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SEM studies of acellular glomerular basement membrane in human diabetic glomerulopathy.

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THE ANATOMICAL RECORD 216:349-358 (1986)
SEM Studies of Acellular Glomerular Basement
Membrane in Human Diabetic Glomerulopathy
EDWARD C.CARLSON AND KRISTENE K. SURERUS
Department of Anatomy, University of North Dakota School of Medicine,
Grand Forks. ND 58202
ABSTRACT
Previous transmission electron microscopic studies have demonstrated glomerular basement membrane (GBM) thickening and mesangial matrix
(MM) expansion in chronic stages of diabetes. It is difficult, however, to achieve a n
appreciation of GBM surface features and distribution of MM in planar views. In
the current study, autopsy human renal cortical tissue from patients with end-stage
diabetic nephropathy were minced and rendered acellular with detergents prior to
fixation, cryofracture, and preparation for light microscopic CM), transmission electron microscopic (TEM), and scanning electron microscopic (SEM) observation in a n
effort to visualize extracellular materials in three dimensions.
Our studies demonstrated that although diabetic glomerular changes vary widely
within and between individuals, most showed alterations primarily affecting peripheral (epithelial) GBM (with MM increased but diffusely distributed), or they exhibited similar GBM changes but with variable nodular MM expansion leading
ultimately to capillary occlusion. Both types showed peripheral GBM thickening
and demonstrated external surface irregularities that by SEM appeared as “cauliflower-like” lobulations. In these glomeruli, GBM lamellation or reduplication was
common with internal layers frequently thrown into lumenward projections.
Glomeruli with diffusely distributed MM generally showed patent capillary channels with little evidence of occlusion. By TEM, highly compact, epithelial GBMs
were clearly distinguishable from the electron-lucent MM. In these preparations the
matrix was concentrated in relatively small discrete masses sometimes covered by
a finely fibrillar material, which extended intermittently onto lumenal surfaces of
epithelial GBMs.
In more advanced stages of MM involvement, glomeruli typically exhibited smoothsurfaced nodules that were increased at the expense of capillary surface area. By
TEM, MM nodules were comprised of a meshwork of very fine (20-A) fibrils surrounding a variety of detergent-resistant structures including collagenous fibrils
and non-collagenous 30-nm circular fibrils with 16-nm subunits. By SEM, GBM and
MM nodules were not distinguishable and merged to form substantial barriers to
capillary blood flow. In those capillary channels remaining patent, inwardly projecting folds and ridges were common GBM features, and frequently thin fenestrated
layers, distinctly separate from epithelial GBMs, formed sieve-like linings for the
channels. These three-dimensional observations provide unique views of the processes leading to diabetic glomerular occlusion and suggest a potential for this
technique in the study of renal BM disease.
Morphological changes associated with end-stage diabetic nephropathy in humans have been described in
numerous reports detailing light and electron microscopic features of the disease (Dachs et al., 1964; Kimmelsteil et al., 1966; Bloodworth, 1978; Huang, 1980;
Mauer et al., 1983a; Osterby, 1983).In these studies, the
most commonly reported alterations relate to increased
accumulations of peripheral glomerular basement membrane (GBM) and a n expansion of the extracellular matrix surrounding mesangial cells (mesangial matrix,
MM) in centrolobular regions (see Osterby, 1983, for
review). This latter change Ieads ultimately to reduced
0 1986 ALAN R. LISS, INC.
renal perfusion, renal decompensation, and ultimately,
renal failure (Bloodworth, 1978; Mauer, 1983b).
Most morphological studies have been carried out on
intact renal tissues derived from biopsy, autopsy, or
nephrectomy. These were useful because they demonstrated the relationships of glomerular cells to GBMs
and MM matrix in chronic stages of diabetic nephropaReceived February 28, 1986; accepted June 19,1986.
Address reprint requests to Dr. Edward C. Carlson, Department of
Anatomy, School of Medicine, University of North Dakota, Grand
Forks. ND 58202.
350
E.C. CARLSON AND K.K. SURERUS
Fig. 1. Light micrograph of section through detergent-treated specimen of normal adult human renal cortex. The in vivo histoarchitecture
of all major renal BM types including tubular CTBM), glomerular
(GBM), peritubular capillary PTCBM), and Bowman’s capsule (BCBM)
is preserved by this procedure. In this section, the GBM is continuous
with the BCBM (arrows) near the vascular pole. MM, mesangial matrix. ~ 5 2 5 .
