Keywords glomerulonephritis - classification - membranoproliferative glomerulonephritis - C3
glomerulonephritis - complement
Dysregulation of the alternative pathway of complement can result in two most prominent
disease states, involving the kidney, C3 glomerulopathy and atypical hemolytic uremic
syndrome (aHUS). The classification, pathology, and kidney biopsy finding of these
entities is reviewed. Other forms of glomerulonephritis such as immunoglobulin A (IgA)
nephropathy and antineutrophil cytoplasmic antibody-associated glomerulonephritis
where the alternative pathway of complement may also be involved are not discussed
in this review.
Classification of Glomerulonephritis
Classification of Glomerulonephritis
Proliferative glomerulonephritis results from deposition of Ig/immune complexes (IC)
and/or complement factors in mesangium and/or along glomerular capillary walls.[1 ] The deposition of Ig and complement factors results in an inflammatory response
from the following:
Proliferation of indigenous glomerular cells such as mesangial cells, endothelial
cells, parietal epithelial cells and/or infiltration and proliferation of leukocytes,
and
Synthesis of matrix material such as mesangial matrix material, basement membrane
material, and fibrin.
Glomerular deposition of Ig/IC originates from three basic pathogenic mechanisms:
Deposition of monoclonal Ig as a result of a monoclonal gammopathy due to a plasma
cell or B cell disorder,[2 ]
[3 ]
[4 ]
Deposition of antigen–antibody IC as a result of an infection,[5 ] and
Deposition of IC as a result of an autoimmune disease.[6 ]
[7 ]
Immunofluorescence studies can often confirm the underlying pathogenic mechanism of
Ig/IC deposition based on the type of Ig detected. Complement factors are also noted
along with the Ig/IC due to activation of the classical and terminal pathway by the
Ig/IC. On the contrary, glomerular deposition of complement factors alone or in the
presence of scant Ig results from dysregulation of the alternative pathway of complement.
The term C3 glomerulopathy is used to define this entity. On the basis of these findings,
proliferative glomerulonephritis has recently been classified into Ig/IC-mediated
glomerulonephritis and complement-mediated glomerulonephritis.[8 ] Thus, immunofluorescence studies of the kidney biopsy are the key to the classification
of proliferative glomerulonephritis into Ig/IC mediated or complement mediated.
Pathology of C3 Glomerulopathy
Pathology of C3 Glomerulopathy
Deposition of complement factors in the mesangium and/or along the glomerular capillary
walls results in a proliferative glomerulonephritis. The term “C3 glomerulopathy”
is now used to define the entity of a glomerulonephritis characterized by C3 accumulation,
with absent or scanty Ig deposition.[9 ]
[10 ]
[11 ]
[12 ] C3 glomerulopathy encompasses the entities of C3 glomerulonephritis and dense deposit
disease (DDD).[9 ]
[11 ]
[13 ]
On kidney biopsy, C3 glomerulonephritis and DDD present as a proliferative glomerulonephritis.
The most common pattern on light microscopy pattern for both C3 glomerulonephritis
and DDD is that of a membranoproliferative glomerulonephritis. Other patterns of injury
include diffuse proliferative glomerulonephritis, mesangial proliferative glomerulonephritis,
or even a necrotizing and crescentic glomerulonephritis.[14 ] Two or more patterns of injury may be seen on the same biopsy. On immunofluorescence
studies, both C3 glomerulonephritis and DDD are characterized by bright mesangial
and capillary wall staining for C3. In DDD, C3 staining may also be seen along the
tubular basement membranes. Tubular basement membrane staining for C3 is uncommon
in C3 glomerulonephritis. The main differentiating factor between C3 glomerulonephritis
and DDD lies in the electron microscopy findings. In C3 glomerulonephritis, the complement
deposits are discrete and are located in the mesangium and along the capillary walls
in subendothelial region of the glomerular basement membrane. Subepithelial and few
intramembranous deposits are also often present. The deposits often assume a lobular
shape and have a waxy appearance with ill-defined margins. On the contrary, in DDD
the deposits are intensely osmiophilic and are located in the mesangium and within
the glomerular basement membranes (intramembranous deposits) often forming large dense
ribbon-/sausage-shaped bands that can completely transform the glomerular basement
membranes. On the basis of the pathology, it is not possible to differentiate between
the acquired and hereditary causes of C3 glomerulonephritis or DDD. [Fig. 1 ] (A–E) shows a representative case of C3 glomerulonephritis. Electron microscopy
of a case of DDD is also shown for comparison (panel F).
