The glomerulus consists of three main cell types - mesangial cells, endothelial cells, and podocytes. The glomerular basement membrane anchors the glomerular capillaries into the mesangial region. The glomerular filtration barrier is made up of the podocyte, glomerular basement membrane, and endothelial cell. Electron, light, and immunofluorescence microscopy can be used to examine the glomerulus and identify abnormalities of the filtration barrier that can indicate different renal pathologies. Proper handling and preparation of samples is important for optimal examination and diagnosis.

1. RENAL PATHOLOGY

2. STRUCTURE OF GLOMERULUS

3. Glomerulus
• Mesangial Cells
• Mesangial cells are modified smooth muscle cells with
phagocytic capability that can influence intracapillary
blood pressure.
• Endothelial Cells
• Endothelial cell nuclei protrude into the glomerular
capillary lumen and these should not be included in the
assessment for mesangial cellularity. The endothelial cells
are fenestrated with fenestrae that measure 70-100 nm in
diameter.
• Visceral Epithelial Cells (Podocytes)
• Podocytes are a terminally differentiated cell with complex
processes that intertwine with adjacent podocytes along
the glomerular basement membranes (GBM).
• Parietal Epithelial Cells
• The 4th cell type (parietal epithelial cell) lines Bowman
capsules, but the lesser known parietal podocyte can also
be found lining Bowman capsules and are more common
near the vascular pole.
The average human glomerulus is
up to 200 microns in diameter.
Glomerular size is harder to assess
without morphometry. One quick
method that works for most
microscopes – the glomerulus
should not be more than half of the
field of view when viewing with
the 40X objective lens.
The glomerular tuft consists of three
cells types (mesangial, endothelial,
and visceral epithelial cells – also
known as podocytes).

4. GBM
• The GBM width typically averages
between 300-350 nm and is
contiguous with Bowman capsule.
• The GBM anchors into the mesangial
region (arrowhead).
• Detachment of this anchor results in
2 separate glomerular capillaries
becoming one larger capillary, which
occurs with mesangiolysis.
• The glomerular filtration barrier
consists of the podocyte, glomerular
basement membrane, and
endothelial cell.

5. Scanning electron micrograph (400×) showing a vascular
cast of two juxtamedullary glomeruli (rat). Each capillary
tuft is supplied by an afferent arteriole (AA) which, on
the surface of the tuft, immediately divides into several
branches. Efferent arterioles (EA) emerge out of the
centre of the tuft.

8. • Part of a glomerular lobule (rat), showing the
arrangement of structures in the glomerular tuft.
• The capillary (C) is outlined by a flat fenestrated
endothelium (E). The podocyte layer (PO) and the
glomerular basement membrane (GBM) do not
encircle the individual capillary completely, they
form a common surface cover around the lobule.
• In the peripheral portion of the capillary the
filtration barrier is formed.
• Two subdomains of the GBM are delineated from
each other by mesangial angles (arrows): the
pericapillary GBM (cGBM) faced by the podocyte
layer and the endothelial layer, and the
perimesangial GBM (mGBM) bordered by the
podocyte layer and the mesangium.
• Within the mesangium two types of cells are
shown: contractile mesangial cells (M) and a cell (*)
which is probably a macrophage that has invaded
the mesangium.
• Note the intimate relationships between the
endothelium and the mesangium (arrowheads).
US = urinary space. 6100×.

9. • Longitudinal section through the
glomerular vascular pole showing the
juxtaglomerular apparatus with both
arterioles (rat).
• At the entrance into the glomerulus, the
afferent arteriole (AA) immediately
branches into capillaries (C).
• The efferent arteriole (EA) usually arises
deeper in the tuft and can be identified by
the high number of endothelial cells (E) at
the exit from the glomerulus.
• The macula densa (MD) of the thick
ascending limb is in contact with the
extraglomerular mesangium (EGM) and the
glomerular arterioles. The media of the AA
contains granular cells (G). M, mesangial
cells; PE, parietal epithelium; PO,
podocytes; US, urinary space.
• Transmission electron micrograph (1300×).

10. • Scanning electron micrograph (3300×)
of rat glomerular capillaries.
• The urinary side of the capillary is
covered by the highly branched
podocytes.
• The interdigitating system of primary
(P) and secondary (F) processes lines
the entire surface of the glomerular
basement membrane an proceeds also
beneath the cell bodies.
• In between the interdigitating foot
processes (F) of neighbouring cells the
filtration slits are spared

11. .
• Filtration barrier.
• The peripheral part of the glomerular
capillary wall comprises the fenestrated
endothelial layer (E), the glomerular basemen
membrane, and the interdigitating foot
processes (F).
• The filtration slits between the foot processes
are bridged by thin diaphragms (long
arrows).
• Arrowhead point to the endothelial pores.
• The glomerular basement membrane shows
lamina densa (2) bounded by the lamina rara
interna (l) and the lamina rar externa (3).
• In this picture, tannic acid staining allows
discrimination between the alternating foot
processes of two neighbouring podocytes: the
more densely stained processes belong to one
cell, and the others to the neighbouring cell C
= capillary lumen. 60,000×.

