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The morphogenesis of glomerular crescents in rapidly progressive glomerulonephritis
Kidney International, Vol. 5 (1974), p. 47—56
The morphogenesis of glomerular crescents in rapidly progressive glomerulonephritis K. W. MIN, F. GYöRKEY, P. GYöRKEY, J. J. Yjurvi and G. EKNOYAN Department of Pathology and the Renal Section, Department of Medicine, Veterans Administration Hospital and Baylor College of Medicine, Houston, Texas
The morphogenesis of glomerular crescents in rapidly progressive glomerulonephritis. Kidney tissue obtained by needle biopsy from eight patients with rapidly progressive glomerulonephritis was studied. Crescents were present in more than
épithCliale. Ces constations suggèrent que les FDL de Ia mem-
brane basale glomerulaire sont initialement associées a des alterations a point de depart immunologique dues soit 1. un complexe immun ou a une maladie glonierulaire anti-membrane basale. La glomCrulonephrite rapidement progressive serait un
8O% of the glomeruli examined. Variations in the cellular composition of the crescents were present in each biopsy specimen, but were mostly epithelial in four and fibroepithelial in the others. Immunofluorescent staining of glomeruli for IgG, and
syndrome resultant de l'apparition de FDL avec, par voie de consequence, formation de croissants avec une frequence plus élevée que dans les autres situations oi les mêmes lesions peuvent exister.
/I1C revealed a linear pattern of distribution in four, a focal nodular distribution primarily in the mesangial areas in three and no staining in one. Positive staining for fibrinogen in the
Diffuse glomerular crescent formation characterizes a form of glomerulonephritis associated with rapidly deteriorating renal function often resulting in uremia and death within several months. This clinicopatholog-
epithelial crescents was present in all. A consistent finding on electron microscopy was the association of the crescents with a focal disruptive lesion (FDL) of the glomerular basement mem-
brane. Intravascular contents appeared in various stages of extravasation through these focal defects with concomitant fibrin precipitation and epithelial cell proliferation. These findings suggest that FDL of the glomerular basement mem-
ical entity which is now well recognized [1—161 has in
the past been designated as subacute glomerulone-
brane is usually associated with immunologically initiated alterations due to either immune-complex or antiglomerular basement
phritis [1], rapidly progressive type I [2], acute oliguric glomerulonephritis [9, 101 and, more recently, rapidly progressive glomerulonephritis (RPGN) [11—16]. It is the larger size and higher frequency with which crescents are present in this condition which differen-
membrane glomcrular disease, and that rapidly progressive glomerulonephritis is a syndrome probably resulting from FDL
with consequent crescent formation, occurring in higher frequency than in other conditions in which the same lesions may be present.
tiates it from other forms of acute oliguric glomerulonephritis occurring with or without crescents. The pathogenesis of crescent formation, however, is not clearly understood. Fibrin and red blood cells have
Morphogenese des croissants glomérulaires dans Ia gloméru-
Ionéphrite rapidement progressive. Le tissu renal obtenu par biopsie a l'aiguille chez huit malades atteints de glomérulonéphrite rapidement progressive a été étudié. Des croissants ont été
been repeatedly observed within the crescents by
observes dans plus de 8O°/ des glomérules examines. IDes variations dans la composition cellulaire des croissants ont été observées d'une biopsie a l'autre, rnais cette composition était principalement épithéliale dans quatre cas et fibro-epithéliale
several investigators [2, 11—16]. This has led to the assumption that crescents may represent a reaction of the epithelial cells to the products of exudation into the urinary space, particularly fibrin and red blood
dans les autres. L'immunofiuorescence des glomérules pour ]'EgG et le $1C a montré une distribution linéaire dans quatre cas, une distribution focale principalement dans les zones
cells [11]. Vassali and McCluskey have presented evidence for a role of the coagulation process in the
mésangiales pour trois cas et l'absence de fixation dans un cas. Du fibrinogène a été observe dans tous les croissants épithéliaux.
Une constation uniforme en microscopie électronique a été
pathogenesis of experimental glomerulonephritis pro-
l'association aux croissants de lesions disruptives focales (FDL) de la membrane basale glomerulaire. Le contenu intravasculaire est diversement extériorisé a travers ces interruptions focales avec lesquelles coincident les dépôts de fibrine et la proliferation
duced in rabbits by the inoculation of anti-rabbit
kidney serum. In their experimental model, the course of the disease and microscopic changes of the kidney
were in some respects similar to those of RPGN in humans, including diffuse crescent formation and
Received for publication June 8, 1973; and in revised form September 5, 1973. © 1974, by the International Society of Nephrology.
fibrin precipitation in the crescent [17]. In a subsequent
47
48
Mm
et a!
presenting with the clinical features of RPGN. Some of the clinical and laboratory data and course of these
study, they demonstrated that this experimentally induced renal disease could be prevented by treating the animals with anticoagulants prior to the administration of anti-kidney sera [18]. Additional evidence to
patients are summarized in Table 1. All had antistreptolysin (ASO) titers of less than 200. Findings of chest roentgenograms were negative except for initial
incriminate the clotting mechanism in the pathogenesis of this condition has been presented by the encouraging results reported with the use of anticoagulant therapy in some patients with RPGN [19, 20].
congestive changes in some patients. Complement levels
were normal. Antinuclear antibody (ANA) titers and
lupus erythematosus (LE) cell preparations were
Al though these observations and experimental
negative. No patients gave a history of hemoptysis or demonstrable skin lesions.
