Biol. Neonate 36: 111 281 (1979)
Urinary Excretion o f Glomerular Basement Membrane Antigens in Premature Infants and the Newborn G. Lubec and H. Coradello Department o f Paediatrics, University o f Vienna, Vienna
Key Words. Glomerular basement membrane • Premature infant • Newborn • Urinary antigen excretion
Introduction Little information is given on the develop ment of the glomerular basement membrane. Morphologically, the development and matura tion of the glomerulus has been described by Potter (1965): The terminal ampulla, limited by a well-marked basement membrane (BM) is surrounded by a blastemic cap of nephrogenic cells. The ampulla becomes pushed in on one side and arch shaped. The compact mass of nephrogenic cells changes into an oval renal vesicle with a limiting membrane. The vesicle fuses together with the extremity of the collect ing duct, a unique BM results. A sulcus forms
on the lateral side of the vesicle, the sulcus becomes a cleft - the classical S-shaped body is completed and the differentiation o f the epithe lial layers begins. The glomerular basement membrane (GBM) which separates epithelium and endothelium derives from the thin BM which surrounds the primitive nephrogenic ves icle. The localization of the GBM is determined by the cleft o f the S-shaped body. It has been widely accepted that the devel opment of new glomeruli stops at birth or soon after. In several respects, however, the kidney and glomeruli are not completely developed at birth. In the process of histological maturation of glomeruli, McDonald and Emery (1959)
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Abstract. 25 premature infants, 8 mature newborns and 25 children between 5 and 15 years of age were examined for urinary excretion of glomerular basement membrane (GBM) antigens. For the characterization o f the excreted GBM antigen, immunoelectrophoresis was applied. In the group of 25 premature infants 23 showed a -1-mobility, in the group o f 8 mature newborns all showed a-l-mobility, and in the group o f the 25 children aged 5-1 5 years 24 showed migration into the a-2-zone. Differentiating, whether the difference between the immature and mature GBM is quantitative or qualitative, the immunoelectrophoretical difference points to the interpretation that the premature GBM shows a unique chemical composition.
Lubec/Coradello
distinguish three phases: ( l ) a nephrogenic phase, during which new glomeruli are formed, ending during the 36th week of gestation, sometimes not until the 44th week after con ception; (2) a phase during which all the glomer uli are present, but immature, lasting until the age o f 3 -5 years, and (3) a final phase o f maturation lasting from the age o f 3 to 12 years. Vernier and Birch-Andersen (1962) per formed ultrastructural studies on the fetal and newborn GBM finding significant differences o f that structure: in the 5th month of gestation the GBM measures 700 A , at birth about 1,000 A and with 3 years of age about 3,000 A. To differentiate whether the difference be tween the immature and mature GBM is quanti tative or qualitative, we performed immunoelectrophoretic studies showing that the imma ture structure indeed reveals a different immu nochemical composition.
Patients, Materials and Methods Investigations o f urinary excretion o f GBM anti gens were made in 25 premature infants (28th—36th week o f gestation, birth weight 980-2,430 g), 8 new born infants (mature neonates 3,000-3,760 g), and 25 children between 5 and 15 years o f age. In the infants examined no renal diseases were found. Preparation o f Antigen for Immunisation 8 normal human kidneys from persons aged be tween 20 and 40 years were obtained at necropsy, washed and perfused with cold phosphate-buffered saline (PBS) and comminuted carefully. This homo genate was forced through a 115 mesh sieve (metal) allowing the glomeruli to pass, while adding several litres o f cold PBS. A second filtration followed through a sieve (mesh 150) holding back the glomer uli. The glomeruli were put in 300 ml cold (4 °C) PBS and collected by centrifugation in an MSE refrigerated centrifuge at 350 g for 15 min; the supernatant was
discarded. The pellet was resuspended and sedimented four times. Aliquots o f the suspension were sonicated in an ice bath by an MSE ultrasonic disintegrator, applying several bursts o f 1 min each o f 1.6 A , 220 V at 5-min intervals. The disrupted material was sus pended in 1 M NaCl and centrifuged at l,4 0 0 g for 15 min. The GBM were washed three times in 1 M NaCl and three times in distilled water; the prepara tion was controlled by phase contrast microscopy and stored in the lyophilized state (Mahieu and Winand, 1970). Immunisation Schedule. A t 2-week intervals rab bits were injected intracutaneously with 10 mg GBM mixed with aluminium hydroxide and complete Freund’s adjuvant (Difco). 2 months later the animals were bled by cardiac puncture. The sera were absorbed eight times by glutaraldchyde insolubilized human plasma (Avrameas and Ternynck, 1969), platelets (twice), erythrocytes (three times) and white blood cells (three times). The absorbed sera were allowed to run against normal human serum in the double-diffu sion method o f Ouchterlony, whereby no precipita tion lines were found. Antibody activity against red blood cells was excluded applying a hemagglutination test, and reactivity against platelets as well as white blood cells by agglutination assays. The anti-GBM serum was tested by indirect immunofluorescence, producing a titre o f 1:256 using human kidney ob tained by biopsy. Absorption experiments as described by McPhaul and D ixon (1969) were made to investi gate the relationship to the GBM .
