The appearance of new antigens in the embryo during differentiation has been investigated by a number of authors. Among the proteins studied were myosin (Holtzer, 1961; Ebert, 1962), lens crystallin (Ten Cate & Van Doorenmaalen, 1950), chick embryo haemoglobin (Wilt, 1962), and keratin during feather formation in chick embryo (Ben-Or & Bell, 1965). The development of liver proteins in the chick embryo was studied by D’Amelio, Mutolo & Piazza (1963).

Okada & Sato (1963) and Okada (1965) studied the appearance of a ‘kidneyspecific’ antigen in the developing mesonephros. Lahti & Saxen (1966) demonstrated the appearance of mouse kidney-specific tubule antigens during development both in vivo and in vitro.

‘Kidney-specific’ antigens are found in the metanephric proximal secreting tubules of various mammals (Hill & Cruickshank, 1953; Weiler, 1956; Groupe & Kaplan, 1967; Nairn, Ghose & Maxwell, 1967), including man (Nairn, Ghose, Fothergill & McEntegart, 1962), and in the mesonephric tubules of birds and fish (Nairn et al. 1967). In addition cross-reacting membrane antigens (Dumonde, 1966; Weinberger & Boss, 1966) have been demonstrated in the basement membranes and reticular fibres of many organs, including kidney (Cruickshank & Hill, 1953). These are implicated in the glomerulonephritis induced by antikidney serum (Masugi, 1933). The appearance of kidney antigens in the human foetus has not previously been investigated.

The purpose of the present investigation was to relate the appearance of new antigens to the morphological differentiation of the metanephrogenic mesenchyme of the human kidney using immunodiffusion and immunofluorescence techniques.

Antigen

Human foetal and postnatal kidneys were used. Foetal kidneys from thirty-six foetuses were obtained within 1 h after legal abortion performed by caesarian section. The crown-rump length of the foetuses was 2·5–12 cm. Kidneys from children and adults ( = postnatal kidneys) were obtained at post-mortem within 12 h after death or occasionally as operation specimens.

Small tissue blocks were taken for immunohistology (see below). The main bulk of the tissues was then roughly minced with scissors in saline to remove as much blood as possible. The tissue specimens not used for immunohistology were frozen (−20°C) and thawed (room temperature) 2 or 3 times to rupture the cell membranes. They were then homogenized in an equal amount of cold distilled water in an ice bath with a Ultra Turrax homogenizer for a total of 5 min, with frequent intervals to avoid heating. The homogenates were centrifuged at 100 rev/min for 10 min to remove tissue debris. The supernatant was used for immunization. Material for immunodiffusion was prepared from the supernatant after centrifugation at 10000 g for 30 min. The supernatant was lyophilized and stored in a desiccator at room temperature until used. The pellets were stored at −20°C to be used as absorption material. Both pooled and individual homogenates were used.

Tissue blocks for immunofluorescence studies were prepared by freezedrying or cryostat sectioning. Blocks about 2×3 mm were cut for freezedrying, and somewhat larger blocks for cryostat sectioning, then frozen in liquid nitrogen. Freeze-dried blocks were embedded in paraffin wax (Gurr), m.p. 45°C, or in Polyester wax (B.D.H.) m.p. 37°C and sectioned in a serial microtome at 2−−4 μ. Paraffin was removed from sections with xylene. The sections were then passed through ethanol and dried at room temperature.

After drying on the slide cryostat sections were rinsed in buffered saline (PBS: 0-85 M-NaCl buffered at pH 7-2 with 0 01 M phosphate buffer) for 20 min at 4°C to remove material smeared at sectioning. The sections were then fixed while still wet in 2% formol in PBS, acetone, methanol or ethanol at 4°C or in a mixture of acetone, methanol and ethanol. After this the sections were rinsed in PBS for 15 min at 4°C, and used in immunofluorescence experiments.

Immunization and antisera

Adult rabbits of mixed stock were used. Material for immunization was prepared as described in the previous section, and mixed with an equal amount of Freund’s complete adjuvant (Difco). Fresh material was prepared for each injection from pooled or individual kidneys. Two rabbits were immunized with foetal kidney homogenate (one with 0·1 ml/injection, one with 0·5 ml/injection), and four with postnatal kidney homogenate (two with 0·1 ml/injection, two with 0·5 ml/injection). The rabbits were given monthly intracutaneous and subcutaneous injections in the inguinal or scapular regions. The rabbits were bled every second month, generally 10 days after the usual booster containing adjuvant. The antibody response was tested by double diffusion. The number of precipitin lines increased during the first year of immunization. Rabbits receiving the larger dose of antigen at each injection produced potent antisera in a somewhat shorter time. Some individual variations in the numbers of pre cipitin lines produced by different antisera against the same antigenic material were observed. This was not associated with differences in immunofluorescent staining properties. Immunization was continued for 2−3 years.

A commercial preparation of sheep anti-rabbit globulin (Mann Research Laboratories, New York) was used in immunofluorescence studies after labelling (see below). The preparation of antiserum against α-foetoprotein has been previously described (Linder & Seppälä, 1968).

Absorption of immune sera

To increase the selectivity of the anti-kidney sera they were absorbed with whole homogenate or occasionally with water-soluble or insoluble tissue fractions or with pooled plasma or serum. Autopsy and tumour material was prepared as described for kidney. Nephroblastoma tissue was obtained immediately after surgical removal of the tumour. The tumour usually used for absorption of the antisera was T2. The tumour material is described in a separate paper (Linder, 1969 a). Plasma, serum and the soluble portion of the homogenate after high-speed centrifugation were lyophilized, while the sedimented tissue fractions were stored at −20°C as described previously. The minimum amount of antigen necessary to obtain an effective absorption was determined by adding increasing amounts of antigen (10, 20, 40, 80, 100, 150 and 200 mg/ml antiserum). The mixtures were incubated at room temperature for 2 h and then at 4°C for 1−4 days. After incubation the mixture was centrifuged for 30 min at 5000 rev/min in the cold to remove the precipitate. Absorbed antisera were tested by double diffusion against different dilutions of antigen, usually 40, 80 and 100 mg/ml. The tested supernatants were stored at 4°C.