studies have established a significant body of evidence
The renal tissue was bisected longitudinally and one-half was immediately frozen at — 70°C by immersion in isopentane which had been precooled in a mixture of acetone and dry ice. Fresh frozen sections were cut at 4 and 6 and stained with hematoxylineosin, periodic-acid Schiff, Fetrot and Congo red. For immunochemistry, fresh frozen sections (4 to 6 .t) were incubated for 15 mm with fluorescein isothiocyanate conjugated anti-human IgG, 1gM, IgA, fllC and fibrinogen. The anti-human IgG, 1gM, IgA and
in favor of the pathogenic role of the coagulation process in RPGN, in certain cases the luxuriant crescents far exceed the amount of demonstrable fibrin suggesting the possibility that additional stimuli might be operative [113. Moreover, while the presence
of fibrin has been attributed to exudation, it is only recently that specific lesions have been demonstrated which would explain the presence of fibrin in this
condition in contrast to other forms of glomerulonephritis associated with otherwise comparable changes in the glomerular tufts and mesangial region
/3lC sera (produced in goat) and the anti-human
[12, 13].
fibrinogen (produced in rabbit) were purchased from Hyland Laboratories, Los Angeles, California. After incubation, the sections were washed in three changes of phosphate-buffered physiological saline for five mm, mounted with buffered glycerine and examined with the fluorescent microscope (Zeiss) using excitor filter UG 2 and barrier filter 440. The specificity of the positive staining was checked with the blocking technique by pretreating sections with the unconjugated specific serum of each before fluorescent staining. The other half of the biopsy specimen was cut into
This is a report of the immunofluorescent, light and electron microscopic observations on serial sections of renal biopsy specimens from eight patients with RPGN which elucidates the morphogenesis of the crescents and demonstrates an association and possible etiologic
role of a focal disruptive lesion of the glomerular capillary wall in crescent formation. Methods
small pieces, less than 1 mm in diameter, fixed in
Needle biopsy specimens of kidney tissue were obtained under local anesthesia from eight patients
glutaraldehyde [21] and postfixed in osmium tetroxide
Table 1. Clinical and laboratory data
Patient
No.
Age
Sex
Duration of illness before hospitalization, weeks
Protein
Presenting clinical picture
Blood pressure,
Blood urea
mm Hg
mq/100 nil
mg/100 ml
2
15
Creatinine,
excretion,
nitrogen,
g/24 hr
I
49
M
4
NS
180/110
56
2
21
F
3
NS
190/130
120
9
16
ARF5
200/120
152
27
2
160/90
172
21
6
15
1.6
3
60
M
3
4
26
M
1—2
ARF
5
54
M
2-3
ARF
120/90
100
6
49
M
2—3
ARF
180/110
144
7
16
M
8
NS
130/105
130
8
51
M
4
ARF
130/85
113
Clinical course and treatment
Treated with prednisone and imuran; died after three months Peritoneal dialysis, twice; prednisone and imuran therapy; died after two months Chronic hemodialysis; died four montht later due to septicemia
Repeated peritoneal dialysis; died five
5
months later Peritoneal dialysis with slow deterioration for one year; maintained on intermittent dialysis Peritoneal dialysis initially then prednisone
10
12
Following peritoneal dialytis two times,
18
2
7.3
and imuran therapy; improved; BUN,
60 mg/lOO ml
. NS nephrotic syndrome. b ARF = acute renal failure.
improved; lost to follow-up Treated with prednisone, imuran, and cytoxan with some improvement; died three years later in renal failure
49
Morphogenesis of glomerular crescents Table 2. Summary of biopsy findings
Patient No.
glomeruli 1
2 3
4 5
18 5 5
14
6
6
12
7
5
8
14
Character of crescent Epithelial Fibroepithelial No crescents Fibroepithelial Epithelial Fibroepithelial Sclerosed Fibroepithelial Sclerosed Fibroepithelial Sclerosed No crescents Epithelial Sclerosed No crescents Epithelial No crescents Epithelial Fibroepithelial Sclerosed No crescents
Electron microscopy
1mmunofluorescence
Light microscopy Number of
'C
IgG
1gM
12 Linear
+
+
ab
5 Linear I Linear
+ +
+ ÷
—
+
x
++
a
a
+
+
++
++
+++
++
a
++
+
"Humps"
+
a
+
+
Subendothelial deposits
Pattern,
2 4 3 I
9 Linear 5 3
+++ —
—
2
IgA
a
x
Fibrinogen
+
+
No electron dense deposits
+
No electron dense deposits No electron dense deposits
No electron dense deposits No electron dense deposits
I
8 Mesangial, 2 Lumpy
4 Lumpy I
5 Spotty, 3 Mesangial, 3
Subendothelial and mesangial electron dense deposits
3 Lumpy
Fluorescence is graded Ito 4+ on the basis of intensity and extent of distribution. b Refers to no staining for fluoresence performed.
[22J for electron microscopy. After dehydration in
graded alcohol solutions containing uranyl acetate, the specimens were embedded in epoxy resin (EponAraldite) [23]. Sections 1 to 2 in thickness were cut, stained with toluidine blue and scanned for glomeruli. From each biopsy specimen at least three glomeruli
were serially cut through the entire diameter of the glomerulus with an ultratome (LKB 8800). Ribbons of
glomerular tufts on a random section were normocellular. Serial screening, however, disclosed one or more segmental areas of hypercellularity and necrotizing glomerulitis in all of the specimens. This focal lesion often served as the nidus for crescent formation (Fig. 1) and is considered by us to be important in the morphogenesis of glomerular crescents. We will describe, in detail, this type of lesion, including its ultrastructural
sections were mounted on 200 mesh coated copper grids (Formvar), stained with uranyl acetate and lead citrate [24, 25] and studied with an electron micro-
changes.
scope (RCA 3G).
linear pattern of staining with anti-IgG revealing the
Immunohistochemistry. The results of immuno-
fluorescent staining are summarized in Table 2. Diffuse
contour of the glomerular basement membrane (GBM) Results
Histopathology. The histopathological changes were strikingly uniform and characterized by an exuberant proliferation of extracapillary cells forming crescents in the majority of glomeruli. From 80 to l00% of all glomeruli were involved in a given biopsy specimen (Table 2). The site of cellular proliferation was distinctly within Bowman's space (Fig. 1). The crescents were either epithelial or fibroepithelial. Although there was
some variation of the cellular composition of the crescents from one glomerulus to another in a given biopsy, the crescents were mostly epithelial in four patients and fibroepithelial in the others. Scierosed crescents were present in five.