Fig. 2. Light micrograph of section through specimen similar to that
shown in Figure 1, but from a patient with end-stage diabetic glomerulopathy. Bowman’s capsule BM (BCBM) is massively thickened.
Glomerular BM peripheral loops (GBM) are likewise thickened and
show numerous irregularities. Centrolobular regions show large concentrations of mesangial matrix material (MM), which encroach upon
peripheral glomerular channels. X525.
thy (Farquhar et al., 1959; Osterby, 1975; Watanabe and
Emoto, 1981; Mauer et al., 1983a). Moreover, careful
morphometric analyses of these preparations yielded
valuable statistical data on the increased GBM thickness and MM expansion at various stages in the progression of the disease (Osterby, 1975, 1983; Butcher et al.,
1977;Gundersen and Osterby, 1977;Mauer et al., 1983a).
The presence of glomerular cells in these preparations,
however, inhibited careful morphological analyses of the
three-dimensional distributions of noncellular materials
and prevented the demonstration of their topographical
(surface) features by scanning electron microscopy
(SEMI.
The development of a nondisruptive method involving
sequential detergent washings for isolating BMs Weezan et al., 1975; Carlson et al., 1978) provided opportunity for morphological studies of intact BM. In an effort
t o investigate the relationships of the various structural
components of the extracellular matrix, we recently applied this procedure to renal tissue blocks from several
species, including humans (Carlson and Kenney, 1980a,
1982; Carlson and Chatterjee, 1983; Carlson and Hinds,
1983; Carlson et al., 1986). Our studies showed that
following detergent solubilization of cells, the histoarchitecture of the renal cortical stroma, including all
major BM types, was preserved and available for study
by light microscopy (LM), transmission electron microscopy (TEM), and SEM (Carlson and Kenney, 1980a).
Topographical (SEMI views of acellular GBMs were particularly interesting and showed retention of spherical
Fig. 3. Scanning electron micrograph of cryofractured acellular renal
tissue from diabetic human. The acellular preparation is composed
primarily of a labyrinth of renal tubular BMs (TBM), but occasional
glomerular BMs (GBM) are fully or partially fractured. X 110.
Fig. 4. Higher magnification scanning electron micrograph of diabetic renal specimen similar to that in Figure 3 and showing external
surface of highly lobulated glomerular BM (GBM). X3,000.
Fig. 5. Scanning electron micrograph of cryofractured acellular diabetic glomerular BM similar to that shown in Figure 4. In this altered
glomerulus, increased mesangial matrix (MM) is diffusely distributed
and appears continuous with ridges on internal surfaces of thickened
and irregular glomerular BM (GBM). ~ 3 , 9 0 0 .
Fig. 6. Transmission electron micrograph of acellular diabetic glomerular BM similar to that shown in Figure 5. Wide Bowman’s capsule BM (BCBM) surrounds the irregularly thickened glomerular BM
(GBM). Mesangial matrix (MM) is expanded but localized primarily in
centrolobular regions. x 1,800.
SEM OF BASEMENT MEMBRANES
351
352
E.C. CARLSON AND K.K. SURERUS
conformations despite the apparent lack of supporting
collagenous fibrils (Carlson and Kenney, 1980a, 1982;
Carlson and Chatterjee, 1983; Carlson et al., 1986).
In the present study the detergent solubilization technique is applied specifically to human kidneys from
patients with end-stage diabetic nephropathy in an effort to demonstrate the distribution and topographical
features of altered GBM and associated MM in this
disease. We show, by correlated LM, SEM, and TEM
analyses of acellular renal tissue blocks, fresh new views
demonstrating specific morphological features of GBM
and MM in diabetics and the relationships of expanded
MM to thickened GBMs.
MATERIALS AND METHODS
Twenty kidneys from insulin-dependent diabetic male
patients (aged 60-69) and more than 30 normal kidneys
were received from the National Diabetes Research Interchange, from the Hennepin County Medical Center
in Minneapolis, and from the Pathology Department at
the University of North Dakota. The tissues were derived from autopsy and transplant or nephrectomy patients and were received within 72 hours following
surgical removal or death. All tissues used in the current study were from patients with end-stage diabetic
nephropathy or age- and sex-matched controls. Sections
of kidney cortex were shipped to our laboratory in Collin's medium or Tris-buffered saline with protease inhibitors (0.025 M ethylenediamine tetraacetic acid [EDTA],
0.001 M benzamidine hydrochloride, 0.001 M phenylmethyl sulfonyl fluoride [PMSF], 0.01 M N-ethylmaleimide "EM] on wet ice.
