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Kidney Int. 2024 Mar 4:S0085-2538(24)00169-8. doi:10.1016/j.kint.2024.02.013
Apolipoprotein E is enriched in dense deposits and is a marker for dense deposit disease in C3 glomerulopathy.
Benjamin Madden, Raman Deep Singh, Mark Haas, Lilian M P Palma, Alok Sharma, Maria J Vargas, LouAnn Gross, Vivian Negron, Torell Nate, M Cristine Charlesworth, Jason D Theis, Samih H Nasr, Karl A Nath, Fernando C Fervenza, Sanjeev Sethi.
PMID: 38447879
Introduction
Many years ago we knew an entity known as membranoproliferative glomerulonephritis (MPGN) with three subtypes (D’Agati and Bomback, Kidney Int 2012). Historically, dense deposit disease (DDD) was classified as MPGN type II, and what we now know as C3 glomerulopathy was a nebulous entity hidden within the old nomenclatures, MPGN I and MPGN III. With the development of immunofluorescence, its classification has evolved. C3 glomerulopathy is characterized by bright C3 glomerular staining on immunofluorescence. This, combined with the absence or paucity of immunoglobulins and with clinical features of glomerulonephritis, is required to make its diagnosis.
C3 glomerulopathy itself is a group of diseases triggered by dysregulation in the alternative complement pathway. It consists of two major subtypes: dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). These rare but devastating glomerulopathies present a significant challenge in terms of pathophysiology and diagnostics, limiting the development of mechanism-directed therapies. Patients with DDD are faced with a grim prospect of end-stage renal failure within 10 years of diagnosis, as well as a very high risk of disease recurrence and allograft failure in transplant patients. Expanding our understanding of DDD is crucial for the development of targeted treatment. As the saying goes, knowing your enemy is half the battle. The present study (Madden et al, Kidney Int 2024) sheds further light on DDD, offering new insights into its diagnostic potential with apolipoprotein E (ApoE).
The complement system is a complex concert of multiple proteins that defend the body against pathogens. Majority ( >90%) of C3 glomerulopathy cases are caused by dysregulation in the alternative complement cascade (Smith et al, Nat Rev Nephrol 2019), while studies have shown the involvement of classical/lectin pathways in some (Michels et al, Front Immunol 2021). The alternative pathway is spontaneous and continuous, generating large amounts of C3b. This combines with components such as Factors B and D to generate C3 convertase, a major driver of the complement response. A key aspect of this pathway is the amplification loop, which exponentially generates substrate for C3 convertase. The binding of C3b created by the other pathways to C3 convertase generates C5 convertase. The latter cleaves C5 to C5b, which combines with the terminal complements (C6-C9) to form the membrane attack complex (MAC), causing lysis of cells.
The image below shows the various complement cascades.
Image from Cytologics
Genetic mutations at the level of C3 convertase or the Factor H gene, the most relevant of the complement regulatory proteins, occur in approximately 25% of cases (Smith et al, Nat Rev Nephrol 2019). These mutations can lead to overactivation of C3 convertase and abnormal deposition of C3b and other complement proteins in the glycocalyx, which coats the luminal side of glomerular capillaries. This eventually leads to a cascade of inflammatory reactions.
Acquired drivers of C3 glomerulopathy include autoantibodies against various components of the complement cascade. C3 nephritic factor and C5 nephritic factor are the most common autoantibodies that cause dysregulation of C3 and C5 convertase, respectively, and are present in 50-80% (Smith et al, Nat Rev Nephrol 2019) of the population with C3 glomerulopathy.
Other etiologies include monoclonal gammopathy in older adults.
Figure showing differential diagnosis of C3 dominant glomerulopathy from Smith JH et al., Nature Reviews Nephrology 2019
DDD and C3GN have overlapping features. Both can present as a mesangial proliferative, endocapillary proliferative, or membranoproliferative pattern of glomerular injury with C3 dominant immune complex deposition. Electron microscopy (EM) is essential to differentiate between these two subtypes. In DDD, EM reveals highly electron-dense, sausage-shaped osmiophilic deposits that transform the glomerular basement membrane.
