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American Journal of Kidney Diseases

B7-1 Immunostaining in Proteinuric Kidney Disease

Published:September 30, 2014DOI:https://doi.org/10.1053/j.ajkd.2014.07.023
      To the Editor:
      B7-1–Positive proteinuric kidney disease is a recently described subcategory of primary podocytopathy in which immunostaining is reported to detect B7-1 in the podocyte cytoplasm.
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      Through inactivation of β1 integrin, podocytes upregulate B7-1 in many forms of experimental kidney diseases with proteinuria, including immune-mediated, drug-induced, genetic, and bacterial toxin–induced diseases.
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      • Reiser J.
      • von Gersdorff G.
      • Loos M.
      • et al.
      Induction of B7-1 in podocytes is associated with nephrotic syndrome.
      • Doria A.
      • Niewczas M.A.
      • Fiorina P.
      Can existing drugs approved for other indications retard renal function decline in patients with type 1 diabetes and nephropathy?.
      A recent report observed podocyte B7-1 immunostaining in >50% of randomly selected kidney biopsy specimens from patients with proteinuric kidney disease.
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      This staining was thought to reflect disease-related B7-1 induction that might be a useful companion diagnostic for treatment of glomerulopathies with abatacept, a CTLA4 agonist that inhibits B7-1. This report generated significant excitement because it suggested an additional treatment for a disease that currently has limited therapeutic options. However, subsequent correspondence raised doubts about the validity of the immunohistochemical procedures used to detect B7-1, as well as the frequency of response to this agent.
      • Benigni A.
      • Gagliardini E.
      • Remuzzi G.
      Abatacept in B7-1-positive kidney disease.
      • Alachkar N.
      • Carter-Monroe N.
      • Reiser J.
      Abatacept in B7-1 positive proteinuric kidney disease.
      We sought to determine the prevalence of B7-1–positive proteinuric kidney disease in a large cohort using 2 different staining techniques.
      Sixty biopsy specimens were stained for B7-1 during the study period. All patients had nephrotic syndrome at the time of the biopsy. Clinical and demographic details are shown in Table 1. All cases were subjected to an immunoperoxidase staining assay performed on paraffin-embedded tissue after retrieval and a previously described immunofluorescence assay on fresh tissue,
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      provided tissue was available for both assays (see Item S1).
      Table 1Clinical Characteristics of Cases Stained With B7-1, by Biopsy Diagnosis
      CharacteristicValue
      Primary FSGS, NOS28 (47)
       Age (y)40.3 ± 21.9
       Proteinuria (g/24 h)6.7 ± 6.5
       Native vs transplant biopsy22:7
      FSGS, tip variant5 (8)
       Age (y)53.2 ± 6.4
       Proteinuria (g/24 h)7.7 ± 4.7
       Native vs transplant biopsy4:1
      Collapsing glomerulopathy5 (8)
       Age (y)49.6 ± 15.6
       Proteinuria (g/24 h)5.4 ± 3.5
       Native vs transplant biopsy5:0
      Minimal change disease19 (32)
       Age (y)35.3 ± 23.4
       Proteinuria (g/24 h)6.8 ± 3.6
       Native vs transplant biopsy19:0
      Membranous glomerulopathy3 (5)
       Age (y)46.0 ± 24.3
       Proteinuria (g/24 h)3.6 ± 0.4
       Native vs transplant biopsy3:0
      Note: Continuous values given as mean ± SD, categorical values given as number (percentage).
      Abbreviations: FSGS, focal segmental glomerulosclerosis; NOS, not otherwise specified.
      For both staining techniques and in all cases, B7-1 was undetectable within podocytes. By immunofluorescence, membranous glomerulopathy cases showed strong granular capillary staining, but in each case, there was judged to be identically intense staining on the negative control (no primary antibody) slide, suggestive of staining by the secondary antibody. Similarly, many podocytopathy cases showed dusty granular podocyte staining that was identical to that seen on the negative control. These cases were not scored as positive because podocyte staining in the B7-1–stained section did not exceed that present in the negative control. In all cases, including those with secondary antibody staining by immunofluorescence, the immunoperoxidase stain did not detect B7-1–positive podocytes. Focal interstitial inflammatory cells stained positive for B7-1 in most cases and served as an internal positive control (Fig 1).
      Figure thumbnail gr1