Preparation of Acellular Tissue Blocks
Renal sections were sliced ( < 2 mm thick), and the
cortex was removed from each slice with a razor blade.
Medullary tissue was discarded and the cortical strips
were further minced to < lmmd. The resultant cortical
tissue blocks were either fixed for LM and EM studies
or rendered acellular by sequential treatment with detergents as described previously (Carlson, 1980; Carlson
and Kenney, 198Oa). In brief, tissues were exposed to 10
mM EDTA (8-12 hours, 4"C), followed by 3% Triton X100 (6-8 hours, 25"C), and 4% sodium deoxycholate (68 hours, 25°C). Extensive distilled water rinses were
carried out following each step. All solutions contained
0.1% sodium azide, and treatments were carried out
with intermittent shaking. Acellular tissue blocks resulting from this procedure were fixed for microscopy as
described below.
Light and Transmission Electron Microscopy
ded EpodAraldite (Carlson and Hinds, 1981). Epoxy
blocks were cured 24 hours at 37°C and an additional
48 hours at 60°C. Sections 1pm thick were cut, mounted
on glass slides, and stained with toluidine blue (1%in
1% sodium borate). These were observed and photographed with an Olympus BH-2 compound light microscope. Thin sections were cut on a Sorvall-Dupont MT2B
ultramicrotome provided with a diamond knife, mounted
on naked 200-mesh copper grids and stained with lead
citrate (Venable and Coggeshall, 1965) and uranyl acetate (5%in 50% ethanol). These were observed and photographed with a JEOL 100s transmission electron
microscope at original magnifications of 4,000-15,000
diameters.
Scanning Electron Microscopy
Samples to be studied by SEM were cryofractured
prior to observation as described previously (Carlson
and Hinds, 1983). In brief, acellular tissue blocks were
transferred from absolute ethanol to freon 22 (cooled
with liquid N2) and then plunged directly into liquid N2.
These were cleaved with a cold, single-edgedrazor blade
and returned to absolute ethanol prior to critical-point
drying in a Tousimis Sam-Dri dryer. Dehydrated tissue
samples were mounted on aluminum specimen stubs
with double-sided cellophane tape and coated with a
thin layer of gold in a Hummer VI sputter-coater. Coated
preparations were observed and photographed in an Hitachi S-800field emission scanning electron microscope
at original magnifications of 100-10,000 diameters.
RESULTS
Sequential treatment with detergents renders renal
tissue blocks completely acellular, while structural components of the extracellular matrix, composed principally of BMs and associated collagenous fibrils, are left
intact. These structures are easily resolvable at the level
of light microscopy (Fig. 11, which shows that in normal
tissues all major BM types are preserved by the detergent procedure. By this technique, GBM shape is maintained despite the lack of cells or interstitial collagenous
support. Moreover, the relative distributions of MM and
peripheral GBM are easily assessed.
When tissues from end-stage diabetic kidneys are rendered acellular, GBMs show striking changes (Fig. 2).
The extent of the alterations varies greatly within and
Fig. 7. Higher magnification of acellular glomerular BM (GBM)
similar to that shown in Figure 6. Two peripheral blood channels
(PBC) are shown. The channel on the left is bounded by GBM that
appears normal. The GBM on the right is thickened and shows external surface irregularities. The epithelial GBM appears bilaminar with
the internal layer thrown into folds (F). Discrete masses of mesangial
matrix (MM) are covered partially by a thin layer of fine fibrillar
material 0.
X5,ZOO.
Primary fixation was carried out in paraformaldehyde-glutaraldehyde (Karnovsky, 19651, buffered at pH
7.4 with 0.2 M sodium cacodylate/HCl for 1-3 hours at
room temperature. Tissue samples were postfixed for 90
minutes (4°C) in 2% Os04 buffered with 0.2 M sodium
cacodylateMC1. Fixed tissue samples were rinsed ( x 3)
Fig. 8. Transmission electron micrograph of portion of acellular
with distilled water and stained en bloc with 1%tannic renal
glomerulus in advanced stage of diabetic glomerulopathy. Peacid (60 minutes) followed by 90 minutes in 0.5% uranyl ripheral glomerular BM (GBM) shows deep invaginations (I
some
),of
which are filled with fibrous matrix. Large nodules of mesangial maacetate.
Dehydration was carried out in graded ethanols, fol- trix (MM) reduce peripheral GBM surface area. The matrix is covered
by a thin layer of fibrillar material (FM), which also intermittently
lowing which randomly chosen samples were set aside covers
internal surfaces of GBM. Collagenous fibrils (arrows) and other
for SEM studies. Remaining tissues were carried circular fibrils (CF) are embedded in the MM. F, folds in internal layer
through several changes of propylene oxide and embed- of GBM. ~4,400.
