3 f 5.0 222.6 f 9.7 64.8 i 2.5 23.7 f 1.3 34.7 f 3.5 17.0 f 0.9 28.3 f 1.6 58.1 i 3.1 8.2 f 1.4 28.7 f 0.8
*Residues per 1000 amino acid residues, mean+SD. t Glomeruli sonicated three times. Table 2. Ratios of 3-hydroxyproline to 4-hydroxyproline and 4-hydroxyproline
to proline in GBM from normal and diseased kidneys
Control CNF 1 CNF 2 CNF 3
0.161 (0.128-0.174) 0.098 (0.097-0.099) 0.091 (0.090-0.092) 0.1 03 (0.1 0 1-0.1 05)
1.094 (1.029-1.287) 0.828 (0.826-0.830) 0.707 (0.703-0.71 1) 0.888 (0.881-0.895)
The ratios are calculated from the values shown in Table 1. 3-Hyp = 3Hydroxyproline, 4-Hyp = 4-Hydroxyproline, Pro = Proline.
microscope and they proved to be completely pure except for few occasional small tubular fragments and small tubular segments attached to some glomeruli. The GBM was usually prepared after 8 min sonication [ l o ] , but because the glomeruli from the diseased ludneys were found to be more resistant to sonication than the controls, one sample (CNF 3) was sonicated for 24 min. The purity of the membranes was determined by measuring the DNA content of the preparations as an indicator of cellular contamination and it was found to be less than 0.5% in the controls, 0.9% in CNF 2 and 0.6% in CNF 3. The DNA content in CNF 1 was not measured.
CNF 3 , which can be considered very pure GBM, although the magnitudes of the changes were somewhat smaller. Calculation of the ratio of 3-hydroxyproline to 4hydroxyproline indicated that this value was decreased by about 40% in a l l three patients (Table 2). The ratio of 4-hydroxyproline to proline was likewise definitely decreased (Table 2). The concentration of 4-hydroxyproline was also determined directly in whole glomeruli from normal and diseased kidneys (Table 3). This value was found t o be reduced by about 35% in the diseased kidneys. Table 3. Hydroxyproline content in whole glomeruli from normal
and CNF kidneys
Amino acid composition Results of amino acid analysis of GBM in normal and CNF kidneys are shown in Table 1. In CNF 1 and CNF 2 the relative amounts of hydroxylysine, 3- and 4-hydroxyproline and glycine were decreased, whereas the relative amounts of lysine, arginine, alanine, half-cystine and valine were increased. Similar changes were found in
Hydroxyproiine (pg/rng glomeruli)* Normal (n = 3) CNF (n = 5)
16.1 f 2.3 10.4 f 0.9 *Mean f SD.
GLOMERULAR BASEMENT MEMBRANE
179
Table 4. Carbohydrate composition of human GBM in normal and CNF kidneys*
Glucose Galactose
Normal kidneys (n = 7)
CNF 1 (n = 2)
CNF 2 (n = 3)
CNF 3 t (n = 3)
2.21a0.23 3.04i0.26
1.80+0.10
2.46t0.60
2.26i0.05 2.50i0.10
2.10k0.26 2.73i0.05
*&lo0 fig, meaniSD.
t Glomeruli sonicated three times. Carbohydrate composition Results of assays for glucose and galactose content in GBM are shown in Table 4. There was no change in the glucose content of the GBM in CNF, whereas a slight decrease in galactose content was noticed. The ratio of galactose to glucose was 1.38 in the normal GBM, 1.37 inCNF 1 , l . l l i n C N F 2 , a n d 1.30inCNF3. Discussion
The filtration barrier of the glomerulus is a complicated system which consists of endothelium, GBM and epithelial pedicels with their interval slit pores. The exact site of ultrafiltration is under debate [16]. In a number of glomerular diseases there is a change in the permeability for macromolecules of the GBM and in many cases the GBM undergoes morphological changes. It would therefore be likely that there are changes in the chemical composition of the GBM in diseased states. A main problem in studying the GBM is to obtain pure membrane and to determine the degree of purity. A widely used criterion of purity and the criterior used in this study is the DNA content of the GBM which indicates the contamination by cellular material [ l o , 171. On the basis of this criterion GBM isolated from control kidneys and from CNF 3 was quite pure, whereas a small amount of contamination was probably present in CNF 1 and CNF 2. As an additional criterion of purity the amino acid composition of the GBM preparations can be compared with those previously reported. Our values for GBM in healthy subjects are similar to those previously reported [18, 191, and indicates the purity of the GBM studied here. The GBM consists of a collagen component and noncollagenous glycoproteins [ 18, 201. All collagens have a high content of glycine, and contain hydroxyproline and hydroxylysine, the two latter amino acids being found almost exclusively in this protein [21-231. The collagen of basement membranes is called type IV collagen and i t is characterized by higher contents of hydroxyproline and hydroxylysine than other collagens [18, 201. In addition, over 10% of the hydroxyproline is in the 3-hydroxy isomer form, whereas only 1-2% is present in this form in the interstitial collagens [18,20, 241. Accordingly, a change in the ratio of 3hydroxyproline to 4-hydroxyproline in a tissue may reflect a change in the ratio of type IV collagen to other types of collagen [25].
