ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 1 Copyright © 1978, Institute for Clinical Science

Light Microscopical Examination of Glomerular Basement Membrane in Systemic Lupus Erythematosus

J. V. KLAVINS, M.D., Ph.D .,*f Z. WESSELY, M .D.f and P. PICKETT*

Departments o f Pathology, Queens Hospital Center, Long Island Jewish-Hillside Medical Center Affiliation,

Jamaica, NY 11432 and

Duke University Medical Center, Durham, NC 27710

ABSTRACT

To obtain as much information as possible from light-microscopy about changes in the glomerular basem ent-mem brane, a thin section microtome attachm ent is necessary in order to produce 1 to 3 micron sections.

The most informative staining m ethod of paraffin em bedded tissue is the Müller-Mowry-Masson trichrome (M-M-M) combination stain. The deposits, e.g., in systemic lupus erythematosus (SLE), stain red, the basem ent m em ­brane light orange and the endothelial cell cytoplasm light pink. W ith this stain, as w ith the Müller-Mowry stain, normal glomeruli show an uninter­rupted line of blue acid mucopolysaccharides along the basem ent mem­brane, w here it is jo ined by foot processes of the epithelial cells. In contrast, in glomeruli from patients with SLE, focal loss of acid mucopolysaccharides is noted.

Introduction

Light microscopy, when utilizing spe­cial staining techniques and a th in section microtome attachment, can provide sig­nificant information about the glomerular basem ent m em brane, e.g., in system ic lupus erythematosus (SLE).

D uring th ese s tu d ie s , a new techn ique—the M üller-M owry-M asson trichrome combination—was developed.

* Address for reprint requests: J. V. Klavins, M.D., Ph.D., Department of Pathology, The Cath­olic Medical Center of Brooklyn-Queens, 88-25 153rd St., Apt. 2-J, Jamaica, NY 11432.

Materials and Methods

Fifty cases clinically classified as SLE were studied. The paraffin blocks were at first sectioned with the conventional mi­crotome and approximately five to six mi­cron thick sections w ere stained w ith hematoxylin and eosin (H&E). These sec­tions includedthe entire tissue sample that was em bedded in paraffin. Areas for more detailed studies were then selected from the preliminary H&E slides and were cut out from the paraffin blocks to provide a field approximately 5 x 5 mm. Such small blocks were necessary to obtain uniformly

74

STAINING O F BASEM ENT MEMBRANE 7 5

F ig u r e 1. Mü'ller- Mowry-Masson trichrome stain x 1380. Portion of glomerulus from patient with system ic lupus erythematosus. Red de­posits can be differenti­ated from a light orange basem ent membrane, blue podocyte area and light pink cytoplasm of endothelial cells.

th in sections on su b seq u en t cutting w ith a th in section attachm ent to the m icrotom e, according to the m ethod by C hurg and G rishm an.2 T hese sections m easured a p ­proxim ately 1 to 3 ¡x in thickness.

M u l l e r -M o w r y -M a s s o n T r ic h r o m e C o m b in a t io n S t a in (M -M -M )

This was done by first u tiliz ing steps 1 through 6 o f the M uller-M ow ry colloidal

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/ * <■ ^-F igure 2. Muller-Mowry colloidal iron stain x 800. Portion of normal glomerulus. Acid mucopoly­

saccharides are present uninterrupted along the entire basement membrane, where on electron-microscopic examination the foot processes of the epithelial cells join the basement membrane.

76 KLAVINS, W ESSELY AND PIC K ETT

iron stain7 followed by a modification of the original M asson’s trichrome stain,6 namely:

(1) Staining with Ponceau acid fuchsin solution for 5 minutes; (2) Rinsing in 5 percent water; (3) Placing in 10 percent phosphomolybdic acid for 5 minutes; (4) Rinsing in acidified water; (5) Staining in light green solution for5 minutes; (6) Rins­ing in acidified water for 3 to 4 m inutes; (7) Dehydrating in 95 percent alcohol, abso­lute alcohol and xylol and (8) Mounting.

R e a g e n t s

1. Ponceau acid fuchsin solutionA. O negofP onceaudexy led ineand

1 g of acid fuchsin in 100 ml distil­led water.

B. Exactly 0.5 g of azophloxine G.A. in 100 ml of distilled water.

C. Precisely 0.5 ml of acetic acid in 100 ml of distilled water.

2. Working solution of Ponceau acidfuchsinA. A d d l0 m lo fso lu tionA and2m lo f

solution B to 88 ml of solution C.3. Light green solution

A. One goflight green to 100mlof0.5 percent acetic acid solution.

4. Acidified waterA. Exactly 0.5 g of glacial acetic acid

to 100 ml of distilled water.In addition, the following staining pro­

cedures were applied:Masson trichrome stain;6 W ilder’s re­

ticulum stain;5 Muller-Mowry colloidal iron m ethod ;7 P e rio d ic ac id Schiff technique (PAS);4 Feulgen technique;4 and Periodic acid-m ethenam ine-silver- Masson trichrome sequence (PA-MS-M).1

F i g u r e 3. Mtiller-Mowry colloidal iron stain x 1600. Portion of glomerulus from patient with systemic lupus erythematosus. Focal absence of acid mucopolysaccharides over the glomerular basement membrane.