SEM OF BASEMENT MEMBRANES
353
354
E.C. CARLSON AND K.K. SURERUS
between individuals, but, in general, MM material is
more prominent, BCBMs and peripheral GBMs appear
thickened, and the latter show surface irregularities
that stand in contrast to the smooth, evenly contoured
GBMs seen in normal tissues (Fig. 1).
Acellular renal cortical tissue blocks that have been
cryofractured, critical-point-driedand prepared for SEM,
maintain their histoarchitecture and exhibit labyrinths
of BMs (Fig. 3). These consist primarily of tubular BMs,
but peritubular capillary BMs and GBMs are also present. GBMs usually appear as spheres with surface convolutions somewhat reminiscent of sulci and gyri in
miniature cerebral hemispheres. Occasionally they are
completely or partially cryofractured (Fig. 3) and allow
comparative observations of internal (endothelial-mesangial) and external (epithelial) surfaces.
SEM shows that in diabetic glomerulopathy, external
surfaces of GBM peripheral channels are frequently lobulated (Fig. 4).Corresponding surface irregularities are
observed by LM (Fig. 2) and by TEM (Fig. 6). Although
this common external feature of diabetic GBMs correlates well with internal alterations in glomerular structure (principally GBM thickening and MM expansion),
it is not a reliable indicator of the type or extent of the
disease.
Cryofracture of some peripheral GBMs in diabetic BM
disease shows that although they are somewhat irregular, peripheral channels remain patent and unobstructed (Fig. 5). Moreover, MM material, though
variably expanded in these glomeruli, does not appear
to decrease significantly the cross-sectional area of peripheral channel lumens.
By TEM, these diffusely altered glomeruli show thickened peripheral (epithelial) GBMs with numerous external irregularities (Fig. 6). The GBMs occasionally appear
partially lamellated with the internal layers thrown
into folds. MM material, which in normal tissues appears flocculent and loosely arranged, is organized in
discrete masses with smooth surfaces.
At higher magnifications, substructural features of
diabetic GBMs are clearly elucidated (Fig. 7). Epithelial
GBMs occasionally delaminate to form two or more layers. Highly compact, electron-dense, and lobulated external layers show sharply demarcated urinary surfaces.
Inner layers are ultrastructurally similar, but their surfaces are not as crisply defined, and often they fold upon
themselves to form lumenal projections that are not
related directly to external lobulations. MM material is
the least electron-dense component of acellular glomeruli, though it frequently exhibits striated collagen fibrils and other fibrillar structures. In axial regions it is
located in smooth-surfaced masses covered by an interrupted finely fibrillar coat. Occasionally the coat extends onto the internal surfaces of peripheral GBMs
where it probably represents the endothelial-mesangial
BM.
Although relatively diffuse changes, such as those seen
in Figure 7, are present in some glomeruli, others show
considerably more nodular invclvement (Fig. 8). In the
latter, epithelial GBMs are thicker and more irregular,
and the finely fibrillar layer that surrounds MM nodules
forms a distinct layer, which intermittently lines their
internal surfaces. External GBM surfaces remain crisp
but show numerous invaginations. These external infoldings frequently are filled with fibrous material that
is not solubilized by detergent extraction. In these glomeruli, MM frequently comprises large nodular masses
and is easily distinguishable from peripheral GBM.
The MM in advanced diabetic glomerulopathy is morphologically complex and differs markedly from its normal counterpart (Carlson and Chatterjee, 1983). This
complexity is particularly striking in detergent-treated
specimens, which contain numerous striated collagenous fibrils and other unidentified fibrils, many of which
appear to form circular or whorled patterns (Fig. 8).
Higher magnification shows that the latter are approximately 30 nm in diameter and are composed of 16 nm
repeating subunits (Fig. 9). MM enclosed by circular
fibrils is coarsely granular and more electron-densethan
the remainder of the matrix. Other nonperiodic fibrils,
“lamellated bodies,” and dense granules- are embedded
in a background of very fine fibrils (20 A in diameter),
which are heterogeneously distributed.
When acellular glomeruli in advanced stages of diabetic BM disease (Fig. 8) are cryofractured and prepared
for SEM, lobulated external surfaces of glomerular
channels, thickened peripheral GBMs, and nodular
masses of MM are displayed in dramatic relief (Fig. 10).