The morphologic overlap between these two entities is demonstrated below:
Case of dense deposit disease with PAS stain demonstrating a membranoproliferative pattern of glomerular injury, mesangial and capillary loop C3 deposition by immunofluorescence, and electron-dense deposits along the subendothelial surface and mesangium. The diagnostic feature is electron-dense transformation of the GBM (arrows).
Case of C3 glomerulonephritis with similarities in light and immunofluorescence microscopy. Bulky electron-dense immune type deposits are present without electron-dense transformation within the GBM.
The deposits are typically present along the glomerular, tubular basement membranes, Bowman’s capsule, and mesangium (Pickering et al, Kidney Int 2013). They may also be found in the subendothelial region or as subepithelial humps. In contrast, in C3GN, the deposits are ill-defined and non-dense, blending with the surrounding extracellular matrix. There is a lack of electron-dense transformation of the glomerular basement membrane, with deposits in various regions, including subepithelial, subendothelial, and intramembranous.
Electron microscopy images showing: dense osmiophilic deposits in DDD (a,b), amorphous less dense deposits, subepithelial deposits/humps in C3GN (c,d)
Figure from: Pickering MC et al., Kidney International 2013
Laser capture microdissection and mass spectrometry (LCM/MS) have various applications in kidney pathology, particularly for subtype classification of amyloidosis and membranous nephropathy. It has emerged as a valuable tool to confirm the accumulation of complement proteins in C3GN and DDD. This technique is used to isolate cells of interest for various downstream applications, including proteomic analysis. Multiple glomeruli can be obtained from each tissue section for the comprehensive analysis of proteins, which is then compared to controls.
The authors through this paper provide a comparative analysis of both complement and non-complement proteins in DDD and C3GN. Importantly, this study highlights the prominence of ApoE within the dense deposits of DDD and its potential as a crucial diagnostic tool.
The Study
Methods
LM: Light microscopy, IF: Immunofluorescence, EM: Electron microscopy
LCM/MS: Laser capture microdissection/Mass spectrometry, IHC: Immunohistochemical
The pattern of histological injury that was noted in the study amongst patients diagnosed with DDD was consistently membranoproliferative glomerulonephritis (MPGN). Whereas in C3GN, histology was variable between focal sclerosing, focal crescentic and mesangial proliferative and sclerosing patterns of glomerular injury. The flowchart below provides an evaluation scheme for a membranoproliferative pattern of glomerular injury with C3 glomerulopathy as a differential.
Figure from Sethi S et al, CJASN 2011
Various analyses were performed, including LCM/MS, immunohistochemical (IHC) staining for ApoE, and immunofluorescence (IF) confocal staining for co-localization analysis.
Figure depicting the process of LCM from Curran S et al, Mol Pathol 2000
LCM/MS:
LCM is used to isolate target cells from a heterogeneous sample. The laser beam temporarily melts the thermoplastic film that covers the cap, causing it to adhere to the selected cells. The chosen tissue remains adherent to the cap after removing it from the slide. The isolated cells undergo protein extraction and digestion to peptides (proteomic analysis), which facilitates subsequent mass spectrometry (MS) analysis. MS measures the mass-to-charge ratio of ions generated from the peptides, allowing for their identification and quantification. The MS data acquired are processed and analyzed using bioinformatics tools. The proteins present in the sample are identified by comparing the experimental MS data against reference databases.
IHC staining for ApoE:
IHC staining for ApoE was done using the Leica Bond RX stainer.
IF and confocal studies:
In traditional immunofluorescence, antibodies labeled with fluorescent dyes bind to specific target proteins within the tissue sample. Confocal microscopy is a powerful imaging technique that allows for high-resolution, three-dimensional imaging of fluorescently labeled samples. It produces clear images with improved contrast and resolution compared to traditional fluorescence microscopy. Combining the two techniques enables researchers to study the localization and expression patterns of specific proteins within tissue samples with exceptional detail and clarity.
