Glomerular Disease Pathology in the Era of Proteomics: From Pattern to Pathogenesis
Authors: Mark Haas
Journal: Journal of the American Society of Nephrology (2017)

Among glomerular diseases, even the very uncommon ones, fibrillary GN (FGN) has always been among the most poorly understood. From an early description as "Congo red-negative amyloidosis-like glomerulopathy,"1 its ultrastructural appearance has both captivated and confused renal pathologists. In the 1990s, it was hotly debated whether FGN was a variant of immunotactoid glomerulopathy (or vice versa) or a separate entity,2,3 despite clear differences between the two lesions with regard to the size, shape, and pattern of the fibrils by electron microscopy (EM) and in their association with hematologic malignancies, the latter being far more commonly associated with immunotactoid glomerulopathy.4 Although immuno-EM studies have suggested that the fibrils in FGN contain immune complexes rather than matrix components,5 its pathogenesis, including the antigen(s) within the immune complexes, has remained unclear, and perhaps as a result, attempts at treatment of FGN have been largely unsuccessful.4 Use of the sequential methods of laser capture microdissection followed by liquid chromatography and mass spectroscopy (ref. 6 reviews the methodologies) has enabled investigators to investigate the proteins within glomeruli isolated from formalin-fixed, paraffin-embedded tissue sections of renal biopsies. This method of proteomic analysis has allowed for the discovery of new forms of renal amyloidosis7 as well as the identification of individual components of the alternative pathway of complement in glomeruli from biopsies showing a membranoproliferative pattern of GN,8 contributing to the reclassification of membranoproliferative GN from a morphologic pattern–based diagnosis to a pathogenesis-based diagnosis, the latter including C3 GN. In this issue of the Journal of the American Society of Nephrology (JASN), groups from the Mayo Clinic and the University of Washington, working independently and each using glomeruli isolated from formalin-fixed, paraffin-embedded tissue by laser capture microdissection and a proteomic approach involving analysis by liquid chromatography and tandem mass spectroscopy (LC-MS/MS), each found DnaJ heat shock protein family member B9 (DNAJB9) to be abundantly present in glomeruli from biopsies with FGN but not present in normal glomeruli and glomeruli from biopsies showing other glomerular diseases, including different types of amyloidosis, immunotactoid glomerulopathy, diabetic nephropathy, idiopathic (smoking-related) nodular glomerulosclerosis, and light-chain deposition disease.9,10 Each group also subsequently showed by immunofluorescence and immunohistochemistry that antibodies to DNAJB9 specifically labeled glomeruli from biopsies with FGN but not these other lesions and that staining for DNAJB9 colocalized with that for IgG in FGN glomeruli. The 100% sensitivity and specificity of DNAJB9 immunostaining for FGN indicate that such staining can be used diagnostically in lieu of EM in identifying patients with cases of FGN, a useful finding in cases where glomeruli for EM study are not available and in centers around the world that do not have access to EM or do not routinely perform EM in renal biopsy analysis. It is also possible that serum and/or urine levels of DNAJB9 or immune complexes containing this protein could serve as a useful biomarker for assessing disease activity and treatment efficacy in FGN. The findings of the two groups9,10 strongly suggest that the glomerular deposits in FGN contain, at minimum, DNAJB9 and IgG antibodies that are most likely directed against DNAJB9 plus complement in most cases. Where these immune complexes are formed (i.e., in the circulation, in the glomerulus, or both) remains unknown, as are the reasons why the deposits form fibrils and why they are localized primarily (although not exclusively in all cases) in glomeruli. The answers to the latter questions as well as an increased knowledge of the biology of DNAJB9 will be important to a greater understanding of the pathogenesis of FGN and more specifically directing treatment for this relatively uncommon form of GN. Perhaps more importantly, these studies illustrate the immense power of laser capture microdissection followed by proteomic analysis using LC-MS/MS as a method for improving our understanding of glomerular diseases, especially those involving deposition of proteins not normally present or present in only very small amounts within glomeruli. The use of these methods has become standard diagnostic practice for many centers in identifying amyloid subtypes in biopsies where available immunofluorescence and immunohistochemical stains are not diagnostic, as is typical with more unusual amyloid subtypes and may be seen in cases of light-chain amyloid where alteration of the protein structure of the light chain in the process of fibril formation renders it undetectable by commercially available antibodies. Also, as noted above, our current understanding that many cases of membranoproliferative GN, including essentially all cases of dense deposit disease, are related to complement abnormalities evolved in part from studies identifying alternative and terminal pathway complement proteins within isolated glomeruli by mass spectroscopy.8 Some other glomerular diseases in which a proteomic approach using laser capture microdissection of glomeruli may potentially contribute to our understanding of their pathogenesis are shown in Figure 1. Candidate antigens have been reported for poststreptococcal GN11; these findings can be confirmed using a proteomic approach. Furthermore, this approach can potentially be used to identify pathogenic antigens in poststaphylococcal GN. Identification of such antigens could further allow for an understanding of why immune