12. Different Microscopy Technique

13. Optical Microscope
• It is a type of microscope that commonly uses visible
light and a system of lenses to generate magnified
images of small objects.
• All modern optical microscopes designed for viewing
samples by transmitted light share the same basic
components of the light path.
• Components:
• Eyepiece (ocular lens) (1)
• Objective turret, revolver, or revolving nose piece (to
hold multiple objective lenses) (2)
• Objective lenses (3)
• Focus knobs (to move the stage)
• Coarse adjustment (4)
• Fine adjustment (5)
• Stage (to hold the specimen) (6)
• Light source (a light or a mirror) (7)
• Diaphragm and condenser(8)
• Mechanical stage (9)

14. Immunofluorescence
• Immunofluorescence is a technique
used for light microscopy with
a fluorescence microscope and is used
primarily on microbiological samples.
• This technique uses the specificity
of antibodies to their antigen to
target fluorescent dyes to
specific biomolecule targets within a
cell, and therefore allows visualization
of the distribution of the target
molecule through the sample.

15. Immunofluorescence
• In order to probe the tissue with antibodies, two techniques are available:
immunofluorescence uses labelled antisera or antibodies (which require native tissue
without fixation) and immunohistochemistry (which can be done with formalin-fixed
tissue, whilst more aggressive fixatives destroy the epitopes and preclude
immunohistochemical investigations.
• The workhorses for immunofluorescence/ immunohistochemistry are antisera or
monoclonal antibodies against immunoglobulins (IgA, IgG and IgM) and components of
the classical or alternative complement pathway (C1q, C3c and C4) as well as - and -light
chains, albumin and fibrinogen.
• The pathologist should not only report whether the reaction is positive, but should also
comment on the pattern of staining, e.g. mesangial vs capillary staining pattern, linear (or
pseudolinear) vs granular staining.
• If possible, he should also describe where the deposits are located, e.g. in a
subendothelial, intramembranous or subepithelial position.
• In renal transplant biopsies, immunostaining for the C4d fragment of the complement
pathway has become extremely popular.

16. Electron Microscopy
• An electron microscope is a microscope that uses
a beam of accelerated electrons as a source of
illumination.
• As the wavelength of an electron can be up to
100,000 times shorter than that of visible
light photons, electron microscopes have a
higher resolving power than light
microscopes and can reveal the structure of
smaller objects.
• A scanning transmission electron microscope has
achieved better than 50 pm resolution in annular
dark-field imaging mode and magnifications of
up to about 10,000,000× whereas most light
microscopes are limited by diffraction to about
200 nm resolution and useful magnifications
below 2000×.

17. Electron Microscopy
• EM is valuable for defining the morphology of
• The basement membranes, which is abnormal in some forms of hereditary nephropathy (e.g.,
Alport syndrome and thin basement membrane nephropathy),
• For identifying fibrils (e.g., in amyloidosis)
• Tubuloreticular intracellular structures (e.g., in lupus nephritis).
• EM is also useful for localizing the site of immune deposits, which are usually
homogeneous and electron dense.
• Electron-densevdeposits are seen in the mesangium or along the capillary wall on the
subepithelial or subendothelial side of the GBM.
• Infrequently, the electron-dense material follows a linear pattern within the GBM.
• The sites of immune deposits are helpful in the classification of the types of GN.
• For some renal diseases, the definite diagnosis requires electron microscopy, such as
Alport’s disease, thin basement disease, immunotactoid disease, minimal change
nephropathy.

18. Sample Handling and
Transportation

19. Clinical Information
• The absence of clinical information is a sore point in many partnerships between
clinicians and pathologists.
• In an ideal world, the pathologist obtains information on the clinical history, recent
laboratory values in particular urine (proteinuria, haematuria, leukocyturia, cylindruria)
and serum [urea, creatinine, cholesterol, total protein, creatine clearance, C3, C4, ANA,
ANCA, Anti GBM], presence of diabetes mellitus or hypertension or other systemic
diseases, other parameters of interest (if available) and current therapy

20. Light Microscopy
• Fixative
• The most commonly used fixative for LM is buffered, 10% aqueous formaldehyde solution
(formalin).
• Formalin is stable at room temperature, provides acceptable morphology, and allows IHC or
molecular studies to be performed.
• Some laboratories prefer alcoholic Bouin’s, Duboscq-Brasil or Zenker’s fixatives that provide
better preservation of certain morphologic details. However, these fixatives limit recovery of
material for EM, IP or molecular studies, and require additional precautions and handling.
• Bouin’s and Duboscq-Brasil fluid contain picric acid that is highly explosive when dry and can
be a disposal problem.
• Processing and sectioning
• To avoid specimen loss during processing, the specimen for LM should be gently wrapped in
lens paper that has been prewetted with fixative.
• Neither sponges nor plastic embedding bags should be used because mechanical artefact
almost always occurs.
• Tissues can be processed overnight using a protocol appropriate for small biopsy samples.

21. Light Microscopy
• Sectioning and staining
• Serial sections of 2 µm thickness are cut and at least two sections should be placed on each
slide.
• There are many acceptable staining protocols; most include staining alternating slides with
hematoxylin and eosin stain (H&E) periodic acid–Schiff reaction (PAS), silver methenamine
and trichrome stains.

22. Immunofluorescence
• Preparation
• IF is best performed on unfixed, frozen sections.
• Tissues can be transported to the laboratory fresh on saline-soaked gauze or in Michel’s fixative
(Zeus medium’).
• 15 Serial sections are cut at 2–4 µm in a cryostat.
• IP staining requires no special tissue preparation in that the same formalin-fixed, paraffin-
embedded material used for LM is also used for IP.
• Staining
• The antigens that should be routinely examined include: immunoglobulins (primarily IgG,
IgM and IgA), complement components (primarily C3, C1q, and C4), fibrin, and kappa and
lambda light chains.
• Additional antibodies may be required in specific circumstances, for example, amyloid
speciation, collagen IV alpha chains in hereditary nephritis,IgG subclasses, virus identification,
lymphocyte phenotyping in allografts in suspected cases of PTLD, C4d in allograft biopsies,
etc.
• In the absence of appropriate tissue in the IF sample, paraffin-embedded material can be
examined using IP techniques.

