Sealfon et al.1 recently reported the molecular characterization of membranous nephropathy (MN) using the Nephrotic Syndrome Study Network (NEPTUNE) cohort and the European Renal cDNA Bank (ERCB) cohort. They took a machine-learning approach to identify molecular characteristics of MN, but we believe that in silico–based approaches can also provide a clue to discover new MN antigens.
Eight MN antigens have been identified to date, starting with PLA2R,2 but others may exist. Rather than the NEPTUNE and ERCB databases used by Sealfon et al., we used Nephroseq, a web-based dataset that provides information on genome-wide gene expression,3 to examine transcription patterns in various types of kidneys. We applied Nephroseq according to the following protocol.
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(1)
Using the Ju CKD Glom dataset, place the genes with higher expression in MN than in other diseases in order, from the top rank to 100. By doing so, we unexpectedly observed that three of the eight known MN antigens were ranked in the top 0.6% when we compared MN with other renal diseases: NELL1 (ranked first, P=7.9 × 10−10), PCDH7 (ranked 27th, P=2.2 × 10−6), and PLA2R (ranked 110th, P=4.7 × 10−5).
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(2)
Check where in the cell each gene is expressed, and identify and retain genes encoding secreted and membrane-bound proteins as candidates; exclude others.
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(3)
Using the Ju CKD Glom dataset, exclude genes whose expression levels are lower in MN compared with in healthy living donors or in patients with minimal change nephrotic syndrome.
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(4)
Using the Lindenmeyer Normal Tissue Panel dataset, compare expression levels in glomeruli to those in tubulointerstitium, and exclude genes whose expression is higher in tubulointerstitium than in glomeruli.
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(5)
Search the encoded proteins’ characteristics and check the staining patterns in the kidney in the Human Protein Atlas, as done by Sealfon et al.1
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(6)
Perform immunofluorescence staining. After this procedure, we observed that eight of the top 100 genes remaining (TSPAN6, FAM189A2, PAMR1, AMIGO2, ANXA1, LHFP, PON2, P4HTM) encode proteins in the glomerulus. We performed immunofluorescence staining for these candidate proteins in MN tissue samples (n=19) that were negative for PLA2R, THSD7A, and NELL1. All eight candidates were negative in immunohistochemistry, but, notably, five (TSPAN6, PAMR1, AMIGO2, ANXA1, PON2) were also listed in the Sealfon group’s MN-specific gene list.
This type of unprecedented approach has the potential to open new avenues to MN antigen exploration.
Disclosures
K. Yamagata reports receiving honoraria from Astellas, AstraZeneca, Kyowa-Kirin, Mochida, Otsuka, and Tanabe-Mitsubishi; having consultancy agreements with Chugai Co. Ltd. and Japan Medtronic Co. Ltd.; and receiving research funding from Kyowa-Kirin and Tanabe-Mitsubishi. All remaining authors have nothing to disclose.
Funding
This work was supported by Japan Society for the Promotion of Science KAKENHI grants JP22J20394, JP19KK0216, and JP21H02822.
Acknowledgments
We are deeply grateful to Ms. Mikie Daigo and Ms. Mami Kobayashi (University of Tsukuba, Tsukuba, Japan) for their technical support.
Footnotes
Published online ahead of print. Publication date available at www.jasn.org.
Author Contributions
M. Takahashi-Kobayashi, J. Usui, and K. Kawanishi were responsible for data curation; M. Takahashi-Kobayashi and J. Usui conceptualized the study, wrote the original draft, and were responsible for formal analysis, funding acquisition, investigation, and methodology; M. Takahashi-Kobayashi, J. Usui, and K. Yamagata were responsible for project administration; and all authors reviewed and edited the manuscript.
References
- 1.Sealfon R, Mariani L, Avila-Casado C, Nair V, Menon R, Funk J, et al. : Molecular characterization of membranous nephropathy. J Am Soc Nephrol 33: 1208–1221, 2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Beck LH Jr, Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. : M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 361: 11–21, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Nephroseq v5. University of Michigan, Ann Arbor, MI, USA. Available at: https://nephroseq.org. Accessed December 15, 2021