Changes in the chemical composition of the GBM have previously been studied in human subjects with some diseases of the kidney. Westberg & Michael [26] reported a decrease in 3- and 4-hydroxyproline, hydroxylysine and glycine in GBM of patients with membraneous proliferative glomerulonephritis, idiopathic membranous glomerulonephritis and chronic pyelonephritis, whereas no changes were found in chronic glomerulonephritis. Changes in the content of some other amino acids, such as an increase in alanine in all four diseases, were also found, whereas the glucose or galactose content was decreased only in chronic membranoproliferative glomerulonephritis [26] . The glomeruli in diabetic patients contain increased amounts of collagen [ 2 7 ] , and it has also been reported that there are specific increases in hydroxylysine and hydroxylysine-linked disaccharide units of collagen in this disease [28]. However, subsequent reports were unable to confum the latter findings [29,30]. The present results indicate several changes in the amino acid composition of the GBM in CNF. One characteristic features was a decrease in amino acids characteristic for collagen; 3-hydroxyproline, 4-hydroxyproline, hydroxylysine and glycine. The decrease in hydroxyproline was also apparent in the ratio of 4-hydroxyproline to proline and in direct assays of 4-hydroxyproline in whole glomeruli. All these changes suggest a decrease in the collagen content of the GBM in this disease. An additional change was a marked decrease in the ratio of 3-hydroxyproline to 4-hydroxyproline. This might be due either to an impairment of the 3-hydroxylation of proline or to the presence of other types of collagen in addition to type IV collagen in glomeruli of patients with CNF. The other collagens have a low 3-hydroxyproline content, and they contain much more alanine than type IV collagen [18]. The increase of alanine found here would be consistent with the latter possibility. In addition t o the decrease in amino acids characteristic for collagen our data indicate slight increases in the relative amounts of some amino acids. It is possible that those changes in part reflect relative increases in other protein components of the GBM, but the presence of more specific changes can not be excluded. Because GBM consists of several protein components, additional studies on isolated protein components are required to detect possible minor changes in their structure. It may be also noted that Westberg & Michael found decreases in amino acids characteristic for collagen in GBM in three diseases (see above), and our calculation
180
KARL TRYGGVASON
from their data shows a decrease of about 20% in the ratio of 3-hydroxyproline to 4hydroxyproline in GBM of patients with chronic glomerulonephritis, chronic membranous proliferative glomerulonephritis and chronic pyelonephritis. It therefore seems possible that the changes observed here are not specific for CNF but are characteristic of several diseases affecting the kidneys. However, these changes are clearly different from those reported for the GBM in two non-Finnish infants with the congenital nephrotic syndrome, in which an increase was found in amino acids characteristic of collagen [8] . Acknowledgments
The present work was supported in part by grants from the Medical Research Council of the Academy of Finland and the Icelandic Science Foundation. The author gratefully acknowledges the comments of Professor Kari I. Kivirikko and Dr Niilo-Pekka Huttunen and the expert technical assistance of Miss Helmi Konola. References 1 Tryggvason K., Huttunen N.-P. & Kivirikko K.I. (1976) Glomerular basement membrane (GBM) in the congenital neprotic syndrome of the Finnish type (CNF). Abstr., Eur. Fed. Conn. Tissue Clubs, Vth meeting, Likge, 1976. 2 Huttunen N.P. (1976) Congenital nephrotic syndrome of Finnish type. Arch Dis Child 5 1, 344-348. 3 Norio R. (1966) Heredity in the congenital nephrotic syndrome. Ann Paediatr Fenn 12, Suppl. 