Immunodiffusion

Double diffusion in agar gel was carried out in a Petri dish in 1°/0 lonagar (Oxoid) or Agarose (l’industrie Biologique Française) in PBS. A modfication of Wadsworth’s (1957) microtechnique was used. The thickness of the agar layer (0-25 mm) was regulated by inserting a nylon line between the matrix and the supporting glass. The nylon line was removed when the agar had solidified, and the diffusion chamber was kept at 4°C for at least 2 days before use. After filling the wells with reactants the dish was kept for 2 h at room temperature and subsequently transferred to 4°C. The diffusion chambers were examined daily. Precipitin lines were usually visible within 24 h, but new lines sometimes appeared after 7 days. In addition to the conventional arrangement of wells filled with reactants the cross-diffusion system of Abelev (1960) was used in some experiments.

Immunofluorescence

Both the direct and indirect fluorescent antibody techniques were used (Coons & Kaplan, 1950). Antiserum globulin was prepared either by sodium sulphate precipitation according to Kiraly & Jobbagy (1966), or by DEAE-Sephadex A 50 chromatography (Dedmon, Holmes & Deinhardt, 1965).

Antiserum globulin from anti-kidney and anti-rabbit gamma globulin sera were conjugated with fluorescein isothiocyanate (FITC), crystallized and chromatographically pure (Baltimore Biological Co.) The procedure was that of Lewis, Jones, Brooks & Cherry (1964), but using 0·2 mg FITC per ml of phosphate buffer (pH 10·5), instead of 0·625 mg/ml, for adding to one ml of 1% globulin solution. The labelling, for 5 h at room temperature, resulted in a conjugate with an FlTC/protein molar ratio of 2−3 determined by the formula of Mekler et al., quoted by Wagner (1967). Free fluorescein was removed using a Sephadex G-25 column equilibrated with PBS (Killander, Ponten & Roden, 1961). Over-labelled protein molecules were removed by passage through a DEAE Sephadex A-50 column, eluting with 0·01 M phosphate, pH 7·2 containing 0·21 M-NaCl (Dedmon et al. 1965). This absorption reduced the FlTC/protein ratio by only 0·2 or less. The eluted solution was passed through a bacterial filter and then stored at 4°C.

Procedure

Tissue sections were taken from the rinsing solution after fixation and incubated in a moist chamber with continuous agitation at room temperature. When the direct method was used sections were incubated with fluorescent antiserum for 2 h, then rinsed for 15 min in three changes of PBS. In the direct method the sections were incubated with unlabelled antiserum or chromatographically purified globulin from it for 2 h. This was followed by rinsing for 20 min in three changes of PBS. Fluorescent anti-rabbit γ-globulin serum, prepared as described, was added for 45 min. The protein concentration of this antiserum was about 0·3%. After incubation the slides were rinsed in PBS and kept in this solution until examined. The slides could be re-examined after 5 days without loss of specific fluorescence.

Photomicrography

The slides were examined under a Wild fluorescence microscope equipped with a high-pressure mercury vapour lamp and a bright-field quartz condenser using Schott primary filter UG 1 (2 mm) and secondary filter GG 13. For photography using Kodak Super XX film, Schott primary filter BG 12 (3 mm) and secondary filter OG 1 gave better results. Immunodiffusion slides were photographed in dark-field illumination on Agfa Agepe 35 mm document film.

Differentiation of kidney antigens was studied with absorbed antisera against postnatal kidney and the distribution of foetal kidney antigens was studied with absorbed antisera against foetal kidney.

The effect of absorptions was tested against the tissue material used in both immunodiffusion and immunofluorescence. The following controls for specificity of the immunofluorescence studies were performed:

  • Direct method. (1) Labelled globulin solution prepared from pre-immunization serum (0-serum). No fluorescence. (2) Blocking by pre-incubation of slides with unlabelled immune serum; no fluorescence in the proximal tubule. (3) Pre-incubation with O-serum; no effect on the tubule fluorescence.

  • Indirect method. (1) Fluorescent anti-rabbit γ-globulin serum only; no fluorescence. (2) O-serum in the first step instead of immune serum; occasional staining of vessel media and a very faint collective tubule fluorescence (Fig. 4 c). (3) Fluorescent anti-human γ-globulin serum in the final step instead of fluorescent anti-rabbit γ-globulin serum; no staining. (4) The γ-globulin fraction of immune serum instead of whole serum; no change in specific fluorescence; the fluorescence of vessel media was not altered and there was a bright fluorescence of elastic fibre. (5) γ-globulin from O-serum. A bright elastic fibre fluorescence was seen in addition to the fluorescence of vessel media.

Foetal kidney antigens

Immunodiffusion

Anti-foetal kidney serum gave precipitin lines against foetal kidney that were not seen when it reacted against postnatal kidney (Fig. 1a). These lines will be called a and b. The foetal kidney antigens were also demonstrated in kidneys from newborn infants (K1) (Fig. 1b).

Fig. 1

Comparison of two antigen-antibody systems using cross-diffusion, (a) Anti-kidney serum (AK) against pooled adult kidney (K), and anti-foetal kidney serum (AFK) against pooled foetal kidney (FK). (b) AK against pooled newborn kidney (K1) and AFKagainst FK. Antigens present in K are also found in FK and However, a number of precipitin lines are formed against FK and K1 but not against K. All activity against normal human serum (NHS) was absorbed from antisera.

Fig. 1

Comparison of two antigen-antibody systems using cross-diffusion, (a) Anti-kidney serum (AK) against pooled adult kidney (K), and anti-foetal kidney serum (AFK) against pooled foetal kidney (FK). (b) AK against pooled newborn kidney (K1) and AFKagainst FK. Antigens present in K are also found in FK and However, a number of precipitin lines are formed against FK and K1 but not against K. All activity against normal human serum (NHS) was absorbed from antisera.

