Biol. Neonate 35: 325 -3 2 7 (1979)
Immunochemical Characterization of Mature and Immature Glomerular Basement Membrane Gert Lubec and Hermann Coradello Department of Paediatrics, University of Vienna, Vienna
Key Words. Glomerular basement membrane • Immune electrophoretical characterization • Maturation of kidney ■Human glomerulus Abstract. 20 kidneys from premature infants (27th to 38th weeks of gestation), 5 kidneys of mature newborns and 5 kidneys of children between 5 and 15 years of age were obtained at necropsy and glomerular basement membranes (GBM) isolated. The isolated GBMs were degraded by papain and the degradation products were characterized by immunoelectrophoresis applying an antihuman GBM antiserum from the rabbit. GBMs of children between 5 and 15 years showed in each case 3 precipitation lines distributed from the a-7 -zone. The examined newborns and 17 premature infants presented two precipitation lines only, moving with -mobility and /3-'y-interregion. 3 premature infants showed a pattern with 2 —4 different precipitation lines of different mobility, maybe interpretable as a result of bacterial digestion. On the grounds of these findings we postulate that the GBM is, from an immunochemical point of view, immature at birth and in the late fetal life becoming mature in the child with about 5.
The glomerular basement membrane (GBM) separating epithelium and endothelium derives from the basement membrane which surrounds the primitive nephrogenic vesicle. Its localiza tion and distinction from the other membranes is determined by the cleft of the S-shaped body ( 2 ).
Like other glomerulus constituents the GBM is thought to be immature at birth and even in early infancy, a fact shown by morphological
investigations ( 2 , 8 ) and few physiological and clinical findings ( 1, 6 , 7, 9). Neither chemical nor immunological characterization of this renal structure in its premature state has been performed. By means of an immunoelectrophoretical method applied on isolated GBMs of kidneys obtained at necropsy, the immunochemical dif ferences between mature and immature GBMs could be detected.
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Introduction
Lubec/Coradello
326
Materials and Methods 20 kidneys from premature infants without renal
disease (from the 27th week of gestation, birth weight 800 g, to the 38th week of gestation, birth weight 1,660 g), 5 kidneys from mature newborn infants and 5 kidneys of children between 5 and 15 years of age were obtained at necropsy and stored frozen until preparation of GBMs. isolation o f GBM was performed according to the method of Maliieu and Winand (5) and degradated by papain: 5 mg of isolated GBM in 0.2 ml sodium chloride solution, 0.85 g/100 ml containing EDTA in a concentration of 0.05 M and cysteine 0.1 M were incubated for 6 h at 37 °C in a water bath, pH was adjusted to pH 7.0. The enzymatic degradation was inhibited by addition of mercury chloride (II) 0.02 ml of a 0.1 M solution, the precipitation was discarded and the supernatant was applied on immunoelectrophoresis in an LKB producer (Inula) at 50 mA and 340 V, in 1% agar purum (Behringwerke, Marburg), using 1/15 M veronal-sodium acetate buffer pH 8.4. Immunoelectrophoresis was run for 1 h at room temperature, diffusion took place for 24 h. As anti body we used rabbit-antihuman GBM serum which was characterized as described in a previous paper (4).
Results
Applying this immunochemical method of characterization we found characteristic dif ferences between the panels. GBMs of children
Discussion
The immunochemical results reveal qualita tive differences between the mature and im mature GBM. Biochemical interpretation of the findings of 3 precipitation lines in the mature and 2 in the immature structure point to different susceptibility to papain degradation as possibly could have occurred by differences in the chemical composition, either in the protein or the carbohydrate or sialic acid content. The latter explanation being supported by histochemical techniques performed by Klinger and Geyer (3) who found staining differences inter pretable as decreased sialic acid content of the immature structure. Quantitative differences of mature and immature GBMs could be shown by Vernier and Birch-Andersen ( 8 ) measuring the membrane on electron microscopy: dividing glomerular development into three stages they measured the width of GBM in stage I with 500 A, stage II with 1,000 A and stage III with 1,000-3,000 A, reaching a width of 3,000 A at 3 years of age. Physiological evidence for the immaturity of the GBM was described by Vernier and BirchAndersen (9) studying the permeability of the GBM applying perfusion of the kidneys with either colloidal carbon or ferritin (9). In the
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Fig. 1. Result of immunochemical characteriza tion: 3 precipitation lines spread over the a-7-zone were detected in the panel of children presenting the presumably ‘mature’ GBM (above). 2 precipitation lines were found in the ‘immature’ GBM preparation of premature and newborn infants.
