Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats

ORIGINAL ARTICLE

Presence of immunoglobulin M antibodies around the glomerularcapillaries and in the mesangium of normal and passive Heymannnephritis rats

Arpad Z. Barabas, Chad D. Cole, Arpad D. Barabas, Jord M. Cowan, Chang Soon Yoon, David M.

Waisman and Rene Lafreniere

Department of Surgery, University of Calgary, Health Sciences Centre, Calgary, Alberta, Canada

I N T E R N AT I O N A LJOURNAL OFE X P E R I M E N TA LPAT H O L O G Y

Summary

Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M

(IgM) around the glomerular capillary blood vessels, and a more intensely staining larger

deposition in the mesangium, were observed on the kidney sections of normal rats. As

glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect

of the glomerular basement membrane (GBM), especially at the soles of foot processes

and at the slit pores, it was assumed that the IgM antibodies were directed against these

antigens. Investigation by immunofluorescent antibody double-staining techniques of rat

kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the

nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the

mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM

antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but

preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly

for rat IgM, indicating that at these sites the antigenic material was virtually saturated,

while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that

at these locations free nephritogenic epitopes were still available for reaction with the

anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG

and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-

derived antigen with a molecular weight of 70,000. These experimental findings collec-

tively demonstrate that the heterologous IgG and autologous IgM antibodies are directed

against the same nephritogenic antigen, which is found in the glomeruli, the mesangium

and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible phy-

siological role but, in addition, there is evidence of active immunophagocytic events,

manifested in a rapid and continuous entrapment and expulsion of macromolecules after

their processing by the mesangial cells of normal and passive Heymann nephritis rats.

Keywords

autoimmunity, IgM autoantibody, nephritogenic antigen, passive Heymann nephritis,

pathogenic autoantibody

Received for publication:

17 September 2003

Accepted for publication:

14 June 2004

Correspondence:

Dr Arpad Zsigmond Barabas

Department of Surgery,

Health Sciences Centre 2802,

3330 Hospital Dr NW, Calgary,

Alberta, Canada T2N 4N1.

Tel.: +1 403 220 8901;

Fax: +1 403 270 8795;

E-mail: [email protected]

Int. J. Exp. Path. (2004), 85, 201–212

� 2004 Blackwell Publishing Ltd 201

It is well documented that the nephritogenic antigen designated

as megalin/gp330 is abundantly present in the brush border

(BB) region of the proximal convoluted tubules (Kerjaschki &

Farquhar 1982; Kerjaschki et al. 1984; Bergeron et al. 1996;

Jung et al. 1998) and is also found on the epithelial side of

the glomerular basement membrane (GBM) (Kerjaschki &

Farquhar 1982; Tsukada et al. 1994). Investigators have

demonstrated the presence of this nephritogenic autoantigen

around the glomerular capillaries as small, diffuse, beaded

deposits by immunofluorescent antibody tests (Van Damme

et al. 1978; Cornish et al. 1984; Makker & Makker 1986).

When heterologous antibody directed against FX1A antigen

(Kerjaschki & Farquhar 1982; Tsukada et al. 1994) is injected

by the intravenous route into susceptible strains of rats, an

immediate localization of the heterologous immunoglobulin G

(IgG) antibody is observed in the glomeruli (Fleuren et al.

1978), revealing the presence of a target antigen at these sites.

In vitro tests in thoroughly washed-out kidneys of susceptible

normal rats have similarly showed immediate localization of the

intravenously injected rabbit anti-FX1A IgG antibody in the

glomeruli, in the mesangium and in blood vessel walls (Couser

et al. 1978; Makker & Moorthy 1981). The Kerjaschki group

(Kerjaschki & Farquhar 1982) has characterized the nep-

hritogenic antigen and designated it to be a gp330 receptor-

associated protein (Farquhar et al. 1995; Huang & Makker

1995; Farquhar 1996; Raychowdhury et al. 1996). They have

shown that this antigen is locally produced by the epithelial cells

and, after release, is distributed along the epithelial cell surfaces,

coated pits, soles of the foot processes and areas between slit

pores. Others have also characterized the antigen and iden-

tified different but related nephritogenic antigens (Kamata

et al. 1985; Natori et al. 1986; Singh & Makker 1986;

Tsukada et al. 1994).

Since the description of Heymann nephritis (HN) by

Heymann et al. (1959), the role of the nephritogenic antigen

in the initiation and maintenance of immune complex glo-

merulonephritis (ICGN) has been investigated extensively

(Edgington et al. 1967a; Edgington et al. 1968; Kerjaschki &

Farquhar 1982; Bhan et al. 1985; Kamata et al. 1985; Tsukada

et al. 1994; Raychowdhury et al. 1996). How immunopatha-

logical processes could be halted by various means has also

been well studied (Barabas et al. 1969; Barabas et al. 1970b;

Matsukawa et al. 1992; Schiller et al. 1998; Hasegawa et al.