27. 4 Hallman N., Norio R. & Rapola J. (1973) Congenital nephrotic syndrome. Nephron 11,101-1 10. 5 Hallman N., Norio R., Kouvalainen K., Vilska J . & Kojo N. (1970) Das kongenitale nephrotische Syndrome. Ergeb Inn Med Kinderheilkd 30, 3-67. 6 Norio R. (1974) Congenital nephrotic syndrome of Finnish type and other types of early familiar nephrotic syndromes. Birth Defects: Original Article, Series 10, 69-77. 7 Tryggvason K. & Kouvalainen K . (1975) Number of nephrons in normal human kidneys and kidneys with the congenital nephrotic syndrome. Nephron 15,62-68. 8 Monnens L., Mahieu P. & van Haelst V. (197') Congenital nephrotic syndrome. Biochemical studies of glomerular basement membrane in two infants. Abstr., Eur. Soc. Pediat. Nephrology, Cambridge. 9 Misra R.P. (1972) Isolation of glomeruli from mammalian kidneys by graded sieving. A m J Clin Pathol58, 135-1 39. 10 Misra R.P. & Berman L.B. (1966) Studies on glomerular basement membrane. 1 . Isolation and chemical analysis of normal glomerular basement membrane. Proc Soc Exp BiolMed 122, 705-710.
11 Grant M.E., Kefalides N.A. & Prockop D.J. (1963) The biosynthesis of basement membrane collagen in embryonic chick lens. I. Delay between the synthesis of polypeptide chains and the secretion of collagen by matrix-free cells. J Biol Chem 247,3539-3544. 2 Moore S. (1963) On the determination of cystine as cysteic acid. J Biol Chem 238,235-237. 3 Tengstrom B. (1966) Enzymatic determination of glucose and galactose in urine. Scand J Clin Lab Invest 18, Suppl. 92, 104-1 13. 4 Kivirikko K.I., Laitinen 0. & Prockop D.J. (1967) Modifications of a specific assay for hydroxyproline in urine. Anal Biochem 19,249-255. 15 Croft D.N. & Lubran M. (1965) The estimation of deoxyribonucleic acid in the presence o f sialic acid: Application t o analysis of human gastric washings. Biochem J 95, 612620. 16 Schneeberger E.E. (1974) Glomerular permeability t o protein molecules-its possible structural basis. Nephron 13, 7-21. 1 7 Spiro R.G. (1967) Studies o n the renal glomerular basement membrane. JBiol Chem 242, 1915-1922. 18 Kefalides N.A. (1973) Structure and biosynthesis of basement membranes. Int Rev Connect Tissue Res 6,63-104. 19 Westberg N.G. & Michael A.F. (1970) Human glomerular basement membrane. Preparation and composition. Biochemistry 9,3837-3846. 20 Kefalides N.A. (1975) Basement membranes: structural and biosynthetic considerations. J Invest Dermatol65,85-92. 21 Kuhn K . & Fietzek P.P. (1975) The structure of collagen. Collagen Metabolism in the Liver (ed. by H. Popper and K. Becker), pp. 15-28. Stratton, New York. 22 Gallop P.M. & Paz M.A. (1975) Posttranslational protein modifications, with special attention t o collagen and elastin. Physiol Rev 55,418-487. 23 Kivirikko K.I. & Risteli L. (1976) Btosynthesis of collagen and its alterations in pathological states. Med Biol 54, 159186. 24 Kefalides N.A. (1975) Basement membranes: current concepts of structure and synthesis. Dermatologira 150,4-15. 25 Man M. & Adams E. (1975) Basement membrane and interstitial collagen content of whole animals and tissues. Biochem Biophys Res Commun 66,9-16. 26 Westberg N.G. & Michael A.F. (1973) Human glomerular basement membrane: chemical composition in glomerulonephritis and pylonephritis. Acta Med Scand 194,49-57. 27 Klein L., Butcher D.L., Sudilovsky O., Kikkawa R. & Miller M. (1975) Quantification of collagen in renal glomeruli isolated from human nondiabetic and diabetic kidneys. Diabetes 24, 1057-1065. 28 Beisswenger P.J. & Spiro R.G. (1970) Human glomerular basement membrane: chemical alteration in diabetes mellitus. Science 168,596-598. 29 Kefalides N.A. (1974) Biochemical properties of human glomerular basement membrane in normal and diabetic kidneys. J Clin Invest 53,403-407. 30 Westberg N.G. & Michael A.F. (1973) Human glomerular basement membrane: chemical composition in diabetes mellitus. Acta Med Scand 194, 39-47.