23. Immunofluorescence
• Controls
• IHC controls should include a negative control (no antibody applied) and a positive control
(albumin can serve this purpose) for each run.
• Internal controls exist for many of the antigens in routine use.
• For example, IgA is generally present in tubular casts, IgG in protein droplets and C3 in blood
vessels.
• Each time a new vial of antibody is opened, the correct dilution should be determined using
known positive slides.

24. Electron Microscopy
• The tissue for EM may be fixed in 2–3% glutaraldehyde or 1–4% paraformaldehyde.
• Adequate fixation can also be obtained when tissue is fixed in buffered formalin.
• EM cannot be performed on tissues exposed to mercury-based fixatives (Zenker’s, B-5).
• The tissue sent in Michel’s transport medium would have poor ultrastructural preservation since
this is not a fixative solution.
• Rapid placement of the sample into fixative will provide the best outcome.
• Tissue can be reprocessed from the paraffin block, or the frozen block if no glomeruli are available
in the EM sample. Although severe cellular artefacts may result when frozen and paraffin-
embedded tissue are processed for EM, the GBM and immune deposits are usually sufficiently
intact for evaluation.
• Toluidine blue-stained, 1 mm thick, so-called ‘thick’ sections, are examined to identify appropriate
structures for thin sectioning and examination with the electron microscope.
• In general, one or two glomeruli are examined ultrastructurally. Low-, medium- and high-
magnification photographs are taken to include both capillary loops and mesangial areas.
• The tubulointerstitium and vessels are also examined and pertinent photographs taken to illustrate
any abnormalities in these areas.

25. Stains

26. Hematoxylin-Eosin Stain
• Hematoxylin-eosin (H&E) staining is the most common stain used in pathology,
including renal pathology.
• Hematoxylin is a dark blue or purple stain that is basic and binds acidic substances such
as DNA or RNA, thereby staining nuclei blue or purple.
• Eosin is a pink stain that is acidic and binds positively charged substances such as
cytoplasmic protein.
• Eosin will stain cytoplasm in a reddish pink color and collagen in a paler pink color.
• It nicely stains red blood cells, so thrombi may be easier to identify
• H&E stain is most useful for evaluating the integrity and cellularity of a glomerulus and
can provide information on renal tubule epithelial integrity, mesangial cellularity and
sclerosis, and degree of interstitial infiltrate.
• The hematoxylin phloxine saffron stain is an excellent stain to identify fibrinoid necrosis
in vessels and capillaries and is a better stain to quantify collagen in areas of fibrosis

28. Periodic Acid Schiff
• Periodic acid–Schiff stain is used to
identify polysaccharides.
• The reaction involves oxidation of
hydroxyl groups (vicinal diols) in
glycogen, glycoproteins, and
glycolipids, resulting in a purple
staining pattern.
• Because these carbohydrates are more
commonly found in basement
membranes, periodic acid–Schiff stain
is useful to judge increased thickness
of tubular and glomerular basement
membranes.

29. Methenamine
Silver Stain
• Jones stain is a methenamine silver stain
with an H&E counterstain that is also used
to assess basement membranes.
• Areas of the glomerular basement
membrane that show spikes and holes on
Jones-stained sections are suggestive of
membranous glomerulopathy.

30. Trichrome Stain
• Trichrome stain uses 2 or more
dyes that are used to selectively
differentiate basic tissue
components.
• The use of multiple dyes allows
for cytoplasm to be visualized
as red and collagen as blue.
• In kidney tissue, the presence of
blue staining areas on
trichrome-stained sections will
indicate the presence of
collagen in areas of fibrosis.
• In addition, immune complexes
may appear as red granules
with trichrome stain.

31. Stains Summary

32. Diagnostic Process

33. Diagnostic Labels
• Multiple terms may be used to describe one condition.
• Diagnostic labels are based variously on clinical presentation, immunology, histology,
and pathogenesis.
• For example,
• Immunological - ANCA-positive vasculitis
• Clinically - presents as RPGN
• Histological - pauci-immune necrotizing glomerulonephritis
• It is important to appreciate the overlap of these different diagnostic labels; in this
example, ANCA-positive vasculitis is just one cause of RPGN and a necrotizing
glomerulonephritis.

34. Single Morphology- Different Conditions
• The kidney has a limited number of ways to respond to injury and a single morphology
may be seen in a number of very different conditions.
• For example, a nodular glomerulosclerosis may be seen in diabetic nephropathy, light
chain deposition disease and in idiopathic nodular sclerosis.
• In chronic kidney disease, whatever the primary insult, the final common pathway is
renal fibrosis with glomerulosclerosis and tubular atrophy.
• At this stage, histological changes are frequently non-specific and the challenge for the
pathologist is to identify clues to the underlying disease process.
• Equally, one condition can produce diverse morphologies. This is particularly true for
lupus nephritis and IgA nephropathy, which can result in almost any pattern of
glomerular disease.

35. Spectrum of Glomerular Disease
• For glomerulonephritides, there is a link between the target of injury, the morphology, and
clinical manifestation.
• Those conditions that selectively damage the permeability barrier (such as membranous
nephropathy and MCD) produce the nephrotic syndrome, with little or no glomerular
inflammation or proliferation.
• Necrotizing glomerular injury with rupture of capillary walls (as in vasculitic
glomerulonephritis and anti-GBM disease) results in haematuria and a marked reduction in
glomerular filtration rate, producing a RPGN. The exudation of fibrin and cytokines results in
reactive extracapillary proliferation, producing a crescentic morphology.
• Those conditions (such as lupus nephritis and IgA nephropathy) associated with mesangial or
subendothelial immune deposits produce glomerular inflammation with the nephritic
syndrome and a mesangial or endocapillary proliferative morphology.
• This latter group show the most variation in morphology and clinical manifestations; when
there is associated damage to the permeability barrier there may be heavy proteinuria and the
nephrotic syndrome, and if there is necrosis they may present with a RPGN.

