Glomerular membrane attack complex is not a reliable marker of ongoing C5 activation in lupus nephritis

Abstract

Complement plays an important role in the pathogenesis of lupus nephritis (LN). With the emergence of therapeutic complement inhibition, there is a need to identify patients in whom complement-driven inflammation is a major cause of kidney injury in LN. Clinical and histopathological data were obtained retrospectively from 57 biopsies with class III, IV, and V LN. Biopsies were stained for complement components C9, C5b-9, C3c, and C3d and for the macrophage marker CD68. C9 and C5b-9 staining were highly correlated (r = 0.92 in the capillary wall). C5b-9 staining was detected in the mesangium and/or capillary wall of both active and chronic proliferative LN in all but one biopsy and in the capillary wall of class V LN in all biopsies. C5b-9 staining intensity in the tubular basement membrane correlated with markers of tubulointerstitial damage, and more intense capillary wall C5b-9 staining was significantly associated with nonresponse to conventional treatment. Glomerular C5b-9 staining intensity did not differ between active and chronic disease; in contrast, C3c and CD68 staining were associated with active disease. Evaluation of serial biopsies and comparison of staining in active and chronic LN demonstrated that C5b-9 staining persisted for months to years. These results suggest that C5b-9 staining is almost always present in LN, resolves slowly, and is not a reliable marker of ongoing glomerular C5 activation. This limits the utility of C5b-9 staining to identify patients who are most likely to benefit from C5 inhibition.

Keywords: complement; glomerulonephritis; renal pathology; systemic lupus erythematosus.

Graphical abstract

Figure 1

Glomerular immunostaining for complement C5b-9 in control biopsies. Representative images of C5b-9 staining in (a) membranous nephropathy and (b) a renal biopsy reported as within normal parameters. Grade 3 capillary wall staining is seen in membranous nephropathy. No staining is evident in the normal biopsy. Original magnification ×20. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 2

Glomerular immunostaining for complement C9 and C5b-9. Representative images of (a) capillary wall and (b) mesangial staining using antibodies to either C9 or C5b-9. Serial sections from the same biopsy were used. Images depict grade 0, 1, 2, and 3 staining. Staining intensity correlated for each antibody (capillary wall: r = 0.92, P < 0.0001 and mesangium: r = 0.86, P < 0.0001). Arrows indicate vascular staining. Original magnification ×20. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 3

Correlation of glomerular complement C3c staining intensity and maximum glomerular CD68 count. C3c staining intensity in the (a) capillary wall and (b) mesangium correlated significantly with maximum glomerular CD68 count (r = 0.57, P = 0.0003 and r = 0.51, P = 0.002, respectively).

Figure 4

Correlation of glomerular complement C3c staining intensity and maximum glomerular CD68 count with serum C3, serum C4, and anti–double-stranded DNA (dsDNA) titer. Mesangial C3c staining intensity correlated significantly with serum C3 (r = −0.53, P < 0.001), serum C4 (r = −0.44, P = 0.004), and dsDNA (r = 0.53, P = 0.003). Capillary wall C3c staining intensity correlated significantly with serum C3 (r = −0.49, P = 0.001), serum C4 (r = −0.56, P = 0.0001), and dsDNA (r = 0.43, P = 0.017). Maximum glomerular CD68 count correlated significantly with serum C3 (r = −0.64, P < 0.00001), serum C4 (r = −0.61, P < 0.0001), and dsDNA (r = 0.78, P < 0.000001).

Figure 5

Correlation of complement C3c staining intensity, maximum glomerular CD68 count, and serum complement markers with renal biopsy features. The percentage of glomeruli with active disease correlated significantly with capillary wall C3c staining intensity (r = 0.66, P < 0.00001), maximum glomerular CD68 count (r = 0.75, P < 0.0000001), serum C3 (r = −0.65, P < 0.00001), and serum C4 (r = −0.62, P < 0.00001). The percentage of glomeruli with crescents correlated significantly with capillary wall C3c staining intensity (r = 0.48, P = 0.002), maximum glomerular CD68 count (r = 0.45, P = 0.003), serum C3 (r = −0.38, P = 0.01), and serum C4 (r = −0.38, P = 0.01). The percentage of glomeruli with chronic disease correlated significantly with capillary wall C3c staining intensity (r = −0.41, P = 0.01), maximum glomerular CD68 count (r = −0.61, P < 0.0001), serum C3 (r = 0.68, P < 0.000001), and serum C4 (r = 0.69, P < 0.000001).

Figure 6

Capillary wall C5b-9 and mesangial C3c staining intensities and treatment response. There was a significant difference in capillary wall C5b-9 staining intensity between treatment responses at 1 year. Patients with more intense C5b-9 staining were more likely to be nonresponders (P = 0.01). There was a significant difference in mesangial C3c staining intensity between treatment responses at 1 year. Patients with more intense C3c staining were more likely to be responders (P = 0.04). CR, complete response; NR, nonresponse; PR, partial response.

Figure 7

Representative images of tubular basement membrane C5b-9 staining. (a) Grade 0 staining in a case with 0% interstitial fibrosis and tubular atrophy (IFTA). (b) Grade 1 staining in a case with 15% IFTA. (c) Grade 2 staining in a case with 30% IFTA. The presence of IFTA positively correlated with the intensity of C5b-9 staining (r = 0.50, P = 0.0001). Original magnification ×20. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 8

Glomerular C5b-9 staining in serial biopsies. Representative C5b-9 staining in sequential renal biopsies pretreatment (biopsy 1) and posttreatment (biopsy 2), with varying interbiopsy intervals. Original magnification ×20. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.