The antibodies in anti-foetal kidney serum that reacted with adult kidney homogenate in double diffusion could be absorbed by 40 mg of this lyophilized kidney tissue (soluble fraction) (Fig. 2). The absorbed antiserum gave lines a and b. Absorption with 50 mg/ml pooled foetal organs (excluding kidney) or foetal serum (50 mg/ml) prevented the formation of a lines. After these absorptions the anti-foetal kidney sera still formed one or two lines (marked b) when reacted with foetal kidneys. However, anti-kidney serum also produced theline(s) b when reacting with foetal kidney (Fig. 1 a and b). The antigen(s) responsible for the production of line(s) b were demonstrated in some postnatal kidneys in small amounts. This explains why anti-kidney serum produced line b. The amount of antigen in pooled material was, however, not sufficient to absorb the antibodies producing lines b from anti-foetal kidney serum. A reaction of identity was seen between a lines and an antiserum against a-foetoprotein.

Fig. 2

Foetal kidney antigens. Reactions between FK and AFK absorbed with NHS and increasing amounts of AK: 20 mg/ml (2), 40 mg/ml (3) or 80 mg/ml (4). Additional absorption with foetal serum (20 mg/ml) abolished a number of precipitin lines (designated a), but did not affect two lines designated b (5). Absorption with 100 mg/foetal serum per ml did not affect the formation of lines b (6). Abbreviations as in Fig. 1.

Fig. 2

Foetal kidney antigens. Reactions between FK and AFK absorbed with NHS and increasing amounts of AK: 20 mg/ml (2), 40 mg/ml (3) or 80 mg/ml (4). Additional absorption with foetal serum (20 mg/ml) abolished a number of precipitin lines (designated a), but did not affect two lines designated b (5). Absorption with 100 mg/foetal serum per ml did not affect the formation of lines b (6). Abbreviations as in Fig. 1.

Immunofluorescence

Foetal kidney antigens were studied by immunofluorescence using antisera against foetal kidneys absorbed with pooled adult kidneys and normal human serum. The antiserum had an affinity for the undifferentiated foetal kidney mesenchyme. The fluorescence had the same distribution as the interstitial connective tissue (Fig. 5). The tubules and glomeruli were not stained. After additional absorption with foetal serum the antiserum had no specific affinity for foetal kidney. Thus the staining of undifferentiated kidney mesenchyme seems to be due to antigens marked a.

Antigens appearing during differentiation of the metanephrogenic mesenchyme Immunodiffusion

Antisera against postnatal kidney absorbed with normal human serum still gave about twenty precipitin lines in double diffusion against postnatal kidney. Precipitin lines produced against postnatal kidney also formed against foetal kidney. Antisera further absorbed with tumour gave 6−8 precipitin lines against foetal or postnatal kidneys (Fig. 3). Antisera further absorbed with lung left six precipitin lines, with liver four or five and with small intestine two lines against kidney.

Fig. 3

Reaction between K and AK absorbed with tumour (T) and NHS. Eight precipitin lines are formed against kidney, but none against the tumour used for absorption. One line deviates slightly by another tumour (T1). Abbreviations as in Fig. 1.

Fig. 3

Reaction between K and AK absorbed with tumour (T) and NHS. Eight precipitin lines are formed against kidney, but none against the tumour used for absorption. One line deviates slightly by another tumour (T1). Abbreviations as in Fig. 1.

Fig. 4

Foetal kidney from 80 mm foetus (rump-head length), (a) Haematoxylin-eosin, (b) treated with anti-adult kideny serum absorbed with NHS and tumour. Staining present in all parts of the nephron, (c) control section treated with pre-immunization serum.

Fig. 4

Foetal kidney from 80 mm foetus (rump-head length), (a) Haematoxylin-eosin, (b) treated with anti-adult kideny serum absorbed with NHS and tumour. Staining present in all parts of the nephron, (c) control section treated with pre-immunization serum.

Fig. 5

Initiation of morphological differentiation of the metanephrogenic mesenchime—the condensation stage. Fluorescence is seen in the undifferentiated mesenchyme (mes.) but not in condensed areas (c) at the tips of the collecting tubules (col.)

Fig. 5

Initiation of morphological differentiation of the metanephrogenic mesenchime—the condensation stage. Fluorescence is seen in the undifferentiated mesenchyme (mes.) but not in condensed areas (c) at the tips of the collecting tubules (col.)

Immunofluorescence

Absorption of antisera against postnatal kidneys with whole homogenates of a number of normal tissues and some nephroblastomas resulted in an antiserum that stained different parts of the nephron but not the undifferentiated kidney mesenchyme (Table 1). All of these absorptions removed the activity against undifferentiated kidney mesenchyme. Normal tissues were more efficient than tumour tissue as judged by double diffusion and by a more widespread fluorescence in the nephron.

Table 1

Immunohistochemical localization of kidney antigens using various absorbed antisera

Immunohistochemical localization of kidney antigens using various absorbed antisera
Immunohistochemical localization of kidney antigens using various absorbed antisera

Antisera against postnatal kidneys absorbed with normal human serum and the soluble fraction of tumour homogenate, localized diffusely in postnatal kidney. It reacted with undifferentiated kidney mesenchyme but failed to stain the condensed areas of mesenchyme in differentiating foetal kidney (Fig. 5). Additional adsorption with the insoluble fraction of tumour homogenate abolished the fluoresecence of the interstitial connective tissue and undifferentiated mesenchyme, but did not affect that of the different parts of the nephron (Fig. 4b). Fluorescence was seen also in the basement membranes of the glomeruli and the tubules. (Figs. 6b, 7, 14, 16a). Staining of vessel media was also present (Fig. 14). The effect of absorption with AB red blood cells was tested in each new kidney studied. This absorption abolished staining of red blood cells when present and weakened the collecting tubular fluorescence. If an effect due to this absorption was noticed, absorption with red blood cells was used in further experiments.