Preparation o f the Infant's G B M Antigen(s) The urine o f infants was collected in plastic bottles containing a pea-sized thymol crystal and kept in a refrigerator at 10 °C. After ultrafiltration in an Amicon stirred cell (diaflo U F 2000) applying a membrane permeable for molecular weights below 20,000, they were dialyzed against 0.1 M phosphate buffer o f pH 7.4. Urines were concentrated 200 times and the pro tein content adjusted to a standard concentration o f 20 mg/ml. Immunoelectrophoresis was performed on an LKB productor, at 50 mA and 340 V in 1% agar purum in a 1/15 M veronal-sodium acetate buffer, pH 8.4. Electrophoresis was run for 50 min at room temperature. Diffusion took place during a 48-hour period anti the precipitations were photographed.
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278
Antigen Excretion in Premature Infants
279
f T-
Results In the group o f 25 premature infants 23 infants showed the precipitation line in the a -1-zone. 2 infants presenting 2 or 3 precipita tion lines had birth weights o f 980 and 2,400 g. In the panel o f 8 newborn infants the pattern of a -1-migration was detected without excep tion. In the group of 25 children between 5 and 15 years of age, 24 showed a single precipita tion line moving in the a-2-region as described for healthy young children of that age group and adults. In 1 case a double precipitation line was present.
Discussion Several papers reported about the urinary excretion of kidney antigens in men (Gilman, 1935; Antoine and Neveu, 1968; Lubec, 1978; McPhaul and Dixon, 1969; McPhaul and Lerner, 1968). As pointed out in ‘ Results’ , we detected different immunoelectrophoretical patterns in premature infant and newborn GBM
immature antigen with a-l-m obility, below the mature GBM ag migrating into the a-2-zone.
antigens on the one hand and in the children of the age group o f 5—15 years on the other. The excretion pattern with 2 or 3 precipitation lines could be interpreted as microbial degradation o f the urine samples. The results correlate well with the findings o f authors who performed morphological stud ies and clinical observations. The morphological studies o f Vernier and Birch-Andersen (1962) on the glomerulum revealed 3 stages of develop ment. In stage I the visceral epithelium is separated from the endothelial cells by a very thin continuous double membrane. In its sim plest form, this membrane appears to consist of the opposed limiting plasma membrane of endo- and epithelium only. Little, if any dense material is present within the 500 A space, separating the 2 membranes. A t stage II the GBM is about 1,000—1,300 A in width. The space between the two plasma membranes now contains a moderate amount o f electron-dense material which is rather loosely organized into a continuous band in the center o f the space. At higher magnification the dense part is seen to be composed of intermeshed short fine fila-
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
Fig. 1. Results o f the immunoelectrophoretic char acterization o f excreted GBM antigen. Above the
280
agreement with morphological and functional studies by other authors and could possibly explain the clinical signs as, e.g., proteinuria in infants. Additionally, we could show that the difference between the mature and immature GBM is not only quantitative but immunochemically qualitative.