Composition of the glomerular basement membrane in the congenital nephrotic syndrome of the Finnish type* KARL TRYGGVASON, Department of Medical Biochemistry and Department of Pediatrics, University of Oulu, Oulu, Finland Received 23 August 1976
Abstract. The composition of the glomerular basement membrane (GBM) was studied in three patients with the congenital nephrotic syndrome of the Finnish type (CNF). A decrease was found in the relative amounts of amino acids characteristic of collagen, i.e. 3-hydroxyproline, 4-hydroxyproline, hydroxylysine and glycine. The decrease in hydroxyproline was also apparent in the ratio of 4-hydroxyproline to proline and in direct assays of 4-hydroxyproline in whole glomeruli. The ratio of 3-hydroxyproline to 4-hydroxyproline was decreased by about 40%. Slight increases were found in the amounts of some amino acids. No significant change was found in the glucose content of the GBM whereas the galactose content was slightly decreased. The results suggest a decrease in the relative amount of the collagen component in the GBM of CNF patients, but a decrease in the relative amount of 3-hydroxyproline indicates that other changes also exist.
logically normal and improved clinically during 9 months of haemodialysis, but died after an unsuccessful transplantation [4]. This suggests that clinically the CNF gene affects the kidneys alone. The number of glomeruli is increased in kidneys of CNF patients by about 70% [ 7 ] , but the role of this finding in the pathogenesis is uncertain. A fault in the chemical structure of the GBM might be important in the pathogenesis of the disease [3] and studies of the GBM in two non-Finnish patients with the congenital nephrotic syndrome revealed an increase in the content of hydroxylysine and 3- and 4-hydroxyproline as well as glucosylgalactosylhydroxylysine [8] . It is possible that the clinical symptoms of the CNF are due to a faulty structure in the GBM, and it is the purpose of this study to examine and compare the chemical properties of the GBM in CNF and normal kidneys. Materials and Methods
Key words. Basement membrane, collagen, kidney,
nephrotic syndrome. Introduction
The congenital nephrotic syndrome of the Finnish type (CNF) seems to form a distinct entity among the rare congenital nephrotic syndromes (CNS). The incidence of the disease in Finland is exceptionally high or 12.4: 100 000 births [2] . The disease is inherited as an autosomal recessive disease [3] and it is characterized by a large placenta, early onset of massive selective proteinuria and oedema, steroid resistance and a fatal outcome after 1-3.5 years. A great many of the glomeruli are fibrotic or hyalinized but by electron microscopy the glomerular basement membrane (GBM) looks mostly normal; in some places i t may be thickened but does not contain any dense aggregations and there is a general fusion of the pedicels [4-6]. The pathogenesis of the disease is unknown but an apparently important finding is that a patient who was nephrectomized at the age of 4 months became sero*Part of these results were presented as a preliminary communication [ 1 ] . Correspondence: Dr Karl Tryggvason, Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, SF-90220 Oulu 22, Finland.