Control pre-immunization sera sometimes stained the vessels, but no other part of the sections. Unless otherwise stated the differentiation of kidney antigens was studied using antiserum against postnatal kidneys absorbed with normal human serum, and whole homogenate of tumour. The sera were absorbed further with homogenates of various normal organs in order to distinguish between different antigens in the nephron (Table 1).

The antigens appearing during differentiation could be classified on the basis of their distribution and their staining with antisera absorbed with different normal tissues. There were five groups of antigens, (a) Basement membrane antigens. The fluorescence was abolished by absorption with placenta, but only partially abolished by absorption with spleen, (b) Glomerular epithelium antigen(s). Stained after absorption with placenta, (c) Glomerular endothelium antigen(s). Did not stain after placental absorption, (d) Proximal secreting tubules contained at least two specific antigens. One of these was present also in loops of Henle (but nowhere else in the kidney). The fluorescence was abolished by absorbing antiserum with small intestine, (e) The proximal secreting tubule fluorescence was still present after this absorption, but was abolished after absorption with kidney.

Mesonephros

Mesonephric glomeruli and tubules were stained before metanephric development had led to the differentiation of the parts of the nephron (Fig. 6). At early stages of morphogenesis the fluorescence in the metanephros was restricted to the basement membranes of the branching ureteric bud—the prospective collecting tubules.

Fig. 6

Section through 20 mm embryo showing early metanephros (mt) and degenerating mesonephros (ms). In addition to basement membrane staining, seen in both mesonephros and metanephros, there is bright staining of mesonephric tubules and glomeruli. In the mesonephric duct (d) there is only basement membrane fluorescence. Note weak intestinal brush border staining at (g).

Fig. 6

Section through 20 mm embryo showing early metanephros (mt) and degenerating mesonephros (ms). In addition to basement membrane staining, seen in both mesonephros and metanephros, there is bright staining of mesonephric tubules and glomeruli. In the mesonephric duct (d) there is only basement membrane fluorescence. Note weak intestinal brush border staining at (g).

Fig. 7

Renal vesicle (rv) surrounded by a fluorescent limiting membrane. Note fusion of this membrane with the basement membrane of the collecting tubule (col.).

Fig. 7

Renal vesicle (rv) surrounded by a fluorescent limiting membrane. Note fusion of this membrane with the basement membrane of the collecting tubule (col.).

Metanephros

1 Basement membranes

Fluorescence of basement membranes was seen at epitheliomesenchymal borders and in glomerular capillary tufts. The renal vesicle was surrounded by a fluorescent limiting membrane (Fig. 7), which became more intensely stained during the development of the S-shaped body. The membrane of the renal vesicle bordering the tubule fused with the membrane of the collecting tubule (Fig. 7). Concurrently the membrane fluorescence began to split as the S-shaped body developed (Fig. 8). The cells of the future Bowman’s capsule were surrounded by a less intensely stained membrane than the upper and medial segments. At the time of early cleft formation there was a marked increase in the intensity of the basement membrane fluorescence of the medial and lower segments of the S-shaped body facing the cleft (Fig. 8).

Fig. 8

S-shaped body. Shows appearance of proximal secreting tubule antigens (pr). The basement membrane is doubled next to the collecting tubule.

Fig. 8

S-shaped body. Shows appearance of proximal secreting tubule antigens (pr). The basement membrane is doubled next to the collecting tubule.

The capillary basement membrane fluorescence was never clear and well demarcated in the primitive glomerulus (Fig. 12). Instead the fluorescence of the endothelial cells forming the primitive glomerular tuft was continuous with that of the capillary walls. Gradually, during the maturation of the glomerulus, the staining became more distinct and more like membrane staining.

Absorption with placenta homogenate abolished all the membrane fluorescence. After absorption with spleen there was still a faint fluorescence in some basement membranes of the tubules, but the basement membranes of the glomerulus were not stained.

2 Glomeruli

Antigens were demonstrated in both epithelial and endothelial cells of the glomeruli.

Epithelial cells

Antigenic differentiation was seen when the single layers of cells of the presumptive glomerulus epithelium began to proliferate, and to form ingrowths between the cells filling the primitive glomerular tuft. At this stage the future Bowman’s capsule cells, which constitute the outer layer of the bowl-like primitive glomerulus anlage, are flat and contain a reduced amount of cytoplasm (Fig. 9).

Fig. 9

Formation of glomerular epithelium antigens (ep) and proximal secreting tubule antigens (pr) seen in the luminal part of tubule cells.

Fig. 9

Formation of glomerular epithelium antigens (ep) and proximal secreting tubule antigens (pr) seen in the luminal part of tubule cells.

No capillary structures could be seen in the cells constituting the glomerular tuft (Figs. 10, 11, 14). In the primitive elongated epithelial cells the fluorescence seemed to be associated with the cell membranes (Fig. 13). However, as proliferation proceeded the fluorescence was clearly seen to be associated with the cytoplasm (Fig. 15). At this stage the capillary lumina of the tuft had formed, and the epithelial cells were arranged in septa between the primitive capillary loops.

Fig. 10

Beginning of proliferation of glomerular epithelium cells towards the primitive glomerular tuft. Endothelial and epithelial glomerulus antigens located at the epithelio-endothelial border.

Fig. 10

Beginning of proliferation of glomerular epithelium cells towards the primitive glomerular tuft. Endothelial and epithelial glomerulus antigens located at the epithelio-endothelial border.

Fig. 11

Beginning of proliferation of glomerular epithelium cells towards the primitive glomerular tuft. Endothelial and epithelial glomerulus antigens located at the epithelio-endothelial border.

Fig. 11

Beginning of proliferation of glomerular epithelium cells towards the primitive glomerular tuft. Endothelial and epithelial glomerulus antigens located at the epithelio-endothelial border.

Fig. 12

Early lumen formation in primitive glomerular capillary tuft. Fluorescence in both endothelial and epithelial cells of the glomerulus is seen.

Fig. 12

Early lumen formation in primitive glomerular capillary tuft. Fluorescence in both endothelial and epithelial cells of the glomerulus is seen.