References Antoine, B .L . and Neveu, T .: Pathological urinary excretion o f tissue macromolecules (histuria). J . Lab. clin. Med. 71: 101-112 (1968). Arant, B .S., jr.: Developmental patterns o f renal func tional maturation compared in the human neonate. J. Pediat. 92: 705 712 (1978). Avramcas, S. and Ternynck, T .: The cross linking o f proteins with glutaraldehyde and its use for the preparation o f immunoabsorbents. Immunochemistry 6: 53-6 6 (1 9 6 9 ). Bloom, P .: Width o f glomerular basement membrane in man at various ages. Anat. Rec. 133: 251 267 (1959). Fetterman, G .H .; Shuplock, N .A .; Philipp, F .J ., and Gregg. H .S.: The growth and maturation o f human glomeruli and proximal convolutions from term to adulthood. Pediatrics, Springfield 35: 601-619 (1965). Gilm an, G .: Urinary proteins. The appearance o f kidney proteins in the urine o f some cases o f severe chronic glomerular nephritis. J . Urol. 34: 727- 731 (1935). Klinger, G . und Geyer, G .: Histochemische Untcrsuchungen an renalen Basalmcmbranen wahrend der Entwicklung. Acta histochem. 21: 261-267 (1965). Lubec, G .; Balzar, E .; Weissenbacher, G ., and Syre, G .: Urinary excretion o f glomerular basement mem brane antigens in Alport’s syndrome. A new diag nostic approach. Archs Dis. Childh. 53: 401 406 (1978). Mahieu, P. and Winand, R .J.: Chemical structure o f tubular and glomerular basement membranes o f human kidney. Eur. J . Biochem. 12: 410-418 (1970).
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merits. Stage III is classified by the appearance of ‘adult glomeruli’ differing from adult glomer uli in that their GBM is very thin, measuring about 1,000—1,300 A, though the lamina densa is more compact than in stage II. Higher magni fication reveals fine filaments o f GBM sub stance, many of them appearing to bridge the lamina rara and to contact the limiting mem brane of the epithelial foot processes. At 3 years o f age the GBM width reaches 3,000 A 0Bloom, 1959). That the glomerulum is not mature at deliv ery after a normal gestational span was reported and confirmed by Fetterman et al. (1965) measuring the size o f the newborn glomerulum (average diameter 85 instead o f about 200 /rm in adults): This morphological finding being congruent with our immunochemical studies revealing the immature pattern of moving into the a-1-region. Klinger and Geyer (1965) per formed histochemical studies on human fetal glomeruli reporting evidence for staining differ ences of the GBM possibly indicating decreased sialic acid content, a fact which could interpret the GBM antigen mobility towards the a -1-zone by the altered charge of the protein. Vernier and Birch-Andersen (1963) studied the permea bility of the GBM applying perfusion of the kdiney with either colloidal carbon or ferritin. In the mature glomeruli the lamina densa ap peared to function as a filter and only a few particles penetrated the structure, in the im mature glomerulum ferritin was frequently found in the urinary space, which indicated functional immaturity as well. Smith (1945) reported that in the urine of the newborn infant the protein content was increased, this finding being confirmed by the investigations of Rhodes et al. (1962) and Arant (1978). In conclusion, we describe the characteriza tion of immature GBM antigens which are in
Lubec/Coradello
Antigen Excretion in Premature Infants
Archs Path. 80: 241-256 (1965). Rhodes, P .G .; Hammel, C .H ., and Berman, L.B.: Uri nary constituents o f the newborn infant. J . Pediat. 60: 18 26 (1962).
Smith, A .C .: The physiology o f the newborn infant, pp. 39 41 (Thomas, Springfield 1945). Vernier, R .L . and Birch-Andersen, A .: Studies on the human fetal kidney. I. Development o f the glomer ulus. J . Pediat. 60: 754 768(1962). Vernier, R .L . and Birch-Andersen, A .: Studies on the human fetal kidney. II. Permeability characteristics o f the developing glomerulus. J . ultrastruct. Res. 8: 66 88 (1963).
Gert Lubec, M D, University o f Vienna, Department o f Paediatrics, Währinger Gürtel 7 4 -7 6 , A -1090 Vienna (Austria)
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
McDonald, S. and Emery, J.L .: The late intrauterine and postnatal development o f human renal glomer uli. J. Anat. 93: 331-340 (1959). McPhaul, J . J . , jr. and Dixon, E .J.: lmmunoreactive basement membrane antigens in normal human urine and serum. J . exp. Med. 130: 1395-1409 (1969). McPhaul, J . J . , jr. and Lerner, R .A .: Glomerular base ment membrane antigen excretion in normal and nephritic men. Abstract. Fed. Proc. 27: 544 (1968). Potter, E .L .: Development o f the human glomerulus.