2001; Spicer et al. 2001). But in spite of the vast collection of

knowledge and information in the medical literature, it is not

yet certain what role the native autoantigen plays in normal

and disease states. For example, there are those who have suggested

that the native antigens are present in the circulation also (presum-

ably derived from the tubules) and that they contribute to the

presence of the glomerular-localized antigens, forming immune

complexes (ICs) in the glomeruli with the developing pathogenic

autoantibodies during the development of autoimmune disease

(Edgington et al. 1968; Glassock et al. 1968; Miyakawa et al.

1976; Naruse et al. 1976; Abrass et al. 1980; Abrass 1986; Singh

& Makker 1986; Singh & Schwartz 1986; Hori & Abrass 1990).

At present, however, local production of the nephritogenic antigen

by glomerular epithelial cells is held to occur in susceptible strains

of rats (Kerjaschki & Farquhar 1982; Farquhar 1996).

With experimental findings which demonstrate that the

nephritogenic antigen is indeed present in the circulation, our

data establishes that there is a continuous low-level produc-

tion of naturally occurring IgM autoantibody that is released

into the circulation and directed against antigens of the renal

tubular BB regions. We also show that this IgM autoantibody

is also present in the glomeruli as well as in the mesangium.

Immunofluorescence microscopy demonstrates a beaded dis-

tribution pattern for this autoantibody.

We used a high-titred heterologous anti-rat FX1A antibody

to identify the presence of the nephritogenic antigen on rat

kidney sections and also to ascertain whether the tissue-

localizedand circulating IgM autoantibody was directed against

the same antigen. This article describes our results and discusses

the significance of the presence of IgM autoantibody in the

circulation, in the glomeruli and in the mesangium.

Materials and methods

Preparation of kidney tubular antigen FX1A

Adult normal Sprague Dawley rats were euthanized by IF

injections of Euthanyl (MTC Pharmaceuticals, Cambridge,

Ontario, Canada) and bled out and their kidneys flushed

out with cold physiological saline. FX1A fraction was obtained

by differential centrifugation according to the method of

Edgington et al. (1967b). The liquid FX1A was lypholysed

and stored at �30 �C.

Preparation of kidney tubular antigen rKF3

As for FX1A antigen, Sprague Dawley rat kidneys were used

to prepare the rat kidney fraction 3 (rKF3). The kidneys were

flashed out with cold physiological saline. Renal cortices were

homogenized into a fine suspension by a Virtishear Cyclone

(Virtis) and in a Potter–Elverjhem homogenizer prior to

obtaining the rKF3 fraction by differential centrifugation

according to a method previously described (Barabas et al.

2003). The protein content of the preparation was determined

by the biuret technique (Weichelbaum 1946) and subsequently

stored at �35 �C till use. The antigenic components of pre-

paration, FX1A and rKF3 antigens, are just about the same.

202 A. Z. Barabas et al.

� 2004 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 85, 201–212

Both preparations contain multiple renal tubular antigenic

components with similar molecular weights (MWs).

Production of rabbit anti-rat FX1A IgG antibody

A pathogenic IgG antibody to FX1A antigen was raised in two

rabbits by multiple intramuscular injections of 5 mg of FX1A

incorporated into Freund’s complete adjuvant, followed by

two subcutaneous injections of the same aqueous antigen

prior to obtaining the active serum.

Production of rat anti-rat KF3 IgM antibody

The low level of circulating naturally occurring rat anti-rat

KF3 nonpathogenic IgM autoantibody can be boosted by

repeated intraperitoneal injections of an aqueous KF3 prepara-

tion. About 0.2 ml of 50 mg rKF3 antigen was administered by

weekly intraperitoneal injections to 10 adult Sprague Dawley

rats for 4 weeks. Four days after the last administration of the

antigen, rats were bled for sera, and individual serum samples

were tested in an indirect fluorescent antibody test on normal

rat kidney sections. Sera with high antibody titres against

the BB region of the proximal convoluted tubules (between

1 : 80 and 1 : 220) were pooled, bottled and stored at �35 �C

until use.

Immunofluorescence study

Kidney biopsy samples and blood for sera were obtained in

this study from each rat at the same time as described below.

Direct fluorescent antibody test

Kidney biopsy samples obtained from rats were cut at 2 mthickness on a Microm HM 500M cryostat. Sections were

left in 0.9% saline for 20 min before being fixed in ether:

alcohol 50 : 50. Following fixation, sections were washed and

stained as required with suitable dilutions of Alexa Fluor� 488

goat anti-rat IgG (H+L), Alexa Fluor� 488 goat anti-rat IgM

(m chain), Alexa Fluor� 488 goat antirabbit IgG (H + L) and

Alexa FluorTM 546 goat antirabbit IgG (H+L) (Molecular

Probes/Cedarlane Inc., Eugene, OR, USA) for rat IgG and

IgM and rabbit IgG.