Six kidneys were obtained from healthy individuals (4, 28 and 32 years of age) who had died in accidents and six kidneys from three children with CNF up to 3 years of age. The glomeruli were isolated by graded sieving as described elsewhere [ 7 , 9 ] and the GBM was prepared after sonication of the glomeruli for 8 min in a MSE sonicator with 1.5 amperes [ 101 . Amino acids were determined after hydrolysis in 6 mol/l HCl at 110°C for 2 0 h on a Jeol JLC-SAH automatic amino acid analyser. The 3-hydroxyproline was determined separately [ 111 using equal colour values for 3 - and 4-hydroxyproline. The cysteine was determined as cysteic acid and methionine as methionine sulfone [I21 The neutral sugars were isolated [13] and assayed for glucose and galactose with glucose- and galactoseoxidase reagents (Glox, Galax, AB Kabi, Sweden). The 4-hydroxyproline content in whole glomeruli was assayed by a specific procedure [14]. The GBM was examined for purity by determining DNA content ~ 5 1 . Results
Preparation of glomeruli The glomeruli were examined for purity in a phase 177
178
KARL TRYGGVASON Table 1. Amino acid composition of GBM from normal and diseased kidneys*
Normal kidneys (n = 6) Hydroxylysine Lysine Histidine Arginine 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonine Serine Glutamatic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
CNF 2 (n = 2)
CNF 1 (n = 2)
16.5 i 1.9 33.6 f 1.8 17.1 f 1.5 51.4 f 3.3 4.6 f 0.1 50.4 f 1.1 74.4 f 2.4 41.5 f 0.9 52.8 f 0.6 99.3 f 4.1 71.3 f 0.4 194.7 f 1.6 67.8 f 1.7 22.1 f 1.1 43.2 f 0.1 18.0 f 0.6 32.4 f 0.2 66.2 i 2.8 13.0 f 0.7 29.7 f 0.1
18.3 i 0.1 40.7 f 0.8 20.5 f 0.7 57.3 f 2.4 5.4 i 0.5 55.4 f 5.8 86.4 f 0.8 44.1 f 0.8 54.6 f 1.3 94.4 t 5.0 66.9 f 4.0 163.1 f 5.2 67.9 f 1.1 25.6 f 0.3 41.8 i 0.1 18.1 f 0.2 32.0 f 0.1 68.2 f 0.7 10.8 f 0.8 28.5 f 0.2
23.4 f 1.7 25.5 ?I 2.2 15.5 f 1.0 42.2 f 1.5 12.4 i 2.3 77.2 i 2.4 72.5 f 2.5 37.9 f 2.5 49.0 f 1.5 89.1 f 1.5 70.6 i 1.6 227.6 f 3.1 59.8 f 3.7 19.8 f 1.0 31.2 f 1.3 15.0 i 0.7 28.1 k 0.4 61.9 f 1.0 12.9 i 3.4 28.4 f 1.2
CNF 3 t (n = 2) 19.9 f 2.1 25.8 f 1.3 13.8 f 0.4 47.8 i 4.1 7.0 f 0.9 66.9 f 3.3 78.3 f 3.3 40.1 f 4.0 48.0 f 0.2 91.0 f 1.5 75.3 f 5.0 222.6 f 9.7 64.8 i 2.5 23.7 f 1.3 34.7 f 3.5 17.0 f 0.9 28.3 f 1.6 58.1 i 3.1 8.2 f 1.4 28.7 f 0.8
*Residues per 1000 amino acid residues, mean+SD. t Glomeruli sonicated three times. Table 2. Ratios of 3-hydroxyproline to 4-hydroxyproline and 4-hydroxyproline
to proline in GBM from normal and diseased kidneys
Control CNF 1 CNF 2 CNF 3
0.161 (0.128-0.174) 0.098 (0.097-0.099) 0.091 (0.090-0.092) 0.1 03 (0.1 0 1-0.1 05)
1.094 (1.029-1.287) 0.828 (0.826-0.830) 0.707 (0.703-0.71 1) 0.888 (0.881-0.895)
The ratios are calculated from the values shown in Table 1. 3-Hyp = 3Hydroxyproline, 4-Hyp = 4-Hydroxyproline, Pro = Proline.
microscope and they proved to be completely pure except for few occasional small tubular fragments and small tubular segments attached to some glomeruli. The GBM was usually prepared after 8 min sonication [ l o ] , but because the glomeruli from the diseased ludneys were found to be more resistant to sonication than the controls, one sample (CNF 3) was sonicated for 24 min. The purity of the membranes was determined by measuring the DNA content of the preparations as an indicator of cellular contamination and it was found to be less than 0.5% in the controls, 0.9% in CNF 2 and 0.6% in CNF 3. The DNA content in CNF 1 was not measured.