281
Urinary Excretion o f Glomerular Basement Membrane Antigens in Premature Infants and the Newborn G. Lubec and H. Coradello Department o f Paediatrics, University o f Vienna, Vienna
Key Words. Glomerular basement membrane • Premature infant • Newborn • Urinary antigen excretion
Introduction Little information is given on the develop ment of the glomerular basement membrane. Morphologically, the development and matura tion of the glomerulus has been described by Potter (1965): The terminal ampulla, limited by a well-marked basement membrane (BM) is surrounded by a blastemic cap of nephrogenic cells. The ampulla becomes pushed in on one side and arch shaped. The compact mass of nephrogenic cells changes into an oval renal vesicle with a limiting membrane. The vesicle fuses together with the extremity of the collect ing duct, a unique BM results. A sulcus forms
on the lateral side of the vesicle, the sulcus becomes a cleft - the classical S-shaped body is completed and the differentiation o f the epithe lial layers begins. The glomerular basement membrane (GBM) which separates epithelium and endothelium derives from the thin BM which surrounds the primitive nephrogenic ves icle. The localization of the GBM is determined by the cleft o f the S-shaped body. It has been widely accepted that the devel opment of new glomeruli stops at birth or soon after. In several respects, however, the kidney and glomeruli are not completely developed at birth. In the process of histological maturation of glomeruli, McDonald and Emery (1959)
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
Abstract. 25 premature infants, 8 mature newborns and 25 children between 5 and 15 years of age were examined for urinary excretion of glomerular basement membrane (GBM) antigens. For the characterization o f the excreted GBM antigen, immunoelectrophoresis was applied. In the group of 25 premature infants 23 showed a -1-mobility, in the group o f 8 mature newborns all showed a-l-mobility, and in the group o f the 25 children aged 5-1 5 years 24 showed migration into the a-2-zone. Differentiating, whether the difference between the immature and mature GBM is quantitative or qualitative, the immunoelectrophoretical difference points to the interpretation that the premature GBM shows a unique chemical composition.
Lubec/Coradello
distinguish three phases: ( l ) a nephrogenic phase, during which new glomeruli are formed, ending during the 36th week of gestation, sometimes not until the 44th week after con ception; (2) a phase during which all the glomer uli are present, but immature, lasting until the age o f 3 -5 years, and (3) a final phase o f maturation lasting from the age o f 3 to 12 years. Vernier and Birch-Andersen (1962) per formed ultrastructural studies on the fetal and newborn GBM finding significant differences o f that structure: in the 5th month of gestation the GBM measures 700 A , at birth about 1,000 A and with 3 years of age about 3,000 A. To differentiate whether the difference be tween the immature and mature GBM is quanti tative or qualitative, we performed immunoelectrophoretic studies showing that the imma ture structure indeed reveals a different immu nochemical composition.
Patients, Materials and Methods Investigations o f urinary excretion o f GBM anti gens were made in 25 premature infants (28th—36th week o f gestation, birth weight 980-2,430 g), 8 new born infants (mature neonates 3,000-3,760 g), and 25 children between 5 and 15 years o f age. In the infants examined no renal diseases were found. Preparation o f Antigen for Immunisation 8 normal human kidneys from persons aged be tween 20 and 40 years were obtained at necropsy, washed and perfused with cold phosphate-buffered saline (PBS) and comminuted carefully. This homo genate was forced through a 115 mesh sieve (metal) allowing the glomeruli to pass, while adding several litres o f cold PBS. A second filtration followed through a sieve (mesh 150) holding back the glomer uli. The glomeruli were put in 300 ml cold (4 °C) PBS and collected by centrifugation in an MSE refrigerated centrifuge at 350 g for 15 min; the supernatant was
discarded. The pellet was resuspended and sedimented four times. Aliquots o f the suspension were sonicated in an ice bath by an MSE ultrasonic disintegrator, applying several bursts o f 1 min each o f 1.6 A , 220 V at 5-min intervals. The disrupted material was sus pended in 1 M NaCl and centrifuged at l,4 0 0 g for 15 min. The GBM were washed three times in 1 M NaCl and three times in distilled water; the prepara tion was controlled by phase contrast microscopy and stored in the lyophilized state (Mahieu and Winand, 1970). Immunisation Schedule. A t 2-week intervals rab bits were injected intracutaneously with 10 mg GBM mixed with aluminium hydroxide and complete Freund’s adjuvant (Difco). 2 months later the animals were bled by cardiac puncture. The sera were absorbed eight times by glutaraldchyde insolubilized human plasma (Avrameas and Ternynck, 1969), platelets (twice), erythrocytes (three times) and white blood cells (three times). The absorbed sera were allowed to run against normal human serum in the double-diffu sion method o f Ouchterlony, whereby no precipita tion lines were found. Antibody activity against red blood cells was excluded applying a hemagglutination test, and reactivity against platelets as well as white blood cells by agglutination assays. The anti-GBM serum was tested by indirect immunofluorescence, producing a titre o f 1:256 using human kidney ob tained by biopsy. Absorption experiments as described by McPhaul and D ixon (1969) were made to investi gate the relationship to the GBM .