between 5 and 15 years of age showed in each case, 3 precipitation lines distributed from the a- to 7 -zone. The examined 5 newborn GBMs presented like 17 premature infants two preci pitation lines, one moving with (*i -mobility, the second moving into the 0 -7 -region. 3 GBM preparations of premature infants with birth weights of 1,350, 1,440 and 2,000 g showed a pattern with 2—4 precipitation lines of dif ferent mobility, maybe interpretable as a result of bacterial or fungal degradation.
Fetal Basement Membrane
327
2 du Bois, A.M.: in Rouiller and Muller, The kidney, vol. I (Academic Press, New York 1969). 3 Klinger, G. und Geyer, G.: Histochemische Unter suchungen an renalen Basalmembranen während der Entwicklung. Acta histochem. 21: 261-267 (1965). 4 Lubec, G.; Baizar, E.; Weissenbacher, G.,and Syre, G.: Urinary excretion of glomerular basement membrane antigens in Alport’s syndrome: a new diagnostic approach. Archs Dis. Childh. 53: 401-406 (1978). 5 Mahieu, P. and Winand, R.J.: Chemical structure of tubular and glomerular basement membranes of human kidney. Eur. J. Biochem. 12: 410-418 (1970). 6 Rhodes, P.G.; Hammel, C.H., and Berman, L.B.: Urinary constituents of the newborn infant. J. Pediat. 60: 18-26 (1962). 7 Smith, A.C.: The physiology of the newborn infant, pp. 39-41 (Thomas, Springfield 1945). 8 Vernier, R.L. and Birch-Andersen, A.: Studies on Acknowledgement the human fetal kidney. I. Development of the glomerulus. J. Pediat. 60: 754-768 (1962). I am highly indebted to Dr. Leidolt, Institute of 9 Vernier, R.L. and Birch-Andersen, A.: Studies on Pathology, University of Vienna, for supplying the the human fetal kidney. II. Permeability character kidneys. istics of the developing glomerulus. J. ultrastruct. Res.
Immunochemical Characterization of Mature and Immature Glomerular Basement Membrane Gert Lubec and Hermann Coradello Department of Paediatrics, University of Vienna, Vienna
Key Words. Glomerular basement membrane • Immune electrophoretical characterization • Maturation of kidney ■Human glomerulus Abstract. 20 kidneys from premature infants (27th to 38th weeks of gestation), 5 kidneys of mature newborns and 5 kidneys of children between 5 and 15 years of age were obtained at necropsy and glomerular basement membranes (GBM) isolated. The isolated GBMs were degraded by papain and the degradation products were characterized by immunoelectrophoresis applying an antihuman GBM antiserum from the rabbit. GBMs of children between 5 and 15 years showed in each case 3 precipitation lines distributed from the a-7 -zone. The examined newborns and 17 premature infants presented two precipitation lines only, moving with -mobility and /3-'y-interregion. 3 premature infants showed a pattern with 2 —4 different precipitation lines of different mobility, maybe interpretable as a result of bacterial digestion. On the grounds of these findings we postulate that the GBM is, from an immunochemical point of view, immature at birth and in the late fetal life becoming mature in the child with about 5.
The glomerular basement membrane (GBM) separating epithelium and endothelium derives from the basement membrane which surrounds the primitive nephrogenic vesicle. Its localiza tion and distinction from the other membranes is determined by the cleft of the S-shaped body ( 2 ).