Proliferative changes in the glomerular tufts were much less evident. Significant alterations in the glom-
erular tufts consisting of diffuse hypercellularity were present in only two of the biopsy specimens. In
the remainder of the biopsies, the majority of the
Fig. 1. Glomerulus from patient 1. Note the proliferative changes
forming an epithelial crescent centered around necrotic segments (arrows) of glomerular tufts (periodic-acid Schiff stain [PAS] >< 160).
50
Mm et a!
was present in four patients with RPGN (Fig. 2). The immune complex type of "lumpy" irregular staining was noted in three patients (Fig. 3). In one patient no staining was demonstrable.
Positive staining for human fibrinogen in the epithelial crescents (Fig. 4) was a constant finding in the renal biopsy specimen of all patients. This was present in clumps in the crescentic areas while staining
was spotty and only occasional in the glomerular
ing pattern, in four specimens, a classical linear pattern of immunofluorescence was seen and the focal disruptive lesion was associated with focal exudation of polymorphonuclear leukocytes and destruction of the GBM without electron dense deposits. In these biopsies, the GBM was diffusely thickened with moderate
irregularity of the contour. The intensity of staining varied resulting in a mottled appearance. in some areas,
capillaries. Fibrotic crescents stained faintly for fibrino-
the subendothelial space was markedly widened and the endothelial cells were detached from the basement
gen but it was clearly present in the outline of the
membrane. In such areas the basement membrane
crescents.
showed increased electron density on the endothelial
Electron microscopy. A consistent finding in all
side.
biopsy specimens was focal disruption of the GBIvI, spilling of the capillary contents into the urinary space and inflammatory exudate. These findings were associated with focal electron dense deposits in the mesangium or subepithelial space or both in three biopsies showing a lumpy irregular immunofluorescent stain-
We regard the "focal disruptive lesion" (FDL) of the GBM as a trigger mechanism for the initiation of crescent formation. The ultrastructural alterations in
Fig. 2
Fig. 3
relation to this lesion are, therefore, presented in detail.
The Focal Disruptive Lesion (FDL). In glomeruli
Fig. 2. Glomerulus (patient 4) stained for human lgG globulin. Note the linear distribution of fluorescence along the glomerular basement membrane (human lgG, x 500). Fig. 3. Gloinerulus (patient 7) stainedfor human JgG globulin showing lumpy nodular fluorescence along the GBM outlining the glomerular loops (human lgG, x 500).
Fig. 4. Glomerulus (patient 2) showing positive fluorescence for human fibrinogen in the crescent (human fibrinogen, x 1000).
Fig. 4
Morphogenesis of glomerular crescents
51
with epithelial crescents, the necrotic nidus observed by light microscopy (Fig. 1) was the site of FDL as observed with electron microscopy. The GBM in focal areas was disrupted and blood cells appeared in various stages of extravasation through focal defects of the GBM (Fig. 5). In the urinary space near the FDL,
associated with cellular crescents indicating that
clumps of fibrin precipitates with the typical periodicity
of fibrin were seen among the proliferating epithelial cells. Occasionally, layered fibrin precipitates appeared
serum [17]. The glomerulonephritis in these animals resembled human RPGN in its clinicopathological features including fibrin precipitates in the crescents. More importantly, the epithehial cell proliferation
vascular or urinary space. Glomerular capillary tufts proximal or adjacent to the FDL also showed fibrin in the subendothelial space and capillary lumina. In some
could be prevented by anticoagulants prior to the administration of anti-serum, while deposition of antibody in the basement membrane was not [18]. These observations lend support to the concept that
areas, the GBM appeared intact and the fibrin
fibrin precipitation initiates epithelial cell proliferation.
deposit in the capillary lumen mimicked a thrombotic occlusion. Even though FDL could not be made out initially in such areas, additional serial sectioning led to the identification of FDL which could have been missed with less extensive sectioning. In these areas fibrin precipitates were seen to be freely present among the proliferating epithelial cells. In glomeruli with fibrous crescents, FDL could only be identified with difficulty. More careful and extensive serial sectioning, however, again invariably disclosed focal defects in the GBM, usually at the axis of the crescent (Fig. 6) where mesangial cells were intermingled with fibroblastlike cells and epithelial cells.
Fibrin precipitate was either absent or much less
The reason for fibrin precipitation, however, has not been demonstrated. Our study suggests that the FDL could probably be an initiating event which leads to fibrin precipitation. Our findings are for the most part in agreement with the electron microscopic changes observed in RPGN by Bacani and associates [14]. However, they failed to detect the FDL. We found it necessary to examine serial sections of the entire glomerulus in order to identify the otherwise elusive FDL. Disruption of the basement membrane similar to the one described here has been observed by others in glomerulonephritis of different etiologies and morphologic types including RPGN [12, 13, 31]. Detailed studies of its association
evident in fibrous crescents.
with crescent formation, however, have not been
to seal off the FDL (Fig. 5). The precipitation of fibrin, however, was not restricted to the extra-
In biopsy specimens with epithelial crescents, occasional glomeruli showed focal hypercellularity without crescent formation. Such glomeruli revealed
fibrin precipitation occurred early in the pathological process and might have initiated the cell proliferation. Vassali and McCluskey described an animal model of proliferative glomerulonephritis in rabbits induced
by the injection of heterologous nephrotoxic anti-
reported.