36. The spectrum of glomerular diseases. It is useful to contrast the pathological processes causing
proteinuria (‘nephrosis’) on the left, from those causing glomerular haematuria (‘nephritis’) on the
right

37. Pathogenesis of
Glomerular Injury

38. Pathogenesis of Glomerular Injury
• Immune mechanisms underlie most forms of
primary glomerulopathy and many
secondary glomerular disorders.
• Two forms of antibody-associated injury have
been established:
• Injury by antibodies reacting in situ
within the glomerulus, either binding to
insoluble fixed (intrinsic) glomerular
antigens or extrinsic molecules planted
within the glomerulus, and
• Injury resulting from deposition of
circulating antigen-antibody complexes in
the glomerulus.

40. Mechanism of Glomerular Injury
• Whatever the antigen may be, antigen-antibody complexes formed or deposited in the
glomeruli may elicit a local inflammatory reaction that produces injury.
• The antibodies may activate complement and engage Fc receptors on leukocytes and
perhaps glomerular mesangial or other cells, leading to inflammation.
• The glomerular lesions may exhibit leukocytic infiltration and proliferation of mesangial
and endothelial cells.
• Electron microscopy reveals electron-dense deposits, presumably containing immune
complexes, that may lie in the mesangium, between the endothelial cells and the GBM
(subendothelial deposits), or between the outer surface of the GBM and the podocytes
(subepithelial deposits).

41. Mechanism of Glomerular Injury
• Deposits may be located at more than one site in a given case.
• By immunofluorescence microscopy, the immune complexes are seen as granular
deposits along the basement membrane, in the mesangium, or in both locations.
• Once deposited in the kidney, immune complexes may eventually be degraded, mostly
by infiltrating neutrophils and monocytes/macrophages, mesangial cells, and
endogenous proteases, and the inflammatory reaction may then subside. Such a course
occurs when the exposure to the inciting antigen is short-lived and limited, as in most
cases of PSGN.
• However, if immune complexes are deposited repeatedly for prolonged periods, as may
be seen in SLE or viral hepatitis, many cycles of injury may occur, leading to a more
chronic membranous or membranoproliferative type of glomerulonephritis.

42. Mechanism of Glomerular Injury
• Several factors affect glomerular localization of antigen, antibody, or immune complexes.
• The molecular charge and size of these reactants are clearly important.
• Highly cationic antigens tend to cross the GBM, and the resultant complexes eventually
reside in a subepithelial location.
• Highly anionic macromolecules are excluded from the GBM and are trapped
subendothelially or are not nephritogenic at all.
• Molecules of neutral charge and immune complexes containing these molecules tend to
accumulate in the mesangium.
• Large circulating complexes are not usually nephritogenic, because they are cleared by
the mononuclear phagocyte system and do not enter the GBM in significant quantities.

43. Mechanism of Glomerular Injury
• The distinct patterns of localization of immune complexes is a key determinant of the
injury response and the histologic features that subsequently develop.
• Immune complexes located in subendothelial portions of capillaries and in mesangial
regions are accessible to the circulation and more likely to be involved in inflammatory
processes that require interaction and activation of circulating leukocytes.
• Diseases in which immune complexes are confined to the subepithelial locations and for
which the capillary basement membranes may be a barrier to interaction with circulating
leukocytes, as in the case of membranous nephropathy, typically have a noninflammatory
pathology.

44. Localization of immune complexes
in the glomerulus:
(1) Subepithelial humps, as in acute
glomerulonephritis;
(2) Membranous deposits, as in
membranous nephropathy and
Heymann nephritis;
(3) Subendothelial deposits, as in
lupus nephritis and
membranoproliferative
glomerulonephritis;
(4) Mesangial deposits, as in IgA
nephropathy.
EN, Endothelium; EP, epithelium; GBM,
glomerular basement membrane; LD, lamina
densa; LRE, lamina rara externa; LRI, lamina rara
interna; MC, mesangial cell; MM, mesangial
matrix.

45. Glomerular Lesions

46. Distribution of lesions
• The terms focal and diffuse are used to describe the proportion of glomeruli involved,
whereas segmental and global refer to the extent of involvement within individual
glomeruli.
• FOCAL lesion is one involving < 50% of glomeruli,
• And a DIFFUSE lesion is one involving most glomeruli, > 50%.
• SEGMENTAL lesion is one involving < 50% of a glomerular tuft
• And a GLOBAL lesion one involving > 50% of the glomerulus.

47. Proliferation
• Proliferation is used to describe an increase in glomerular cells that may result from
infiltrating leucocytes or proliferation of endogenous glomerular cells.
• For this reason, the term hypercellularity is sometime more appropriate than
proliferation.
• Hypercellularity/proliferation is subclassified according to the part of the glomerulus
involved.
• The site of proliferation gives an indication of the underlying cause and is of prognostic
and therapeutic importance.

48. Mesangial
Hypercellularity
More than three mesangial cells
in peripheral mesangial area in a
standard 2–3-micron thick
paraffin section.
The central stalk of the tuft
should not be used for assessing
cellularity.
Thicker sections give an
artefactual impression of
hypercellularity.
(PAS) IgA nephropathy showing mesangial
hypercellularity in which there are ≥ 4 mesangial cells in a
peripheral mesangial area.

