B7-1 Is Not Induced in Podocytes of Human and Experimental Diabetic Nephropathy

Abstract

The incidence of progressive kidney disease associated with diabetes continues to rise worldwide. Current standard therapy with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers achieves only partial renoprotection, increasing the need for novel therapeutic approaches. Previous studies described B7-1 induction in podocytes of patients with proteinuria, including those with FSGS and type 2 diabetic nephropathy (DN). These findings sparked great excitement in the renal community, implying that abatacept, a costimulatory inhibitor that targets B7-1, could be a novel therapy for diabetic renal disease. Given previous concerns over the value of B7-1 immunostaining and the efficacy of abatacept in patients with recurrent FSGS after renal transplantation, we investigated B7-1 expression in human and experimental DN before embarking on clinical studies of the use of B7-1 targeting strategies to treat proteinuria in DN. Immunohistochemical analysis of kidney specimens using different antibodies revealed that B7-1 is not induced in podocytes of patients with DN, independent of disease stage, or BTBR ob/obmice, a model of type 2 diabetes. These results do not support the use of abatacept as a therapeutic strategy for targeting podocyte B7-1 for the prevention or treatment of DN.

Keywords: B7–1; diabetic nephropathy; podocyte.

Figure 1.

B7-1 is not expressed in glomeruli of patients with diabetes and overt nephropathy. (A–C) Representative images of B7–1 immunofluorescence staining (red; with polyclonal goat anti–human B7–1 antibody) in glomeruli of control subjects and patients with diabetes. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), and renal structures were stained with fluorescein wheat germ agglutinin (WGA; green). (A′–C′) The images are also shown without WGA to highlight the B7–1 signal, although it is scarce. (A and A′) Glomerular B7–1 was absent in the control subjects. (B and B′) The 55% of patients with diabetes and overt nephropathy displayed glomeruli that were completely negative for B7–1 staining. (C and C′) In the glomeruli of the remaining patients’ biopsy samples (45%), few B7–1 signal spots were observed. (D–F) Representative images of B7–1 immunoperoxidase staining (with the monoclonal mouse anti–human B7–1 antibody) in kidney biopsies of control subjects and patients with diabetes. (D) Glomerular B7–1 staining was not detectable in control subjects. (E) In patients with diabetes, glomeruli were completely negative, whereas interstitial inflammatory cells (arrows) and tubular epithelial cells (asterisks) showed a strong B7–1 signal. (F) B7–1-positive inflammatory infiltrate (arrows) and tubular epithelium (asterisks) found in diabetic kidney biopsies. Scale bar, 50 μm.

Figure 2.

B7-1 is not expressed in glomeruli of BTBR WT and BTBR ob/ob mice euthanized at 20-22 weeks of age. (A and B) Representative images of glomerular B7–1 staining (red; with the polyclonal goat anti–mouse antibody) in kidney sections of BTBR WT and BTBR ob/ob mice. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), and renal structures were stained with fluorescein wheat germ agglutinin (WGA; green). (A′ and B′) Each picture is also shown without WGA to point out B7–1 signal. (A and A′) Faint B7–1 glomerular expression was found in renal sections of BTBR WT mice comparable with the B7–1 expression observed in glomeruli of BTBR ob/ob mice (B and B′). (C and C′) Costaining for B7–1 (red) and PDX (green) in BTBR ob/ob glomeruli detected as just a few dots of colocalization (Inset) between the two markers. (D–F) Representative micrographs of B7–1 immunostaining (red) in BTBR murine renal samples using the monoclonal hamster anti–mouse B7–1 antibody. B7–1 glomerular staining was not detectable in glomeruli of (D) BTBR WT and (E) diabetic BTBR ob/ob mice. (E and F) Occasional B7–1-positive inflammatory cells were found in diabetic renal samples (arrows). Scale bar, 20 μm. PDX, podocalyxin.

Figure 3.

B7-1 is not expressed in glomeruli of C57BL/6 mice with STZ-induced diabetes euthanized at 16-20 weeks after disease induction. (A and B) Representative micrographs of B7–1 immunostaining (red; with the polyclonal goat anti–mouse antibody) in renal samples. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), and renal structures were stained with fluorescein wheat germ agglutinin (WGA; green). (A′ and B′) Each picture is also shown without WGA to highlight the B7–1 signal. (A and A′) Spots of B7–1 staining were observed in glomeruli of control C57BL/6 mice, and almost the same glomerular B7–1 expression was detected in C57BL/6 mice after STZ injection (B and B′). (C and C′) Double staining for B7–1 (red) and PDX (green) in glomeruli of STZ–injected C57BL/6 mice showed very few spots of colocalization (Inset) between the two markers. (D–F) Representative micrographs of B7–1 immunostaining (red) in C57BL/6 murine renal samples using the monoclonal hamster anti–mouse B7–1 antibody. No glomerular B7–1 staining was observed in (D) control or (E) diabetic C57BL/6 mice, whereas (F) B7–1-positive inflammatory cells were detected in the renal interstitium (arrow). Scale bar, 20 μm. PDX, podocalyxin.