Preparation o f the Infant's G B M Antigen(s) The urine o f infants was collected in plastic bottles containing a pea-sized thymol crystal and kept in a refrigerator at 10 °C. After ultrafiltration in an Amicon stirred cell (diaflo U F 2000) applying a membrane permeable for molecular weights below 20,000, they were dialyzed against 0.1 M phosphate buffer o f pH 7.4. Urines were concentrated 200 times and the pro tein content adjusted to a standard concentration o f 20 mg/ml. Immunoelectrophoresis was performed on an LKB productor, at 50 mA and 340 V in 1% agar purum in a 1/15 M veronal-sodium acetate buffer, pH 8.4. Electrophoresis was run for 50 min at room temperature. Diffusion took place during a 48-hour period anti the precipitations were photographed.
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
278
Antigen Excretion in Premature Infants
279
f T-
Results In the group o f 25 premature infants 23 infants showed the precipitation line in the a -1-zone. 2 infants presenting 2 or 3 precipita tion lines had birth weights o f 980 and 2,400 g. In the panel o f 8 newborn infants the pattern of a -1-migration was detected without excep tion. In the group of 25 children between 5 and 15 years of age, 24 showed a single precipita tion line moving in the a-2-region as described for healthy young children of that age group and adults. In 1 case a double precipitation line was present.
Discussion Several papers reported about the urinary excretion of kidney antigens in men (Gilman, 1935; Antoine and Neveu, 1968; Lubec, 1978; McPhaul and Dixon, 1969; McPhaul and Lerner, 1968). As pointed out in ‘ Results’ , we detected different immunoelectrophoretical patterns in premature infant and newborn GBM
immature antigen with a-l-m obility, below the mature GBM ag migrating into the a-2-zone.
antigens on the one hand and in the children of the age group o f 5—15 years on the other. The excretion pattern with 2 or 3 precipitation lines could be interpreted as microbial degradation o f the urine samples. The results correlate well with the findings o f authors who performed morphological stud ies and clinical observations. The morphological studies o f Vernier and Birch-Andersen (1962) on the glomerulum revealed 3 stages of develop ment. In stage I the visceral epithelium is separated from the endothelial cells by a very thin continuous double membrane. In its sim plest form, this membrane appears to consist of the opposed limiting plasma membrane of endo- and epithelium only. Little, if any dense material is present within the 500 A space, separating the 2 membranes. A t stage II the GBM is about 1,000—1,300 A in width. The space between the two plasma membranes now contains a moderate amount o f electron-dense material which is rather loosely organized into a continuous band in the center o f the space. At higher magnification the dense part is seen to be composed of intermeshed short fine fila-
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
Fig. 1. Results o f the immunoelectrophoretic char acterization o f excreted GBM antigen. Above the
280
agreement with morphological and functional studies by other authors and could possibly explain the clinical signs as, e.g., proteinuria in infants. Additionally, we could show that the difference between the mature and immature GBM is not only quantitative but immunochemically qualitative.