Indirect fluorescent antibody test

Blood collected from the two anti-rat FX1A antibody-

producing rabbits and from individual normal and anti-rat

FX1A antibody-injected rats at the time of kidney biopsies

were tested on normal rat kidney sections. Dilutions of sera

were tested for antibody activity against renal tubular cell

components. All the sections were counterstained with appro-

priately labelled antibodies including Alexa Flour� 488 goat

antirabbit IgG (H+L), Alexa Flour� 488 goat anti-rat IgG

(H+L) and Alexa Flour� 488 goat anti-rat IgM (m chain).

The last dilution of sera still showing staining of the proximal

tubular cytoplasm in the indirect fluorescent antibody test was

considered to be the highest titre giving positive results.

Sandwich technique

The presence of antibody activity against the glomerular-fixed

nephritogenic antigen was ascertained to find out if hetero-

logous and autologous antibodies reacted with the same or

different antigenic sites in the glomeruli and mesangium. Sec-

tions of normal rat kidneys were first reacted with dilutions of

rabbit anti-rat FX1A antibody and then stained with Alexa

FluorTM 546 goat antirabbit IgG and Alexa Fluor� 488 goat

anti-rat IgM (m chain). Using rabbit anti-rat FX1A antibody at

1 : 40 dilution was found to give good and reproducible results.

Kidney sections of rats injected intravenously with 1 ml of

rabbit anti-rat FX1A antibody were obtained 1, 5 and 24 h post-

injection and similarly stained to find out if in vivo contact with

the glomerular and mesangial localized antigens would result in

more effective occupation of available nephritogenic antigens.

Control kidney sections were included and stained to

exclude possible nonspecific stainings.

Experimental design

Ten normal Sprague Dawley male rats were kidney

biopsied under isoflurane anaesthesia using standard surgical

procedures and bled for sera at the same time on days 0, 14,

28 and 42.

From an additional four normal Sprague Dawley male rats,

kidney biopsies and sera were obtained before any procedures.

Three to five days later, the animals were injected intrave-

nously with 1 ml rabbit anti-rat FX1A antibody and biopsied

and bled for sera 1, 5 and 24 h postinjection. Testing of the

biopsy and serum samples were carried out as described in the

immunofluorescence studies.

Photomicroscopy

The variously stained kidney sections were viewed with a Zeiss

Axioscop microscope, and digital pictures were taken using a

spot digital camera (Diagnostic Instruments Inc., Sterling

Heights, MI, USA) and filed in a Micron computer. On the

double-stained kidney sections, areas that were stained for

both rat IgM (green) and rabbit IgG (red) were identified and

IgM antibodies in the kidney 203


brought into a photo-enhancing program and superimposed.

The superimposed areas which stained for both rat IgM and

rabbit IgG turned into a bright orange/yellow colour. Using a

pixel sample tool the orange/yellowish areas were enhanced by

changing their colours to bright blue. These blue areas identi-

fied the common sites where both rat IgM and rabbit IgG

antibodies reacted with the same nephritogenic epitopes in

the glomeruli and in the mesangium.

Western blot analysis

Rat FX1A fraction prepared from renal cortical tubules was

diluted with SDS-PAGE sample buffer and subjected to SDS-

PAGE. Proteins were transferred to nitrocellulose membrane

(Bio-Rad, Hercules, CA, USA) at 4 �C for 1 h. The membrane

was blocked in TTBS (Tris-buffered saline containing 0.1%

Tween-20) with 5% skim milk at room temperature for 1 h

and then incubated with primary antibody in TTBS with 5%

skim milk at room temperature for 1 h. The following dilu-

tions of primary antibody were used: 1 : 1500 dilution of

polyclonal rabbit anti-rat kidney FX1A IgG antibody and

1 : 1000 dilution of polyclonal rat antirKF3 IgM antibody.

The membrane was then washed with TTBS at least six

times (10 min each) at room temperature and then incubated

in 1 :2000 dilution of antirabbit or anti-rat horseradish peroxidase-

conjugated secondary antibodies in TTBS containing 5% skim milk

at room temperature for 1h. The membrane was then washed with

TTBS at least 10 times (10min each) at room temperature and then

developed with the ECL plus Western Blotting Detection System

(Amersham Biosciences, Little Chalfont, Buckinghamshire, UK)

and visualized by chemiluminescence.

Results

Direct fluorescent antibody test

Normal rat kidney sections stained for the presence of rat IgG

and IgM showed tissue-localized IgM only. In all of the rat

kidney sections, there was a diffuse faint and small beaded

deposition of rat IgM around the glomerular capillaries and a

more intense staining with larger deposits in the mesangium

(Figure 1). Image enhancement was needed to fully appreciate

the presence of the faint, diffuse, often multilayered deposition

of rat IgM around the glomeruli. The more intense, obviously

more abundant entrapment of IgM-reactive components in the

mesangium needed no enhancement. When these normal sec-

tions were incubated with dilutions of the animals’ own sera

prior to staining them for rat IgM, brighter staining of both the

glomerular and mesangial deposits was observed. This might

have been due to access by IgM autoantibodies to additional

antigenic sites (Figure 2).