Fig. 1 C3 glomerulopathy (A–E). Representative kidney biopsy findings in C3 glomerulonephritis.
(A) Light microscopy showing features of both membranoproliferative glomerulonephritis
and diffuse proliferative glomerulonephritis (Periodic acid Schiff stain ×40), immunofluorescence
microscopy showing (B) negative IgG staining and (C) bright mesangial and capillary
wall staining for C3 (B and C ×10), electron microscopy showing (D) numerous mesangial
and subendothelial electron dense deposits and (E) a subepithelial electron dense
deposit (white arrow). (F) Electron microscopy showing numerous intramembranous deposits
(black arrow) in a case of dense deposit disease as comparison with C3 glomerulonephritis
shown in earlier panels. (D–F ×10,900). IgG, immunoglobulin G.
Proteomics of C3 Glomerulonephritis and DDD
Proteomics of C3 Glomerulonephritis and DDD
Both C3 glomerulonephritis and DDD are diseases resulting from dysregulation of the
alternative pathway of complement. Why are the deposits discrete in C3 glomerulonephritis
and intramembranous in DDD? Recent studies using the technique of laser microdissection
of glomeruli followed by mass spectrometry showed accumulation of complement factors
of the alternative pathway and terminal complement pathway in both the conditions.
Large spectra numbers of C3 and C9 were noted in both C3 glomerulonephritis and DDD,
while smaller spectra numbers of C5, C6, C7, and C8 were also present ([Fig. 2 ]).[15 ]
[16 ] Complement regulating proteins such as vitronectin and clusterin were also present
in large spectra numbers. In addition, large spectra numbers of complement factor
H–related protein (FHR)-1 and FHR-5 were present. There was little or no significant
accumulation of complement factors of the classical complement pathway, such as C1,
C2, or C4. In addition, there was little or no Ig present. There was also no factor
B present, indicating absence of C3 and C5 convertase in the glomeruli, suggesting
that activation of alternative and terminal pathway occurs in the fluid phase rather
than resulting from local disturbance of the alternative pathway in both C3 glomerulonephritis
and DDD. Thus, based on proteomic studies, it appears that the complement profile
in both C3 glomerulonephritis and DDD is similar. As whole glomeruli are dissected,
the proteomic studies are not absolutely quantitative, even though higher spectra
numbers are indicative of greater abundance and typically yield greater amino acid
sequence coverage. Thus, it is possible that the relative amounts of complement factors
and their breakdown products might be responsible for the difference in appearance
on electron microscopy studies. It should be pointed out, some cases do show crossover
electron microscopy findings, with C3 glomerulonephritis showing few dense intramembranous
deposits, and DDD showing few discrete subendothelial and mesangial deposits (Sethi,
unpublished data, 2014).
Fig. 2 Proteomics of C3 glomerulopathy. Representative mass spectrometry data of three cases
of C3 glomerulonephritis and three cases of dense deposit disease. Mass spectrometry
shows spectra of complement factors of the alternative pathway and terminal complement
pathway, in particular C3 and C9 in all cases. The “spectra” value indicates the total
number of mass spectra collected on the mass spectrometry that matched the protein
in question utilizing the proteomics software. A higher number of mass spectra is
indicative of greater abundance and will typically yield greater amino acid sequence
coverage. A higher mass spectra value also indicates a higher confidence in the protein
identification. (Reproduced with permission from Sethi S, Vrana JA, Theis JD, Dogan
A. Mass spectrometry based proteomics in the diagnosis of kidney disease. Current
Opinion in Nephrology and Hypertension 2013;22:273–280).[15 ]
Postinfectious Glomerulonephritis and C3 Glomerulonephritis
Postinfectious Glomerulonephritis and C3 Glomerulonephritis
Postinfectious glomerulonephritis is characterized by a proliferative glomerulonephritis
on light microscopy, staining for granular IgG and C3 on immunofluorescence (IF) microscopy,
and mesangial, subendothelial, and subepithelial “hump”-like deposits on electron
microscopy. However, in some cases while the electron microscopy shows the characteristic
“hump”-like subepithelial deposits, IF studies show only dominant C3 with scant or
no Ig. Thus, in this setting the IF findings are similar to C3 glomerulonephritis.