Fig. 13

Glomerular epithelium fluorescence demonstrated with antiserum absorbed with spleen. No fluoresence visible in endothelial cells and basement membranes.

Fig. 13

Glomerular epithelium fluorescence demonstrated with antiserum absorbed with spleen. No fluoresence visible in endothelial cells and basement membranes.

Fig. 14

Fluorescence in the endothelium but not the epithelium of the primitive glomerular tuft.

Fig. 14

Fluorescence in the endothelium but not the epithelium of the primitive glomerular tuft.

Fig. 15

Appearance of the glomerular epithelium fluoresecence after absorption of antiserum with spleen. The stage of development is that of Fig. 12.

Fig. 15

Appearance of the glomerular epithelium fluoresecence after absorption of antiserum with spleen. The stage of development is that of Fig. 12.

As the glomerulus matured and the intercapillary spaces were reduced there was still an irregular fluorescence associated with the membranes of glomerular epithelium cells. Absorption with spleen or placenta homogenates did not affect the epithelial fluorescence (Figs. 13,15). The fluorescence was however abolished by absorption with lung homogenate.

Endothelial cells

At the stage when the glomerular epithelium antigen was first demonstrated a new antigen was seen in cells of the primitive tuft bordering the epithelial layer. At about the time of formation of cavities in the cell mass the endothelium antigen was seen in all the cells of the tuft (Fig. 14). The fluorescence was homogeneous in the walls of the newly formed cavities (Fig. 12). It was continuous with the capillary basement membrane fluorescence and formed a continuous strand in the mature glomeruli.

Antibodies against the endothelial antigen(s) could be absorbed by spleen homogenate (Figs. 13, 15).

3 Tubules

Tubule fluorescence was demonstrated in all parts of the nephron (Fig. 4). Fluorescence of the proximal secreting tubule was the most prominent, and could also be demonstrated by the indirect fluorescent antibody technique. The fluorescence was very intense in the brush border of the cells, which often filled the whole lumen of the tubules. In addition to the brush border fluorescence there was a distinct fluorescence in the cytoplasm of the cells. This was usually more obvious in distal parts of the proximal secreting tubules. Both the cytoplasmic and the brush border fluorescence extended into the Bowman’s space (Fig. 1 b). During maturation of the kidney the fluorescence extending into the glomerulus became gradually less prominent and the fluorescence was confined to the brush border. Distally, where the proximal secreting tubule is continuous with the loops of Henle, there was an abrupt change in the character of the fluorescence. The narrow loops of Henle had a uniform cytoplasmic staining. This was most obvious in the renal papilla where the loops of Henle contrasted clearly with the weakly stained collecting tubules. In the cortical region the weakly stained tubules were either collecting tubules or distal tubules. The collecting tubule fluorescence was continuous with that of the epithelium of the renal pelvis.

The antigens of the proximal secreting tubule in the medial segment of the S-shaped body were demonstrated at about the same developmental stage as glomerulus antigens (Fig. 9). Sometimes the proximal secreting tubule antigens were seen a little earlier than glomerular epithelium antigens (Fig. 8).

Non-identity of glomerulus and tubule antigens was demonstrated by absorption experiments. Absorption with placenta and lung homogenates abolished the staining of the glomeruli and basement membranes (Fig. 16), but not that of the proximal secreting tubules and loops of Henle.

Fig. 16

In (a) the proximal tubule, glomerulus and basement membrane antigens are stained. In (b) only proximal secreting tubule fluorescence is seen after absorption of antiserum with lung and placenta. Note the extension of proximal secreting tubule cells to form a part of the parietal Bowman’s capsule.

Fig. 16

In (a) the proximal tubule, glomerulus and basement membrane antigens are stained. In (b) only proximal secreting tubule fluorescence is seen after absorption of antiserum with lung and placenta. Note the extension of proximal secreting tubule cells to form a part of the parietal Bowman’s capsule.

Absorption with small intestinal homogenate abolished the staining of loops of Henle, but reduced the fluorescence of the cytoplasm of the proximal secreting tubules only slightly, and the fluorescence of the brush border not at all. Absorption with whole kidney homogenate or washed sediment abolished all of the staining, but absorption with the lyophilized soluble fraction did not affect the staining.

Antigenic differentiation of the metanephrogenic mesenchyme

Condensation of the undifferentiated metanephrogenic mesenchyme at the tips of the ureteric buds involved the first immunohistochemically demonstrated change—loss of connective tissue and foetal antigens. Development of the renal vesicle was accompanied by surrounding fluorescence. This fluorescence appeared to be associated with a basement membrane, although no basement membrane can be seen in the renal vesicle at this stage with the light microscope. This fluorescence fused with that of the basement membrane of the collecting tubule. As the S-shaped body was formed proximal secreting tubule antigens appeared in the medial segment. Endothelium and epithelium antigens became demonstrable at the interface between the cells of the tuft and those of the lower segment of the S-shaped body after formation of the primitive glomerular tuft. As the primitive vascular spaces developed, the fluorescence in the endothelium and in the vascular membrane basement became indistinguishable. During maturation of the glomerulus the epithelial and endothelial cells of the intercapillary spaces gradually degenerated and the antigens become closely associated with the capillary basement membranes.

Connective tissue and foetal antigens were found in the undifferentiated kidney mesenchyme. These antigens were lost during the initial step in differentiation—condensation of mesenchyme cells at the tips of the ureteric buds. It is not possible to decide whether these antigens are cellular or intercellular. If they are intercellular their absence in the condensed areas may be the result of increased cellular adhesion, leading to a gradual disappearance of the intercellular spaces (Saxén & Wartiovaara, 1966).

The reactions with foetal kidney antigens might be due to antibodies against a-foetoprotein, since absorption with foetal serum removed the activity. This interpretation is supported by the fact that antiserum against a-foetoprotein became localized in the same areas as anti-foetal kidney serum (Linder & Seppälä, 1968).

