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Elevated serum concentrations of DNAJB9 in fibrillary glomerulonephritis: another step toward understanding a progressive disease
commentary
Elevated serum concentrations of DNAJB9 in fibrillary glomerulonephritis: another step toward understanding a progressive disease Nicole K. Andeen1 DnaJ homolog subfamily B member 9 (DNAJB9) is a sensitive and specific marker of fibrillary glomerulonephritis (FGN) in kidney biopsies. In this issue, Nasr and Dasari et al. demonstrate significantly elevated concentrations of DNAJB9 in serum from patients with FGN. This advances our understanding of DNAJB9 as a biomarker in FGN and reframes questions about pathogenesis and potential clinical applications of DNAJB9 serum testing. Kidney International (2019) 95, 1025–1026; http://dx.doi.org/10.1016/j.kint.2019.01.034 Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see technical notes on page 1269
I
n 2017, DnaJ homolog subfamily B member 9 (DNAJB9) was identified as a sensitive and specific marker of fibrillary glomerulonephritis (FGN) in kidney biopsies. Its specific abundance was initially discovered with laser capture microdissection of glomeruli followed by liquid chromatography and mass spectrometry; sensitivity and specificity were further demonstrated with immunohistochemistry.1–3 Since its discovery, new questions have arisen regarding the role of this protein in the pathogenesis of FGN. Does DNAJB9 represent an antigen to which an autoantibody develops? DNAJB9 is involved in endoplasmic reticulum stress and the unfolded protein response pathway, and it binds aggregation-prone peptides.4 Could initial deposition of aggregationprone peptides—of as yet undetermined etiology— followed by DNAJB9 binding contribute to the pathogenesis of FGN? DNAJB9 staining in renal biopsies co1 Department of Pathology, Oregon Health & Science University, Portland, Oregon, USA
Correspondence: Nicole K. Andeen Oregon Health & Science University, Department of Pathology, 3181 SW Sam Jackson Park Road, Mail code: L113, Portland, Oregon 97239 USA. E-mail: [email protected] Kidney International (2019) 95, 1012–1026
localizes with IgG deposition within glomerular and extraglomerular deposits; now that a more widely accessible immunohistochemistry-based technique is available, to what extent might these patients have detectable extrarenal immune complex deposition? In the Technical Note in this issue of Kidney International, Nasr and Dasari et al.5report the development of an immunoprecipitation-based multiple reaction monitoring (MRM) test to detect serum levels of DNAJB9 protein in patients with FGN, non-FGN patients, and controls. The test consists of an enrichment step (adding beadbound anti-DNAJB9) followed by a mass spectrometry–based quantification step, using selected peptide sequences. Using this assay, they demonstrate that serum DNAJB9 concentrations are significantly increased in patients with FGN, compared with normal controls, and with patients with either non-FGN glomerular disease, Ig light-chain amyloidosis, or multiple myeloma. Identification of elevated serum concentrations of DNAJB9 in patients with FGN is a novel development that has potential to enhance diagnosis and
management of such patients. From a clinical standpoint, the authors demonstrate statistical sensitivity and specificity of this test, but questions of clinical utility remain. Namely, as shown in Figure 1a of the report,5 6 of the 12 FGN patients have DNAJB9 serum concentrations within the range of non-FGN glomerular disease patients, 4 of which are also within the spectrum of normal controls. Conversely, approximately 8 each of Ig light-chain amyloidosis patients (n ¼ 13), non-FGN glomerular disease patients (n ¼ 23), and controls (n ¼ 30) all have serum DNJAB9 levels within the range of values reported for FGN patients. The authors5 also note an inverse relationship between estimated glomerular filtration rate (eGFR) and serum DNAJB9 levels in FGN patients, and raise the possibility of decreased clearance of this protein in chronic kidney disease. This possibility is pertinent, as eGFR differs significantly between studied FGN patients and normal controls (Table 1 of the report5), suggesting that elevation of serum DNAJB9 protein concentrations in FGN and other patients may be due in part to a lower eGFR. The FGN cohort has the lowest median eGFR (35 ml/min per 1.73m2), but this rate is not statistically significantly different from that of non-FGN glomerular disease (42 ml/min per 1.73m2) or Ig light-chain amyloidosis patients (50 ml/min per 1.73m2). To address the biologic challenge of meaningfully assessing serum levels of a protein which may be at least partially renally eliminated, the authors calculate adjusted odds ratios and remove normal controls from some comparisons. After adjusting for eGFR, DNAJB9 levels remain statistically significantly higher in patients with FGN than other disease cohorts. Thus, the authors5 provide convincing evidence that serum DNJAB9 concentrations are significantly higher in FGN patients even after adjusting for the lower eGFR generally seen in FGN patients. The absolute serum DNAJB9 concentration differences among groups are modest, and the ranges have considerable overlap in this discovery cohort 1025
commentary
Figure 1 | DnaJ homolog subfamily B member 9 (DNAJB9) is a biomarker in fibrillary glomerulonephritis (GN).