Rats injected intravenously with rabbit anti-rat FX1A anti-

body and biopsied 1, 5 and 24 h later showed linear and/or

beaded depositions of rabbit IgG around the glomerular capil-

laries with intense fluorescence. The Bowman’s capsules (BCs)

25 µm

Figure 1 Direct fluorescent antibody test. Part of a glomerulus of a

normal rat kidney section showing glomerular capillary loops

with small, diffuse, faint beaded stainings for rat IgM (red arrow).

Mesangial areas stain with heavier beaded deposits (white arrow).

25 µm

Figure 2 Indirect fluorescent antibody test. Part of a glomerulus of

a normal rat kidney section reacted with 1 : 10 dilution of its own

serum prior to being stained for rat IgM. As compared to Figure 1,

there is a more pronounced staining of the glomerular capillary

loops with fine beaded deposits and equally more obvious heavy

mesangial staining.



and tubular basement membranes (TBMs) were stained in

places with beaded depositions and the BB region of the

renal tubular cytoplasm with a patchy distribution (Figure 3).

Kidney sections were negative for rat IgG but stained for rat

IgM just like in normal rats.

Indirect fluorescent antibody test

Sera of 10 normal rats analysed for the presence of both

pathogenic IgG and nonpathogenic IgM autoantibodies

against the BB regions of the proximal convoluted tubules

revealed a low level of circulating IgM autoantibodies only

in an indirect fluorescent antibody test on normal rat

kidney sections. These autoantibodies stained the BB regions

of the proximal convoluted tubules with a fine linear staining

pattern (Figure 4) in a patchy distribution. The IgM auto-

antibody levels of individual serum samples directed against

the BB antigens fluctuated minimally between 1 : 20 and

1 : 130 dilutions from one analysis to the next at 0, 14, 28

and 42 days.

Sera of four rats injected with 1 ml of rabbit anti-rat FX1A

antibody with an antibody titre of 1 : 64000 were analysed for

circulating IgM levels before injection and 1, 5, and 24 h after

administration of the antibody. At day 0 and 1 h after antibody injection, circulating IgM autoantibody titres were simi-

lar to findings described for the 10 normal rats. By 5 h

following the rabbit anti-rat FX1A antibody injection, IgM

autoantibody levels had dipped by an average of 30%, and by

24 h by 50%, below the original level of circulating IgM

autoantibodies. The decrease in the level of circulating IgM

autoantibodies was probably due to their reaction with

released nephritogenic antigens (from the damaged tubules

into the circulation) and their resultant sudden removal.

The level of retained circulating rabbit anti-rat FX1A anti-

body was also analysed in the rats’ sera. On average, it was

positive against tubular cytoplasm of normal renal tissues

up to 1 : 3600 dilution at 1 h after the injection of the

antibody and up to 1 : 2500 and 1 : 1300 dilutions at 5 and

24 h, respectively.

Fluorescent antibody test results by the sandwich

technique

Normal rat kidney sections stained by the sandwich technique,

first by 1 : 40 dilution of the rabbit anti-rat FX1A antibody

and subsequently by suitably labelled anti-rat IgM and anti-

rabbit IgG antibodies for the presence of rat IgM and rabbit

IgG, revealed similarities and dissimilarities in their distri-

bution in the glomeruli and the mesangium. Four pictures

(Figure 5a–d) clearly illustrate areas where rat IgM dominates

(especially in the mesangium) (Figure 5a) and areas where

25 µm

Figure 4 Indirect fluorescent antibody test. Normal rat kidney

section incubated with its own 1 : 10 dilution of serum and then

stained with Alexa Fluor-labelled anti-rat IgM. Note the fine

linear staining of the brush border regions of the proximal

convoluted tubules (red arrow).

25 µm

Figure 3 Direct fluorescent antibody test. Part of a glomerulus,

Bowman’s capsule and renal proximal tubules showing staining for

rabbit IgG, 5 h after the injection of the rabbit anti-FX1A antiserum.

The glomerular capillary loops are stained with heavy linear

deposits and in places with beaded deposits and the Bowman’s

capsule and the tubular basement membrane with a beaded pattern

of fluorescence (white arrows). Brush border regions of the proximal

convoluted tubules also stain at places (red arrow).



rabbit IgG is more abundant (especially around glomerular

capillary loops) (Figure 5b). Figure 5c shows the combined

areas of activity of rat IgM and rabbit IgG in the glomeruli

and mesangium with their respective locations of dominance

emphasized, while Figure 5d illustrates the areas in the glo-

merular capillary loops and mesangium occupied by both

antibodies at once (blue staining). In most of the glomerular

capillary loops, both rat IgM and rabbit IgG are present at

shared antigenic sites. Mesangial areas mainly stain for rat

IgM (green staining), but at the entry into the mesangial

space, double staining is observed, indicating reactivity with

free nephritogenic antigenic sites by both rat IgM and rabbit

IgG antibodies.