Our study suggests that FDL may be associated with either one of the two patterns of immunological
segmental areas in which the GBM was partially disrupted and herniated and showed inflammatory cells (Fig. 7). In these glomeruli, there was no evi-
tissue damage [32—36]. In four biopsy specimens, FDL was associated with a linear fluorescent staining pattern without electron dense deposits in accordance with the
dence of spilling of the intravascular content, nor was fibrin precipitate seen in the urinary space, and epithe-
current concept of an anti-GBM-antibody pathogen-
hal proliferation was absent. Conceivably, such changes could represent the precrescent lesion. Discussion
The coagulation process has been implicated in the pathogenesis of certain forms of glomerulonephritis
because of the frequent association of fibrin with glomerular lesions [2, 11, 12, 14, 16, 26—30]. Attention
has been focused on the possible pathogenetic role of fibrin in RPGN since Ellis [2] first described the presence of fibrin-like material among the cells forming crescents in patients with rapidly progressive type I nephritis. Bacani et al [14] and Lewis et al [15] also identified fibrin in the crescents in all of their patients with RPGN. The fibrin precipitates were frequently
esis, while in three biopsy specimens, FDL was associated with subendothelial or subepithelial electron dense
deposits or both with an irregular lumpy fluorescent staining pattern consistent with an antigen-antibody complex pathogenesis. Independent of the pathways
followed, it has been shown that tissue-localized interaction of complement and antigen-antibody com-
plexes release leukotactic agents, and the attracted polymorphonuclear leukocytes may actively destroy the glomerular basement membrane [37, 38]. In all the samples examined, FDL was invariably associated with focal inflammation of the glomerular tufts. The defec-
tive margins of the GBM in FDL were usually in direct contact with polymorphonuclear leukocytes. By studying several glomeruhi on serial sections it is
possible to reconstruct a developmental sequence
which may occur in the formation of crescents.
Mlnctal
52
I.
-
'
•vBS p
-
S
-
Pb t Gla,nernlns from patient 6 Mowlqjbcal tilenqatien of GBM Qietwesi arrows) throqh which hatawcqillley contents extromente. Fibdn (F) and epitSial cells (EP) seal off the disrupted capillary wall (x 13,000).
53
Wfl!%i
*r
•
•
t
7vlorphogenesis of glomerular crescents
Fig. 6. A glomerulus from patient 4 showing a Jcal disruptive lesion in a glomerulus with fibroepithelial crescent. Note the GBM defect (between arrows). The urinary space is now occupied by epithelial cells (EP) and fibroblasts (FB). Note the presence of collagen (C) between the crescentic cells (x 13,000).
54
Mm
et al
it -
_
Fig. 7. Segment of the glomeru/ar tuft showing a relatively normal basement membrane (BM) for comparison adjacent to a focal disruptive lesion (FDL). Note the beginning of the altered basement membrane (small arrows) becoming progressively attenuated and terminating in a break (between arrow heads) with herniation of two inflammatory cells (PMN) at the site of FDL. This probably represents the precrescent formation stage >< 21,000).
55
Morphogenesis of glomerular crescents
Admittedly, this reconstruction is not based upon
Schönleiri purpura, systemic lupus erythematosus,
observation of progression of the lesion in sequential biopsy specimens from the same patients, but rather it represents a logical sequence of the changes observed in this series of eight patients. It would appear that the earliest lesion consists of one or more focal hyper-
bacterial endocarditis, poststreptococcal acute glomerulonephritis and hypersensitivity angiitis. Since the FDL and associated crescents appear to be
cellular segments due to infiltration of polymorphonuclear leukocytes attracted by the leukotactic factors released by either one of the two pathways of immunoglobulin deposition within the kidney. The
the result of a combined immune and coagulative process, it appears that therapy in this condition could best be directed at both processes rather than at one in preference to the other. The answer to this problem can come only from findings of carefully controlled clinical and pathological studies.
leukocytes then penetrate and weaken the GBM resulting in partial disruption and herniation. At this
Acknowledgments
stage, there are no discontinuities in the basement membrane nor are there fibrin precipitates in the urinary space (Fig. 7). Later, the GBM becomes
This report was presented in part at the 61st Annual Meeting of the International Academy of Pathology,
focally disintegrated and intravascular contents escape into the urinary space with precipitation of fibrin (Fig. 5). At this stage, the fibrin precipitate actually appears to seal off the capillary leakage (Fig. 5). Following the appearance of fibrin, there is proliferation of the epithelial cells and crescent formation begins. Conceivably, proliferation of the parietal and visceral cells with crescent formation may be the only mode of reaction to the basement membrane damage
and ensuing exudation or leakage of fibrin and red blood cells. Subsequently, the amount of fibrin precipitate decreases, and the cells forming the crescents are now fibroblastic (Fig. 6). Even in the glomeruli with completely fibrotic crescents, focal disruption of the GBM with free communication between intra- and extracapillary spaces is present (Fig. 6). Crescents in the various stages of development just described were often present in the same specimen (Table 1) indicating the continuing and progressive nature of the disease.
The diversity in the patterns observed by us on immunohistochemistry and electron microscopy is similar to that found by others [14, 15]. Our observations, however, suggest that RPGN is a characteristic syndrome associated with FDL and consequent diffuse crescent formation. They also explain the association of this lesion with renal diseases of diverse pathogenetic mechanisms in which immunological damage may be
due to either immune complex or anti-GBM antibody deposits. It should be noted, however, that FDL may occur in the absence of any demonstrable localized immunoglobulins, as was the case in patient 5 (Table
2). It is, perhaps, the degree of "severity" of the sequence of events that ensues complement activation
which in some cases results in the disruption of the basement membrane by the leukocytes and development of the FDL. Such a mechanism may be operative in other renal diseases. This would explain the presence of crescents, albeit in fewer number and size, in con-
ditions such as Goodpasture's syndrome, Henoch-
San Francisco, California, March 12, 1972. This investigation was supported in part by Public Health Service Clinical Research Center grant FR-00134 from the National Institutes of Health, and by a Veterans Administration Pathology research grant. Reprint requests to Dr. G. Eknoyan, Department of Medicine, Baylor College of Medicine, Houston, Texas 77025, U.S.A.
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P, SIMON G, ROUILLEX C: Electron microscopy
38.
