Volume 92, Issue 1, Pages 79-88 (July 2017) Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression  Becker M.P. Law, Ray Wilkinson, Xiangju Wang, Katrina Kildey, Mae Lindner, Melissa J. Rist, Kenneth Beagley, Helen Healy, Andrew J. Kassianos  Kidney International  Volume 92, Issue 1, Pages 79-88 (July 2017) DOI: 10.1016/j.kint.2017.02.006 Copyright © 2017 Terms and Conditions

Figure 1 Identification of natural killer (NK) cell subsets in human kidney tissue. Gating strategy used to identify total NK cells (CD3− CD19− CD56+ lymphocytes) and NK cell subpopulations (CD56dim and CD56bright NK cells) in human kidney tissue. Representative flow cytometric data from 1 of 16 individual fibrotic renal biopsy specimens are shown. An identical gating strategy was used for healthy kidney tissue and nonfibrotic renal biopsy specimens. DN, double negative; FSC-A, forward-scatter area; MNC, mononuclear cells; SSC-A, side-scatter area. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 2 Significantly increased natural killer (NK) cell numbers in diseased biopsy specimens with interstitial fibrosis. Absolute numbers of total (a) NK cells, (b) CD56dim NK cells, and (c) CD56bright NK cells in healthy kidney tissue and diseased biopsy specimens without and with fibrosis. Values for individual donors are presented; bars represent means. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal-Wallis test. Spearman correlation analyses of absolute numbers of (d) total NK cells, (e) CD56dim NK cells, and (f) CD56bright NK cells versus percentages of interstitial fibrosis in diseased biopsy specimens. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 3 CD56bright natural killer (NK) cell numbers correlate significantly with loss of kidney function. Absolute numbers of total (a) NK cells, (b) CD56dim NK cells, and (c) CD56bright NK cells in healthy kidney tissue and diseased biopsy specimens with an estimated glomerular filtration rate ≥60 (chronic kidney disease I–II) and <60 (chronic kidney disease III–V). Values for individual donors are presented; bars represent means. Absolute numbers of total (d) NK cells, (e) CD56dim NK cells, and (f) CD56bright NK cells in healthy kidney tissue and diseased biopsy specimens with glomerular immune-mediated, glomerular nonimmune-mediated, and nonglomerular primary diagnoses. Values for individual donors are presented; bars represent means. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test. Glom, glomerular. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 4 Significantly increased proportion of CD56bright natural killer (NK) cells with loss of kidney function. Frequency of CD56bright NK cells among total NK cells in healthy kidney tissue and (a) diseased biopsy specimens without and with fibrosis and (b) diseased biopsy specimens with an estimated glomerular filtration rate (eGFR) ≥60 (chronic kidney disease [CKD] I–II) and <60 (CKD III–V). Values for individual donors are presented; bars represent means, with mean values presented in parentheses. *P < 0.05, Kruskal-Wallis test. (c) Contour plots (gated on CD3− CD19− double-negative lymphocytes) highlighting the relative proportions of CD56dim and CD56bright NK cell subsets from representative diseased biopsy specimens with an eGFR ≥60 (CKD I–II) (top panel) and an eGFR <60 (CKD III–V) (bottom panel). Percentage values of CD56bright NK cells among total NK cells are presented. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 5 Human CD56bright natural killer (NK) cells in fibrotic kidney tissue display an activated phenotype. Surface expression of (a) CD69 on CD56dim NK cells and (b) CD56bright NK cells in healthy kidney tissue and diseased biopsy specimens without and with fibrosis. Values of median fluorescence intensity (MFI) for individual donors are shown; bars represent means, with mean values presented in parentheses. *P < 0.05, Kruskal-Wallis test. (c) Relative expression of CD69, NKp46, CD117, CXCR3, and CX3CR1 by CD56bright NK cells compared with CD56dim NK cells, T cells, and isotype control in fibrotic kidney tissue. Representative flow cytometric data from 8 individual experiments are shown. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 6 Colocalization of human natural killer (NK) cells with proximal tubular epithelial cells (PTEC). Immunofluorescent staining of frozen kidney sections from diseased biopsy specimens without (left panel) and with (middle/right panels) interstitial fibrosis stained for NKp46 (red), PTEC marker Aquaporin-1 (white), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). NKp46+ cells are circled (right panel). Bars = 100 μm (left/middle panels) and 10 μm (right panel). Representative results for 3 individual donor experiments are shown. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 7 Human natural killer (NK) cells produce proinflammatory cytokine interferon-γ (IFN-γ) in fibrotic kidney tissue. Immunofluorescent labeling of frozen fibrotic kidney tissue stained for NKp46 (red; left panel) and IFN-γ (green; middle panel). Colocalization is visualized by the yellow merge of red and green (right panel). NKp46+ IFN-γ+ cells are circled. Bars = 100 μm for large frames and 10 μm for insets. Representative results for 3 individual donor experiments are shown. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure 8 Human CD56bright natural killer (NK) cells are a key source of interferon-γ (IFN-γ) in fibrotic kidney tissue. Immunofluorescent labeling of frozen fibrotic kidney tissue stained for NKp46 (red; panel on right), CD117 (orange; second panel) and IFN-γ (green; third panel). Colocalization is visualized by merging the 3 colors (fourth panel). NKp46+ CD117+ IFN-γ+ cells are circled. Bars = 100 μm for large frames and 10 μm for insets. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions

Figure S1 Natural killer (NK) cell numbers do not significantly correlate with levels of inflammatory activity in diseased biopsy specimens. Absolute numbers of total NK cells (A), CD56dim NK cells (B), and CD56bright NK cells (C) in healthy kidney tissue and diseased biopsy specimens grouped based on histologic scoring of interstitial inflammation (Nil, not present; Minimal, ≤10%; Mild, 10%–25%; Moderate, 26%–50%, and Severe, >50%). Values for individual donors are presented; bars represent means. Kidney International 2017 92, 79-88DOI: (10.1016/j.kint.2017.02.006) Copyright © 2017 Terms and Conditions