Abatacept in B7-1-positive proteinuric kidney disease

Abstract

Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize β1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.

Figure 1. Blockade of Disease-Associated Podocyte Migration by Abatacept

Panel A shows Western blot analysis of B7-1 protein in podocytes with stable transfection of vector control (pcDNA), B7-1, or B7-1Δtail before the addition of lipopolysaccharide (LPS), which was the control condition, and after the addition of LPS. Panel B shows B7-1 protein in normal podocytes, non-silencing short hairpin RNA (shRNA)–expressing podocytes (scrambled), B7-1 knockdown podocytes (B7-1kd), scrambled shRNA-expressing α3^−/− podocytes (α3^−/−/scrambled), and B7-1 knockdown α3^−/− podocytes (α3^−/−/B7-1kd). In the analyses shown in Panels A and B, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Panel C shows the results of quantitative analysis of podocyte migration. P values were calculated with the use of Bonferroni’s multiple-comparison test; see the Supplementary Appendix for details. The T bars indicate standard deviations.

Figure 2. Disruption of the Binding of Talin to β1 Integrin, but Not to β3 Integrin, by B7-1

As shown in Panel A, endogenous talin coprecipitated with activated β1 integrin in normal podocytes but not in α3−/− podocytes. Immunoprecipitation (IP) with anti–green fluorescent protein (GFP) antibody served as a negative control. Input refers to protein extracts that served as starting material from which endogenous proteins were immunoprecipitated. As shown in Panel B, FLAG–B7-1 did not bind to talin (GFP–talin-HN, left lane); HN denotes head N-terminal domain. GFP–B7-1-tail but not GFP–B7-1Δ tail blocked the interaction of talin (GFP–talin-HN) with β1 integrin (FLAG–β1ΔEC) in cotransfected HEK293 cells. Instead, B7-1-tail coprecipitated with β1 integrin. As shown at the top of Panel C, immobilized β1 integrin (GST–β1ΔEC) but not the GST control bound directly to purified talin (FLAG–talin-HN). In the presence of increasing amounts of FLAG–B7-1ΔEC, the binding of talin-HN to GST–β1ΔEC was gradually lost, whereas the binding of B7-1 to β1 integrin could be detected. As shown at the bottom of Panel C, Coomassie-stained sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE) analysis showed the purity of recombinant proteins. As shown in Panel D, co-expression of GFP–B7-1-tail did not block the interaction of talin (GFP–talin-HN) with β3 integrin (FLAG–β3ΔEC) in triple-transfected HEK293 cells. Instead, both GFP–B7-1-tail and GFP–talin-HN coprecipitated with FLAG–β3ΔEC. No binding was found with the fusion protein GFP–sui (negative control).