Miho Karube

Tubulointerstitial nephritis without glomerular lesions in three patients with myeloperoxidase-ANCA-associated vasculitis

BackgroundMyeloperoxidase–antineutrophil cytoplasmic antibody (MPO–ANCA)-associated vasculitis frequently induces crescentic glomerulonephritis. However, a few cases have so far been reported to have only tubulointerstitial (TI) nephritis without any apparent glomerular lesions. We recently treated three similar cases. Therefore, their pathological features as well as clinical manifestations were studied in detail.MethodsThe pathological study was performed with immunohistochemical staining using various antibodies to the vascular endothelial cell surface markers, von Willebrand factor, type IV collagen, cytokeratin, E-cadherin, and MPO in addition to the routine histochemical examination.ResultsThe study disclosed the loss of CD34 endothelial cell surface markers with and without the destruction of type IV collagen (capillary basement membrane) in the peritubular capillaries, even though the glomeruli showed good staining of these factors. Electron microscopy showed breaks in the capillary basement membrane. The loss of CD34 staining was associated with the infiltration of a few mononuclear cells and neutrophils in the lumen of peritubular capillaries and the surrounding interstitial tissues. The cytokeratin staining in the tubular epithelial cells was also diminished around these areas. Tubulitis was demonstrated with or without the destruction of the tubular basement membrane. The clinical manifestations of these three cases were only a few red blood cells and granular casts in the urinary sediment as well as slightly increased β2-microglobulin in the urine, but no proteinuria.ConclusionBased on these findings, the loss of CD34 vascular endothelial markers occurs in the early phase of the disease because of the MPO, which is presumed to have burst out from the infiltrated, activated neutrophils. This MPO, which releases proteolytic enzymes and radical oxygen species, acts on tissue destruction, namely the lysis of endothelial cell membranes as well as vascular basement membranes in the peritubular capillary. This mechanism eventually proceeds to the destruction of the peritubular capillary walls (vasculitis). This pathogenesis is thought to play an important role in the pathogenesis of TI nephritis, which is associated with MPO–ANCA vasculitis.

Tubulointerstitial immune complex nephritis in a patient with systemic lupus erythematosus: role of peritubular capillaritis with immune complex deposits in the pathogenesis of the tubulointerstitial nephritis

The correct name of the fourth author should be given as Yoshihiro Arimura, not Yoshiro Arimura.

Lipoprotein glomerulopathy-like disease in a patient with type III hyperlipoproteinemia due to apolipoprotein E2 (Arg158 Cys)/3 heterozygosity

A 77-year-old woman developed nephrotic syndrome associated with type III hyperlipoproteinemia (III HLP) and increased apolipoprotein E (apo E). Apo E analysis disclosed E2/E3 heterozygosity in phenotypic and genotypic expressions, without any other mutations. A renal biopsy showed intraluminal and subendothelial thrombus-like deposits in the dilated capillary loops of the glomerulus that stained positive for lipids and apo E. Electron microscopy revealed tiny granular particles in the capillary lumina, as well as between the glomerular basement membrane and the endothelial cells. It was therefore concluded that III HLP associated with apo E2/E3 heterozygosity could induce lipoprotein glomerulopathy-like disease and nephrotic syndrome.

SAT0516 Two distinct subsets of low density granulocytes in anca associated vasculitis