(see Figure 1 of Nasr and Dasari et al.5). Although the differences are statistically significant, this finding leaves diagnostic uncertainty, particularly for a patient who has a serum DNAJB9 concentration in the lower range for FGN and the upper range for non-FGN glomerular disease (such as 0.4–1.0nM) or one who has, for example, a serum DNAJB9 concentration of 0.46 nM, which would fall within the interquartile range for all 5 cohorts of diseases and normal controls. Thus, this assay would not be expected to obviate the need for a diagnostic kidney biopsy in patients with proteinuria, renal insufficiency, or elevated serum DNAJB9. The work by Nasr and Dasari et al.5 is a step forward in understanding DNAJB9 as a biomarker and reframes some scientific questions about pathogenesis. What do elevated concentrations of DNAJB9 in serum mean from a mechanistic standpoint? What process and/or organ is the major driver of serum DNAJB9 levels? Do elevated protein levels represent a pathogenic mediator of disease or a secondary phenomenon? In glomeruli, abundance of DNAJB9 in FGN was not
1026
accompanied by overexpression of other proteins in the unfolded protein response pathway.1 This finding provides evidence for the diagnostic specificity of DNAJB9, and against the possibility that it represents a nonspecific marker of endoplasmic reticulum stress in FGN. In the current investigation,5 serum was enriched for detection of DNAJB9, and other proteins in the unfolded protein response were not evaluated. A question remains as to whether there is a specific abundance of DNAJB9 in serum of FGN patients, or if this represents an epiphenomenon of alterations in endoplasmic reticulum stress or the unfolded protein response. Future investigations with animal models with elevated serum DNAJB9 levels, or human serum proteome studies in GN patients, would help contextualize the findings. Further quandaries include: To what degree is FGN a systemic or autoimmune disease? Is there a circulating anti-DNAJB9 antibody? The histogenesis of a variety of glomerular, vascular, and tubulointerstitial diseases has centered on the pathogenic effects of auto-antibodies. In view of elevated serum DNAJB9 levels, this remains an important gap in our understanding of FGN, given the codeposited IgG in kidney biopsies. Since its description more than 30 years ago,6 our knowledge of this immune complex–mediated GN with unique fibrillary substructure has evolved. Despite an early period of doubt regarding the existence of FGN as a distinct entity, recent progress has accelerated, with establishment of DNAJB9 as a robust diagnostic immunohistochemical marker for kidney biopsies and now a demonstration of elevated concentrations of the heat shock protein in the blood of affected
patients. Moving forward, could we better assess and adjust attempts at therapeutic intervention by monitoring DNAJB9 levels, as is now done with PLA2R antibody titers in primary membranous nephropathy? Could therapies targeting DNAJB9 affect disease progression? Our field is advanced by the findings described by Nasr and Dasari et al.5 in this issue of Kidney International. Future studies in larger cohorts and perhaps with less technically complex methodology—like an enzyme-linked immunosorbent assay (ELISA)—could potentially validate a clinical utility for DNAJB9 serum testing in diagnosis and management of FGN. DISCLOSURE
The author declared no competing interests.
ACKNOWLEDGEMENTS
The author thanks Megan Troxell, MD, PhD, for critical reading of this commentary. REFERENCES 1. Andeen NK, Yang HY, Dai DF, et al. DnaJ homolog subfamily B member 9 is a putative autoantigen in fibrillary GN. J Am Soc Nephrol. 2018;29:231–239. 2. Dasari S, Alexander MP, Vrana JA, et al. DnaJ heat shock protein family B member 9 is a novel biomarker for fibrillary GN. J Am Soc Nephrol. 2018;29:51–56. 3. Nasr SH, Vrana JA, Dasari S, et al. DNAJB9 is a specific immunohistochemical marker for fibrillary glomerulonephritis. Kidney Int Rep. 2018;3:56–64. 4. Behnke J, Mann MJ, Scruggs FL, et al. Members of the Hsp70 family recognize distinct types of sequences to execute ER quality control. Mol Cell. 2016;63: 739–752. 5. Nasr SH, Dasari S, Lieske JC, et al. Serum levels of DNAJB9 are elevated in fibrillary glomerulonephritis patients. Kidney Int. 2019;95:1269–1272. 6. Alpers CE, Rennke HG, Hopper J Jr, Biava CG. Fibrillary glomerulonephritis: an entity with unusual immunofluorescence features. Kidney Int. 1987;31:781–789.