Immunization with unfractionated kidney material resulted in antibodies against many tissue antigens. After absorption with nephroblastoma tissue a number of cross-reacting antibodies were removed. This was seen as a decrease in the number of precipitin lines in double diffusion, and as a distinct localization of fluorescent antiserum in various parts of the nephron and vessel walls.

The antigens with which these antisera reacted were not detected by immunofluorescence in the undifferentiated foetal kidney mesenchyme. The absorbed antiserum could therefore be used to study the appearance of a number of tissue antigens during morphogenesis. Further absorptions with a number of normal tissues made the anti-kidney sera more specific. Antisera could then distinguish between common glomerular epithelium and endothelium antigens, tubule antigens, cross-reacting and ‘kidney-specific’ proximal secreting tubule antigens.

Formation of the renal vesicle or pretubular structure was associated with a fluorescence like that of basement membranes. No basement membrane could be seen with the light microscope at this stage, but using the electron microscope Kurtz (1958) and Jokelainen (1963) observed some amorphous material, resembling primitive basement membrane, surrounding the renal vesicle.

Wartiovaara (1966 a, b) used the electron microscope to study the early phase of basement membrane formation during differentiation of mouse metanephrogenic mesenchyme in vitro. He demonstrated small fibrils embedded in a homogeneous matrix surrounding the pretubular structures, and provided evidence suggesting that pretubular cells participated in the formation of this membrane. The possibility that mesenchyme was also involved could not be excluded. This agrees with the observation of Kallman & Grobstein (1965), who showed that mesenchyme participates in membrane formation around salivary epithelium. Absorption of antiserum with spleen abolished almost all of the basement membrane fluorescence. This indicates that the fluorescence was principally due to antigenic material of mesenchymal origin. A faint fluorescence remained surrounding some tubules and may have been due to the presence of antigens of epithelial origin (Pierce, 1966).

The cells of the glomerular tuft were shown to acquire antigenic properties lacking in the cells of the stromal mesenchyme. The new antigen(s) seemed to become associated with the walls of the early capillaries as development of the glomerular capillary tuft proceeds. This agrees with the observations of Jokelainen (1963). He showed that during the formation of the S-shaped body stromal mesenchyme cells invade the cleft separating the lower and middle limbs, and suggested that these cells are the progenitors of the endothelial cells of the glomerular tuft. He demonstrated that the development of the lumens of the glomerular capillaries is initiated by a gradual widening of the intercellular spaces in the compact mass of endothelial cells. The cells containing the endothelial antigen(s) could not be seen to be directly derived from the mesenchyme cells, and there may be an alternative to the postulated transformation of mesenchymal cells to epithelial cells proposed by Jokelainen. It cannot be excluded that a few cells of endothelial origin accompany the ‘row of erythrocytes’ observed by Jokelainen, even if he could not see any capillary wall surrounding these cells. The studies of Potter (1965) and Osathanodh & Potter (1966) indicate that at least from a functional point of view there is a capillary already present during early cleft formation and that this vessel, by gradual broadening and fenestration, will give rise to the glomerular capillaries. This developmental sequence seems to reflect rapid proliferation of endothelial cells. Scott & Rowell (1967) observed similar fluorescence to that described here, in the primitive capillary tuft of the developing rat glomerulus. They used an antiserum against splenic reticulum or isolated glomeruli. This agrees with the finding in the present paper that absorption of antiserum with spleen abolished the endothelial fluorescence. Scott (1957) also observed fluorescence in the glomerular epithelium. He showed that the antigen was not organ-specific by obtaining identical fluorescence with antiserum against synovial membrane and against glomeruli. The nature of the glomerular epithelium antigen(s) was not investigated further in the present study. However, the epithelial basement membrane antigen (Pierce, 1966) does not seem to be responsible for the immuno-fluorescent staining of these cells, since absorption with placenta abolished the fluorescence of basement membranes but not that of the glomerular epithelium.

The proximal secreting tubule antigens were demonstrated as a lumen fluorescence in the medial segment of the S-shaped body. It is interesting that there was no demonstrable difference between the time of appearance of the ‘kidneyspecific’ and of the cross-reacting proximal secreting tubule antigens. Okada (1965) studied the appearance of ‘kidney-specific’ proximal secreting tubule antigens in the developing chick mesonephros. These antigens ‘became demonstrable coinciding with an initiation of the epithelial architecture of the cell’, and there was a weak specific fluorescence in the cells of the condensed nephrogenic mesenchyme. Lahti & Saxén (1966) demonstrated ‘kidney-specific’ antigens in differentiating mouse metanephrogenic mesenchyme in vitro at a rather late stage in tubule formation (Wartiovaara, 1966b).

The antigens of kidney tubules cross-react with those of lung and liver (Okada, 1962; Okada & Sato, 1963). However, even after absorption with these tissues the antisera are not necessarily kidney-specific, since Nairn et al. (1967) observed reactivity with seminal vesicle and Edgington et al. (1967) isolated two proximal tubule antigens which cross-react with brush border antigens of small intestinal mucosa. In addition there is some cross-reaction between the antigens of proximal secreting tubules and those of the tubules of the epididymis (Linder, 1969b). Absorption with small intestine neutralized antibodies reacting with epididymis, gut and seminal vesicle but not those reacting with the brush border of the proximal tubules. Therefore these brush border antigens may be kidney-specific.

Differentiation of antigens in the metanephrogenic mesenchyme of the human foetal kidney was studied by immunofluorescence and double diffusion in agar gel using antisera against foetal and postnatal kidneys.

Absorption with nephroblastoma tissue removed antibodies against common or cross-reacting antigens. The anti-kidney sera then did not react with undifferentiated kidney mesenchyme and could be used to detect kidney antigens appearing during differentiation.

Foetal kidney antigens were demonstrated with anti-foetal kidney sera after absorption with normal human serum and adult kidney. The kidney antigens appearing during differentiation were classified in five groups on the basis of their histological distribution: (1) basement membranes, (2) glomerular epithelium, (3) glomerular endothelium, (4) proximal secreting tubules, (5) loops of Henle.