Background Low density granulocyte (LDG), a proinflammatory population of neutrophils, was first described in systemic lupus erythematosus (SLE) and has been shown to contribute to lupus pathogenesis. It has been suggested that LDGs have a pathogenic role in ANCA associated vasculitis (AAV) based on the data of gene expression signature in AAV and the ability of excessive neutrophil extracellular traps (NETs) formation by LDGs in AAV. However, more detailed analysis of LDG in AAV patients has not been reported. Objectives In this study we investigated the characteristics of LDG in AAV patients using flow cytometry and proteomics approach and examined the correlations with disease activity. Methods We examined the presence of LDGs in peripheral blood of 10 AAV patients before treatment and followed them for 4 months with immunosuppressive therapy. Normal-density granulocytes (NDGs) were isolated by dextran sedimentation and PBMCs were isolated by Ficoll gradient. LDGs were assessed using cell surface expression of CD14 and CD15 by flow cytometry and isolated by magnetic bead procedure from PBMCs. We performed comparative proteomic analysis among LDGs and autologous NDGs and healthy controls (HC)-NDGs. Results LDG frequencies were 9.8-fold higher in AAV patients before treatment relative to HC. There were two distinct populations of LDGs showing different cell surface expressions of CD10, CD14, and CD15 in AAV patients. One population of LDGs was mainly CD10 positive and another one was CD10 negative. Although the frequency of CD10 positive-LDG decreased along with decrease of disease activity, the frequency of CD10 negative-LDG increased in 4 months (17.9-fold higher than before treatment). Comparative proteomic analysis revealed these two populations of LDGs were distinct from each other and had common differences from autologous NDGs and HC-NDGs. LDG frequencies were 9.8-fold higher in AAV patients before treatment relative to HC. There were two distinct populations of LDGs showing different cell surface expressions of CD10, CD14, and CD15 in AAV patients. One population of LDGs was mainly CD10 positive and another one was CD10 negative. Although the frequency of CD10 positive-LDG decreased along with decrease of disease activity, the frequency of CD10 negative-LDG increased in 4 months (17.9-fold higher than before treatment). Comparative proteomic analysis revealed these two populations of LDGs were distinct from each other and had common differences from autologous NDGs and HC-NDGs. Conclusions We identified two distinct subsets of LDGs in AAV. It is possible that they play different roles in the pathogenesis of AAV. References [1] Denny MF, et al. A distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type I Interferons. J Immunol2010;184(6):3284–3297. [2] Grayson, et al. Neutrophil-Related Gene Expression And Low-Density Granulocytes Associated with Disease Activity and Response to Treatment in ANCA-Associated Vasculitis. Arthritis Rheumatol2015;67:1922–1932. Disclosure of Interest None declared

FRI0247 The Release of Nets from MPO-AAV Neutrophils was Increased by Anti-MPO Antibody and Correlated with Disease Activity

Background NETosis, a unique form of cell death of neutrophils, is characterized by the active release of chromatin fibers called NETs, that trap and kill invading microbes extracellularly. Although NETosis plays a crucial role in host defense, excessive NETs formation becomes self-defeating by promoting tissue injury and organ damage. It has been known that NETs are implicated also in the pathogenesis of autoimmune vasculitis such as SLE, RA and ANCA –associated vasculitis (AAV). Objectives We observed NETs formation of neutrophils from MPO-AAV patients which is a majority type of AAV in Asia, examined the effects of anti-MPO antibody and immunosuppressive therapy on the release of NETs in order to investigate the role of NETs and MPO-ANCA in MPO-AAV.We also observed the damage of human renal glomerular endothelial cells (HRGECs) by neutrophils in order to investigate the role of NETs in AAV. Methods Isolated peripheral blood neutrophils from healthy donors, pre/post-treatment MPO-AAV patients were incubated with phorbol myristate acetate (PMA), which is known as a strong inducer of NETs, PMA plus anti-MPO antibody or PMA plus anti-PR3 antibody. Neutrophils were stained with Hoechst 33342, SYTOX Green and the percentage of NETs producing cells were calculated. Brd-U labeled HRGECs stimulated by PMA were co-cultured with neutrophils. A cellular DNA fragmentation ELISA was used to quantitatively determine HRGECs damage. Results Neutrophils from pre-treatment MPO-AAV patients produced much more fiber-like NETs than neutrophils from controls and the shape of fiber-like NETs were different from controls. Anti-MPO antibody increased the release of fiber-like NETs both in controls and MPO-AAV patients. The release of fiber-like NETs decreased in tandem with the decrease of ANCA titer after initial treatment and increased with the rise of ANCA titer in some MPO-AAV patients. Although PMA-stimulated HRGEC were damaged in the presence of neutrophils, HRGEC without PMA stimulation were not damaged. Conclusions Neutrophils from pre-treatment MPO-AAV patients released different type of fiber-like NETs and the amount of fiber-like NETs correlated with the activity of AAV. HRGECs stimulated with PMA were damaged in the presence of neutrophils. These data suggest the release of fiber-like NETs by neutrophils at vascular endothelium is important for the pathogenesis of AAV. References Jennette JC et al: 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides, 65: 1-11, 2013. Arimura Y et al: Serum myeloperoxidase and serum cytokines in anti-myeloperoxidase antibody-associated glomerulonephritis, Clin Nephrol, 40: 256-264, 1993. Kessenbrock K et al: Netting neutrophils in autoimmune small-vessel vasculitis, Nat Med, 15: 623-625, 2009. Gupta AK et al: Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death, FEBS Lett, 584: 3193-3197, 2010. Kambas K et al: Tissue factor expression in neutrophil extracellular traps and neutrophil derived microparticles in antineutrophil cytoplasmic antibody associated vasculitis may promote thromboinflammation and the thrombophilic state associated with the disease, Ann Rheum Dis, 73: 1854-1863, 2014. Disclosure of Interest None declared