Many of these cases with the “hump”-like subepithelial deposits and bright C3 staining
were deemed postinfectious glomerulonephritis in the past. Terms such as “resolving”
or “persistent” or “chronic” postinfectious glomerulonephritis were used when hematuria
and proteinuria persisted or when there was deterioration of kidney function, as the
postinfectious glomerulonephritis typically resolves within weeks. Recently, it was
shown that such cases with “hump”-like subepithelial deposits and bright C3 staining
and scant/no Ig and persistent hematuria/proteinuria, previously diagnosed as “resolving”
postinfectious glomerulonephritis, were associated with abnormalities of the alternative
pathway of complement. The term “atypical” postinfectious glomerulonephritis, similar
to the terminology of aHUS was introduced to highlight the underlying alternative
pathway abnormalities in these patients.[17 ] The key differentiating feature between postinfectious glomerulonephritis and “atypical”
postinfectious glomerulonephritis is the presence of both Ig and C3 in postinfectious
glomerulonephritis, while there is only C3 with scant or no Ig in “atypical” postinfectious
glomerulonephritis, even though subepithelial humps are common to both entities. It
is postulated that an infection activates the alternative pathway of complement in
atypical postinfectious glomerulonephritis. However, due to an underlying defect in
the regulatory mechanisms, there is persistent activation of the alternative pathway
of complement with resultant deposition of complement factors and ensuing inflammation
in the glomeruli. It should be pointed out that similar findings, that is, subepithelial
“humps” are also seen in DDD.[18 ] Thus, it is conceivable that DDD and C3 glomerulonephritis may be triggered by an
infection. It is the underlying regulatory defect of the alternative pathway of complement
that then drives the glomerular inflammation even after the infection is controlled.
The term “atypical” DDD or C3 glomerulonephritis may thus be used to signify that
the underlying pathophysiology is that of an alternative pathway abnormality, even
though the triggering event may be an infection as evidenced by the presence of subepithelial
“humps.”
Recurrent C3 Glomerulopathy
Recurrent C3 Glomerulopathy
There is a high recurrence rate of DDD and C3 glomerulonephritis in the transplant.
With regard to DDD, there is a 60 to 85% rate of recurrence in the transplant, resulting
in allograft failure in 45 to 50% within 5 years.[19 ]
[20 ]
[21 ] There is limited data with regard to recurrent C3 glomerulonephritis. In a recent
study, there was recurrence of C3 glomerulonephritis in 66.7% of patients, with graft
loss in 33% of patients within 5 years.[22 ] Kidney biopsy of early recurrent C3 glomerulonephritis, particularly those detected
on routine protocol biopsies shows a mesangial proliferative glomerulonephritis on
light microscopy, mesangial C3 deposition on IF studies, and mesangial electron dense
deposits on electron microscopy. On the contrary, membranoproliferative glomerulonephritis
is more common during the later stages or when the biopsy is done for clinical indications.[22 ] This is similar to the findings of recurrent membranoproliferative glomerulonephritis
in general.[23 ] The kidney biopsy findings of recurrent C3 glomerulonephritis are shown in [Fig. 3 ].
Fig. 3 Recurrent C3 glomerulonephritis. Representative kidney biopsy findings in recurrent
C3 glomerulonephritis. Top panel shows a case of early recurrent C3 glomerulonephritis.
(A) Light microscopy showing a mild mesangial proliferative glomerulonephritis (hematoxylin
and eosin, ×40), (B) immunofluorescence microscopy showing mild mesangial C3 staining
(×20), and (C) electron microscopy showing few mesangial electron dense deposits (×17,900).
Bottom panel shows a case of florid/late recurrent C3 glomerulonephritis. (D) Light
microscopy showing a membranoproliferative and diffuse proliferative pattern of injury
(silver methenamine, ×40), (E) immunofluorescence microscopy showing bright C3 staining
in the mesangium and along capillary walls, and (F) electron microscopy showing numerous
mesangial electron dense deposits and infiltrating leukocytes (asterisk) (×3,100).
Arrow points at deposits.
