Seeing through the density of dense deposit disease

Little is known about what constitutes the dense deposits of dense deposit disease (DDD), apart from components of the complement pathway. This study presents the novel finding that large accumulations of apolipoprotein E are present in the deposits of DDD, as revealed by mass spectroscopy and confirmed by both confocal microscopy and immunohistochemistry. The findings suggest a new modality for diagnosis of DDD and introduce potential new mechanisms for understanding DDD pathophysiology. Little is known about what constitutes the dense deposits of dense deposit disease (DDD), apart from components of the complement pathway. This study presents the novel finding that large accumulations of apolipoprotein E are present in the deposits of DDD, as revealed by mass spectroscopy and confirmed by both confocal microscopy and immunohistochemistry. The findings suggest a new modality for diagnosis of DDD and introduce potential new mechanisms for understanding DDD pathophysiology. Sethi and colleagues have been at the forefront in the application of laser capture microdissection/mass spectroscopy studies for the identification of key peptide components of a variety of kidney diseases, including prior studies that identified components of the complement pathway in dense deposit disease (DDD). In this issue, they build on this earlier work and offer a compelling study that presents the novel finding that the presence of an abundance of apolipoprotein E (ApoE) is a distinguishing component of the deposits in DDD.1Madden B. Singh R.D. Haas M. et al.Apolipoprotein E is enriched in dense deposits and is a marker for dense deposit disease in C3 glomerulopathy.Kidney Int. 2024; 105: 1077-1087Abstract Full Text Full Text PDF Google Scholar This finding comes from an initial study cohort of 36 kidney biopsies, equally divided into those diagnosed as DDD, those diagnosed with the related but distinct glomerular disease of C3 glomerulonephritis (C3GN), which together with DDD comprises the broad category of C3 glomerulopathy, and time-zero donor kidney biopsies as normal controls. Case assignment depended on electron microscopic identification of the uniquely dark deposits of DDD, currently the only definitive diagnostic for separating DDD from C3GN. The authors found a 9-fold increase in ApoE abundance in DDD, compared to that in C3GN and the control biopsies, and found it to be similarly scanty or absent in a survey of glomerular diseases previously studied by this group in other studies using similar techniques. The authors then utilized both confocal microscopy and conventional immunohistochemistry (IHC) to localize the ApoE accumulations to glomerular mesangium and glomerular and tubular basement membranes in distributions congruent with the dense deposits of DDD. The IHC findings recapitulated the separation of DDD and C3GN identified by mass spectroscopy, leading to the clinically important suggestion that in an appropriate context, IHC of ApoE could serve as a highly specific diagnostic test for DDD. The authors also show in the present study that no meaningful differences were present in accumulations of numerous components of the complement system in DDD versus C3GN, other than greater accumulations of the terminal complement pathway moiety C5b-9 in DDD versus C3GN. This finding currently remains unexplained. At least 2 major advances emerge from this study:(i)identification of ApoE accumulation in kidney structures as a potentially useful clinical and pathologic diagnostic marker that can more specifically distinguish DDD from other related disease entities; and(ii)identification of ApoE accumulation as a biologic process that with further directed studies may help us better understand the largely opaque pathogenesis of DDD. Beginning with the clinical advance, a particular strength of this study is a rigorous evaluation of the diagnostic utility of immunohistochemical localization of large accumulations of ApoE to establish a diagnosis of DDD. To accomplish this, validation studies were performed on a second cohort of 31 C3 glomerulopathy biopsies from another institution, which showed that accumulation of ApoE demonstrable by IHC distinguishes most (but not all!) cases of DDD from C3GN, as the authors detail in their Supplementary Table S2.1Madden B. Singh R.D. Haas M. et al.Apolipoprotein E is enriched in dense deposits and is a marker for dense deposit disease in C3 glomerulopathy.Kidney Int. 2024; 105: 1077-1087Abstract Full Text Full Text PDF Google Scholar Based on the positive ApoE staining, DDD was confirmed in 12 of the 15 cases of DDD (80%) in the validation cohort. Based on the absence of significant ApoE staining, C3GN was confirmed in 13 of 16 cases of C3GN (81.3%) in the validation cohort. Overall, 80.6% of the cases were correctly diagnosed as DDD or C3GN, based on ApoE staining correlated with electron microscopy findings as a gold standard. Based on the validation studies, the positive predictive value for ApoE IHC to diagnose DDD is 80%, and the negative predictive value is 81.25%. This concordance is strong, but not perfect, and indicates that ApoE IHC can serve currently as a useful adjunct for making the diagnosis of DDD but is