49. Endocapillary
Hypercellularity
An increased number of cell
within glomerular capillary
lumina.
These may be endothelia cells or
intravascular leucocytes.
(H&E) Post-infectious glomerulonephritis showing
endocapillary hypercellularity, in which the
capillary lumina are filled with infiltrating
leucocytes

50. Extracapillary
proliferation
Hypercellularity/proliferation withi Bowman’s
space, producing more than two cell layers between
the capillary tufts and Bowman’s capsule.
This appearance is commonl referred to as a cellular
crescent which is usually a resul of necrosis with
exudation of fibrin and cytokines.
A collapsing glomerulopathy may be associated
with extracapillary proliferation producing a similar
appearance to a cellular crescent.
(H&E silver) Anti-GBM disease showing
extracapillary proliferation or cellular crescent, in
which there is partial tuft collapse and proliferation
of cells within Bowman’s space.

51. Crescent formation. In early crescent formation, cytokines and growth factors cross the glomerular basement membrane (GBM) to
initiate proliferation of the parietal epithelial cells. Small breaks in the GBM occur secondary to injury from oxidants and proteases
from neutrophils and macrophages, thus allowing the macrophage to enter Bowman space, where it can proliferate. Breaks in
Bowman capsule secondary to the periglomerular inflammation also occur, allowing the entrance of more inflammatory cells as
well as fibroblasts. The proliferation of parietal and visceral epithelial cells and macrophages is associated with fibrin deposition,
slowly choking the glomerular tuft until filtration becomes impossible. In the late stages, the crescent becomes fibrotic and the
glomerulus end stage. Alternatively, in less severe cases, complete restitution of the glomerular tuft can occur.

52. Necrosis
Disruption of the glomerular
basement membrane (best
appreciated on a silver-stained
section) with fibrin exudation
and Karyorrhexis.
The latter may not be evident
and the minimum requirement
for the definition of a necrotizing
lesion is extracapillary fibrin
exudation.
(H&E silver) ANCA-associated vasculitis,
showing necrosis with capillary wall rupture and
fibrin exudation.

53. Membrano
proliferative pattern
Also termed mesangiocapillary pattern
Mesangial hypercellularity with thickening of capillary walls.
This produces a lobular appearance to the glomerulus.
Capillary wall thickening is due to duplication of the
glomerular basement membrane, as a reaction to interposed
cells and subendothelial or intramembranous deposits.
H&E and silver: Membranoproliferative pattern, showing a lobular appearance
of the glomerular tuft with mesangial hypercellularity and thickened capillary
walls, with GBM duplication evident on the silver stain.

54. Sclerosis
• Sclerosis is an increase in extracellular matrix within the glomerulus.
• In segmental and global glomerulosclerosis, the excess matrix is associated with obliteration of
capillary lumina.
• Globally sclerosed glomeruli that are expanded and solidified by matrix are seen in advanced
diabetic glomerulopathy or in a chronic glomerulonephritis.
• In mesangial sclerosis and nodular glomerulosclerosis, capillaries are patent.
• An adhesion is continuity with matrix material between glomerular basement membrane and
Bowman’s capsule that is separate from an area of sclerosis, that is, capillaries associated with
an adhesion are patent.
• Glomerular obsolescence is collapse of the glomerular tuft with fibrosis in Bowman’s space, and
is typical of ischaemic injury.
• Hyalinosis is the accumulation of non-matrix proteins, that is, insudation of plasma proteins,
between the endothelium and the glomerular basement membrane. The hyaline has an
amorphous eosinophilic appearance that is best appreciated in PAS or silver/ H&E-stained
sections.

55. (A) (H&E silver) Glomerular tip lesion, showing adhesion and hyalinosis at the tubular pole. This lesion reflects
severe proteinuria and may be seen in a number of nephrotic conditions, including primary FSGS,
membranous nephropathy and diabetic nephropathy. Patients with tip lesions and otherwise normal glomeruli
by light microscopy have a clinical course similar to minimal change disease.
(B) (H&E silver) Collapsing lesion, showing tuft collapse with proliferation and swelling of visceral epithelial cells,
an indicator of severe podocyte injury.

56. (C) (PAS) Perihilar segmental sclerosis, typical of secondary FSGS, associated with hyperfiltration
injury.
(D) (H&E) Segmental sclerosis, not otherwise specified, typical of primary nephrotic FSGS.

57. (E) (H&E) A broad-based bland segmental scar, typical of sclerosis following segmental necrosis in
vasculitic glomerulonephritis.
(F) (H&E) Nodular glomerulosclerosis in diabetic nephropathy, showing patent capillary loops
around the mesangial nodules of matrix

58. Tubulointerstitial Lesions

59. In the normal renal cortex tubules are back to back without significant intervening space. The
interstitium comprises peritubular capillaries and a delicate network of myofibroblasts that are not
appreciated without immunohistochemistry (IH).
(a, b) Normal renal tubules on H&E/silver and PAS stains. The proximal tubules have abundant
eosinophilic cytoplasm. PAS stain demonstrates the brush border on the luminal surface. The tubular
basement membranes are closely apposed with very little interstitium evident.

60. Tubular atrophy
• Atrophic proximal tubules have
thick irregular basement
membranes and decreased
diameter.
• Atrophic distal tubules are
frequently increased in diameter
and contain Tamm–Horsfall
protein casts.
• Sheets of tubules with this
appearance are sometimes
described as ‘thyroidization’
due to their resemblance to
thyroid follicles.
(c, d) (H&E and PAS) Focal tubular atrophy, typically seen in chronic
glomerular disease. Atrophic proximal tubules are shrunken and have thick
basement membranes, highlighted on PAS stain.