Kidney sections of the rabbit anti-rat FX1A antibody-injected

rats were also stained for rabbit IgG and rat IgM. Kidney

sections stained for rat IgM (Figure 6a) revealed fine diffuse

beaded depositions around the glomerular capillary loops,

except around one loop (red arrow) where the deposition was

massive. Mesangial areas stained with heavy depositions for rat

IgM (bright green areas), except one mesangial space, which did

not stain (white arrow). Kidney sections staining for rabbit IgG

(Figure 6b) showed beaded deposition around the glomerular

capillary loops and staining in parts of a reduced number of

mesangial areas (compare to Figure 6a). The mesangial space

that did not stain for rat IgM (Figure 6a, white arrows) stained

for rabbit IgG. Figure 6c shows the combined areas of activity

of rat IgM and rabbit IgG in the glomeruli and mesangium,

while Figure 6d illustrates where the rat IgM and rabbit IgG

antibodies are both present at once around the glomerular

capillary loops and in the mesangium (blue staining). Around

the glomerular capillary loops, rabbit IgG dominates but rat

IgM is also present. The glomerular capillary loop that stained

25 µm

25 µm

25 µm

25 µm

(a)

(c) (d)

(b)

Figure 5 Double-stained normal rat kidney section. Section was stained with 1 : 40 dilution of rabbit anti-FX1A antiserum followed by

Alexa Fluor� 546-labelled antirabbit IgG and then by Alexa Fluor� 488-labelled anti-rat IgM. (a) Part of a glomerulus staining for rat

IgM. Note beaded depositions of rat IgM around the glomerular capillary loops mainly with faint stainings (red arrow) and with heavy

depositions in the mesangium (white arrow). (b). Diffuse beaded stainings of the glomerular capillary loops for rabbit IgG (green arrow).

In most mesangial areas, rabbit IgG, the rabbit anti-FX1A IgG antibody, is sparsely present (white arrow). (c) Superimposition of

Figure 5a,b showing dominant localization of rabbit IgG (orange) around the glomerular capillary loops and an obvious presence of rat

IgM (green) in the mesangium. (d) Superimposition of Figure 5a,b showing, after computer enhancement (as described in the Materials

and methods), the presence of antigenic sites where both rat IgM and rabbit IgG are present (blue). The glomerular capillary loops more

readily share common antigenic sites than the mesangium, of which only areas at the entry share common antibody hits.



heavily for rat IgM (red arrow) showed minimal presence of

rabbit IgG. In at least two locations (Figure 6d, large white

arrows), the mesangium primarily stained red, indicating that

recently emptied mesangial spaces were now occupied by

immune complexes associated mainly with rabbit anti-rat

FX1A antibody.

Anatomical localization and detection of the nephritogenic

antigen

The nephritogenic antigen was detected in the proximal con-

voluted tubules of normal rat kidney sections when reacted with

dilutions of normal rat serum (containing nonpathogenic IgM

autoantibodies to BB-related autoantigens) and counterstained

with suitably labelled anti-rat IgM antibody (Figure 4); the

antigen was detected also in the glomerular capillary loops and

in the mesangium when stained by direct and indirect fluores-

cent antibody tests for rat IgM (Figures 1 and 2). These results

were confirmed with double-stained normal rat kidney sections

showing shared antibody activity against the same nephrito-

genic epitopes residing around the glomerular capillaries and

in the mesangium (Figure 5a–d). By similar tests, nephritogenic

antigen was observed in the BC and TBM (Figure 3) in a beaded

pattern on rat kidney sections of rabbit anti-FX1A-injected

25 µm

25 µm

25 µm

25 µm

(a)

(c) (d)

(b)

Figure 6 Kidney section double-stained for rat IgM and rabbit IgG, from a rat injected with rabbit anti-FX1A antiserum 24 h earlier. (a)

Part of a glomerulus. Diffuse beaded deposition of rat IgM is observed around the glomerular capillary loops with faint stainings (insert)

except around one loop (red arrow) where massive deposition is present. Mesangial areas show massive diffuse and beaded staining

(yellow arrows). One mesangial space does not stain for IgM (white arrow). (b) The same section as Figure 5a. Note diffuse linear-like

and beaded depositions of rabbit IgG around the glomerular capillary loops (insert). Only certain mesangial areas stain for rabbit IgG.