Antisera absorbed with various normal tissues could discriminate between these groups, except that glomerular endothelial antigens could not be differentiated from capillary basement membrane antigens.

The antigens of the proximal secreting tubules were of two types: cytoplasmic and brush border. The latter were both cross-reacting and kidney-specific.

During the condensation stage, which initiates the differentiation of the metanephrogenic mesenchyme, foetal and interstitial connective tissue antigens decreased and became undetectable. New antigen(s) could be seen in the limiting membrane of the renal vesicle. Proximal secreting tubule antigens were demonstrated in the medial segment of the S-shaped body. Formation of the primitive glomerular tuft was associated with the appearance of both epithelium and endothelium antigens in the glomerulus.

The present results show that tissue specificity is acquired during organogenesis in the human metanephros. The findings do not support the view that initiation of histogenesis is preceded by the synthesis of new molecular species.

Différenciation d’antigènes du rein chez le fœtus humain

La différenciation d’antigènes dans le mésenchyme métanéphritique fœtal humain a été étudié par immunofluorescence et double diffusion dans un gel d’agar en utilisant des antiserums à l’égard des reins fœtaux et postnataux.

L’absorption par du tissu lisse de blastème rénal a enlevé les anticorps vis à vis des antigènes communs ou à réaction croisée. A la suite de cela, les sérums anti-rein n’ont pas réagi avec du mésenchyme rénal indifférencié et n’a pas ou être utilisé pour la détection d’antigènes rénaux qui auraient pu apparaître au cours de la différenciation.

Des antigènes rénaux fœtaux ont été démontrés en utilisant des sérums antirein fœtal absorbés avec du sérum humain normal et du rein adulte. Les antigènes rénaux apparaissant au cours de la différenciation ont été classés en cinq groupes sur la base de leur distribution histologique: (1) membranes basales, (2) épithélium glomérulaire, (3) endothélium glomérulaire, (4) tubes proximaux, (5) anses de Henle.

Des antisérums absorbés par des tissus normaux variés ont pu faire une discrimination entre ces groupes, sauf que l’antigène glomérulaire endothélial n’a pu être distingué d’antigènes de la membrane basale des capillaires.

Les antigènes des tubes proximaux étsient de deux types: cytoplasmiques et de bordure en brosse. Ces derniers montraient à la fois une réaction croisée et une réaction rénale spécifique.

Pendant le stade de condensation qui marque le début de la différenciation du mésenchyme métanéphritique, les taux d’antigènes des tissus conjonctifs fœtal et intersticiel diminuent au point que ces antigènes n’ont plus pu être détectés. De nouveaux antigènes (ou antigène) ont pu être décelés dans la membrane limitante des vésicules rénales. Des antigènes de tubule sécréteur proximal ont été démontrés dans le segment moyen du corpuscule en S. La formation de la houppe glomérulaire primitive s’est montrée associée à l’apparition des deux antigènes épithélial dans le glomérule.

Ces résultats démontrent que la spécificité tissulaire est acquise pendant l’organogénèse du métanéphros humain. Les faits constatés ne sont pas en faveur de l’idée que l’initiation de l’histogénèse puisse être précédée de la synthèse de nouvelles espèces de protéines.

