Electron Microscopy in Glomerulonephritis

Electron microscopy has been in use as a research tool for many years, and as such has helped to demonstrate and elucidate the fine structure of many organs, including that of the kidney. The intricate pedicel structure of the renal glomerular epithelial cell, the glomerular endothelial fenestrae (Fig. I) and the varying cytological arrangements in the tubular part of the nephron were all unknown before the advent of electron microscopy. As a development of this anatomical function of the microscope, the technique began to be applied to diseased tissues in an attempt to analyse further the relation of disordered structure to disordered function. This type of investigation held the inherent difficulty that human tissues to be examined in this way had, of necessity, to be biopsy material, since autolytic changes in post-mortem material arc so gross that cytological ultrastructure is significantly destroyed. Copyright Royal Medical Society. All rights reserved. The copyright is retained by the author and the Royal Medical Society, except where explicitly otherwise stated. Scans have been produced by the Digital Imaging Unit at Edinburgh University Library. Res Medica is supported by the University of Edinburgh’s Journal Hosting Service: http://journals.ed.ac.uk ISSN: 2051-7580 (Online) ISSN: 0482-3206 (Print) Res Medica is published by the Royal Medical Society, 5/5 Bristo Square, Edinburgh, EH8 9AL Res Medica, Spring 1967, 5(3): 33-39 doi: 10.2218/resmedica.v5i3.469 ELECTRON M ICRO SCO PY IN

E lectron m icroscopy has been in use as a research tool for m any years, and as such has helped to dem onstrate and elucidate the fine structure o f m any organs, including that of the kidney.T h e intricate pedicel structure of the renal glom erular epithelial cell, the glom erular endothelial fenestrae (Fig. I) and the varying cytological arrangem ents in the tubular part of the nephron were all unknown before the advent of electron m icroscopy.A s a develop ment of this anatom ical function of the m icro scope, the technique began to be applied to diseased tissues in an attem pt to analyse further the relation of disordered structure to disorder ed function.T h is type of investigation held the inherent difficulty that hum an tissues to be examined in this way had, of necessity, to be biopsy m aterial, since autoly tic changes in post mortem m aterial arc so gross that cytological ultrastructure is significantly destroyed.
Investigations were carried out in m any human diseases w here biopsy material was available, and in particular several diseases of the renal glom eruli becam e capable of m ore thorough description, classification and clinicopathological correlation.It is probably not too much to say that the concept of glom erulo nephritis itself was revolutionised.

E A R L Y CONCEPTS OF GLOMERULO NEPHRITIS
B efore the advent of the electron m icroscope and the technique of renal biopsy the usually accepted classification of glom erulonephritis was that o f E llis, proposed in 19 42; this had superseded the old classification elaborated by V olh ard and Falir, which divided the disease into three stages, acute, subacute and chronic.E llis ' classification, m ore clinically acceptable, described two separate disease processes.O ne was nam ed T y p e I glom erulonephritis; this was of acute nature, clinically and patho logically, and usually resolved w ith com plete clinical recovery.It m ight, how ever, progress to a chronic, fibrosing stage in which the patient developed chronic renal failure and died.T h is condition was accepted as being of post-streptococcal nature.T y p e II glom er ulonephritis was a com pletely different disease, in w hich the patient presented w ith a slow ly developing nephrotic syndrom e; this eventually progressed to chronic renal failure in the m ajority of cases, although in a few com plete recovery ensued.T h e pathology of this second type, according to E llis, was basically a thicken ing o f the glom erular capillary walls, although other com plicating and often inflam m atory features were described in som e cases.T h e cause of the capillary wall thickening was quite unknow n, and likewise the aetiology was obscure.It m ust be appreciated that all the histological m aterial on which this extrem ely perspective concept was based was post-mortem in nature.
T h e m ore com plex and subtle types of glom erulonephritis were at that tim e alm ost unrecognised.
M o st cases of glom erular disease were put into one or other of the two main categories if n o generalised system ic condition such as diabetes m ellitus was present.Focal glom erulonephritis was not well apprec iated as a clinical or pathological entity, and th e e x iste n c e o f a fu n c tio n a l d isease o f th e g lo m e ru la r c a p illa ry b a se m e n t m e m b ra n e u n asso ciated w ith gross stru c tu ra l g lo m e ru la r ch an g es w as u n su sp e cte d .
It w as at this h isto ric a l p o in t, in th e m id d le 19 5 0 's, th at th e electro n m ic ro sc o p e b e c a m e fu n c tio n a lly tran s la te d fro m a n a to m y to th e p a th o lo g ic a l field, an d b egan to a n sw er so m e o f th e q u estio n s w h ich lig h t m ic ro sc o p y h ad created b u t had b een u n a b le to so lve ; an d it w as at a p p ro x i m a te ly th e sam e tim e th a t renal b io p sy b e cam e p ra ctic a l, sa fe an d an a c ce p te d p art o f in v esti ga tio n o f ren al disease.The epithelial layer is co ntin uo us over the basement membrane.