complex deposits in the latter condition contain IgA as the predominant Ig.11 Viruses, such as hepatitis B and hepatitis C, have long been associated with glomerular lesions, particularly membranous nephropathy and proliferative GN with a membranoproliferative GN pattern, respectively.12 However, mechanisms by which these viruses produce glomerular lesions remain largely unknown, and identification of viral antigens within glomeruli by immunopathologic methods has remained elusive. A proteomic approach similar to that used in identifying DNAJB9 within microdissected glomeruli from renal biopsies with FGN has the potential for identifying viral antigens in these cases as well as possible extraglomerular antigens in other forms of secondary membranous nephropathy, such as that seen in the context of malignancies. The latter cases were long thought to be related to the formation of immune complexes containing circulating or planted tumor antigens, although this has recently come into question.13 Finally, the potential role of a circulating factor or factors in the pathogenesis of primary FSGS and the identity of such a factor remain enigmatic. The argument for such a factor is particularly strong in cases where FSGS recurs in the renal allograft shortly after transplantation14 and supported by a report where a patient with ESRD due to primary FSGS developed proteinuria shortly after receiving a renal transplant; when the kidney was removed and retransplanted into a recipient with ESRD due to diabetic nephropathy, it functioned well without proteinuria.15 A proteomic approach comparing proteins present within glomeruli isolated from preimplantation biopsies and biopsies from the same allografts after early development of recurrent proteinuria and possibly between glomeruli of nonrejecting grafts with and without early recurrent FSGS may have significant potential for identifying potential FSGS factors should these be proteins present in large-enough amounts and not normally expressed in glomeruli.Figure 1.: Documented (left panels) and potential (right panels) uses for proteomic analysis in the study of glomerular diseases. (Top left panel) Amyloid nephropathy (Congo red stain, polarized). (Middle left panel) C3 GN with a membranoproliferative pattern (Jones silver methenamine stain). (Bottom left panel) FGN (electron micrograph showing deposits composed of nonbranching fibrils at the outer [epithelial] aspect of the glomerular basement membrane). (Bottom right panel) Secondary membranous nephropathy (direct immunofluorescence with fluorescein-conjugated anti-IgG; note the segmental distribution of the glomerular capillary wall deposits that is more typical of secondary than primary membranous lesions). (Middle right panel) Early recurrent FSGS in a renal allograft (electron micrograph showing nearly complete podocyte foot process effacement). (Top right panel) Infection-related (postinfectious) GN (electron micrograph showing the characteristic hump-like subepithelial deposits). The image in the center is one of a mass spectrum.Whenever potentially pathogenic antigens/factors are identified using LC-MS/MS, confirmation by immunopathologic methods is necessary, just as was done by Desari et al.9 and Andeen et al.10 for DNAJB9 in FGN, establishing the specificity and sensitivity of antigen detection for the disease process using methods available in most pathology laboratories. After this is established, the antigen itself and/or immune complexes containing the antigen can be tested as potential serum and urine biomarkers for disease activity and treatment response. The potential of this proteomic approach in improving our understanding of glomerular diseases in which the suspected antigenic target is intrinsic to the glomerulus (e.g., phospholipase A2 receptor–positive membranous nephropathy), where the antigen is likely to itself be an abnormal Ig (e.g., IgA nephropathy) or where an Ig is deposited without a specific antigen (e.g., monoclonal Ig deposition disease), is not nearly as clear. However, even in such cases, there are questions that can potentially be addressed using proteomic analysis of glomeruli, such as whether mucosal antigens might be directly involved in some cases of IgA nephropathy. Whether Igs and/or complement components are, in fact, deposited in renal allografts showing histologic changes suggestive of antibody-mediated rejection (glomerulitis and peritubular capillaritis) in the absence of identifiable donor-specific antibodies or glomerular C4d deposits is also a question that might be addressed using glomerular proteomics. If identified, such Igs could potentially be eluted from the tissue to identify their specificity. Clearly, proteomics has great potential for increasing our understanding of glomerular and other renal diseases, as illustrated by the studies of FGN published in this issue of JASN,9,10 and we have likely just begun to scratch the surface of this potential. Presently, there are few renal pathology laboratories performing laser capture microdissection and LC-MS/MS analysis of renal tissue, and to this end, most centers that desire typing of amyloid in cases where immunofluorescence and immunohistochemistry are not successful in doing so must send tissue to specialized centers performing this analysis. However, it was not long ago that molecular techniques, such as next generation sequencing, were also performed in a small number of centers, and today, molecular analysis of tumors has become standard of care. If proteomic analysis lives up to its great potential, it is anticipated that this will soon become far more prevalent as well. Disclosures M.H. serves as a paid consultant on pathology adjudication committees for two industry-sponsored clinical trials (Shire ViroPharma and AstraZeneca). Neither represents a conflict of interest relevant to any of the material presented in this editorial.