Background Despite optimal treatment, lupus nephritis (LN) remains associated with irreversible kidney damage.¹ A better understanding of the mechanisms underlying LN pathogenesis is needed to develop better treatment targets. As part of the Accelerating Medicines Partnership (AMP), we discovered that urinary PR3, a myeloid degranulation product, correlated with histological activity implicating neutrophil/monocyte degranulation in proliferative LN, the most aggressive type.² PR3 is a serine protease that can mediate kidney damage. Mature neutrophils with classical polylobate nuclei are rare in LN kidney biopsies. However, recent evidence displayed how immature, degranulating myeloid cells have been implicated in the pathogenesis of LN,³ but their role in mediating kidney damage is not completely understood.

Objectives To investigate PR3+ cells in LN kidney, their association with histopathological features, and define their immunophenotype.

Methods We performed multiplexed histology using serial immunohistochemistry (sIHC)⁴ on archival LN kidney biopsies to quantify the expression of PR3 and multiple cell lineage markers (20-plex). Image analysis including deconvolution, cell segmentation, glomerular annotation, and quantitative histology was performed using Indica HALO. The analysis was limited to renal cortex.

Results A total of 11 patients with LN who underwent a clinically indicated kidney biopsy were enrolled: 6 (55%) with pure proliferative LN (ISN/RPS class III or IV) and 5 (45%) with pure membranous LN. PR3+ cells were identified in all LN biopsies (range 343–7625 per sample). Most PR3+ cells did not show a polylobate nucleus. The majority of PR3+ cells were in the tubulointersitium (figure 1A). However, accounting for the smaller glomerular area, there was a higher density of PR3+ cells in the glomeruli (figure 1A-C). PR3+ cell abundance was higher in proliferative LN, especially in the glomeruli (figure 1A-C). Glomerular PR3+ cell density very strongly correlated with histological activity measured by the NIH Activity Index (Pearson’s r=0.97, p=5*10^-5; figure 1D). Preliminary serial IHC analysis showed that PR3+ cells coexpressed MPO and variably coexpressed CD66b and CD14, but not neutrophil elastase, CD3, or CD20.

Conclusions PR3+ cells are abundant in LN. PR3+ cells are increased in proliferative LN and are strongly associated with histological activity thereby characterizing a more aggressive phenotype. This population densely infiltrated the glomeruli emphasizing a potential role in the endothelial pathogenic process. In preliminary analysis, kidney infiltrating PR3+ cells were not polymorphonucleated, did not express neutrophil elastase, and variably expressed CD14 suggesting a phenotype consistent with degranulating monocyte or an immature myeloid population. We previously showed the association between urinary PR3 and histological activity suggesting that intrarenal PR3+ cells are actively degranulating and therefore likely inducing kidney damage. These findings nominate PR3+ cells as a potential therapeutic target. Spatial transcriptomics and proteomic studies are ongoing to define the lineage and function of these cells.

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Abstract 1011 Figure 1

References

1. Mahajan A, et al. Systemic lupus erythematosus, lupus nephritis and end-stage renal disease: a pragmatic review mapping disease severity and progression. Lupus 2020;29:1011–1020

2. Fava A, et al. A neutrophil degranulation signature identifies proliferative lupus nephritis. Arthritis Rheumatol. 2021;73(suppl 9).

3. Mistry P, et al. Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus. Proc. Natl. Acad. Sci. U. S. A. 2019;116:25222–25228

4. Akturk G, Sweeney R, Remark R, Merad M, Gnjatic S. Multiplexed immunohistochemical consecutive staining on single slide (MICSSS): multiplexed chromogenic IHC assay for high-dimensional tissue analysis. Methods Mol. Biol. 2020;2055:497–519.