BURKHOLDER PM: Ultrastructural
1965 demonstration of injury
perforation of glomerular capillary basement in acute proliferative glomerulonephritis. Am Pathol 56:251—265, and
1969
I
The morphogenesis of glomerular crescents in rapidly progressive glomerulonephritis K. W. MIN, F. GYöRKEY, P. GYöRKEY, J. J. Yjurvi and G. EKNOYAN Department of Pathology and the Renal Section, Department of Medicine, Veterans Administration Hospital and Baylor College of Medicine, Houston, Texas
The morphogenesis of glomerular crescents in rapidly progressive glomerulonephritis. Kidney tissue obtained by needle biopsy from eight patients with rapidly progressive glomerulonephritis was studied. Crescents were present in more than
épithCliale. Ces constations suggèrent que les FDL de Ia mem-
brane basale glomerulaire sont initialement associées a des alterations a point de depart immunologique dues soit 1. un complexe immun ou a une maladie glonierulaire anti-membrane basale. La glomCrulonephrite rapidement progressive serait un
8O% of the glomeruli examined. Variations in the cellular composition of the crescents were present in each biopsy specimen, but were mostly epithelial in four and fibroepithelial in the others. Immunofluorescent staining of glomeruli for IgG, and
syndrome resultant de l'apparition de FDL avec, par voie de consequence, formation de croissants avec une frequence plus élevée que dans les autres situations oi les mêmes lesions peuvent exister.
/I1C revealed a linear pattern of distribution in four, a focal nodular distribution primarily in the mesangial areas in three and no staining in one. Positive staining for fibrinogen in the
Diffuse glomerular crescent formation characterizes a form of glomerulonephritis associated with rapidly deteriorating renal function often resulting in uremia and death within several months. This clinicopatholog-
epithelial crescents was present in all. A consistent finding on electron microscopy was the association of the crescents with a focal disruptive lesion (FDL) of the glomerular basement mem-
brane. Intravascular contents appeared in various stages of extravasation through these focal defects with concomitant fibrin precipitation and epithelial cell proliferation. These findings suggest that FDL of the glomerular basement mem-
ical entity which is now well recognized [1—161 has in
the past been designated as subacute glomerulone-
brane is usually associated with immunologically initiated alterations due to either immune-complex or antiglomerular basement
phritis [1], rapidly progressive type I [2], acute oliguric glomerulonephritis [9, 101 and, more recently, rapidly progressive glomerulonephritis (RPGN) [11—16]. It is the larger size and higher frequency with which crescents are present in this condition which differen-
membrane glomcrular disease, and that rapidly progressive glomerulonephritis is a syndrome probably resulting from FDL
with consequent crescent formation, occurring in higher frequency than in other conditions in which the same lesions may be present.
tiates it from other forms of acute oliguric glomerulonephritis occurring with or without crescents. The pathogenesis of crescent formation, however, is not clearly understood. Fibrin and red blood cells have
Morphogenese des croissants glomérulaires dans Ia gloméru-
Ionéphrite rapidement progressive. Le tissu renal obtenu par biopsie a l'aiguille chez huit malades atteints de glomérulonéphrite rapidement progressive a été étudié. Des croissants ont été
been repeatedly observed within the crescents by
observes dans plus de 8O°/ des glomérules examines. IDes variations dans la composition cellulaire des croissants ont été observées d'une biopsie a l'autre, rnais cette composition était principalement épithéliale dans quatre cas et fibro-epithéliale
several investigators [2, 11—16]. This has led to the assumption that crescents may represent a reaction of the epithelial cells to the products of exudation into the urinary space, particularly fibrin and red blood
dans les autres. L'immunofiuorescence des glomérules pour ]'EgG et le $1C a montré une distribution linéaire dans quatre cas, une distribution focale principalement dans les zones
cells [11]. Vassali and McCluskey have presented evidence for a role of the coagulation process in the
mésangiales pour trois cas et l'absence de fixation dans un cas. Du fibrinogène a été observe dans tous les croissants épithéliaux.
Une constation uniforme en microscopie électronique a été
pathogenesis of experimental glomerulonephritis pro-
l'association aux croissants de lesions disruptives focales (FDL) de la membrane basale glomerulaire. Le contenu intravasculaire est diversement extériorisé a travers ces interruptions focales avec lesquelles coincident les dépôts de fibrine et la proliferation
duced in rabbits by the inoculation of anti-rabbit
kidney serum. In their experimental model, the course of the disease and microscopic changes of the kidney
were in some respects similar to those of RPGN in humans, including diffuse crescent formation and
Received for publication June 8, 1973; and in revised form September 5, 1973. © 1974, by the International Society of Nephrology.
fibrin precipitation in the crescent [17]. In a subsequent
47
48
Mm
et a!
presenting with the clinical features of RPGN. Some of the clinical and laboratory data and course of these
study, they demonstrated that this experimentally induced renal disease could be prevented by treating the animals with anticoagulants prior to the administration of anti-kidney sera [18]. Additional evidence to
patients are summarized in Table 1. All had antistreptolysin (ASO) titers of less than 200. Findings of chest roentgenograms were negative except for initial
incriminate the clotting mechanism in the pathogenesis of this condition has been presented by the encouraging results reported with the use of anticoagulant therapy in some patients with RPGN [19, 20].
congestive changes in some patients. Complement levels
were normal. Antinuclear antibody (ANA) titers and
lupus erythematosus (LE) cell preparations were
Al though these observations and experimental
negative. No patients gave a history of hemoptysis or demonstrable skin lesions.