References Antoine, B .L . and Neveu, T .: Pathological urinary excretion o f tissue macromolecules (histuria). J . Lab. clin. Med. 71: 101-112 (1968). Arant, B .S., jr.: Developmental patterns o f renal func tional maturation compared in the human neonate. J. Pediat. 92: 705 712 (1978). Avramcas, S. and Ternynck, T .: The cross linking o f proteins with glutaraldehyde and its use for the preparation o f immunoabsorbents. Immunochemistry 6: 53-6 6 (1 9 6 9 ). Bloom, P .: Width o f glomerular basement membrane in man at various ages. Anat. Rec. 133: 251 267 (1959). Fetterman, G .H .; Shuplock, N .A .; Philipp, F .J ., and Gregg. H .S.: The growth and maturation o f human glomeruli and proximal convolutions from term to adulthood. Pediatrics, Springfield 35: 601-619 (1965). Gilm an, G .: Urinary proteins. The appearance o f kidney proteins in the urine o f some cases o f severe chronic glomerular nephritis. J . Urol. 34: 727- 731 (1935). Klinger, G . und Geyer, G .: Histochemische Untcrsuchungen an renalen Basalmcmbranen wahrend der Entwicklung. Acta histochem. 21: 261-267 (1965). Lubec, G .; Balzar, E .; Weissenbacher, G ., and Syre, G .: Urinary excretion o f glomerular basement mem brane antigens in Alport’s syndrome. A new diag nostic approach. Archs Dis. Childh. 53: 401 406 (1978). Mahieu, P. and Winand, R .J.: Chemical structure o f tubular and glomerular basement membranes o f human kidney. Eur. J . Biochem. 12: 410-418 (1970).
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
merits. Stage III is classified by the appearance of ‘adult glomeruli’ differing from adult glomer uli in that their GBM is very thin, measuring about 1,000—1,300 A, though the lamina densa is more compact than in stage II. Higher magni fication reveals fine filaments o f GBM sub stance, many of them appearing to bridge the lamina rara and to contact the limiting mem brane of the epithelial foot processes. At 3 years o f age the GBM width reaches 3,000 A 0Bloom, 1959). That the glomerulum is not mature at deliv ery after a normal gestational span was reported and confirmed by Fetterman et al. (1965) measuring the size o f the newborn glomerulum (average diameter 85 instead o f about 200 /rm in adults): This morphological finding being congruent with our immunochemical studies revealing the immature pattern of moving into the a-1-region. Klinger and Geyer (1965) per formed histochemical studies on human fetal glomeruli reporting evidence for staining differ ences of the GBM possibly indicating decreased sialic acid content, a fact which could interpret the GBM antigen mobility towards the a -1-zone by the altered charge of the protein. Vernier and Birch-Andersen (1963) studied the permea bility of the GBM applying perfusion of the kdiney with either colloidal carbon or ferritin. In the mature glomeruli the lamina densa ap peared to function as a filter and only a few particles penetrated the structure, in the im mature glomerulum ferritin was frequently found in the urinary space, which indicated functional immaturity as well. Smith (1945) reported that in the urine of the newborn infant the protein content was increased, this finding being confirmed by the investigations of Rhodes et al. (1962) and Arant (1978). In conclusion, we describe the characteriza tion of immature GBM antigens which are in
Lubec/Coradello
Antigen Excretion in Premature Infants
Archs Path. 80: 241-256 (1965). Rhodes, P .G .; Hammel, C .H ., and Berman, L.B.: Uri nary constituents o f the newborn infant. J . Pediat. 60: 18 26 (1962).
Smith, A .C .: The physiology o f the newborn infant, pp. 39 41 (Thomas, Springfield 1945). Vernier, R .L . and Birch-Andersen, A .: Studies on the human fetal kidney. I. Development o f the glomer ulus. J . Pediat. 60: 754 768(1962). Vernier, R .L . and Birch-Andersen, A .: Studies on the human fetal kidney. II. Permeability characteristics o f the developing glomerulus. J . ultrastruct. Res. 8: 66 88 (1963).
Gert Lubec, M D, University o f Vienna, Department o f Paediatrics, Währinger Gürtel 7 4 -7 6 , A -1090 Vienna (Austria)
Downloaded by: King's College London 137.73.144.138 - 1/23/2019 11:52:46 PM
McDonald, S. and Emery, J.L .: The late intrauterine and postnatal development o f human renal glomer uli. J. Anat. 93: 331-340 (1959). McPhaul, J . J . , jr. and Dixon, E .J.: lmmunoreactive basement membrane antigens in normal human urine and serum. J . exp. Med. 130: 1395-1409 (1969). McPhaul, J . J . , jr. and Lerner, R .A .: Glomerular base ment membrane antigen excretion in normal and nephritic men. Abstract. Fed. Proc. 27: 544 (1968). Potter, E .L .: Development o f the human glomerulus.
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