Abelev
,
G. L
(
1960
).
Modification of the agar precipitation method for comparing two antigenantibody systems
.
Folia Biol
.
6
,
56
8
.
Ben-Or
,
S.
&
Bell
,
E.
(
1965
).
Skin antigens in the chick embryo in relation to other developmental events
.
Devi Biol
.
11
,
184
201
.
Coons
,
A. H.
&
Kaplan
,
M. H.
(
1950
).
Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent antibody
.
J. exp. Med
.
91
,
1
13
.
Cruickshank
,
B.
&
Hill
,
A. G. S.
(
1953
).
The histochemical identification of a connective tissue antigen in the rat
.
J. Path. Bact
.
66
,
283
9
.
D’amelio
,
V.
,
Mutolo
,
V.
&
Piazza
,
E.
(
1963
).
A serological study of the cell fractions during the embryonic development of liver in chick
.
Expl Cell Res
.
31
,
499
507
.
Dedmon
,
R. E.
,
Holmes
,
A. W.
&
Deinhardt
,
F.
(
1965
).
Preparation of fluorescein isothiocyanate-labelled globulin by dialysis, gel filtration, and ion exchange chromatography in combination
.
J. Bact
.
89
,
734
9
.
Dumonde
,
D. C.
(
1966
).
Tissue-specific antigens
.
Advanc. Immunol
.
5
,
245
384
.
Ebert
,
J. D.
(
1962
).
The acquisition of biological specificity
.
In The Cell
(ed.
J.
Brachet
and
E. E.
Mirsky
), vol.
I
, pp.
619
93
.
New York, London
:
Academic Press
.
Edgington
,
T. S.
,
Glassock
,
R. J.
,
Watson
,
J. I.
&
Dixon
,
F. J.
(
1967
).
Characterization and isolation of specific renal tubular epithelial antigens
.
J. Immunol
.
99
,
1199
1210
.
Groupe
,
W. E.
&
Kaplan
,
M. H.
(
1967
).
A proximal tubular antigen in the pathogenesis of autoimmune nephrosis
.
Fedn Proc. Fedn Am. Socs exp. Biol
.
26
,
573
.
Hill
,
A. G. S.
&
Cruickshank
,
B.
(
1953
).
A study of antigenic components of kidney tissue
.
Br. J. exp Path
.
34
,
27
34
.
Holtzer
,
H.
(
1961
).
Aspects of chondrogenesis and myogenesis
.
In Synthesis of molecular and cellular structure, Growth Symp. no. 19
(ed.
D.
Rudnick
), pp.
35
87
.
New York
:
Ronald Press
.
Jokelainen
,
P.
(
1963
).
An electron microscope study of the early development of the rat metanephric nephron
.
Acta Anatomica
(Suppl. 47 = 1 ad Vol. 52).
Kallman
,
F.
&
Grobstein
,
C.
(
1965
).
Source of collagen at epithelio-mesenchymal interfaces during inductive interaction
.
Devi Biol
.
11
,
169
83
.
Killander
,
J.
,
Ponten
,
J.
&
Roden
,
L.
(
1961
).
Rapid preparation of fluorescent antibodies using gelfiltration
.
Nature, Lond
.
192
,
182
3
.
Kiraly
,
K.
&
Jobbagy
,
A.
(
1966
).
Control of conjugation of fluorescent protein tracing
.
Path. Microbiol
.
29
,
865
72
.
Kurtz
,
S. M.
(
1958
).
The electron microscopy of the developing human renal glomerulus
.
Expl Cell Res
.
14
,
355
67
.
Lahti
,
A.
&
Saxén
,
L.
(
1966
).
Studies on kidney tubulogenesis. VIII. Appearance of kidneyspecific antigens during in vivo and in vitro development of secretory tubules
.
Expl Cell Res
.
44
,
563
71
.
Lewis
,
W. J.
,
Jones
,
W. L.
,
Brooks
,
J. B.
&
Cherry
,
W. B.
(
1964
).
Technical considerations in the preparation of conjugated fluorescent antibody
.
Appl. Microbiol
.
12
,
343
8
.
Linder
,
E.
(
1969a
).
The antigen structure of nephroblastomas
.
Int. J. Cancer
.
4
,
232
47
.
Linder
,
E.
(
1969b
).
Cross-reacting antigens in kidney and other organs
.
Ann. Med. exp. Fenn
.
47
. (In the Press.)
Linder
,
E.
&
Seppälä
,
M.
(
1968
).
Localization of a-foetoprotein in the human foetus and placenta
.
Acta path, microbiol. scand
.
73
,
565
71
.
Masugi
,
M.
(
1933
).
Über das Wesen der spezifischen Veränderungen der Niere und der Leber durch das Nephrotoxin bzw. das Hepatotoxin. Zugleich ein Beitrag zur Pathogenese der Glomerulonephritis und der eklamptischen Leberkrankung
.
Beitr. path. Anat
.
91
,
82
112
.
Nairn
,
R. C.
,
Ghose
,
T.
,
Fothergill
,
J. E.
&
McEntegart
,
M. G.
(
1962
).
Kidney specific antigen and its species distribution
.
Nature, Lond
.
196
,
385
7
.
Nairn
,
R. C.
,
Ghose
,
T.
&
Maxwell
,
A.
(
1967
).
Distribution of nephric antigens in Australian vertebrates
.
Nature, Lond
.
215
,
867
8
.
Okada
,
T. S.
(
1962
).
Tissue specificity in the soluble antigens in kidney microsomes
.
Nature, Lond
.
194
,
306
7
.
Okada
,
T. S.
(
1965
).
Development of kidney-specific antigens: an immuno-histological study
.
J. Embryol. exp. Morph
.
13
,
285
97
.
Okada
,
T. S.
&
Sato
,
A. G.
(
1963
).
Soluble antigens in microsomes of adult and embryonic kidneys
.
Expl Cell Res
.
31
,
251
65
.
Osathanodh
,
V.
&
Potter
,
E. L.
(
1966
).
Development of the human kidney as shown by microdissection. V. Development of vascular pattern of glomerulus
.
ArchsPath
.
82
,
403
11
.
Pierce
,
G. B.
(
1966
).
The development of basement membranes of the mouse embryo
.
Devi Biol
.
13
,
231
49
.
Potter
,
E. L.
(
1965
).
Development of the human glomerulus
.
Archs Path
.
80
,
241
55
.
Saxén
,
L.
&
Wartiovaara
,
J.
(
1966
).
Cell contact and cell adhesion during tissue organization
.
Int. J. Cancer
.
1
,
271
90
.
Scott
,
D. G.
(
1957
).
A study of the antigenicity of basement membrane and reticulin
.
Br. J. exp. Path
.
38
,
178
85
.
Scott
,
D. G.
&
Rowell
,
N. R.
(
1967
).
Alternations in the antigenic constitution of renal glomerular capillaries accompanying the histological maturation of renal glomeruli in the rat
.
Ann. rheum. Dis
.
26
,
10
17
.
Ten Cate
,
G.
&
Van Doorenmaalen
,
W. J.
(
1950
).
Analysis of the development of the lens in chicken and frog embryos by means of the precipitin reaction
.
Proc. K. ned. Akad. Wet
.
53
,
1
18
.
Wadsworth
,
C.
(
1957
).
A slide microtechnique for the analysis of immune precipitates in gel
.
Int. Archs Allergy appt. Immun
.
10
,
350
60
.
Wagner
,
M.
(
1967
).
Floureszierende Antikörper und ihre Anwendung in der Mikrobiologie
.
Infektionskrankheiten und ihre Erreger, Band
5
.
Jena
:
Gustav Fischer Verlag
.
Wartiovaara
,
J.
(
1966a
).
Studies on kidney tubulogenesis. V. Electron-microscopy of basement membrane formation in vitro
.
Ann. Med. exp. Fenn
.
44
,
140
50
.
Wartiovaara
,
J.
(
1966b
).
Cell contacts in relation to cytodifferentiation in metanephrogenic mesenchyme in vitro
.
Ann. Med. exp. Fenn
.
44
,
469
503
.
Weiler
,
E.
(
1956
).
Antigenic differences between normal hamster kidney and stilboestrolinduced kidney carcinoma. A histological demonstration by means of fluorescing antibodies
.
Br. J. Cancer
10
,
550
63
.
Weinberger
,
N. J.
&
Boss
,
J. H.
(
1966
).
A comparative study of nephrotoxic serum antigens of diverse rat organs
.
Path. Microbiol
.
29
,
324
40
.
Wilt
,
F.
(
1962
).
The ontogeny of chick embryo hemoglobin
.
Proc. natn. Acad. Sei. U.S.A
.
48
,
1582
90
.