studies have established a significant body of evidence
The renal tissue was bisected longitudinally and one-half was immediately frozen at — 70°C by immersion in isopentane which had been precooled in a mixture of acetone and dry ice. Fresh frozen sections were cut at 4 and 6 and stained with hematoxylineosin, periodic-acid Schiff, Fetrot and Congo red. For immunochemistry, fresh frozen sections (4 to 6 .t) were incubated for 15 mm with fluorescein isothiocyanate conjugated anti-human IgG, 1gM, IgA, fllC and fibrinogen. The anti-human IgG, 1gM, IgA and
in favor of the pathogenic role of the coagulation process in RPGN, in certain cases the luxuriant crescents far exceed the amount of demonstrable fibrin suggesting the possibility that additional stimuli might be operative [113. Moreover, while the presence
of fibrin has been attributed to exudation, it is only recently that specific lesions have been demonstrated which would explain the presence of fibrin in this
condition in contrast to other forms of glomerulonephritis associated with otherwise comparable changes in the glomerular tufts and mesangial region
/3lC sera (produced in goat) and the anti-human
[12, 13].
fibrinogen (produced in rabbit) were purchased from Hyland Laboratories, Los Angeles, California. After incubation, the sections were washed in three changes of phosphate-buffered physiological saline for five mm, mounted with buffered glycerine and examined with the fluorescent microscope (Zeiss) using excitor filter UG 2 and barrier filter 440. The specificity of the positive staining was checked with the blocking technique by pretreating sections with the unconjugated specific serum of each before fluorescent staining. The other half of the biopsy specimen was cut into
This is a report of the immunofluorescent, light and electron microscopic observations on serial sections of renal biopsy specimens from eight patients with RPGN which elucidates the morphogenesis of the crescents and demonstrates an association and possible etiologic
role of a focal disruptive lesion of the glomerular capillary wall in crescent formation. Methods
small pieces, less than 1 mm in diameter, fixed in
Needle biopsy specimens of kidney tissue were obtained under local anesthesia from eight patients
glutaraldehyde [21] and postfixed in osmium tetroxide
Table 1. Clinical and laboratory data
Patient
No.
Age
Sex
Duration of illness before hospitalization, weeks
Protein
Presenting clinical picture
Blood pressure,
Blood urea
mm Hg
mq/100 nil
mg/100 ml
2
15
Creatinine,
excretion,
nitrogen,
g/24 hr
I
49
M
4
NS
180/110
56
2
21
F
3
NS
190/130
120
9
16
ARF5
200/120
152
27
2
160/90
172
21
6
15
1.6
3
60
M
3
4
26
M
1—2
ARF
5
54
M
2-3
ARF
120/90
100
6
49
M
2—3
ARF
180/110
144
7
16
M
8
NS
130/105
130
8
51
M
4
ARF
130/85
113
Clinical course and treatment
Treated with prednisone and imuran; died after three months Peritoneal dialysis, twice; prednisone and imuran therapy; died after two months Chronic hemodialysis; died four montht later due to septicemia
Repeated peritoneal dialysis; died five
5
months later Peritoneal dialysis with slow deterioration for one year; maintained on intermittent dialysis Peritoneal dialysis initially then prednisone
10
12
Following peritoneal dialytis two times,
18
2
7.3
and imuran therapy; improved; BUN,
60 mg/lOO ml
. NS nephrotic syndrome. b ARF = acute renal failure.
improved; lost to follow-up Treated with prednisone, imuran, and cytoxan with some improvement; died three years later in renal failure
49
Morphogenesis of glomerular crescents Table 2. Summary of biopsy findings
Patient No.
glomeruli 1
2 3
4 5
18 5 5
14
6
6
12
7
5
8
14
Character of crescent Epithelial Fibroepithelial No crescents Fibroepithelial Epithelial Fibroepithelial Sclerosed Fibroepithelial Sclerosed Fibroepithelial Sclerosed No crescents Epithelial Sclerosed No crescents Epithelial No crescents Epithelial Fibroepithelial Sclerosed No crescents
Electron microscopy
1mmunofluorescence
Light microscopy Number of
'C
IgG
1gM
12 Linear
+
+
ab
5 Linear I Linear
+ +
+ ÷
—
+
x
++
a
a
+
+
++
++
+++
++
a
++
+
"Humps"
+
a
+
+
Subendothelial deposits
Pattern,
2 4 3 I
9 Linear 5 3
+++ —
—
2
IgA
a
x
Fibrinogen
+
+
No electron dense deposits
+
No electron dense deposits No electron dense deposits
No electron dense deposits No electron dense deposits
I
8 Mesangial, 2 Lumpy
4 Lumpy I
5 Spotty, 3 Mesangial, 3
Subendothelial and mesangial electron dense deposits
3 Lumpy
Fluorescence is graded Ito 4+ on the basis of intensity and extent of distribution. b Refers to no staining for fluoresence performed.
[22J for electron microscopy. After dehydration in
graded alcohol solutions containing uranyl acetate, the specimens were embedded in epoxy resin (EponAraldite) [23]. Sections 1 to 2 in thickness were cut, stained with toluidine blue and scanned for glomeruli. From each biopsy specimen at least three glomeruli
were serially cut through the entire diameter of the glomerulus with an ultratome (LKB 8800). Ribbons of
glomerular tufts on a random section were normocellular. Serial screening, however, disclosed one or more segmental areas of hypercellularity and necrotizing glomerulitis in all of the specimens. This focal lesion often served as the nidus for crescent formation (Fig. 1) and is considered by us to be important in the morphogenesis of glomerular crescents. We will describe, in detail, this type of lesion, including its ultrastructural
sections were mounted on 200 mesh coated copper grids (Formvar), stained with uranyl acetate and lead citrate [24, 25] and studied with an electron micro-
changes.
scope (RCA 3G).
linear pattern of staining with anti-IgG revealing the
Immunohistochemistry. The results of immuno-
fluorescent staining are summarized in Table 2. Diffuse
contour of the glomerular basement membrane (GBM) Results
Histopathology. The histopathological changes were strikingly uniform and characterized by an exuberant proliferation of extracapillary cells forming crescents in the majority of glomeruli. From 80 to l00% of all glomeruli were involved in a given biopsy specimen (Table 2). The site of cellular proliferation was distinctly within Bowman's space (Fig. 1). The crescents were either epithelial or fibroepithelial. Although there was
some variation of the cellular composition of the crescents from one glomerulus to another in a given biopsy, the crescents were mostly epithelial in four patients and fibroepithelial in the others. Scierosed crescents were present in five.