Lobulations are not as exaggerated as in less involved
glomeruli, and some surface invaginations appear filled
or partially filled with extracellular matrix. Some channels remain relatively patent, while others are occluded
by MM material. Internal surfaces of some channels
show ridges and folds that appear to coalesce. In others,
these irregularities are covered by a thin interrupted
lining of matrix.
At higher magnifications, internal GBM surface ridges
frequently exhibit a network of interconnecting folds
(Fig. ll),which may be covered by a thin discontinuous
but distinct layer of matrix bridging across their apices
(Fig. 12). By SEM this delicate and highly fenestrated
lining appears to be composed of very fine fibrils with
an intercalated homogeneous matrix.
DISCUSSION
The development of a sequential detergent extraction
technique for isolation of BMs provided a major improvement in their preparation for morphological analyses (Carlson et al., 1978).We have previously employed
this procedure in an effort to demonstrate structural
(Carlson and Kenney, 1980b)and compositional (Carlson
et al., 1981)heterogeneity in BMs isolated from several
organ subfractions. Moreover, when the technique was
modified to remove cellular materials from whole tissues (e.g., renal cortical tissue blocks), all major BM
types retained their in vivo histoarchitectures and
showed unique ultrastructural features that were distinguishable within and between species (see Carlson, 1986,
for review).
Recently, this extraction technique has been applied
to human renal tissue samples to demonstrate GBM
surface alterations in glomerulonephritis (Bonsib,
1985a1, in the nephrotic syndrome (Bonsib, 1985b), in
idiopathic membranous glomerulopathy (Weidner and
Lorentz, 1986a1, and in lupus nephritis (Weidner and
Lorentz, 1986b). Combined with SEM, the technique
permitted direct three-dimensional visualization of GBM
irregularities, discontinuities, small surface defects, and
other alterations, the shapes of which could not be fully
appreciated by LM and TEM images. Moreover, the
Fig. 9. Higher magnification transmission electron micrograph of rows) appear to wall off MM. Dense and moderately dense granules (G)
acellular mesangial matrix (MM)
from specimen similar to that shown may represent collagenous fibrils in various planes of sectioa. All are
in Figure 8. Unidentified circular fibrils (CF) surround coarsely gran- surrounded by a heterogeneous meshwork of very fine 20-A fibrils.
ular MM. These fibrils are -30 nm in diameter and are made up of ~ 5 3 , 0 0 0 .
16-nm repeating subunits. Other continuous fibrillar structures (ar-
small sample size needed for analysis made the procedure easily applicable to biopsy.
In an effort t o study the internal extracellular components of normal human glomeruli, we recently adapted
a cryofracturing technique (Carlson and Hinds, 1983)to
renal cortical tissue blocks rendered acellular with detergents. SEM observations of these preparations showed
differences in the surface morphology of internal and
external sides of normal human peripheral GBMs and
also demonstrated three-dimensional distributions of
MM.
In the current study, our fracturing technique is applied for the first time to renal cortical samples from
patients with end-stage diabetic nephropathy. Our results show that external surfaces of peripheral GBMs
differ dramatically from their normal counterparts.
While normal human GBMs show smooth and evenly
contoured peripheral channels (Carlson and Chatterjee,
1983), those from diabetics exhibit external lobulations
with a “cauliflower-like” appearance. These irregularities correlate well with invaginations and folds seen by
TEM, but their conformations and extent are more fully
appreciated by SEM images.
Wrinkling or folding of GBMs is usually considered an
isochemic change in obsolescent glomeruli that is due to
a variety of conditions including diabetic glomerulopathy (Nagle et al., 1969). Most TEM studies of these
latter tissues do not emphasize this feature, however,
and focus primarily on the recognized “cardinal” diabetic changes of GBM thickening and mesangial expansion (Mauer et al., 1983a; Osterby et al., 1983).
Lobulation, on the other hand, is a more subtle alteration and is best visualized by SEM of acellular glomeruli. Accordingly, it has received little attention (Carlson
et al., 1984).
Cryofractured acellular glomeruli also provide an opportunity for morphological analyses of MM in the diabetic state. Following extraction of cells, the MM stands
out in three-dimensional relief. Its known extension onto
the peripheral GBM (Huang, 1980; Carlson and Hinds,
1983) is particularly well demonstrated, and even in
glomeruli in which the progression of the disease must
be described as diffuse, the MM shows prominent well
defined columns and trabeculae that appear to reach out
from axial regions to form continuities with internal
surfaces of glomerular channels. This is consistent with
the observations of Huang (1980) who showed that in
guanidine-extracted diabetic glomeruli, endothelialmesangial BMs were present as thin coverings of luminal surfaces of thickened epithelial BMs.
