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