Proliferative changes in the glomerular tufts were much less evident. Significant alterations in the glom-
erular tufts consisting of diffuse hypercellularity were present in only two of the biopsy specimens. In
the remainder of the biopsies, the majority of the
Fig. 1. Glomerulus from patient 1. Note the proliferative changes
forming an epithelial crescent centered around necrotic segments (arrows) of glomerular tufts (periodic-acid Schiff stain [PAS] >< 160).
50
Mm et a!
was present in four patients with RPGN (Fig. 2). The immune complex type of "lumpy" irregular staining was noted in three patients (Fig. 3). In one patient no staining was demonstrable.
Positive staining for human fibrinogen in the epithelial crescents (Fig. 4) was a constant finding in the renal biopsy specimen of all patients. This was present in clumps in the crescentic areas while staining
was spotty and only occasional in the glomerular
ing pattern, in four specimens, a classical linear pattern of immunofluorescence was seen and the focal disruptive lesion was associated with focal exudation of polymorphonuclear leukocytes and destruction of the GBM without electron dense deposits. In these biopsies, the GBM was diffusely thickened with moderate
irregularity of the contour. The intensity of staining varied resulting in a mottled appearance. in some areas,
capillaries. Fibrotic crescents stained faintly for fibrino-
the subendothelial space was markedly widened and the endothelial cells were detached from the basement
gen but it was clearly present in the outline of the
membrane. In such areas the basement membrane
crescents.
showed increased electron density on the endothelial
Electron microscopy. A consistent finding in all
side.
biopsy specimens was focal disruption of the GBIvI, spilling of the capillary contents into the urinary space and inflammatory exudate. These findings were associated with focal electron dense deposits in the mesangium or subepithelial space or both in three biopsies showing a lumpy irregular immunofluorescent stain-
We regard the "focal disruptive lesion" (FDL) of the GBM as a trigger mechanism for the initiation of crescent formation. The ultrastructural alterations in
Fig. 2
Fig. 3
relation to this lesion are, therefore, presented in detail.
The Focal Disruptive Lesion (FDL). In glomeruli
Fig. 2. Glomerulus (patient 4) stained for human lgG globulin. Note the linear distribution of fluorescence along the glomerular basement membrane (human lgG, x 500). Fig. 3. Gloinerulus (patient 7) stainedfor human JgG globulin showing lumpy nodular fluorescence along the GBM outlining the glomerular loops (human lgG, x 500).
Fig. 4. Glomerulus (patient 2) showing positive fluorescence for human fibrinogen in the crescent (human fibrinogen, x 1000).
Fig. 4
Morphogenesis of glomerular crescents
51
with epithelial crescents, the necrotic nidus observed by light microscopy (Fig. 1) was the site of FDL as observed with electron microscopy. The GBM in focal areas was disrupted and blood cells appeared in various stages of extravasation through focal defects of the GBM (Fig. 5). In the urinary space near the FDL,
associated with cellular crescents indicating that
clumps of fibrin precipitates with the typical periodicity
of fibrin were seen among the proliferating epithelial cells. Occasionally, layered fibrin precipitates appeared
serum [17]. The glomerulonephritis in these animals resembled human RPGN in its clinicopathological features including fibrin precipitates in the crescents. More importantly, the epithehial cell proliferation
vascular or urinary space. Glomerular capillary tufts proximal or adjacent to the FDL also showed fibrin in the subendothelial space and capillary lumina. In some
could be prevented by anticoagulants prior to the administration of anti-serum, while deposition of antibody in the basement membrane was not [18]. These observations lend support to the concept that
areas, the GBM appeared intact and the fibrin
fibrin precipitation initiates epithelial cell proliferation.
deposit in the capillary lumen mimicked a thrombotic occlusion. Even though FDL could not be made out initially in such areas, additional serial sectioning led to the identification of FDL which could have been missed with less extensive sectioning. In these areas fibrin precipitates were seen to be freely present among the proliferating epithelial cells. In glomeruli with fibrous crescents, FDL could only be identified with difficulty. More careful and extensive serial sectioning, however, again invariably disclosed focal defects in the GBM, usually at the axis of the crescent (Fig. 6) where mesangial cells were intermingled with fibroblastlike cells and epithelial cells.
Fibrin precipitate was either absent or much less
The reason for fibrin precipitation, however, has not been demonstrated. Our study suggests that the FDL could probably be an initiating event which leads to fibrin precipitation. Our findings are for the most part in agreement with the electron microscopic changes observed in RPGN by Bacani and associates [14]. However, they failed to detect the FDL. We found it necessary to examine serial sections of the entire glomerulus in order to identify the otherwise elusive FDL. Disruption of the basement membrane similar to the one described here has been observed by others in glomerulonephritis of different etiologies and morphologic types including RPGN [12, 13, 31]. Detailed studies of its association
evident in fibrous crescents.
with crescent formation, however, have not been
to seal off the FDL (Fig. 5). The precipitation of fibrin, however, was not restricted to the extra-
In biopsy specimens with epithelial crescents, occasional glomeruli showed focal hypercellularity without crescent formation. Such glomeruli revealed
fibrin precipitation occurred early in the pathological process and might have initiated the cell proliferation. Vassali and McCluskey described an animal model of proliferative glomerulonephritis in rabbits induced
by the injection of heterologous nephrotoxic anti-
reported.