Our TEM studies show that in diffuse as well as in
more advanced nodular forms af the disease, reduplications of epithelial GBMs form folds and ridges that project inward toward the capillary lumen. These
Fig. 10. Scanning electron micrograph of portion of cryofractured
acellular glomerulus showing nodular occlusive glomerulopathy. Peripheral glomerular BM (GBM) is irregularly thickened. Some GBM
external invaginations are filled with detergent-resistant matrix (arrows) reducing the overall lobulated appearance. Mesangial matrix
(MM) is present as large nodular masses encroaching on peripheral
channels. Internal surfaces of patent channels show ridges (R)or thin
linings (L) both of which appear continuous with the MM. Higher
magnifications of rectangles A and B are shown in Figures 11 and 12,
respectively. ~ 4 , 8 0 0 .
SEM OF BASEMENT MEMBRANES
357
Fig. 11. Higher magnification of area enclosed by rectangle A in
Fig. 12. Higher magnification of area enclosed by rectangle B in
Figure 10. Ridges located on the internal surface of glomerular BM Figure 10. A delicated fenestrated layer of matrix (FM) composed of a
(GBM) are seen as a system of interconnecting folds. A layer of fibril- meshwork of very fine fibrils (arrows) and intercalated matrix particles
containing matrix (arrows) covers the internal GBM surface. x 18,800. appears continuous with mesangial matrix trabeculae (see Fig. lo),
and lines the epithelial glomerular BM (GEM). x 18,400.
projections do not correlate directly with lobulations on
the external GBM surface, and though they are ultrastructurally indistinguishable from epithelial GBM,
they appear to form separate lamellae. Such BM lamellations are not uncommon in diabetes and have been
described in a variety of tissues including renal tubular
BM (Vracko et al., 1979),muscle capillary BM (Vracko,
1978), and retinal capillary BM (Ashton, 1974). To our
knowledge, however, evidence for similar phenomena in
GBMs has not been shown previously.
Structural heterogeneity of MM in diabetic glomeruli
is well documented in previous TEM studies (Nagle et
al., 1969; Osterby, 1976; Huang, 1980). In these studies,
numerous lamellated bodies, banded fibers, whorled fibers, and other included structures are described. The
current investigation shows that most of these structures persist following detergent extraction. Accordingly, they apparently are not made up of lipid and
probably represent glycoproteinaceouscomponents of the
MM. A new finding in our TEM studies of acellular
glomeruli was a fine layer of fibrillar material covering
MM nodules and extending into glomerular channels as
interrupted linings, distinctly separate from the epithelial GBM. This feature has not been reported by other
investigators. It is possible, however, that it may represent a remnant of the normally flocculent MM and endothelial-mesangial BM (Carlson and Chatterjee, 1983).
SEM images of GBMs with advanced diabetic mesangial hypertrophy show external lobulations that are less
conspicuous than those that are only moderately involved. Surface infoldings appear “filled in” with electron-dense matrix material contributing to a relatively
smooth external contour. This is consistent with our
TEM observations, which show that many such invaginations contain detergent-resistant fibrous deposits.
SEM also shows that with more extensive diabetic
involvement, previously patent GBM channels are partially or completely occluded by MM nodules. This is not
unexpected since it is known that the end result of
mesangial matrix accumulation in the diabetic kidney
is glomerular closure, primarily as a result of MM expansion (Gelman et al., 1959; Bloodworth, 1978; Mauer
et al., 1983b).
Although MM nodules and peripheral GBMs are
clearly disparate at the level of TEM, they are not qualitatively distinguishable by SEM. They appear to merge
to form continuous masses, filling or partially filling
glomerular peripheral channels. Those channels that
remain open are much reduced and frequently show
internal surface modifications consistent with our TEM
observations of similar preparations. The observed network of interconnecting folds is positionally identical
with inward projections noted by TEM and probably
represents epithelial GBM redundancies. Likewise, the
delicate but discrete fenestrated layer of matrix that
covers the folds in some channels is represented in TEM
images by a fine fibrillar layer that also covers MM
nodules. This layer could represent a separation of the
E.C. CARLSON AND K.K. SURERUS
358
endothelial-mesangial BM from its hypertrophic epithelial counterpart.
Most of the topographical features of diabetic acellular
glomeruli described in this report are not new and are
shown or referred to in previous LM and TEM studies of
tissues from renal biopsy and autopsy. It is difficult,
however, to fully appreciate the distribution and structural complexity of these materials in planar views.