not sufficient to serve as a stand-alone test that can replace electron microscopy as the standard for establishing or confirming the diagnosis of DDD. A further caveat is that the differences in ApoE IHC staining are not absolute. The authors found staining for ApoE in some cases of C3GN, albeit of such a small degree that it appears unlikely to confound the discriminating value of ApoE IHC in most cases. These caveats concerning ApoE IHC do not obviate its value as an important adjunctive test. A significant point to note is that diagnostic accumulations of ApoE can be identified with reasonable reliability by commonly available IHC procedures that are relatively low cost and easy to perform. Extrapolating from this validation study, one can surmise that further experience with this assay may allow it to be used to substantiate the diagnosis of DDD in difficult biopsy cases. Such cases include those with sampling issues, limited or atypical manifestations of the disease, overlap with C3GN or other glomerulonephridites with a membranoproliferative pattern of injury, or a lack of access to electron microscopy that currently would preclude such a diagnosis. The biologic implications of these new studies await explication. These studies call attention to ApoE but were not designed to determine whether ApoE is directly or indirectly pathogenic, versus being an innocent bystander in DDD. ApoE is a lipid transport protein, of which the initial biological importance stemmed from its role in lipid and cholesterol metabolism and cardiovascular disease.2Mahley R.W. Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders.J Mol Med (Berl). 2016; 94: 739-746Crossref PubMed Scopus (304) Google Scholar,3Marais A.D. Apolipoprotein E in lipoprotein metabolism, health and cardiovascular disease.Pathology. 2019; 51: 165-176Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar ApoE has 3 major allelic isoforms, termed ApoE2, ApoE3, and ApoE4, and numerous genetic variants of these major isoforms exist.3Marais A.D. Apolipoprotein E in lipoprotein metabolism, health and cardiovascular disease.Pathology. 2019; 51: 165-176Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar ApoE has received subsequent scrutiny for its role in binding to fibrillar structures, such as amyloid, and in the increased genetic risk for the development of Alzheimer's disease conferred in people homozygous for ApoE4 versus other allelic combinations.2Mahley R.W. Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders.J Mol Med (Berl). 2016; 94: 739-746Crossref PubMed Scopus (304) Google Scholar ApoE has a well established role in mediating cholesterol efflux from cells, and hence, it can limit the development and accumulation of foam cells that contribute to atherosclerosis.4Eom M. Hudkins K.L. Alpers C.E. Foam cells and the pathogenesis of kidney disease.Curr Opin Nephrol Hypertens. 2015; 24: 245-251PubMed Google Scholar Preclinical murine strains with deletion or overexpression of ApoE variants accordingly became key model organisms for studies of atherosclerosis, but they generally have had limited use as preclinical models of kidney diseases apart from lipoprotein glomerulopathy, and such models have not been shown to display kidney lesions resembling DDD. This issue poses a major obstacle for future studies designed to understand DDD pathophysiology. The current findings suggest new questions that can direct the exploration of the pathogenesis of DDD. Some of these include the following:(i)Where does the deposited ApoE in DDD come from? Does it get deposited from the circulation? Is it produced locally? Studies have shown that the kidney is a minor site of synthesis of ApoE in humans.3Marais A.D. Apolipoprotein E in lipoprotein metabolism, health and cardiovascular disease.Pathology. 2019; 51: 165-176Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar,5Saito T. Matsunaga A. Fukunaga M. et al.Apolipoprotein E-related glomerular disorders.Kidney Int. 2020; 97: 279-288Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar A search of the Kidney Precision Medicine Project Atlas Explorer (performed February 22, 2024) shows that within the glomerulus, only mesangial cells are a site of ApoE production, as revealed by RNA sequencing studies of both healthy reference tissues and biopsies from patients with kidney disease. Does a trigger lead this production to become dysregulated in DDD?(ii)Additional studies are needed to ascertain whether specific ApoE isoforms or variants can be identified as the principal apolipoproteins in the dense deposits. Mass spectroscopy has been used to identify pathogenic ApoE isoforms in other rare glomerular diseases, such as lipoprotein glomerulopathy.5Saito T. Matsunaga A. Fukunaga M. et al.Apolipoprotein E-related glomerular disorders.Kidney Int. 2020; 97: 279-288Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In the case of lipoprotein glomerulopathy, ApoE isoforms and genetic variants specify differences in pathogenicity and manifestations of this disorder.5Saito T. Matsunaga A. Fukunaga M. et al.Apolipoprotein E-related glomerular disorders.Kidney Int. 2020; 97: 279-288Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,6Katsarou M. Stratikos E. Chroni A. Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy.J Lipid Res. 2018; 59: 2339-2348Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar At least 15 mutations in ApoE isoforms that differ in the ability of ApoE to bind to glomerular structures, and in their predisposition to aggregation, have been identified, and this variation has been linked to variable manifestations in lipoprotein glomerulopathy.3Marais A.D. Apolipoprotein E in lipoprotein metabolism, health and cardiovascular disease.Pathology. 2019; 51: 165-176Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar,5Saito T. Matsunaga A. Fukunaga M. et al.Apolipoprotein E-related glomerular disorders.Kidney Int. 2020; 97: 279-288Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,6Katsarou M. Stratikos E. Chroni A. Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy.J Lipid Res. 2018; 59: 2339-2348Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar We may discover that ApoE deposited in DDD has similar heterogeneity, with implications for pathogenesis.(iii)Additional proteomic or immunohistochemical studies can be done to identify moieties that might be involved in ApoE degradation or modification that contribute to the development or persistence of dense deposits.(iv)ApoE has been studied principally as a binding partner of high-density lipoprotein and very-low-density lipoprotein and as a mediator of cholesterol efflux from cells.4Eom M. Hudkins K.L. Alpers C.E. Foam cells and the pathogenesis of kidney disease.Curr Opin Nephrol Hypertens. 2015; 24: 245-251PubMed Google Scholar Can it be ascertained whether the ApoE or other apolipoproteins identified in this study are binding lipids (which can appear dense on electron microscopy) in the deposits of DDD?7Muda A.O. Barsotti P. Marinozzi V. Ultrastructural histochemical investigations of "dense deposit disease". Pathogenetic approach to a special type of mesangiocapillary glomerulonephritis.Virchows Arch A Pathol Anat Histopathol. 1988; 413: 529-537Crossref PubMed Scopus (0) Google Scholar Deposited lipids might be a major cause of the density of deposits in DDD.(v)Evidence indicates that ApoE binds to heparan sulfate proteoglycans through an N-terminus binding motif.3Marais A.D. Apolipoprotein E in lipoprotein metabolism, health and cardiovascular disease.Pathology. 2019; 51: 165-176Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar,5Saito T. Matsunaga A. Fukunaga M. et al.Apolipoprotein E-related glomerular disorders.Kidney Int. 2020; 97: 279-288Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,8Martínez-Martínez A.B. Torres-Perez E. Devanney N. et al.Beyond the CNS: the many peripheral roles of APOE.Neurobiol Dis. 2020; 138104809Crossref Scopus (61) Google Scholar The authors have speculated that this binding property may contribute to ApoE accumulation in kidney basement membranes, which are rich in heparan sulfate proteoglycans. Why ApoE would bind preferentially to heparan sulfate proteoglycans in the setting of DDD, but not in other disease or non-diseased settings, is not apparent, as heparan sulfate proteoglycans are normally widely distributed within kidney basement membranes.(vi)Very little published evidence links ApoE to the complement system. One intriguing recent study from the neurodegenerative disease literature has found that the isoform ApoE4 can bind to complement Factor H (FH), and that this interaction interferes with complement-mediated phagocytosis and clearance of components of Alzheimer's disease plaques.9Chernyaeva L. Ratti G. Teirilä L. et al.Reduced binding of apoE4 to complement factor H promotes amyloid-β oligomerization and neuroinflammation.EMBO Rep. 2023; 24e56467Crossref PubMed Scopus (3) Google Scholar This finding raises the possibility that one or more isoforms or mutational variants of ApoE that may be present in DDD also may have previously unrecognized properties that allow it to interact with the pathogenic deposits of complement, and that, in turn, this may inhibit clearance of complement components from the kidney basement membranes and hence contribute to the progressive course of DDD. In conclusion, these studies bring illumination to an area where mostly darkness has prevailed. They also illustrate the need for additional luminosity. The possibility of a new technique that improves diagnosis of DDD is exciting and important. Enhancing the glow for pathologists and morphologists is the aesthetic pleasure of knowing an anatomic and biochemical basis for the unexplained unique density of DDD may have been found. All the author declared no competing interests. Apolipoprotein E is enriched in dense deposits and is a marker for dense deposit disease in C3 glomerulopathyKidney InternationalVol. 105Issue 5PreviewC3 glomerulopathy (C3G) is a rare disease resulting from dysregulation of the alternative pathway of complement. C3G includes C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), both of which are characterized by bright glomerular C3 staining on immunofluorescence studies. However, on electron microscopy (EM), DDD is characterized by dense osmiophilic mesangial and intramembranous deposits along the glomerular basement membranes (GBM), while the deposits of C3GN are not dense. Why the deposits appear dense in DDD and not in C3GN is not known.