Pathology of Atypical Hemolytic Uremic Syndrome (aHUS)
Pathology of Atypical Hemolytic Uremic Syndrome (aHUS)
aHUS results from abnormalities in the alternative pathway of complement and is characterized
by a thrombotic microangiopathy that results in a hemolytic anemia, thrombocytopenia,
and multiorgan dysfunction.[24 ]
[25 ] aHUS belongs to a group of disorders in which the underlying characteristic finding
is a thrombotic microangiopathy and includes diarrhea-associated hemolytic uremic
syndrome, frequently resulting from infections with enterohemorrhagic Escherichia coli (EHEC)-HUS, and thrombotic microangiopathy purpura (TTP), due to deficiency of the
enzyme ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1
motif, member 13), an enzyme that cleaves von Willebrand factor.[24 ]
Kidney biopsy findings in aHUS, EHEC-HUS, and TTP are often indistinguishable. Kidney
biopsy shows a thrombotic microangiopathy in which the glomerular capillaries contain
thrombi. However, in the setting of aHUS and EHEC-HUS, the thrombi typically are fibrin
rich, whereas the thrombi in TTP are platelet rich.[26 ] In addition, the glomeruli show mesangiolysis (fluffy mesangial expansion), endothelial
swelling, thickening of the glomerular capillary walls, and schistocytes within the
glomerular capillaries. Arterioles and small arteries may also be occluded by the
microthrombi. As the lesion progresses and becomes chronic, capillary wall remodeling
takes place with formation of new basement membrane material and entrapment of cellular
elements. This results in double contour formation along glomerular capillary walls.
The light microscopy findings at this time may mimic membranoproliferative glomerulonephritis.
However, immunofluorescence studies are negative for Ig/IC and complement factors.
Immunofluorescence studies of thrombotic microangiopathy show positive staining for
fibrinogen with glomerular capillaries and arterioles and small arteries. Electron
microscopy shows subendothelial expansion with fluffy material, endothelial swelling
and loss of fenestrations, and fibrin material within the glomerular tufts but no
deposits. This helps distinguish a thrombotic microangiopathy from a necrotizing glomerulonephritis
in which there is rupture of the glomerular tufts, and fibrin is seen spilling out
into the Bowman space. Double contours are also a characteristic feature of thrombotic
microangiopathy, particularly when the lesion has been present for a while. The kidney
biopsy findings of aHUS are shown in [Fig. 4 ].
Fig. 4 Thrombotic microangiopathy. Representative kidney biopsy findings in aHUS: (A–C)
Fibrin thrombi in the glomerular capillary lumen, (A) hematoxylin and eosin–stained
section—note fragment red blood cells (black arrow), (B) periodic acid Schiff, (C)
Masson trichrome (all ×40, white arrows point at fibrin microthrombi), (D) Microthrombus
in small artery (Trichrome, ×20), (E–G) immunofluorescence microscopy showing (E)
no glomerular staining for C3, (F) fibrinogen staining with glomerular capillaries
indicating fibrin thrombus, and (G) fibrinogen staining in small artery indicating
fibrin thrombus, (H, I) electron microscopy showing fibrin microthrombus in glomerular
capillary lumen (white arrow) and fibrin along the glomerular capillary walls (black
arrow, E–G: ×20, H,I: ×4,200).
Thrombotic microangiopathy may also result from other causes such as connective tissue
disorders, malignant hypertension, eclampsia, exposure to drugs, bone marrow transplantation,
etc. These lesions may be difficult to distinguish from the thrombotic microangiopathy
seen in aHUS/EHEC-HUS/TTP. However, vascular changes such as mucoid hyperplasia of
the intima, hypertrophy of the media, and onion-skinning of the arterial walls is
more likely to be seen in the setting of poorly controlled hypertension/connective
tissue disorders/eclampsia, rather than in aHUS/EHEC-HUS/TTP.
Concluding Remarks
In this review, we have summarized the kidney biopsy findings in disorders of the
alternative pathway of complement, that is, C3 glomerulopathy and aHUS. C3 glomerulopathy
is characterized by a proliferative glomerulonephritis, with bright C3 staining on
IF studies, and mesangial and capillary wall deposits (C3 glomerulonephritis) or dense
intramembranous deposits (DDD) on electron microscopy. On the contrary, aHUS is characterized
by thrombotic microangiopathy as evidenced by thrombi in glomerular capillaries, mesangiolysis,
and capillary wall changes such as endothelial swelling and double contour formation,
with negative immunofluorescence and no deposits on electron microscopy.