One area that does not stain for rat IgM stains for rabbit IgG (white arrow). (c) Superimposition of Figure 6a,b showing dominant

localization of rabbit IgG, the injected anti-FX1A antibody (orange), around the glomerular capillary loops, and a more obvious presence

of rat IgM (green) in the mesangium. (d) Superimposition of Figure 6a,b showing, after computer enhancement, antigenic sites where

both rat IgM and rabbit IgG are present (blue). The glomerular capillary loops more readily share common antigenic sites than the

mesangium, although one capillary loop shows very little common antibody activity (red arrow). Mesangial areas, just as in Figure 5d,

show common antibody hits by rat IgM and rabbit IgG against the nephritogenic antigen especially at the entry to the mesangial spaces.

Two mesangial areas (one seen in insert) are mainly occupied by rabbit IgG-associated ICs (white arrows). This might be due to the

recently emptied mesangium now primarily receiving ICs composed of released tubular nephritogenic antigens (by the injected anti-FX1A

antibody) and circulating rabbit IgG antibodies directed against them.



rats. The latter findings were due to damage and movement of

the nephritogenic antigen from the proximal convoluted tubules

to other sites (Table 1).

Identification of the nephritogenic antigen

In order to identify the renal antigens which were involved in

immunoreactivity with the rabbit anti rat-FX1A IgG and the

rat anti-rat KF3 IgM antibodies, the FX1A preparation was

resolved by SDS page and Western blotted with these anti-

bodies. As can be seen in Figure 7, the major reactivity of both

antibodies was against a 70,000-MW band. When the anti-

body preparations were combined, a single reactive band was

observed confirming that the rabbit anti-FX1A IgG and rat

anti-rat KF3 IgM antibodies indeed reacted against the same

70,000-MW antigen.

Discussion

Classical HN was produced in 1959 by Heymann and col-

leagues in rats given repeated intraperitoneal injections of crude

renal tubular antigens incorporated into Freund’s complete

adjuvant (Heymann et al. 1959). Subsequently, the same disease

was produced by several more refined renal tubular antigens,

also incorporated into adjuvants and administered either by

intraperitoneal or intrafootpad routes (Edgington et al. 1967b;

Edgington et al. 1968; Barabas & Lannigan 1969; Kerjaschki

& Farquhar 1982; Noble et al. 1984; Tsukada et al. 1994;

Raychowdhury et al. 1996; Oleinikov et al. 2000). The patho-

genesis of HN has been well described (Grupe & Kaplan 1969;

Andres et al. 1986; Van Leer et al. 1993), and possible treat-

ment options able to modify its course somewhat at the early or

late phases of the disease have been documented (Barabas et al.

1969; Barabas et al. 1970b; Hasegawa et al. 2001; Spicer et al.

2001). The role of pathogenic autoantibodies in the initiation

and maintenance of the disease is well known (Madaio et al.

1983; Makker 1993). It has also been shown that, in order for

the disease to continue, the presence of the adjuvant-modified

tubular antigen has to be present in the animals (Noble et al.

1984).

Another variant of HN, which is not autoimmune in nature,

is passive Heymann nephritis (PHN), first described by Bara-

bas et al. in 1970 (Barabas et al. 1970a). This ICGN is

initiated by intravenous injection of a heterologous IgG anti-

body directed against the tubular nephritogenic antigen. As

this autoantigen also resides in the glomeruli (Cornish et al.

1984; Makker & Makker 1986), the immediate formation of

ICs following injection of the antibody results in ICGN.

The present communication describes the presence of HN

antigen in the glomeruli and mesangium of normal and PHN

rats, associated with IgM autoantibody. In the mesangium,

IgM was present most often with intense fluorescence staining

parts or the whole of the mesangial tree, with small deposits in

some places and large deposits in others. In the glomeruli, the

deposits were small and stained faintly with a diffuse beaded

pattern of fluorescence. When dilutions of the rats’ own sera

were added to the normal kidney sections prior to staining for

rat IgM, the intensity of fluorescence and the size and number of

fluorescent dots around the glomerular capillaries and in

the mesangium noticeably increased. This was presumably due

to reaction of the serum-containing IgM antibody with free anti-

genic sites. When dilutions of rabbit anti-rat FX1A antiserum

followed by suitably labelled antirabbit IgG and anti-rat IgM

Table 1 Presence of the nephritogenic antigen in the kidney of

normal and passive Heymann nephritis (HN) rats (determined by

the direct and indirect fluorescence antibody tests)

Nephritogenic antigen in the Gl Cap L Mes RPT BC TBM

Normal rat kidney + + + – –

Kidney of passive HN rat */+ */+ */+ * *

BC, Bowman’s capsule; Gl Cap L, glomerular capillary loop; Mes,

mesangium; RPT, renal proximal tubule; TBM, tubular basement

membrane; +, rat anti-rat nephritogenic immunoglobulin M (IgM)

antibody; *, rabbit anti-rat nephritogenic IgG antibody. Nephrito-

genic antigen at various sites in the kidney was detected by the fixation

of specific autologous and injected heterologous antibodies.