Our study suggests that FDL may be associated with either one of the two patterns of immunological
segmental areas in which the GBM was partially disrupted and herniated and showed inflammatory cells (Fig. 7). In these glomeruli, there was no evi-
tissue damage [32—36]. In four biopsy specimens, FDL was associated with a linear fluorescent staining pattern without electron dense deposits in accordance with the
dence of spilling of the intravascular content, nor was fibrin precipitate seen in the urinary space, and epithe-
current concept of an anti-GBM-antibody pathogen-
hal proliferation was absent. Conceivably, such changes could represent the precrescent lesion. Discussion
The coagulation process has been implicated in the pathogenesis of certain forms of glomerulonephritis
because of the frequent association of fibrin with glomerular lesions [2, 11, 12, 14, 16, 26—30]. Attention
has been focused on the possible pathogenetic role of fibrin in RPGN since Ellis [2] first described the presence of fibrin-like material among the cells forming crescents in patients with rapidly progressive type I nephritis. Bacani et al [14] and Lewis et al [15] also identified fibrin in the crescents in all of their patients with RPGN. The fibrin precipitates were frequently
esis, while in three biopsy specimens, FDL was associated with subendothelial or subepithelial electron dense
deposits or both with an irregular lumpy fluorescent staining pattern consistent with an antigen-antibody complex pathogenesis. Independent of the pathways
followed, it has been shown that tissue-localized interaction of complement and antigen-antibody com-
plexes release leukotactic agents, and the attracted polymorphonuclear leukocytes may actively destroy the glomerular basement membrane [37, 38]. In all the samples examined, FDL was invariably associated with focal inflammation of the glomerular tufts. The defec-
tive margins of the GBM in FDL were usually in direct contact with polymorphonuclear leukocytes. By studying several glomeruhi on serial sections it is
possible to reconstruct a developmental sequence
which may occur in the formation of crescents.
Mlnctal
52
I.
-
'
•vBS p
-
S
-
Pb t Gla,nernlns from patient 6 Mowlqjbcal tilenqatien of GBM Qietwesi arrows) throqh which hatawcqillley contents extromente. Fibdn (F) and epitSial cells (EP) seal off the disrupted capillary wall (x 13,000).
53
Wfl!%i
*r
•
•
t
7vlorphogenesis of glomerular crescents
Fig. 6. A glomerulus from patient 4 showing a Jcal disruptive lesion in a glomerulus with fibroepithelial crescent. Note the GBM defect (between arrows). The urinary space is now occupied by epithelial cells (EP) and fibroblasts (FB). Note the presence of collagen (C) between the crescentic cells (x 13,000).
54
Mm
et al
it -
_
Fig. 7. Segment of the glomeru/ar tuft showing a relatively normal basement membrane (BM) for comparison adjacent to a focal disruptive lesion (FDL). Note the beginning of the altered basement membrane (small arrows) becoming progressively attenuated and terminating in a break (between arrow heads) with herniation of two inflammatory cells (PMN) at the site of FDL. This probably represents the precrescent formation stage >< 21,000).
55
Morphogenesis of glomerular crescents
Admittedly, this reconstruction is not based upon
Schönleiri purpura, systemic lupus erythematosus,
observation of progression of the lesion in sequential biopsy specimens from the same patients, but rather it represents a logical sequence of the changes observed in this series of eight patients. It would appear that the earliest lesion consists of one or more focal hyper-
bacterial endocarditis, poststreptococcal acute glomerulonephritis and hypersensitivity angiitis. Since the FDL and associated crescents appear to be
cellular segments due to infiltration of polymorphonuclear leukocytes attracted by the leukotactic factors released by either one of the two pathways of immunoglobulin deposition within the kidney. The
the result of a combined immune and coagulative process, it appears that therapy in this condition could best be directed at both processes rather than at one in preference to the other. The answer to this problem can come only from findings of carefully controlled clinical and pathological studies.
leukocytes then penetrate and weaken the GBM resulting in partial disruption and herniation. At this
Acknowledgments
stage, there are no discontinuities in the basement membrane nor are there fibrin precipitates in the urinary space (Fig. 7). Later, the GBM becomes
This report was presented in part at the 61st Annual Meeting of the International Academy of Pathology,
focally disintegrated and intravascular contents escape into the urinary space with precipitation of fibrin (Fig. 5). At this stage, the fibrin precipitate actually appears to seal off the capillary leakage (Fig. 5). Following the appearance of fibrin, there is proliferation of the epithelial cells and crescent formation begins. Conceivably, proliferation of the parietal and visceral cells with crescent formation may be the only mode of reaction to the basement membrane damage
and ensuing exudation or leakage of fibrin and red blood cells. Subsequently, the amount of fibrin precipitate decreases, and the cells forming the crescents are now fibroblastic (Fig. 6). Even in the glomeruli with completely fibrotic crescents, focal disruption of the GBM with free communication between intra- and extracapillary spaces is present (Fig. 6). Crescents in the various stages of development just described were often present in the same specimen (Table 1) indicating the continuing and progressive nature of the disease.
The diversity in the patterns observed by us on immunohistochemistry and electron microscopy is similar to that found by others [14, 15]. Our observations, however, suggest that RPGN is a characteristic syndrome associated with FDL and consequent diffuse crescent formation. They also explain the association of this lesion with renal diseases of diverse pathogenetic mechanisms in which immunological damage may be
due to either immune complex or anti-GBM antibody deposits. It should be noted, however, that FDL may occur in the absence of any demonstrable localized immunoglobulins, as was the case in patient 5 (Table
2). It is, perhaps, the degree of "severity" of the sequence of events that ensues complement activation
which in some cases results in the disruption of the basement membrane by the leukocytes and development of the FDL. Such a mechanism may be operative in other renal diseases. This would explain the presence of crescents, albeit in fewer number and size, in con-
ditions such as Goodpasture's syndrome, Henoch-
San Francisco, California, March 12, 1972. This investigation was supported in part by Public Health Service Clinical Research Center grant FR-00134 from the National Institutes of Health, and by a Veterans Administration Pathology research grant. Reprint requests to Dr. G. Eknoyan, Department of Medicine, Baylor College of Medicine, Houston, Texas 77025, U.S.A.
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