Moreover, SEM studies of diabetic glomeruli are not
particularly instructive since most changes relate to the
GBM and MM, which are restricted from view in cellular preparations.
In the current study, a simple, yet effective extraction
technique is employed to remove cellular materials while
maintaining the in vivo histoarchitecture of renal cortical stroma including all major BM types. This procedure, combined with cryofracture and SEM demonstrates for the first time in diabetic nephropathy, 1)
external lobulation of GBMs, 2) internal GBM surface
modifications including interconnecting ridges, projections, reduplications, and possible lamellation, 3) texture and conformation of MM in diffuse forms of
glomerulopathy, and 4) distribution of MM i n nodular
glomerulosclerosis.
Since studies similar to those reported here have been
carried out on acellular renal tissues from a variety of
normal animal models (Carlson and Kenney, 1982; Carlson and Hinds, 1983; Carlson et al., 1986) including
humans (Carlson and Chatterjee, 1983), it is not likely
that the alterations reported in the current study are
related to shrinkage, retraction, or other artifactual
change. Nevertheless, the results must be considered
preliminary, and further study is required to determine
the relative frequency of the observed alterations and
their relationship to the severity of the disease.
ACKNOWLEDGMENTS
Research in the current study was funded in part by a
grant from the American Diabetes Foundation, North
Dakota Affiliate, Inc., and was assisted substantially by
acquisition of human normal and diabetic renal tissues
from the National Diabetic Research Interchange. We
wish to thank Mr. Dave Hinds, Ms. Nancy Bjork, and
Ms. Janice Audette for expert technical assistance and
Ms. Julie Horn for typing the manuscript.
REFERENCES
Ashton, N. (1974) Vascular basement membrane changes in diabetic
retinopathy. Br. J. Ophthalmol., 58:344-366.
Bloodworth, J.M. (1978) A Re-evaluation of diabetic glomerulosclerosis
50 years afler the discovery of insulin. Hum. Pathol., 4:439-453.
Bonsib, S.M. (1985a) Scanning electron microscopy of acellular glomeruli in nephrotic syndrome. Kidney Int., 27r678-684.
Bonsib, S.M. (1985b) Glomerular basement discontinuities: Scanning
electron microscopic study of acellular glomeruli. Am. J. Pathol.,
119t357-360.
Butcher, D., R. Kikkawa, L. Klein, and M. Miller (1977) Size and
weight of glomeruli isolated from human diabetic and nondiabetic
kidneys. J. Lab. Clin. Med., 89t544-553.
Carlson, E.C. (1980) Histoarchitectural studies of the acellular human
renal cortex. Anat. Rec., 196:28A (Abstract).
Carlson, E.C. (1986) Morphology of isolated renal tubular and glomerular basement membranes. In: Renal Basement Membranes in
Health and Disease. R.G. Price and B.G. Hudson, eds. Academic
Press, New York (in press).
Carlson, E.C., and M.C. Kenney (1980a) Preparation and histoarchitecture of ultrastructurally pure glomerular basement membrane.
Renal Physiol., 3:280-287.
Carlson, E.C., and M.C. Kenney (1980b) Morphological heterogeneity
of isolated renal basement membranes. Renal Physiol., 3:288-297.
Carlson, E.C., and D. Hinds (1981) Native banded collagen fibrils in
the glomerular mesangial matrix of normal human and laboratory
animals. J. Ultrastruct. Res., 77r241-247.
Carlson, E.C., and M.C. Kenney (1982) An ultrastructural analysis of
isolated basement membranes in the acellular renal cortex: A
comparative study of human and laboratory animals. J. Morphol.,
171:195-2 11.
Carlson, E.C., and S.N. Chatterjee (1983) Ultrastructure of isolated
basement membranes in the acellular human renal cortex. Renal
Physiol., 6:197-208.
Carlson, E.C., and D. Hinds (1983) A topographical (SEM) analysis of
acellular glomerular mesangial matrix i n situ. J. Ultrastruct. Res.,
82t96-110.
Carlson, E.C., K. Brendel, J.T. Hjelle, and E. Meezan (1978)Ultrastructural and biochemical analyses of isolated basement membranes
from kidney glomeruli and tubules and brain and retinal microvessels. J. Ultrastruct. Res., 62:26-53.
Carlson, E.C., E. Meezan, K. Brendel, and M.C. Kenney (1981) Ultrastructural analyses of control and enzyme treated isolated renal
basement membranes. Anat. Rec., 2OOr421-436.