a: Coomassie- blue

b: Western Blotting1: Rat IgM2: Rabbit IgG3: IgM + IgG

staining

(a)kDa

11888

55

24

(b)

1 2 3

Figure 7 Identification of the major immunoreactive species in the

FX1A fraction. The rat tubular FX1A fraction was prepared as

described above and analysed by SDS-PAGE (a) and Western blot

analysis (b). A major immunoreactive protein band of MW

70,000 was detected by the polyclonal rat anti-rKF3 IgM

antibody (1), polyclonal rabbit anti-rat kidney FX1A IgG

antibody (2) and the mixture of both antibodies (3). Standard

MW markers are shown at the left.



antibodies stained the normal rat kidney sections, localization of

IgM antibodies mainly in the mesangium and rabbit IgG anti-

bodies mainly in the glomerular capillary blood vessels was

observed. However, there were common antigenic sites in the

mesangium and in the glomeruli that stained for both antibodies.

Similar staining of the glomeruli and mesangial areas for

rabbit IgG was observed on the rat kidney sections 1, 5 and

24 h after the injections of the rabbit anti-rat FX1A antibody.

The massive linear and beaded diffuse depositions of ICs

around the glomerular capillaries were intensely fluorescent,

signifying a very rapid uptake and localization of the injected

antibody to free nephritogenic antigens. When the same sec-

tions were stained for rat IgM also, they showed both mesan-

gial and glomerular-beaded staining. While a few beaded

deposits stained for both rabbit IgG and rat IgM, most depos-

its stained for either rabbit IgG or rat IgM. These findings

clearly reveal multiple and continuous events taking place

during the normal life span of the rat and following the injec-

tion of the heterologous anti-rat FX1A antibody. The most

striking observation was the heterologous anti-FX1A antibody

and the autologous IgM autoantibody reacting with the same

nephritogenic antigen sites in the glomeruli and in the mesan-

gium (with one antibody often excluding the other in saturat-

ing the antigen sites), indicating that both antibodies are

directed against the same targets.

In Sprague Dawley and in many other strains of rats

(Stenglein et al. 1978; Salant & Cybulsky 1988), nephrito-

genic autoantigen is present in the glomeruli. This antigen is

locally produced (Kerjaschki & Farquhar 1982), but accord-

ing to some observations, it can also be present in the circula-

tion (Glassock et al. 1968; Naruse et al. 1976; Abrass et al.

1983; Singh & Makker 1986; Singh & Schwartz 1986). The

antigen is also found in the mesangium (Makker & Moorthy

1981), and in this case because of its anatomical locality it

must come from the circulation.

In our study, all the normal rats had low levels of naturally

occurring IgM autoantibodies in their circulation directed

against the BB regions of the proximal convoluted tubules.

The IgM presence in the circulation is more than likely due

to the continuous stimulation of IgM-producing cells by

released tubular antigens. Its physiological role, as described

by Weir and others (Weir et al. 1966; Casali & Notkins 1989;

Chen et al. 1995), is to clear the released intracytoplasmic

antigens, in our case tubular antigens, from the circulation

through a complement-dependent removal of ICs by phagocy-

tic cells of the reticuloendothelial system (RES), including the

mesangial cells. In this manner, IgM autoantibodies prevent

accumulation of released intracytoplasmic components and

circumvent toxicity or alteration of these components that

could lead to production of pathogenic autoantibodies. The

function of the pathogenic IgG antibody (once it is formed

following immune response to altered self) is to damage and

perpetuate injury to the target organ (Andres et al. 1986),

which contains the specific autoantigen. We saw in this experi-

ment that following injection into rats of rabbit anti-FX1A

IgG-containing antiserum, damage in the glomeruli and renal

tubules occurred. Reaction with the glomerular-fixed nephri-

togenic antigen and with the naturally occurring primary

source autoantigen residing in the renal tubules induced local

damage and release of nephritogenic antigens. The observed

rapid movement and localization of these released antigens to

the BC and the TBM, where they formed ICs with the injected

pathogenic IgG antibodies, has been documented by others as

well (Mendrick et al. 1980).

Autoantigens released into the circulation will be removed

by the cells of the RES. In this regard, the glomerular mesan-

gial cells play a significant roll. The normal physiological

function of the glomerular mesangium is to filter out macro-

molecules and ICs such as complexed nephritogenic autoanti-

gens and specific IgM autoantibodies in order to keep the

glomerular filtration barrier free of large-MW aggregates.

The obvious and abundant presence of IgM autoantibodies

in the mesangium of normal rats, presumably in the form of

ICs, indicates that these ICs are continuously formed and

filtered out (Rosenzweig & Kanwar 1982; Batsford et al.

1985). It is well known that macromolecules and ICs entering

into the mesangial space will be phagocytosed by mesangial

cells and enzymatically degraded and regurgitated back into

the glomerular capillaries as innocuous small-MW substances.