      Figure 1Immunohistochemical detection of B7-1 in a biopsy specimen from a patient with recurrent nephrotic syndrome after transplantation. (A) Interstitial activated B cells and monocytes show positive B7-1 staining. (B) Interstitial inflammatory cells show strong internal positive control staining for B7-1 while glomerulus (arrow) is completely negative.
      The report of B7-1–positive proteinuric kidney disease aroused hope for a new era of personalized medicine in nephrology. However, closer examination reveals several issues with the report by Yu et al
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      that must be addressed before patients are treated based on the results reported. First, the report is very small, with only 5 patients receiving treatment, all of whom showed positive staining for B7-1 within podocytes. It was presumed that the B7-1 staining indicated upregulation of this protein and an increased likelihood of treatment response to abatacept. However, no control group without staining was reported to have been treated, making it impossible to know whether B7-1–positive staining was a marker of disease response with abatacept. Even more disconcerting than the small sample size and lack of treatment controls is the lack of a negative control in the staining procedure, which almost certainly resulted in false-positive staining and reporting of B7-1 staining in podocytes that was the result of the secondary antibody.
      • Benigni A.
      • Gagliardini E.
      • Remuzzi G.
      Abatacept in B7-1-positive kidney disease.
      Of note, Yu et al
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      reported strong staining in membranous glomerulopathy. Many cases in our study showed B7-1 staining by immunofluorescence, but it was identical to staining in the negative control and therefore was determined to be an artifact. In all our podocytopathy cases, the pattern of immunofluorescence staining for B7-1 and the negative control was consistent with the podocyte IgG staining, which commonly is regarded as nonspecific in these cases.
      • D'Agati V.D.
      • Jennette J.C.
      • Silva F.G.
      Non-Neoplastic Kidney Diseases.
      Our membranous cases showed a pattern of staining that also was identical to that typically seen by IgG. Using the immunoperoxidase technique, which does not have this secondary antibody artifact, no podocyte staining was detected.
      Although results of our case series raise significant doubts about the utility of B7-1 staining as an indication that patients may respond to abatacept, this does not address the potential value of abatacept treatment of patients with steroid-resistant nephrotic syndrome. There are data to support upregulation of this gene in glomerulopathy
      • Reiser J.
      • von Gersdorff G.
      • Loos M.
      • et al.
      Induction of B7-1 in podocytes is associated with nephrotic syndrome.
      and a larger trial certainly is warranted given the response in the 5 patients described.
      • Yu C.C.
      • Fornoni A.
      • Weins A.
      • et al.
      Abatacept in B7-1-positive proteinuric kidney disease.
      Future studies should focus on searching for additional biomarkers to guide therapeutic decision making.
      In summary, we report the first large case series of B7-1 staining in kidney biopsies with a podocytopathy. We failed to identify any cases with positive B7-1 podocyte staining after evaluating 60 biopsy specimens using 2 different antibodies. One other recent report also supports the uncommon nature of this B7-1 staining in FSGS.

      Cara-Fuentes G, Johnson RJ, Reiser J, Garin EH. CD80 and suPAR in patients with minimal change disease and focal segmental glomerulosclerosis: diagnostic and pathogenic significance: response [published online ahead of print May 11, 2014]. Pediatr Nephrol. http://dx.doi.org/10.1007/s00467-014-2840-5.

      We conclude that B7-1 immunostaining is unlikely to serve as a useful diagnostic stain supporting the use of abatacept therapy.

      Acknowledgements

      We thank Dr William Couser for reviewing the manuscript and our colleagues at Nephropath for input into the study.
      Support: None.
      Financial Disclosure: The authors declare that they have no relevant financial interests.
      Contributions: Research idea and study design: CPL, NCM; data acquisition: CPL; data analysis/interpretation: CPL, NCM; supervision or mentorship: PDW. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. CPL takes responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted, and that any discrepancies from the study as planned have been explained.

      Supplementary Material

      References

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