REAPPRAISAL OF CLASSIFICATION
x 8.000 As a result of these advances, it became obvious that when all cases which could be placed in other categories were removed, the group of patients with Type II glomerulone phritis consisted of individuals exhibiting clinically a pure nephrotic syndrome and showing on renal biopsy no inflammatory or proliferative changes of any significance in the glomeruli.
Specimens from these patients showed either a diffuse thickening of the glomerular capillary walls, or, surprisingly, no glomerular abnormality of any importance.It became clear that the development of the former lesion was towards increasing capillary wall thickening and obliteration of normal glomerular structure, with glomerular ischaemia and progressive renal failure.
It is perhaps natural that at this time the type of case in which no capillary wall thickening or other glomerular lesion was evident was thought to be simply an early stage of the con dition, and that given time it would progress to a stage at which the capillary wall thickening became visible on light microscopy.

PATHOLOGICAL CHANGES
Electron microscopic investigation of these cases, by this time usually referred to collec tively as early and late stages of Type II glom erulonephritis, now began to throw light on the structural changes involved and on the relationship of the conditions, changing com pletely some of the ideas hitherto accepted without question.T h e hyaline thickening of the capillary walls seen in the so-called late stage of the condition was found to be account ed for by a complicated change in the structure of the capillary walls and in the relationship of their three main components.(Fig. 2).T h e basement membrane itself, which, as will be recalled, is the middle layer of the capillary x 6,000 of pedicel structure m ight be in fact secondary to the proteinuria, con stituting an attem pt to "seal o ff" the points o f leakage in the basem ent membrane.

M EM BRANEOUS and minimal lesion glomerulonephritis
H ow ever, this relatively sim ple concept of membraneous glom erulonephritis was then undermined by a fact which becam e apparent only as the years w en t by, and m ore patients were follow ed up both clinically and by biopsy examination.T h e obvious corollary to the idea that there were histologically distinguishable early and late stages o f m em braneous glom erul onephritis would be the recognition of the transition o f patients from one stage to the other, and up to the present this has sim ply not been proved to occur.In addition, patients in the so-called early stage are found to have proteinuria and oedem a as severe as those in the " late stage" .Patients suffering from the type of disease in w hich no glom erular abnor malities arc visible on light m icroscopy m ay continue for a long tim e excreting protein and yet the ligh t m icroscopy appearances o f the glomerular capillaries do not becom e those of the " late stage" , and in fact change very little, if at all.Likew ise these patients have a good prognosis clinically, and w hile it is not possible to dogm atise on this subject, it appears that they do not progress to renal failure and hypertension as do the patients w ho exhibit histologically the m ore severe lesion.T h ese findings cast grave doubt upon th e advisability of regarding the two types of case as different stages of the sam e condition, and the idea has gradually evolved that they m ight be, in fact, two different diseases.T h e term " m em brane ous glom erulonephritis" has naturally been reserved for the type in w hich the capillary basement m em brane is visibly abnorm al, and the som ew hat awkward nam e " m inim al lesion glom erulonephritis" was coined for the con dition in which glom erular capillary wall thickening is n ot visible on ligh t m icroscopy.These terms are now well established as the nom enclature for the two conditions, and m ore and m ore clinical workers are apprehending the distinction betw een the diseases, and are appreciating the fact that the prognosis in m inim al lesion glom erulonephritis is good, whereas that in m em braneous glom erulone phritis is bad, although the disease m ay be of long duration.