STAINING O F BA SEM EN T MEMBRANE 77

Results

Cross sections of a normal glomerular basem ent membrane were clearly dem on­strated with all stains as a dense uninter­rupted homogeneous line. They were best delineated by staining with m ethenam ine silver.

W hen kidney containing “ wireloops” were cut in five or six micron sections, a sing le th ick m em brane form ing the glomerular loops was apparent with H&E and PAS stains. However, w hen the sec­tions were cut with the th in section at­tachm ent to the microtome, a clear distinc­tion could be made betw een the basem ent m em brane and the deposits which, when using electron microscopy, appeared as more electron-dense osmiophilic m ate­rial. The deposits appeared less intense than the basem ent membrane although they had the same color.

With Masson trichrome stain, the depo­sits appeared red while the basem ent mem brane appeared light orange. When the PA-MS-M sequence was used, the de­posits stained red and the basem ent mem­brane dark brown or black.

In a few instances, the deposits were also F e u lg e n positive . U tiliz in g the M-M-M sequence, the basem ent mem­brane stained light orange, the area of podocytes blue, the deposits red and the endo the lia l cell cytoplasm ligh t p ink (figure 1).

W hen norm al control k idneys w ere stained with the Muller-Mowry colloidal iron m ethod, an unin terrup ted line of acid mucopolysaccharides was seen be­tw een the basem ent membrane and the epithelium (figure 2). This corresponded to the e lec tronm icroscop ic f in d in g s3 which described a layer of colloidal iron positive material that covered the podo- cyte surface exposed to the urinary space. W hen the Muller-Mowry colloidal iron m ethod was applied to the SLE kidneys, frequent absence of this material was en­countered (figure 3).

These findings were also present w ith the M-M-M staining sequence.

A pply ing the M-M-M s ta in in g se ­quence, the deposits were identified in all three predom inant sites of localization in relationship to the glomerular base­m ent membrane: subendothelially, sub- ep ith e lia lly and w ith in the basem en t membrane where the deposits appeared globular or cylindrical in shape. In addi­tion, there was segmental interruption of the deposit by basem ent membrane-like m aterial. O ther changes co n sis ted of th ickening, fragm entation, sp litting or focal loss of basem ent membrane.

Discussion

T he anatom ical m an ifes ta tio n s of SLE, p a rticu la rly those o f the ren a l glomerulus, have been the subject of in­num erable studies by light microscopy, electron microscopy and immunofluores­cence. It has been established by elec­tron m icroscopy and im m unofluo res­cence that antigen-antibody complexes are deposited along the basem ent m em ­brane of the glom erular capillary in a characteristic location, e ither betw een the basem ent membrane and the endo­thelium or betw een the basem ent mem­brane and the ep ithelium , w ith in the basem ent membrane, within the mesan- gium or in any combination of these.

These deposits and their location were demonstrated using a thin section micro­tome attachment and the M-M-M stain­ing sequence, which were developed in our laboratories. This technique provided the most useful information.

In our cases, as in those described ear­lier, the preferential location of the depo­sits occurred in the subendothelial and mesangial areas in active progressive dis­ease. Clinically, inactive lesions w ere more prone to have subepithelial or m e­sangial deposits and basem ent membrane thickening.

78 KLAVINS, W ESSELY AND PIC K ETT

The state of acid mucopolysaccharides (AMP) in kidney disease has not been in­vestigated as widely as the other param et­ers. AMP was present betw een the base­m ent membrane and the epithelium in glomeruli of normal control kidneys, and was stained with a M-M-M combination stain. There was spotty absence of AMP from the epithelial side of the basem ent m em brane in some cases of SLE. Our light microscopic findings in the normal control kidneys reflected the electron- microscopic studies of Jones3 w herein a layer of colloidal iron positive material was described, covering the podocyte surface exposed to the urinary space. To the best of our knowledge, no comparable studies have been done in kidneys of patients afflicted with SLE. From our studies, it is not apparent w hether the absence of his- tochem ically dem onstrable AMP ind i­cates loss of foot processes or some other

alteration in ultrastructurally visible sites of the epithelial cells.

References

1. BURKHOLDER, P. M.: Atlas of Human Glomeru­lar Pathology. New York, Harperand Row, 1974.

2. Churg , J. and G rishman , E.: Application of thin sections to the problems of renal pathology. J. Mt. Sinai Hosp. 24:736-744, 1957.

3. J on es , D. B.: Mucous substance of the glomerulus. Lab. Invest. 21 :119-125, 1969.

4. LlLLIE, R. D.: Histopathologic Technique and Practical Histochemistry, 3rd ed. New York, McGraw-Hill Book Co., 1965.

5. Man u a l o f H is t o l o g ic St a in in g METHODS OF THE AFIP, 3rd ed. American Registry of Pathology. New York, McGraw- Hill Book Co., 1968.

6. MASSON, P.: Some histological methods; tri­chrome staining and their preliminary tech­nique. J. Techn. Methods 12:75-90, 1929.

7. M ow ry , R. W.: Improved procedure for the staining of acidic polysaccharides by Muller’s colloidal (hydrous) ferric oxide and its combina­tion with the Feulgen and the periodic acid Schiff reactions. Lab. Invest. 7 :566-576, 1958.