Results
LCM/MS of 12 cases each of C3GN, DDD, and control samples
Complement proteins in C3GN and DDD, compared to normal kidney (pre-implantation kidney biopsy control samples)
When comparing complement proteins in DDD vs controls, there was a notable increase in C5, C6, C8, C9, C7, ApoE, complement factor H related (CFHR)5, C3, CFHR1 and complement factor H (CFH), respectively in DDD. Comparison of complement proteins in C3GN vs controls, there is an increase in C6, C9, C5, CFHR1, CFHR5, C8, CHF, C3, C7, and ApoE, respectively. See the specific linear fold-change ratios below:
Comparing DDD vs C3GN there was a 6-to-9 fold increase in the complement proteins of the terminal pathway: C7, C8b, C8g, C9, C5 and C6. The abundance of C3 between both was similar with 1.2 fold-change ratio that was not statistically significant (P value 0.82).
Complement-regulating and other proteins in DDD and C3GN
There was a 9-fold increase in the presence of ApoE in DDD vs C3GN. Other proteins with higher presence in DDD were: apolipoprotein A-V (ApoA5), apolipoprotein A-II and vitronectin.
In DDD compared to C3GN, ApoE was the most abundant protein; along with C7, C8 and C9.
Principal component analysis, examines important variables in a cluster analysis (shown below) and plots its variance. This analysis showed a tighter clustering of DDD cases compared to C3GN cases, suggesting less variability of the proteins found in DDD cases vs C3GN cases.
Figure1f, Cluster analysis from Madden M et al, KI 2024.
IHC staining for ApoE in DDD and C3GN
In all DDD cases, ApoE IHC staining was positive along the GBM, mesangium and Bowman’s capsule. The pattern of ApoE along the GBM and Bowman’s capsule was similar to the linear thick sausage-shape/ribbon-like staining of dense deposits. There was also intense positive ApoE staining on sclerosed glomeruli.
Figure 2 Immunohistochemical staining for ApoE from Madden M et al, KI 2024.
In control cases that included MCD, IgAN, FSGS, diabetic nephropathy, LN and membranous nephropathy, IHC staining for ApoE was negative along the GBM and mesangium. However, in C3GN staining was negative or trace to 1+ in a granular pattern.
Confocal microscopy for ApoE in DDD and C3GN
Confocal microscopy was performed to confirm the LCM/MS findings. In DDD it showed bright staining for ApoE along the GBM and Bowman’s capsule; and focal staining along the tubular basement membranes. The negative staining in representative cases of C3GN, membranous nephropathy and time-0 preimplantation kidney transplant biopsy is shown below.
Figure 3 Confocal microscopy for Apo E from Madden M et al, KI 2024.
Validation (blinded) studies
IHC staining for ApoE was performed on 31 cases of C3G; at the time of staining, the diagnosis of C3GN or DDD was not known. The specific diagnosis was based on EM and then confirmed by the pathologist who had contributed to the validation cases. DDD was confirmed in 80% (12/15) of the cases (significant ApoE staining) and C3GN was confirmed in 81.3% (13/16) of the cases (absence of significant ApoE staining). Validation studies showed a 80% positive predictive value (PPV) and a negative predictive value (NPV) of 81.25% for ApoE IHC staining to diagnose DDD.
Discussion
ApoE is a protein involved in the metabolism of fats. It has been implicated in Alzheimer’s (Liu et al, Nat Rev Neurol 2013) and cardiovascular diseases. The authors of this paper now suggest ApoE could be involved in glomerular pathology as well. In current practice, EM is needed to differentiate between C3GN and DDD. The study authors hypothesized differences in C3GN and DDD deposits’ characteristics are due to differences in the complement profile beyond the scope of routine IF studies.
The proteomic profile showed that both DDD and C3GN have increased accumulation of complement and complement-regulatory proteins. However, when DDD is compared with C3GN, there is a 6-9 fold increase in C5 to C9. With this finding, the authors suggest a higher activity of C5 convertase in DDD versus C3GN. In both, C3GN and DDD, there was no significant difference in the accumulation of other complement-regulating proteins (CFH, CFHR1, and CFHR5), including C3.