The GBM and its associated appendages, in particular

the epithelial cell slit pores, limit large-MW substances from

escaping into the urinary space (Rosenzweig & Kanwar 1982).

Yet, the injected heterologous IgG and to a lesser extent the

autologous IgM antibodies still got across and localized to the

nephritogenic autoantigens on the epithelial side of GBM. It is

a possibility that these large-MW substances eased themselves

across the GBM in conjunction with the molecular movements

of smaller MW nephritogenic antigen fragments to reach the

more abundantly present antigens on the epithelial side of

the GBM (Andres et al. 1986). The IgM autoantibodies

presumably trickled through the GBM slowly, resulting in

the occupation of only a few accessible antigenic sites in the

glomeruli, while the IgG antibodies got through more rapidly

and saturated them. This differential access to the localized

nephritogenic antigen sites in the glomerular capillary loop

resulted in a more abundant rabbit IgG localization. Those

sites not first occupied by IgM autoantibodies were saturated

by rabbit IgG, and those partially reacted with IgM antibodies

became fully occupied with the injected rabbit anti-rat FX1A

IgG antibodies. As a result of these events, there were antigenic



sites that were stained for rat IgM or for rabbit IgG only and

then sites that were stained for both. But while rabbit IgG

antibodies were abundantly present at certain locations in

the mesangium, their distribution was sparse and even absent

from some areas presumably because the IgM autoantibodies

occupied most of the accessible nephritogenic antigen sites.

In two mesangial spaces, however, rabbit IgG dominated

(Figure 6d, white arrows), indicating entrapment of ICs made

up of the nephritogenic antigen and rabbit IgG antibody. This

exchange from mainly IgM autoantibody-containing ICs to

rabbit IgG antibody-trapped ICs could only have taken place

following the ejection of degraded mesangial contents back

into the circulation within the 24 h immediately prior to our

observation. These findings support the view that nephrito-

genic autoantigens were released into the circulation and

pathogenic rabbit IgG antibodies formed ICs with them that

were subsequently trapped in the mesangium.

Animals injected with the anti-FX1A antibody had very

high levels of retained circulating IgG antibodies. These circu-

lating antibodies, which were directed against the nephrito-

genic antigens, initiated considerable damage during the first

few days to the BB region of the proximal convoluted tubules,

causing local cytotoxic injury (Mendrick et al. 1980). Hence,

severe ICGN, patchy tubular cell cytoplasm fluorescence, and

beaded BC and TBM fluorescence were observed on the kidney

sections of these rats. Such lesions are seen in HN and mem-

branous glomerulopathy and are considered to be part of the

pathogenic antibody response that causes the disease (Mendrick

et al. 1980; Markowitz et al. 2000).

Five and 24 h after the anti-FX1A antibody injection, we also

observed in the sera of PHN rats a lowered IgM autoantibody

level. This was presumably due to circulating IgM auto-

antibodies reacting with liberated nephritogenic autoantigens

and forming ICs that were subsequently removed by phagocytic

cells.

We also observed that the heterologous rabbit IgG and

autologous IgM antibodies were directed against the same

nephritogenic antigen in the kidney with a MW of 70,000.

This 70,000-MW nephritogenic antigen was also identified by

Singh and Makker and Singh and Schwartz in 1986, as an

immunodominant renal antigen in the circulation and in the

glomeruli. Our study shows that the 70,000-MW antigen is

important for the production of both pathogenic and non-

pathogenic autoantibodies. As gp330, the main nephritogenic

antigen, is very sensitive to endogenous proteolysis (Kerjaschki

& Farquhar 1982), its fragments containing the pathogenic

70,000-MW determinants in the circulation of normal rats

could contribute to immunopathological events that cause

HN. It is tempting to speculate that the 70,000-MW antigen

is anchored in the mesangium and in the glomeruli temporarily

by the nonpathogenic IgM autoantibody prior to its elimina-

tion into the circulation or into the urinary space. Injection of

a heterologous anti-rat FX1A IgG antibody as in PHN or

developing rat anti-rat kidney FX1A IgG antibody as in HN

(both antibodies directed against the temporarily trapped

nephritogenic autoantigen in the glomeruli) could start a per-

petual sequence of events, involving C-3, C5b-9 and the like

components to produce damaging ICs in the glomerular capil-

laries and subsequent proteinuria.

The results of the present study show that the nonpatho-

genic IgM autoantibody is continuously present in the circula-

tion, and in certain anatomical sites in the kidney including the

glomerular capillary loops and mesangium, where it is trapped

in the form of ICs. The overall function of the IgM auto-

antibody, especially during autoimmune disease development

and progression, needs to be elucidated by further studies in

order to establish its possible role in the downregulation of

pathogenic autoimmune events by neutralizing or removing

both altered and unaltered nephritogenic autoantigens from

the circulation.

Acknowledgement

The competent technical assistance of Twyla Boehmer in

carrying out some of the tests is recognized.

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Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats

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