61. Interstitial Fibrosis
• Interstitial fibrosis is an increase in extracellular
matrix that separates the tubules.
• There is pronounced proliferation of
myofibroblasts in early fibrosis that precedes
matrix production.
• The distribution of interstitial fibrosis and
tubular atrophy gives an indication of its
aetiology.
• Sharply delineated segmental atrophy/fibrosis
is seen in reflux nephropathy or following
healing of cortical infarcts.
• A multifocal distribution is typically seen in
chronic glomerulonephritis or small vessel
disease.
• Diffuse peritubular fibrosis follows
inflammatory tubular injury, such as a
tubulointerstitial nephritis (TIN) in native
kidneys or tubulointerstitial rejection in
transplants
(e) (H&E silver) Chronic calcineurin inhibitor toxicity
showing a striped pattern of atrophy and fibrosis,
reflecting the small vessel disease. (f) (H&E) Diffuse
peritubular fibrosis in chronic tubulointerstitial nephritis.

62. Inflammation
• The significance of interstitial inflammation
depends upon the nature of the cells, their
distribution and the presence of tubulitis
(infiltration of tubules by the inflammatory cells).
• Tubular atrophy and interstitial fibrosis are
usually accompanied by an infiltrate of small
lymphocytes, frequently with lymphoid
aggregates, whatever the aetiology of the chronic
damage; such infiltrates are not a marker of TIN,
but may still be contributing to the fibrotic
process.
• Features of an active TIN are a mixed infiltrate,
frequently including plasma cells and eosinophils,
around non-atrophic tubules with an associated
lymphocytic tubulitis. (a) (H&E) A non-specific infiltrate of small
lymphocytes associated with an area of scarring.
(b) (H&E) Tubulointerstitial nephritis, showing a
mixed interstitial infiltrate of lymphocytes, plasma
cells, eosinophils, and neutrophils.

63. Inflammation
• The type of inflammation may give an
indication to the aetiology.
• Plasma cell-rich infiltrates are typically seen
in TIN associated with systemic autoimmune
diseases such as Sjogren syndrome and IgG4-
associated disease. In the latter, there are
numerous IgG4-positive plasma cells and an
expansile fibroproliferative interstitial
reaction that may result in renal mass lesions
on imaging.
(e) IgG4-associated tubulointerstitial nephritis
showing a plasma cell-rich interstitial infiltrate.
IH (F) demonstrates numerous IgG4-positive
plasma cells.

64. Inflammation
• Eosinophils are frequently prominent in
drug-associated TIN, and there may also
be granulomata.
• Multiple, often confluent discrete
epithelioid granulomata are typical of
renal sarcoidosis.
• Occasional intratubular neutrophils are
common in TIN, and do not necessarily
indicate the presence of an infective
nephritis.
• Features of acute infective
nephritis/pyelonephritis are interstitial
oedema with a peritubular infiltrate of
neutrophils, a neutrophilic tubulitis and
plugs of neutrophils within tubular
lumina.
(c) (H&E) Renal sarcoidosis, characterized by non-
necrotizing epithelioid granulomata.
(d) (H&E) A severe neutrophilic tubulitis with
neutrophil casts, indicative of ascending bacterial
infection.

65. Tubular Casts
• Red blood cell casts are a marker of glomerular
haematuria, unless there has been renal
trauma.
• However, rare such casts may also be seen in
acute TIN.
• The red cells are frequently dysmorphic and
show lysis.
• Large numbers of red blood cell casts may
result in acute kidney injury, even in the
absence of severe necrotizing glomerular
lesions.
(H&E) Red cell casts, indicative of
glomerular haematuria, in a patient with
vasculitic glomerulonephritis.

66. Tubular Casts
• Protein casts: the most frequent protein
casts are formed largely by Tamm–
Horsfall protein (uromodulin), produced
in the thick ascending limb of the loop of
Henle.
• These are seen in atrophic distal tubules
or in association with filtered proteins in
proteinuric states.
• These casts have a uniform hyaline
appearance and, as uromodulin is a
glycoprotein, are PAS positive. PAS-positive Tamm–Horsfall protein
(uromodulin) casts in distal tubules.

67. Tubular Casts
• Other frequently seen casts include
• light chain casts in myeloma/light chain cast
nephropathy, which are PAS negative and have a
hard crystalline, fractured appearance with an
associated inflammatory reaction; and
• Myoglobin or haemoglobin casts associated with
rhabdomyolysis and intravascular haemolysis
respectively. These typically have a granular or
beaded appearance.
• Greenish pigmented bile casts with associated ATI
may be present in patients with severe liver
disease.
(B) (PAS) Light chain cast nephropathy, characterized
by PAS-negative fractured casts with associated
inflammation and tubular epithelial injury.
(C) (H&E) Granular eosinophilic haemoglobin casts in
a patient with AKI associated with intravascular
haemolysis.

68. Tubular Crystals
• The most frequently seen intratubular crystals are
calcium oxalate and calcium phosphate.
• Calcium oxalate crystals are often sheave-shaped
and do not take up standard histochemical stains
and are best visualized under polarized light.
• When extensive, they are indicative of
hyperoxalaemia and hyperoxaluria, but are seen in
small numbers following ATI.
• Tubular deposits of calcium phosphate are
strongly haematoxyphilic and do not polarize.
• They are seen in hypercalcaemic states or with
large loads of ingested phosphate, and less
frequently following severe ATI, when
intraluminal cell debris acts as a nidus for
calcification.
(E) (H&E) Calcium phosphate deposits within tubules in a
patient with hypercalcaemia and nephrocalcinosis.
(F) (H&E polarized light) Calcium oxalate crystals in a
patient with AKI associated with enteric hyperoxaluria.