Carlson, E.C., K.K. Surerus, and D. Hinds (1984) Three dimensional
(SEM) studies of the distribution of mesangial matrix in end-stage
diabetic glomerulopathy. Anat. Rec., 208t27A (Abstract).
Carlson, E.C., K.K. Surerus, and D. Hinds (1986) Ultrastructural analysis of major basement membrane types in rhesus monkey, Mucucu
mulluttu acellular renal cortex. Acta Anat. (Basel), 125:14-22.
Dachs, S., J. Churg, W. Mautner, and E. Grishman (1964) Diabetic
nephropathy. Am. J. Pathol., 44r155-168.
Farquhar, M.G., J. Hopper, and H.D. Moon (1959) Diabetic glomerulosclerosis: Electron and light microscopic studies. Am. J. Pathol.,
35r721-753.
Gellman, D.D., C.L. Pirani, J.F. Soothill, R.C. Muehrcke, N. Maduros,
and R.M. Kark (1959) Structure and function in diabetic nephropathy. The importance of diffuse glomerulosclerosis. Diabetes, 8:251256.
Gundersen, H.J.G., and R. Osterby (1977) Glomerular size and structure in diabetes mellitus. 11. Late abnormalities. Diabetologia,
13:43-48.
Huang, T.W. (1980) The nature of basal lamina alterations in human
diabetic glomerulosclerosis. Am. J. Pathol., 100:225-238.
Karnovsky, M.J. (1965) A Formaldehyde-glutaraldehyde fixative of
high osmolality for use in electron microscopy. J. Cell Biol.,
27r137A-138A (Abstract).
Kimmelstiel, P., G. Osawa, and J. Beres (1966) Glomerular basement
membrane in diabetics. Am. J. Clin. Pathol., 45r21-31.
Mauer, S.M., M.W. Steffes, J. Connett, J.S. Najarian, D.E. Sutherland,
and J. Barbosa (1983a) The development of lesions in the glomerular basement membrane and mesangium after transplantation of
normal kidneys to diabetic patients. Diabetes, 32:948-952.
Mauer, S.M., M.W. Steffes, F.C. Goetz, D.E. Sutherland, and D.E.
Brown (198313) Diabetic nephropathy: A perspective. Diabetes,
32:52-55.
Meezan, E., H.T. Hjelle, K. Brendel, and E.C. Carlson (1975) A simple,
versatile, nondisruptive method for isolation of morphologically
and chemically pure basement membrane from several tissues.
Life Sci., 17tl721-1732.
Naale. R.B., P.W. Kohnen. R.E. Bulger, G.E. Striker. and E.P. Benditt
71969) Ultrastructure of human-renal obsolescent glomeruli. Lab.
Invest., 21t519-526.
Osterby, R. (1975) Early phases in the development of diabetic glomerulopathy. Acta Med. Scand. [Suppl.], 574t13-80.
Osterby, R. (1983) Basement membrane morphology in diabetes mellitus. In: Diabetes Mellitus, Theory and Practice. M. Elenberg and
H. Rifiin, eds. Medical Examination Publishing Co., New York,
pp. 323-341.
Osterby, R., H.J.G. Gundersen, A. Holyck, J.P. Kroustrup, G. Nyberg,
and G. Westberg (1983) Diabetic glomerulopathy: Structural characteristics of the early and advanced stages. Diabetes, 325'9-82.
Venable, J.H., and R. Coggeshall(1965) A simplified lead citrate stain
for use in electron microscopy. J. Cell Biol., 25407-408.
Vracko, R. (1978) Effects of aging and diabetes on basal lamina thickness of six cell types. In: Biology and Chemistry of Basement
Membranes. N.A. Kefalides, ed. Academic Press, New York, pp.
483-493.
Vracko, R., D. Thorning, and T.W. Huang (1979) Basal lamina of
alveolar epithelium and capillaries: Quantitative changes with
aging and in diabetes mellitus. Am. Rev. Respir. Dis., 120:973983.
Watanabe, T., and K. Emoto (1981) Mesangial bridges: A mode of
progression in diabetic glomerulosclerosis. Acta Pathol. Jpn.,
31~1045-1051.
Weidner, N.W., and W.B. Lorentz (1986a) Scanning electron microscopy
of the acellular glomerular basement membranes in idiopathic
membranous glomerulopathy. Lab. Invest., 54r84-92.
Weidner, N. W., and W.B. Lorentz (1986b) Scanning electron microscopy
of the acellular glomerular and tubular basement membranes in
lupus nephritis. Am. J. Clin. Pathol., 85:135-145.
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