CNF 3 , which can be considered very pure GBM, although the magnitudes of the changes were somewhat smaller. Calculation of the ratio of 3-hydroxyproline to 4hydroxyproline indicated that this value was decreased by about 40% in a l l three patients (Table 2). The ratio of 4-hydroxyproline to proline was likewise definitely decreased (Table 2). The concentration of 4-hydroxyproline was also determined directly in whole glomeruli from normal and diseased kidneys (Table 3). This value was found t o be reduced by about 35% in the diseased kidneys. Table 3. Hydroxyproline content in whole glomeruli from normal
and CNF kidneys
Amino acid composition Results of amino acid analysis of GBM in normal and CNF kidneys are shown in Table 1. In CNF 1 and CNF 2 the relative amounts of hydroxylysine, 3- and 4-hydroxyproline and glycine were decreased, whereas the relative amounts of lysine, arginine, alanine, half-cystine and valine were increased. Similar changes were found in
Hydroxyproiine (pg/rng glomeruli)* Normal (n = 3) CNF (n = 5)
16.1 f 2.3 10.4 f 0.9 *Mean f SD.
GLOMERULAR BASEMENT MEMBRANE
179
Table 4. Carbohydrate composition of human GBM in normal and CNF kidneys*
Glucose Galactose
Normal kidneys (n = 7)
CNF 1 (n = 2)
CNF 2 (n = 3)
CNF 3 t (n = 3)
2.21a0.23 3.04i0.26
1.80+0.10
2.46t0.60
2.26i0.05 2.50i0.10
2.10k0.26 2.73i0.05
*&lo0 fig, meaniSD.
t Glomeruli sonicated three times. Carbohydrate composition Results of assays for glucose and galactose content in GBM are shown in Table 4. There was no change in the glucose content of the GBM in CNF, whereas a slight decrease in galactose content was noticed. The ratio of galactose to glucose was 1.38 in the normal GBM, 1.37 inCNF 1 , l . l l i n C N F 2 , a n d 1.30inCNF3. Discussion
The filtration barrier of the glomerulus is a complicated system which consists of endothelium, GBM and epithelial pedicels with their interval slit pores. The exact site of ultrafiltration is under debate [16]. In a number of glomerular diseases there is a change in the permeability for macromolecules of the GBM and in many cases the GBM undergoes morphological changes. It would therefore be likely that there are changes in the chemical composition of the GBM in diseased states. A main problem in studying the GBM is to obtain pure membrane and to determine the degree of purity. A widely used criterion of purity and the criterior used in this study is the DNA content of the GBM which indicates the contamination by cellular material [ l o , 171. On the basis of this criterion GBM isolated from control kidneys and from CNF 3 was quite pure, whereas a small amount of contamination was probably present in CNF 1 and CNF 2. As an additional criterion of purity the amino acid composition of the GBM preparations can be compared with those previously reported. Our values for GBM in healthy subjects are similar to those previously reported [18, 191, and indicates the purity of the GBM studied here. The GBM consists of a collagen component and noncollagenous glycoproteins [ 18, 201. All collagens have a high content of glycine, and contain hydroxyproline and hydroxylysine, the two latter amino acids being found almost exclusively in this protein [21-231. The collagen of basement membranes is called type IV collagen and i t is characterized by higher contents of hydroxyproline and hydroxylysine than other collagens [18, 201. In addition, over 10% of the hydroxyproline is in the 3-hydroxy isomer form, whereas only 1-2% is present in this form in the interstitial collagens [18,20, 241. Accordingly, a change in the ratio of 3hydroxyproline to 4-hydroxyproline in a tissue may reflect a change in the ratio of type IV collagen to other types of collagen [25].