While investigating other non complement proteins, ApoE was increased in DDD 9-fold vs C3GN and 32-fold vs controls. These LCM/MS findings were confirmed with IHC staining and confocal microscopy which showed staining of ApoE along the GBM, mesangium and Bowman’s capsule, and focally in the TBM. This coincides with the location of dense deposits in DDD.
ApoE has been also found in fibrillary glomerulonephritis (Dasari et al, JASN 2018), immunotactoid glomerulonephritis (Nasr et al, NDT 2012), monoclonal Ig deposition disease (Sethi et al, Kidney Int Rep 2023), amyloidosis (Vrana et al, Blood 2009), and lipoprotein glomerulopathy (Saito et al, AJKD 1989). However, these diseases can be distinguished from DDD using other characteristic findings on LM, IF (presence of Igs) or EM (substructure and location of deposits).
This appears to be the first time ApoE is described as a major constituent of dense deposits in DDD. However, why is it there? What is the relationship or role with the complement system? ApoE has been identified as one of the component of retinal drusen in age-related macular degeneration (AMD). Drusen deposits in the retina and promotes activation of the complement system resulting in recruitment of macrophages that with microglial activation promote chronic inflammation (Hu M.L, et.al. 2021.) A schematic illustration of this process is shown below.
(a) Deposition of drusen (also known as basal linear deposits or soft drusen) and reticular pseudodrusen (RPD) in the retina. (b) A model for AMD pathogenesis integrating key risk factors.
Figure 1 Schematic illustration of AMD pathomechanism from Hu ML et al, Life 2021.
Based on the role of ApoE in other diseases like AMD, the authors hypothesize that the formation of the dense deposits results from ApoE binding to the heparan sulfate proteoglycans in the GBM and acting as a scavenging or chaperone protein for C5-9, especially when the levels are high due to increased complement activity.
Figure 5: Schematic showing the difference between DDD and C4GN from Madden M et al, KI 2024. The dense deposits of DDD are composed of ApoE and C5-9, along with C3, CFH, and CFHR proteins. The deposits of C3GN are composed of mostly C3, CFH, and CFHR proteins.
Involvement of terminal complement cascade in DDD is clearly demonstrated in this study. This opens the question to novel medications, like C5 inhibitors being a possible therapeutic approach in C3 glomerulopathy, specifically DDD. In mouse models (Williams et al, Kidney Int 2017) with active C3 glomerulopathy and severe proteinuria, anti-C5 mAb prevented death in half of mice studied. Also, it was suggested that C5a receptor (C5aR)-mediated inflammation contributed to C3G and disease severity was reduced when C5aR was deficient. ACCOLADE (Bomback et al, KI Reports 2022, abstract only conference proceeding) was a phase 2 trial in which patients either received avacopan, a C5a receptor antagonist, or matched placebo. Most of the patients had a C3GN diagnosis and only 14% had DDD. The preliminary results showed no changes in the primary outcome which consisted of a histologic index and not clinical endpoints. However, in patients receiving avacopan, there was an improvement in eGFR, proteinuria and reduction of disease chronicity
The authors suggest that serum ApoE levels in DDD, analysis of ApoE variants associated with DDD and complement binding properties of ApoE may be studied in the future to bring more clarity on the role of ApoE in the pathogenesis and diagnosis of this rare disease.
Conclusion
Distinguishing between C3GN and DDD is challenging since it depends on EM findings. However, EM availability is not a given and even if available, in some cases the diagnosis becomes challenging due to overlapping findings. This study shows ApoE is a major component of dense deposits in DDD and IHC staining for ApoE may become a valuable tool to diagnose DDD without or as an adjunct to EM.
Summary by
Anvitha Rangan
Internal medicine resident
Landmark Medical Center/NYMC, RI
Stephanie Torres Rodríguez
Assistant Professor
UT Southwestern, Dallas, TX
NephEdC Interns, Class of 2024
Reviewed by Brian Rifkin, Jade Teakell,
Tiffany Caza, Pallavi Prasad, Sayali Thakare
Header Image created by AI, based on prompts by Evan Zeitler