69. Acute Tubular Injury
• This may be ischaemic or toxic in aetiology .
• In ischaemic ATI, there is tubular dilatation and simplification of the epithelium without
tubular basement membrane thickening.
• In toxic injury, there may be cytopathic changes within tubular epithelium, such as
cytoplasmic swelling and vacuolation.
• In severe injury, degenerated epithelial cells are shed into the lumen.

70. (A)Ischaemic acute tubular injury showing widespread tubular dilatation and epithelial
simplification.
(B)Cytotoxic acute tubula injury, with marked epithelial swelling and degenerative changes, in this
case secondary to gentamicin toxicity.
(C)Isometric tubular epithelial vacuolation in a renal transplant patient with Tacrolimus toxicity.

71. Viral Inclusions
• Certain viral infections, such as polyoma viruses, CMV, and adenovirus, produce
characteristic inclusions with tubular epithelial nuclei.
• These may be associated with evidence of epithelial injury and an inflammatory reaction
(viral nephritis).
• Special immunostains can identify the specific virus.

72. (D–F) Viral infections in renal transplant patients.
Polyoma virus infection (D) with basophilic nuclear inclusions.
Adenovirus infection (E) characteristically produces marked tubular necrosis.
CMV infection (F) with eosinophilic nuclear inclusions.

73. Vascular Lesion

74. Vascular Lesions
• Arcuate arteries at the corticomedullary junction
give off radial branches, the interlobular arteries
that extend towards the renal capsule. From these
branch afferent arterioles, which supply blood to
the glomeruli.
• Arterial and arteriolar lesions may be acute or
chronic; the latter may reflect vascular
remodeling following earlier acute injury.
(A) (H&E) A normal interlobular artery.
(G) (H&E) Normal afferent and efferent arterioles
at the glomerular hilum.

75. Thrombotic
microangiopathy
It describes the morphology of
acute microvascular injury, also
termed malignant vascular injury.
Endothelial activation and injury
result in a subendothelial exudate
that frequently contains fibrin and
red blood cells.
There may be luminal thrombosis,
particularly of arterioles and
glomerular capillaries.
The chronic phase is characterized
by a proliferative response within
the intima (‘onion skin
proliferation’) and progressive
intimal fibrosis.
B–D) Arterial TMA.
In the acute phase (B), endothelial injury results in
marked intimal oedema.
This progresses to a healing phase, initially with a
proliferative intimal reaction (C) and
finally obliterative intimal fibrosis (D, trichrome).

76. Arteriosclerosis
It describes remodelling of the
arterial intima, largely comprising
elastic fibres, also termed
fibroelastosis.
This is a ‘normal’ age-related
change, accelerated in association
with long-standing hypertension.
(E) (elastic van Gieson) Arteriosclerosis (fibroelastosis), associated with
essential hypertension, showing reduplication of the internal elastic lamina.
(H) (H&E) Marked arteriolar hyalinosis in a patient with diabetic nephropathy.

77. Arteritis
It is characterized by focal
necrotizing vascular lesions.
A necrotizing glomerulonephritis,
frequently with crescents, is the
most frequent lesion in renal
involvement by systemic small
vessel vasculitis (GPA, MPA,
EGPA), with arterial involvement
seen in approximately 20% of cases.
Less common is renal involvement
in polyarteritis nodosa that involves
the larger arcuate and segmental
arteries, producing segmental
necrosis and aneurysms.
(H&E) A necrotizing arteritis in a patient with ANCA-
positive vasculitis.

78. Acute v/s Chronic

79. Acute v/s Chronic

80. Acute v/s Chronic
• Chronic changes are generally irreversible and the extent of chronic changes may be as
important as the primary diagnosis and may have an important bearing on the
management of GN.
• Chronic changes are represented in the glomeruli as segmental or global
glomerulosclerosis, interstitium as tubular atrophy and interstitial fibrosis and vessels as
arteriosclerosis and hyaline arteriolosclerosis.
• Chronic changes are strong predictors of renal outcomes in glomerular diseases.
• However, there are only a few diseases that have a scoring system for chronicity,
including the Oxford classification for IgA nephropathy, the modified International
Society of Nephrology/Renal Pathology Society classification for lupus nephritis and the
Berden classification for ANCA-associated GN, which is based only on
glomerulosclerosis.
• There are no defined criteria for chronic changes in most other types of GN.

81. So

82. Scoring of the chronic lesions in
individual renal tissue compartments

83. Grades of chronic
changes based on total
renal chronicity score
(0–10)

84. Immunofluorescent

85. Evaluation of IF finding
• It is of value to decide if immunostaining is specific, because nonspecific deposits are due to plasma
insudation, or leakage of serum protein: C3, IgM, and albumin into sclerosed or necrotic area.
• Non-specific deposition/absorption of immunoreactants may be seen in glomeruli, tubuli or
vessels.
• Glomerular nonspecific staining is observed in sclerosis (global or segmental) – usually IgM/C3 –
linear along GBM (diabetic nephropathy, tubular atrophy), or in the glomerular visceral epithelial
cells (podocytes).
• Nonspecific tubular epithelial cells staining are due to proteinuric states. Staining of tubular casts
(usually IgA, IgM, κ and λ) may be observed in various glomerulopathies.
• Vascular nonspecific staining in arteries/ arterioles usually includes IgM or C3.
• The specific immunostaining should be evaluated quantitatively using computer image analysis, or
semiquantitatively using an intensity score (0 to 4+).
• A report of the renal biopsy examination should consist of description concerning immunostaining
in all renal compartments (glomeruli, vessels, tubuli and interstitium).
• Glomerular immunostaining may be mesangial, along capillary walls, or both, as well as immune
deposits may be observed within Bowman’s capsules.

