The cellular signature of urinary immune cells in Lupus nephritis: new insights into potential biomarkers

Katharina Kopetschke, Jan Klocke, Anna-Sophie Grießbach, Jens Y Humrich, Robert Biesen, Duska Dragun, Gerd-Rüdiger Burmester, Philipp Enghard, Gabriela Riemekasten, Katharina Kopetschke, Jan Klocke, Anna-Sophie Grießbach, Jens Y Humrich, Robert Biesen, Duska Dragun, Gerd-Rüdiger Burmester, Philipp Enghard, Gabriela Riemekasten

Abstract

Introduction: Urinary T cells represent a reliable noninvasive biomarker for proliferative Lupus nephritis (LN). Little is known about the presence of T cell subsets, B cells and macrophages in the urine although they may further improve the validity of urinary cellular biomarkers for LN.

Methods: We analyzed contemporaneous blood and urine samples of patients with active LN (n = 19), other Systemic Lupus Erythematosus (SLE) patients (n = 79) and urine samples of patients with diabetic nephropathy (DN; n = 14) and anti-neutrophil cytoplasmatic antibody (ANCA) associated vasculitis (AAV; n = 11) by flow cytometry.

Results: Numbers of urinary T cells, B cells and macrophages correlated with disease activity and were significantly higher in the active LN group. Urinary T cells showed excellent distinction of patients with active LN, CD8+ T cells (AUC of ROC = 1.000) and CD4+ T cells (AUC = 0.9969) alike. CD19+ B cells (AUC = 0.7823) and CD14+ macrophages (AUC = 0.9066), as well as the clinical standard proteinuria (AUC = 0.9201), failed to reach these high standards. Patients with DN or AAV also showed increased urinary cell counts, although the CD4/CD8-ratio was significantly lower in SLE compared to in DN (p = 0.0006). Urinary CD4+ T cells of active LN patients proved to be mainly of effector memory phenotype and expressed significantly more CD40L and ki67 than corresponding blood cells. Urinary Treg counts correlated with disease activity.

Conclusions: Despite of detectable urinary cell counts for B cells and macrophages, T cells remain the best urinary cellular biomarker for LN. A low CD4/CD8-ratio seems to be characteristic for LN.

Figures

Figure 1
Figure 1
Urinary cells in systemic lupus erythematosus (SLE) patients and in patients with different nephropathies. (A) Examplary dot-plots of the flowcytometric analysis of urinary T cells, B cells and monocytes in a patient with acute proliferative lupus nephritis (LN). (B-E) Urinary cells in patients with acute proliferative LN (B), other SLE patients (C), patients with diabetic nephropathy (DN) (D) and patients with ANCA-associated vasculitis (AAV) (E).
Figure 2
Figure 2
Urinary CD4/CD8-ratio in various renal diseases. All patients with CD3+ cell counts >100/dl were included. SLE, systemic lupus erythematosus; DN, diabetic nephropathy; AAV, ANCA-associated vasculitis.
Figure 3
Figure 3
Subtyping of CD4+ T cells in systemic lupus erythematosus (SLE) patients in urine and peripheral blood. Filled circles = urine; open circles = peripheral blood. (A) Expression of CD40L on CD4+ T cells (n = 7). (B) Effector memory (CD45RO + CCR7-), central memory (CD45RO + CCR7+) and naïve (CD45RO-CCR7+) T helper cells (n = 7). (C) Proliferation rate as measured by ki-67 expression of CD4+ T cells (n = 10). (D) Regulatory (CD127-FoxP3+) CD4+ T cells (Treg) (n = 10). (E) Expression of CD25 and ki-67 in Treg (n = 10).
Figure 4
Figure 4
Receiver operater characteristic (ROC) curve for diagnosing proliferative lupus nephritis (LN) among systemic lupus erythematosus (SLE) patients with various urinary cell types. (A) CD3 + CD4+ T cells; (B) CD3 + CD8+ T cells; (C) CD19+ B cells; (D) CD14+ macrophages. AUC, area under the curve.

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