THE ROLE OF STEROID THERAPY
T h e com m on m ethod o f dealing w ith cases of both m inim al lesion and m em braneous glom erulonephritis is therapy by steroids.It w ould therefore be a m atter o f great difficulty to collect and com pare series of cases of the two conditions in w hich repeated renal biopsy had been perform ed and in which the course of the disease had not been influenced by steroids.In fact, the m ajority o f reports, relat ing to the investigation of such cases by elec tron m icroscopy in recent years, have referred to patients w ho had been given steroids at som e time.It does seem p ro b able, from observation of occasional untreated cases, that the natural history and m icroscopic appearances in m em braneous glom erulonephritis are in general the sam e, w hether or not steroids h ave been given: w hile there is an im pression that such treat m ent m ay slow up the process, the light and electron m icroscopic appearances undoubtedly still show a continuous, although in som e cases very slow , progression towards disruption of the norm al structure of the capillary walls, w ith thickening and eventual obliteration of capil lary lum ina.
In m inim al lesion glom erulonephritis, on the other hand, it is obvious that while some patients show spontaneous recovery w ithout the aid of steroid therapy, in m any individuals proteinuria w ill continue unless steroids are given, and will then cease, often within a veryshort time.
Indeed, som e patients can be shown to be h igh ly sensitive to steroids, the proteinuria appearing and stopping as these drugs are withdrawn and reintroduced.T h e interesting fact in this connection is that in m ost cases electron m icroscopic investigation of the glom eruli, at a tim e when the patien t has no proteinuria due to steroid therapy or to spontaneous cure, shows m ore or less com plete restoration to norm al o f the capillary wall ultrastructure, with re-appearance o f pedicels.
Basically a m ore im portant point, how ever, arising from the widespread use of steroid therapy, is the possibility that it could be this form o f treatm ent which prevents cases of m inim al lesion glom erulonephritis from pro gressing to m em braneous glom erulonephritis.W ith o u t going into any detail, it m ay be said that there is a significant am ount o f evidence m ilitatin g against this concept.
T h e aetiology o f m inim al lesion and m em braneous glom erulonephritis is quite obscure up to the present tim e, and indeed there is no real evidence that they are the result of the same cause, or even related causes.As might be supposed, it has been suggested that the lesion is basically an immune reaction, possibly of " auto-immune" type : there is some positive evidence that this is so.However, no definite concept of the aetiology of these conditions has yet been evolved.

"E LL IS TYPE I " G L O M E R U L O N E P H R IT IS
W hen we turn to the condition which Ellis described as Type I glomerulonephritis, and which was, in his view, a hypersensitive re action of the glomeruli to the streptococcus, we find that here the electron microscope itself has made a less basic and categorical contribution : however, it is true to say that the advent of renal biopsy, with a combination of light and electron microscopic analysis, has transformed our appreciation of the signifi cance of pathological glomerular changes in this condition, or rather, as soon became obvious, this group of conditions.
T h e essential lesion in what was then called Typ e I glomerulonephritis was a generalised proliferation of the endothelial cells of the glomerular capillaries with endothelial swelling, and consequent partial or complete blockage of the capillary lumina.This led to glomerular ischaemia, to which is due many of the import ant clinical features of the disease.This endo thelial cell change is responsible for the fact that the name now usually given to this con dition is proliferative glomerulonephritis.It was realised that many cases of this type of glomerulonephritis recovered completely, and that a few cases continued or recurred, develop ing in a clinically subacute fashion to a con dition of hypertension and chronic renal fail ure, which always proved fatal.It must be appreciated once again that virtually all the pathological material available for examination in this respect was post-mortem in nature.

PATHOLOGICAL CHANGES
W hen it became possible to visualise the changes going on in the kidney during life, some important and hitherto unrecog nised points were discovered, and a much more complex classification began to evolve.In the first place, it was found that in renal biopsies from patients who presented with one or more of the clinical features of an acute inflammatory glomerular lesion, the lesions encountered were very varied.Some cases might show the characteristic, obvious endothelial cell proliferation with p erh ap s polymorph infiltration of the glomeruli : o thers showed only a very slight degree of this lesion and in several cases the endothelial cell prolifer ation was very focal, occurring only in some glomeruli, and only in parts of these glomeruli In addition, some biopsies showed small scattered foci of necrosis in glomeruli; in other cases, endothelial cell proliferation was assoc iated with localised thickening of the c ap illary walls, and occasionally this thickening had a bright, hypereosinophilie, refractile appearance.Other, more subtle characteristics were picked up as more series of biopsies were examined and gradually composite pathological pictures began to be associated with certain clinical conditions.There is no doubt that we are yet far from a complete and satisfactory classifi cation of the acute inflammatory lesions of the renal glomeruli, but we now possess an overall understanding of clinico-pathological corre lation which is much greater than anything we have known before.