86. Patterns of Renal Injury
• Based on immunohistologic findings, glomerulopathies are classified into three categories
that relate to the pathogenesis of inflammation.
• The pattern of immunostaining can be linear or granular.
• Linear glomerular staining of immunoglobulin indicates anti-GBM antibody mediated
injury.
• Granular staining of immunoglobulin and complement component is due to immune
complex-mediated injury.
• If no or scanty glomerular immunostaining of immunoglobulin is noted and light
microscopy shows glomerular necrosis with crescents formation, pathologic lesions are
related to the pauci-immune glomerulonephritis usually with circulating anti-neutrophil
antibodies (ANCA).

87. (a) Linear GBM positivity for IgG in anti-GBM disease.
(b)Granular capillary wall positivity for IgG in membranous nephropathy.
(c) Mesangial positivity for IgA in IgA nephropathy.

88. (d) Mesangial and capillary wall positivity for C3 in C3 glomerulonephritis.
(e) Mesangial and tubular basement membrane positivity for kappa light chains in light
chain deposition disease.
(f) Positivity for lambda light chains in tubular casts in light chain cast nephropathy.

89. Electron microscopy

90. EM
• EM is essential for diagnosis in approximately one-quarter of native renal biopsies.
• EM provides precise localization of glomerular deposits, and reveals internal structure to
deposits. It is essential for the diagnosis of fibrillary and immunotactoid
glomerulonephritis, conditions that are defined by their ultrastructural appearance.
• In addition to demonstration of deposits, EM is used to quantify the extent of podocyte
foot process effacement in proteinuric conditions.
• Abnormalities of the GBM usually require EM for diagnosis. Whilst localized thinning of
the lamina densa may be seen at sites of injury, diffuse thinning is indicative of thin
membrane nephropathy or early stage or a carrier state of Alport syndrome.
• In established renal involvement in Alport syndrome, there is multilayering of the thin
lamina densa, producing a basket-weave appearance.
• Amorphous thickening of basement membranes is a feature of diabetic glomerulopathy.
• EM plays a central diagnostic role in the diagnosis of certain storage diseases, including
Fabry disease and lecithin cholesterol acyltransferase deficiency.

91. (A)The normal capillary wall, comprising lumen at the bottom, lined by a fenestrated endothelium.
The outer aspect of the basement membrane is covered by podocyte foot processes. The podocyte
cell body is at the top.
(B) Minimal change disease, showing diffuse effacement of podocyte foot processes.

92. (C) Paramesangial deposits in IgA nephropathy.
(D) Subepithelial deposits with membrane spikes in membranous nephropathy.

93. (E) Subendothelial deposits in C3 glomerulonephritis.
(F) Intramembranous electron dense deposits in dense deposit disease.

94. (G) Mesangial deposits of IgG with a fibrillary internal structure in fibrillary
glomerulonephritis.
(H) Lipid inclusions within podocytes and endothelial cells in a patient with Fabry disease.

95. Diagnostic Algorithm

96. Diagnostic algorithm

97. Diagnostic algorithm

98. Diagnostic algorithm

99. Diagnostic algorithm

100. Diagnostic algorithm

101. Diagnostic algorithm

102. Reporting

103. Guidelines for Reporting

104. Classification of GN
Pathogenic Type Specific Disease Entity Pattern of Injury: Focal or Diffuse Scores or Class IF Finding
Immune-complex
GN
IgA nephropathy, IgA vasculitis, lupus
nephritis, infection-related GN, fibrillary
GN with polyclonal Ig deposits
Mesangial, endocapillary, exudative,
membranoproliferative, necrotizing,
crescentic, sclerosing, or multiple
Oxford/MEST scores for
IgA nephropathy
ISN/RPS class for LN
Granular deposits of
polyclonal ig on IF
Pauci-immune GN MPO-ANCA GN, proteinase 3-ANCA GN,
ANCA-negative GN
Necrotizing, crescentic, sclerosing,
or multiple
Focal, crescentic, mixed,
or
sclerosing class
(Berden/EUVAS class)
Negative or
Few ig deposits on IF
Anti-GBM GN Anti-GBM GN Necrotizing, crescentic, sclerosing,
or mixed
Linear
Deposits of ig, most often igg,
and frequently,
C3 along the GBM
Monoclonal Ig GN Monoclonal Ig deposition disease,
proliferative GN with monoclonal Ig
deposits, immunotactoid glomerulopathy,
fibrillary GN with monoclonal Ig deposits
Mesangial, endocapillary, exudative,
membranoproliferative, necrotizing,
crescentic, sclerosing, or multiple
Monotypic ig deposits in the
glomeruli
And/or along tubular
basement membranes
C3
glomerulopathy
C3 GN, dense deposit disease Mesangial, endocapillary, exudative,
membranoproliferative, necrotizing,
crescentic, sclerosing, or multipleb
Presence of dominant C3
deposits
In the glomeruli with
minimal or no ig deposits

105. Format of Kidney Biopsy Report

106. Some Examples

107. Some Examples

108. Definition of Glomerular Lesion

109. Patterns of GN

110. Guidelines for LM Description

111. Guidelines for IF Description

112. Guidelines for EM Description

113. Thank
You