Changes in the chemical composition of the GBM have previously been studied in human subjects with some diseases of the kidney. Westberg & Michael [26] reported a decrease in 3- and 4-hydroxyproline, hydroxylysine and glycine in GBM of patients with membraneous proliferative glomerulonephritis, idiopathic membranous glomerulonephritis and chronic pyelonephritis, whereas no changes were found in chronic glomerulonephritis. Changes in the content of some other amino acids, such as an increase in alanine in all four diseases, were also found, whereas the glucose or galactose content was decreased only in chronic membranoproliferative glomerulonephritis [26] . The glomeruli in diabetic patients contain increased amounts of collagen [ 2 7 ] , and it has also been reported that there are specific increases in hydroxylysine and hydroxylysine-linked disaccharide units of collagen in this disease [28]. However, subsequent reports were unable to confum the latter findings [29,30]. The present results indicate several changes in the amino acid composition of the GBM in CNF. One characteristic features was a decrease in amino acids characteristic for collagen; 3-hydroxyproline, 4-hydroxyproline, hydroxylysine and glycine. The decrease in hydroxyproline was also apparent in the ratio of 4-hydroxyproline to proline and in direct assays of 4-hydroxyproline in whole glomeruli. All these changes suggest a decrease in the collagen content of the GBM in this disease. An additional change was a marked decrease in the ratio of 3-hydroxyproline to 4-hydroxyproline. This might be due either to an impairment of the 3-hydroxylation of proline or to the presence of other types of collagen in addition to type IV collagen in glomeruli of patients with CNF. The other collagens have a low 3-hydroxyproline content, and they contain much more alanine than type IV collagen [18]. The increase of alanine found here would be consistent with the latter possibility. In addition t o the decrease in amino acids characteristic for collagen our data indicate slight increases in the relative amounts of some amino acids. It is possible that those changes in part reflect relative increases in other protein components of the GBM, but the presence of more specific changes can not be excluded. Because GBM consists of several protein components, additional studies on isolated protein components are required to detect possible minor changes in their structure. It may be also noted that Westberg & Michael found decreases in amino acids characteristic for collagen in GBM in three diseases (see above), and our calculation
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from their data shows a decrease of about 20% in the ratio of 3-hydroxyproline to 4hydroxyproline in GBM of patients with chronic glomerulonephritis, chronic membranous proliferative glomerulonephritis and chronic pyelonephritis. It therefore seems possible that the changes observed here are not specific for CNF but are characteristic of several diseases affecting the kidneys. However, these changes are clearly different from those reported for the GBM in two non-Finnish infants with the congenital nephrotic syndrome, in which an increase was found in amino acids characteristic of collagen [8] . Acknowledgments
The present work was supported in part by grants from the Medical Research Council of the Academy of Finland and the Icelandic Science Foundation. The author gratefully acknowledges the comments of Professor Kari I. Kivirikko and Dr Niilo-Pekka Huttunen and the expert technical assistance of Miss Helmi Konola. References 1 Tryggvason K., Huttunen N.-P. & Kivirikko K.I. (1976) Glomerular basement membrane (GBM) in the congenital neprotic syndrome of the Finnish type (CNF). Abstr., Eur. Fed. Conn. Tissue Clubs, Vth meeting, Likge, 1976. 2 Huttunen N.P. (1976) Congenital nephrotic syndrome of Finnish type. Arch Dis Child 5 1, 344-348. 3 Norio R. (1966) Heredity in the congenital nephrotic syndrome. Ann Paediatr Fenn 12, Suppl. 27. 4 Hallman N., Norio R. & Rapola J. (1973) Congenital nephrotic syndrome. Nephron 11,101-1 10. 5 Hallman N., Norio R., Kouvalainen K., Vilska J . & Kojo N. (1970) Das kongenitale nephrotische Syndrome. Ergeb Inn Med Kinderheilkd 30, 3-67. 6 Norio R. (1974) Congenital nephrotic syndrome of Finnish type and other types of early familiar nephrotic syndromes. Birth Defects: Original Article, Series 10, 69-77. 7 Tryggvason K. & Kouvalainen K . (1975) Number of nephrons in normal human kidneys and kidneys with the congenital nephrotic syndrome. Nephron 15,62-68. 8 Monnens L., Mahieu P. & van Haelst V. (197') Congenital nephrotic syndrome. Biochemical studies of glomerular basement membrane in two infants. Abstr., Eur. Soc. Pediat. Nephrology, Cambridge. 9 Misra R.P. (1972) Isolation of glomeruli from mammalian kidneys by graded sieving. A m J Clin Pathol58, 135-1 39. 10 Misra R.P. & Berman L.B. (1966) Studies on glomerular basement membrane. 1 . Isolation and chemical analysis of normal glomerular basement membrane. Proc Soc Exp BiolMed 122, 705-710.
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