CLASSIFICATION
In general, proliferative glomerulonephritis can now be divided into diffuse and focal types.M any of the cases of the diffuse type, in which virtually all of the glomerular tissue is involved, arc obviously post-streptococcal in nature from a clinical point of view, and present the usual clinical features of this condition.
Other patients, however, exhibiting similar histo logical features o n renal biopsy, are not clinic ally diagnosable as definite cases of the post streptococcal syndrome.
In this situation, electron microscopy appears to offer some help.'Examination of biopsy material by this means in several centres has suggested that in true post-streptococcal cases, a characteristic feature can be found in the glomerular capillaries when the disease has been present for some time.This consists of a few, scattered, large, localised granular deposits of darkly osmiophilic material lying on the epithelial surface of the basement membrane, pushing deeply into the epithelium which has often lost its pedicel structure in this region.W hether this feature will be found to be sufficiently pathognomonic to be used as a parameter for diagnosis of post-streptococcal disease remains to be seen.
Focal glomerulonephritis has become a fairly common diagnosis in renal biopsy material; patients exhibiting this pathological condition may present with a variety o f clinical features including haematuria, proteinuria, oedema, hypertension.T h e focal lesions in the glomer uli may be simply of endothelial cell prolifer ation, or may include small patches of necrosis or focal capillary wall thickening.Some of these cases are found on examination to be quite definite, but mild, cases of post-strepto coccal glomerulonephritis; but many others turn out to be in fact cases of so-called " col lagen disease" of some type, usually dissemin ated lupus erythematosus or polyarteritis; Henoch-Schonlein disease also can be associat ed with such a histological picture.W hile in many cases electron microscopy is not able to distinguish between these conditions, a diag nosis of disseminated lupus erythematosus may be strongly supported by the finding of thin, dark, layered patches of a finely granular mater ial, lying between the glomerular capillary basement membrane and the endothelium; this feature is very frequently seen in proved cases of the condition.Such supportive evidence  1967 (in press).
for the presence of disseminated lupus erythematosus may be of great help in dealing with a case otherwise difficult to diagnose.

CONCLUSION
There is no doubt that we are still only scratching at the surface of the problem of glomerulonephritis, in spite of the very numer ous specialised techniques now being used in its investigation.T h e cause of minimal lesion and membraneous glomerulonephritis, the complex question of the reaction of the glomer ulus to the streptococcus, the reasons for the varying types of development of cases of acute proliferative glomerulonephritis, the problem of proteinuria in the presence of a basement membrane normal to ultrastructural examin ation -all these, and many more difficulties, must be elucidated before we can claim to have a rational insight into the diseases which affect the glomerulus, and before we can evolve a really intelligent mode of therapy for these conditions.

EX PERIEN CE
" Our own Experience is but a narrow field to walking.M an 's life is short, nor is even that little spent in Medicinal Enquiries, so that it is a pity so useful a part of knowledge should have no wider Bounds; but even limited as it is we tread surer when we rely on it than trust to the Experience of others.W h at a man has himself seen he is much more certain of than what he is told by others; the pleasure which the success or the pain which a disappointment may have given him, strike him more deeply, and make him for the future more bold or more cautious as his case requires."(Extract from one of the earliest Dissertations in the library, dated 17 5 1.) E R U LO N E PH R IT IS M. K. McDONALD, M.B., Ch.B., F.R.C.P.(Ed.)Senior L ecturer in Pathology, U n iversity o f Edinburgh.

T
h e first real im p a c t o f e lectro n m icro sco p y on h u m a n ren al d isease w as in th e field o f w h a t w as th en k n ow n as E llis T y p e II g lo m e ru lo n e p h ritis.It was b e c o m in g o b vio u s th at E llis h ad, in fa c t, in c lu d e d in this gro u p a number o f d if f eren t c o n d itio n s, so m e o f w h ich were b asically in fla m m a to ry ; m an y cases which c lin ic a lly cou ld be p laced in this gro u p showed h isto lo g ic a l featu res w h ich w ere in fact those o f T y p e I g lo m e ru lo n e p h ritis, w ith the add itio n o f so m e g lo m e ru la r c a p illa ry w all thicken ing and fo c al h y a lin isa tio n o f th e tu fts.The d e v e lo p m e n t o f su ch p rogressive cases of acute g lo m e ru lo n e p h ritis co u ld n o w be follow ed by m ean s o f serial ren al b io p sy.I n a d d itio n , in crea sin g k n o w le d g e o f the e ffect on th e k id n eys o f so m e sy ste m ic diseases o f th e so-called " co lla g e n d isease" group, for e x a m p le , d issem in a ted lu p u s erythem atosus, had revealed th a t c lin ica l states ran gin g from' a c u te g lo m e ru lo n e p h ritis to a p u re nephrotic sy n d ro m e cou ld be p ro d u c e d b y these con d itio n s, and a c o rre sp o n d in g v a riety o f histo lo g ical fea tu re s w as p o ssib le , in c lu d in g glom er ular c a p illa ry w all th ick e n in g .

Fig
Fig. I.This figure shows Fig. 3.This shows part of a glom erulus in a case of m in im a l lesion g lom erulonephritis.The basement membrane of the capillaries appears norm al, b ut the epithe liu m has lost its pedicel structure, and lies in a co ntin uo us layer over the basement membrane.x6,000 A C K N O W L E D G E M E N T SThe author wishes to acknowledge the help of Dr. V .A. Ruckley, who is responsible for much of the electron microscopic work upon which this article is based.Dr. Ruckley is a M edical Research Council Fellow.Equipm ent has been provided by generous grants from the Advisory Comm ittee on M edical Research and from the Lawson T ait M emorial Trust.