Kidney International (2019) 95, 1012–1026
Elevated serum concentrations of DNAJB9 in fibrillary glomerulonephritis: another step toward understanding a progressive disease Nicole K. Andeen1 DnaJ homolog subfamily B member 9 (DNAJB9) is a sensitive and specific marker of fibrillary glomerulonephritis (FGN) in kidney biopsies. In this issue, Nasr and Dasari et al. demonstrate significantly elevated concentrations of DNAJB9 in serum from patients with FGN. This advances our understanding of DNAJB9 as a biomarker in FGN and reframes questions about pathogenesis and potential clinical applications of DNAJB9 serum testing. Kidney International (2019) 95, 1025–1026; http://dx.doi.org/10.1016/j.kint.2019.01.034 Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see technical notes on page 1269
I
n 2017, DnaJ homolog subfamily B member 9 (DNAJB9) was identified as a sensitive and specific marker of fibrillary glomerulonephritis (FGN) in kidney biopsies. Its specific abundance was initially discovered with laser capture microdissection of glomeruli followed by liquid chromatography and mass spectrometry; sensitivity and specificity were further demonstrated with immunohistochemistry.1–3 Since its discovery, new questions have arisen regarding the role of this protein in the pathogenesis of FGN. Does DNAJB9 represent an antigen to which an autoantibody develops? DNAJB9 is involved in endoplasmic reticulum stress and the unfolded protein response pathway, and it binds aggregation-prone peptides.4 Could initial deposition of aggregationprone peptides—of as yet undetermined etiology— followed by DNAJB9 binding contribute to the pathogenesis of FGN? DNAJB9 staining in renal biopsies co1 Department of Pathology, Oregon Health & Science University, Portland, Oregon, USA
Correspondence: Nicole K. Andeen Oregon Health & Science University, Department of Pathology, 3181 SW Sam Jackson Park Road, Mail code: L113, Portland, Oregon 97239 USA. E-mail: [email protected] Kidney International (2019) 95, 1012–1026
localizes with IgG deposition within glomerular and extraglomerular deposits; now that a more widely accessible immunohistochemistry-based technique is available, to what extent might these patients have detectable extrarenal immune complex deposition? In the Technical Note in this issue of Kidney International, Nasr and Dasari et al.5report the development of an immunoprecipitation-based multiple reaction monitoring (MRM) test to detect serum levels of DNAJB9 protein in patients with FGN, non-FGN patients, and controls. The test consists of an enrichment step (adding beadbound anti-DNAJB9) followed by a mass spectrometry–based quantification step, using selected peptide sequences. Using this assay, they demonstrate that serum DNAJB9 concentrations are significantly increased in patients with FGN, compared with normal controls, and with patients with either non-FGN glomerular disease, Ig light-chain amyloidosis, or multiple myeloma. Identification of elevated serum concentrations of DNAJB9 in patients with FGN is a novel development that has potential to enhance diagnosis and
management of such patients. From a clinical standpoint, the authors demonstrate statistical sensitivity and specificity of this test, but questions of clinical utility remain. Namely, as shown in Figure 1a of the report,5 6 of the 12 FGN patients have DNAJB9 serum concentrations within the range of non-FGN glomerular disease patients, 4 of which are also within the spectrum of normal controls. Conversely, approximately 8 each of Ig light-chain amyloidosis patients (n ¼ 13), non-FGN glomerular disease patients (n ¼ 23), and controls (n ¼ 30) all have serum DNJAB9 levels within the range of values reported for FGN patients. The authors5 also note an inverse relationship between estimated glomerular filtration rate (eGFR) and serum DNAJB9 levels in FGN patients, and raise the possibility of decreased clearance of this protein in chronic kidney disease. This possibility is pertinent, as eGFR differs significantly between studied FGN patients and normal controls (Table 1 of the report5), suggesting that elevation of serum DNAJB9 protein concentrations in FGN and other patients may be due in part to a lower eGFR. The FGN cohort has the lowest median eGFR (35 ml/min per 1.73m2), but this rate is not statistically significantly different from that of non-FGN glomerular disease (42 ml/min per 1.73m2) or Ig light-chain amyloidosis patients (50 ml/min per 1.73m2). To address the biologic challenge of meaningfully assessing serum levels of a protein which may be at least partially renally eliminated, the authors calculate adjusted odds ratios and remove normal controls from some comparisons. After adjusting for eGFR, DNAJB9 levels remain statistically significantly higher in patients with FGN than other disease cohorts. Thus, the authors5 provide convincing evidence that serum DNJAB9 concentrations are significantly higher in FGN patients even after adjusting for the lower eGFR generally seen in FGN patients. The absolute serum DNAJB9 concentration differences among groups are modest, and the ranges have considerable overlap in this discovery cohort 1025
commentary
Figure 1 | DnaJ homolog subfamily B member 9 (DNAJB9) is a biomarker in fibrillary glomerulonephritis (GN).