Like other glomerulus constituents the GBM is thought to be immature at birth and even in early infancy, a fact shown by morphological
investigations ( 2 , 8 ) and few physiological and clinical findings ( 1, 6 , 7, 9). Neither chemical nor immunological characterization of this renal structure in its premature state has been performed. By means of an immunoelectrophoretical method applied on isolated GBMs of kidneys obtained at necropsy, the immunochemical dif ferences between mature and immature GBMs could be detected.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/16/2019 11:52:53 AM
Introduction
Lubec/Coradello
326
Materials and Methods 20 kidneys from premature infants without renal
disease (from the 27th week of gestation, birth weight 800 g, to the 38th week of gestation, birth weight 1,660 g), 5 kidneys from mature newborn infants and 5 kidneys of children between 5 and 15 years of age were obtained at necropsy and stored frozen until preparation of GBMs. isolation o f GBM was performed according to the method of Maliieu and Winand (5) and degradated by papain: 5 mg of isolated GBM in 0.2 ml sodium chloride solution, 0.85 g/100 ml containing EDTA in a concentration of 0.05 M and cysteine 0.1 M were incubated for 6 h at 37 °C in a water bath, pH was adjusted to pH 7.0. The enzymatic degradation was inhibited by addition of mercury chloride (II) 0.02 ml of a 0.1 M solution, the precipitation was discarded and the supernatant was applied on immunoelectrophoresis in an LKB producer (Inula) at 50 mA and 340 V, in 1% agar purum (Behringwerke, Marburg), using 1/15 M veronal-sodium acetate buffer pH 8.4. Immunoelectrophoresis was run for 1 h at room temperature, diffusion took place for 24 h. As anti body we used rabbit-antihuman GBM serum which was characterized as described in a previous paper (4).
Results
Applying this immunochemical method of characterization we found characteristic dif ferences between the panels. GBMs of children
Discussion
The immunochemical results reveal qualita tive differences between the mature and im mature GBM. Biochemical interpretation of the findings of 3 precipitation lines in the mature and 2 in the immature structure point to different susceptibility to papain degradation as possibly could have occurred by differences in the chemical composition, either in the protein or the carbohydrate or sialic acid content. The latter explanation being supported by histochemical techniques performed by Klinger and Geyer (3) who found staining differences inter pretable as decreased sialic acid content of the immature structure. Quantitative differences of mature and immature GBMs could be shown by Vernier and Birch-Andersen ( 8 ) measuring the membrane on electron microscopy: dividing glomerular development into three stages they measured the width of GBM in stage I with 500 A, stage II with 1,000 A and stage III with 1,000-3,000 A, reaching a width of 3,000 A at 3 years of age. Physiological evidence for the immaturity of the GBM was described by Vernier and BirchAndersen (9) studying the permeability of the GBM applying perfusion of the kidneys with either colloidal carbon or ferritin (9). In the
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/16/2019 11:52:53 AM
Fig. 1. Result of immunochemical characteriza tion: 3 precipitation lines spread over the a-7-zone were detected in the panel of children presenting the presumably ‘mature’ GBM (above). 2 precipitation lines were found in the ‘immature’ GBM preparation of premature and newborn infants.
between 5 and 15 years of age showed in each case, 3 precipitation lines distributed from the a- to 7 -zone. The examined 5 newborn GBMs presented like 17 premature infants two preci pitation lines, one moving with (*i -mobility, the second moving into the 0 -7 -region. 3 GBM preparations of premature infants with birth weights of 1,350, 1,440 and 2,000 g showed a pattern with 2—4 precipitation lines of dif ferent mobility, maybe interpretable as a result of bacterial or fungal degradation.
Fetal Basement Membrane
327
2 du Bois, A.M.: in Rouiller and Muller, The kidney, vol. I (Academic Press, New York 1969). 3 Klinger, G. und Geyer, G.: Histochemische Unter suchungen an renalen Basalmembranen während der Entwicklung. Acta histochem. 21: 261-267 (1965). 4 Lubec, G.; Baizar, E.; Weissenbacher, G.,and Syre, G.: Urinary excretion of glomerular basement membrane antigens in Alport’s syndrome: a new diagnostic approach. Archs Dis. Childh. 53: 401-406 (1978). 5 Mahieu, P. and Winand, R.J.: Chemical structure of tubular and glomerular basement membranes of human kidney. Eur. J. Biochem. 12: 410-418 (1970). 6 Rhodes, P.G.; Hammel, C.H., and Berman, L.B.: Urinary constituents of the newborn infant. J. Pediat. 60: 18-26 (1962). 7 Smith, A.C.: The physiology of the newborn infant, pp. 39-41 (Thomas, Springfield 1945). 8 Vernier, R.L. and Birch-Andersen, A.: Studies on Acknowledgement the human fetal kidney. I. Development of the glomerulus. J. Pediat. 60: 754-768 (1962). I am highly indebted to Dr. Leidolt, Institute of 9 Vernier, R.L. and Birch-Andersen, A.: Studies on Pathology, University of Vienna, for supplying the the human fetal kidney. II. Permeability character kidneys. istics of the developing glomerulus. J. ultrastruct. Res.