(see Figure 1 of Nasr and Dasari et al.5). Although the differences are statistically significant, this finding leaves diagnostic uncertainty, particularly for a patient who has a serum DNAJB9 concentration in the lower range for FGN and the upper range for non-FGN glomerular disease (such as 0.4–1.0nM) or one who has, for example, a serum DNAJB9 concentration of 0.46 nM, which would fall within the interquartile range for all 5 cohorts of diseases and normal controls. Thus, this assay would not be expected to obviate the need for a diagnostic kidney biopsy in patients with proteinuria, renal insufficiency, or elevated serum DNAJB9. The work by Nasr and Dasari et al.5 is a step forward in understanding DNAJB9 as a biomarker and reframes some scientific questions about pathogenesis. What do elevated concentrations of DNAJB9 in serum mean from a mechanistic standpoint? What process and/or organ is the major driver of serum DNAJB9 levels? Do elevated protein levels represent a pathogenic mediator of disease or a secondary phenomenon? In glomeruli, abundance of DNAJB9 in FGN was not
1026
accompanied by overexpression of other proteins in the unfolded protein response pathway.1 This finding provides evidence for the diagnostic specificity of DNAJB9, and against the possibility that it represents a nonspecific marker of endoplasmic reticulum stress in FGN. In the current investigation,5 serum was enriched for detection of DNAJB9, and other proteins in the unfolded protein response were not evaluated. A question remains as to whether there is a specific abundance of DNAJB9 in serum of FGN patients, or if this represents an epiphenomenon of alterations in endoplasmic reticulum stress or the unfolded protein response. Future investigations with animal models with elevated serum DNAJB9 levels, or human serum proteome studies in GN patients, would help contextualize the findings. Further quandaries include: To what degree is FGN a systemic or autoimmune disease? Is there a circulating anti-DNAJB9 antibody? The histogenesis of a variety of glomerular, vascular, and tubulointerstitial diseases has centered on the pathogenic effects of auto-antibodies. In view of elevated serum DNAJB9 levels, this remains an important gap in our understanding of FGN, given the codeposited IgG in kidney biopsies. Since its description more than 30 years ago,6 our knowledge of this immune complex–mediated GN with unique fibrillary substructure has evolved. Despite an early period of doubt regarding the existence of FGN as a distinct entity, recent progress has accelerated, with establishment of DNAJB9 as a robust diagnostic immunohistochemical marker for kidney biopsies and now a demonstration of elevated concentrations of the heat shock protein in the blood of affected
patients. Moving forward, could we better assess and adjust attempts at therapeutic intervention by monitoring DNAJB9 levels, as is now done with PLA2R antibody titers in primary membranous nephropathy? Could therapies targeting DNAJB9 affect disease progression? Our field is advanced by the findings described by Nasr and Dasari et al.5 in this issue of Kidney International. Future studies in larger cohorts and perhaps with less technically complex methodology—like an enzyme-linked immunosorbent assay (ELISA)—could potentially validate a clinical utility for DNAJB9 serum testing in diagnosis and management of FGN. DISCLOSURE
The author declared no competing interests.
ACKNOWLEDGEMENTS
The author thanks Megan Troxell, MD, PhD, for critical reading of this commentary. REFERENCES 1. Andeen NK, Yang HY, Dai DF, et al. DnaJ homolog subfamily B member 9 is a putative autoantigen in fibrillary GN. J Am Soc Nephrol. 2018;29:231–239. 2. Dasari S, Alexander MP, Vrana JA, et al. DnaJ heat shock protein family B member 9 is a novel biomarker for fibrillary GN. J Am Soc Nephrol. 2018;29:51–56. 3. Nasr SH, Vrana JA, Dasari S, et al. DNAJB9 is a specific immunohistochemical marker for fibrillary glomerulonephritis. Kidney Int Rep. 2018;3:56–64. 4. Behnke J, Mann MJ, Scruggs FL, et al. Members of the Hsp70 family recognize distinct types of sequences to execute ER quality control. Mol Cell. 2016;63: 739–752. 5. Nasr SH, Dasari S, Lieske JC, et al. Serum levels of DNAJB9 are elevated in fibrillary glomerulonephritis patients. Kidney Int. 2019;95:1269–1272. 6. Alpers CE, Rennke HG, Hopper J Jr, Biava CG. Fibrillary glomerulonephritis: an entity with unusual immunofluorescence features. Kidney Int. 1987;31:781–789.
Kidney International (2019) 95, 1012–1026