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. 2016 Jul 21;1(11):e87680.
doi: 10.1172/jci.insight.87680.

Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

Yuki Sato  1 , Akiko Mii  1 , Yoko Hamazaki  2 , Harumi Fujita  2 , Hirosuke Nakata  1 , Kyoko Masuda  3 , Shingo Nishiyama  1 , Shinsuke Shibuya  4 , Hironori Haga  4 , Osamu Ogawa  5 , Akira Shimizu  6 , Shuh Narumiya  7 , Tsuneyasu Kaisho  8 , Makoto Arita  9 , Masashi Yanagisawa  10   11 , Masayuki Miyasaka  12   13 , Kumar Sharma  14 , Nagahiro Minato  2 , Hiroshi Kawamoto  3 , Motoko Yanagita  1
Affiliations

Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

Yuki Sato et al. JCI Insight. .

Abstract

Acute kidney injury (AKI) is a common clinical condition defined as a rapid decline in kidney function. AKI is a global health burden, estimated to cause 2 million deaths annually worldwide. Unlike AKI in the young, which is reversible, AKI in the elderly often leads to end-stage renal disease, and the mechanism that prevents kidney repair in the elderly is unclear. Here we demonstrate that aged but not young mice developed multiple tertiary lymphoid tissues (TLTs) in the kidney after AKI. TLT size was associated with impaired renal function and increased expression of proinflammatory cytokines and homeostatic chemokines, indicating a possible contribution of TLTs to sustained inflammation after injury. Notably, resident fibroblasts from a single lineage diversified into p75 neurotrophin receptor+ (p75NTR+) fibroblasts and homeostatic chemokine-producing fibroblasts inside TLTs, and retinoic acid-producing fibroblasts around TLTs. Deletion of CD4+ cells as well as late administration of dexamethasone abolished TLTs and improved renal outcomes. Importantly, aged but not young human kidneys also formed TLTs that had cellular and molecular components similar to those of mouse TLTs. Therefore, the inhibition of TLT formation may offer a novel therapeutic strategy for AKI in the elderly.

Keywords: kidney; tertiary; lymphoid; tissue; aging.

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Figures

Figure 1
Figure 1. Aged mice develop multiple renal tertiary lymphoid tissues (TLTs) after kidney injury.
(A) Periodic acid-Schiff (PAS) staining and (B) fibrosis scores 45 days after 30-minute ischemic reperfusion injury (IRI) in young and aged mice (n = 4 per group). (C, E, and F) Immunofluorescence analysis of (C) lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), α-smooth muscle actin (αSMA), and CD45; (E) CD3ε and B220; (F) Ki67 and αSMA; and (D) immunohistochemical analysis of peripheral lymph node addressin (PNAd) in inflammatory cell aggregates in aged kidneys at day 45 after 37-minute IRI. (G and H) PAS staining of IRI kidneys of (G) aged mice and (H) young mice at day 45, and (I) the TLT sizes (n = 4 or 5 per group). (J) Ifng and Tnfa mRNA levels of IRI kidneys at day 45 with variable ischemic times in young and aged mice (n = 4 per group). (K) Correlation between TLT sizes and mRNA levels of Ifng and Tnfa in aged IRI kidneys at day 45 with variable ischemic times (n = 16). (L) PAS staining of young and aged kidneys in folic acid (FA) nephropathy (day 21) and unilateral ureteral obstruction (UUO) (day 14) model. (M) Serum creatinine (sCr) concentrations in young and aged mice 21 days after FA treatment (n = 12 or 13 per group) and correlation of sCr with TLT size in aged mice (n = 13). *P < 0.001, **P < 0.01, #P < 0.05 versus control. One-way ANOVA with Tukey’s post-hoc analysis was used to analyze data from (J); a 2-tailed Student’s t test was used for other experiments. Correlation was determined by Pearson’s correlation analysis. Scale bars: (A, C–H, and L) 100 μm. The expression levels were normalized to those of Gapdh and expressed relative to those of young mouse kidney at day 0 (IRI). In (B, I, J, and M), the box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values.
Figure 2
Figure 2. Fibroblasts inside tertiary lymphoid tissues (TLTs) produce CXCL13 and CCL19.
(A–C) The mRNA expression of the homeostatic chemokines Cxcl13, Ccl19, and Ccl21 in ischemic reperfusion injury (IRI) kidneys 45 days after variable ischemic time IRI in young and aged mice (n = 4 per group). The expression levels were normalized to those of Gapdh and expressed relative to those of young mouse kidney at day 0 (IRI). *P < 0.001, **P < 0.01 aged versus young (1-way ANOVA with Tukey’s post-hoc analysis). The box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values. (D–F) Correlations between TLT sizes and mRNA levels of Cxcl13, Ccl19, and Ccl21 in aged IRI kidneys 45 days after variable ischemic time IRI (n = 16). Correlation was determined by Pearson’s correlation analysis. (G–R) Immunohistological analysis of aged kidneys 30 days after 37-minute IRI. Immunofluorescence analysis of (G–I) CXCL13, CCL19, and CCL21 with α-smooth muscle actin (αSMA); (J) CCL21 and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1); (K) PDGFRβ; (L) ER-TR7; (M–O) PDGFRβ, ER-TR7, and CD45 with CXCL13; and (P–R) PDGFRβ, ER-TR7, and CD45 with CCL19. Arrows indicate TLT localization. Scale bars: (G–I) 100 μm, (K and L) 50 μm, (J, M–R) 10 μm.
Figure 3
Figure 3. Fibroblasts with distinct phenotypes underlie tertiary lymphoid tissue (TLT) formation in the aged injured kidney.
Immunofluorescence of (A) retinaldehyde dehydrogenase 2 (RALDH2), p75 neurotrophin receptor (p75NTR), and PDGFRβ; (B) β3-tubulin and RALDH2; (C and D) p75NTR; (E) p75NTR and CD21; (F) CD21 and CXCL13; and (G) CD3ε, CD21, and B220 in aged kidneys after ischemic reperfusion injury (IRI). Arrows indicate TLT localization. Arrowheads indicate the localization of the B cell area. Aged kidneys were analyzed 14 (B and C), 30 (A), and 45 (D–G) days after IRI. (H) Retinoic acid (RA) induces p75ntr mRNA expression in C3H10T1/2 mouse embryonic fibroblasts and FACS-sorted PDGFRβ+ cells (n = 3 per group). The data are presented as dot plots (mean ± SD). (I) Activation-induced cytidine deaminase (Aid) mRNA levels of kidneys 45 days after various ischemic time IRI in young and aged mice (n = 4 per group) and correlation with TLT size (n = 16, aged mice only). The expression levels were normalized to those of Gapdh and expressed relative to those of controls or young mouse kidney at day 0 (IRI). *P < 0.001 versus controls. A 2-tailed Student’s t test was used to analyze data from FACS-sorted PDGFRβ+ cells; 1-way ANOVA with Tukey’s post-hoc analysis was used for other experiments. Correlation was determined by Pearson’s correlation analysis. The box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values. Scale bars: (A, C, and D) 100 μm, (E and G) 50 μm, (B and F) 10 μm.
Figure 4
Figure 4. Myelin protein zero-Cre (P0-Cre) lineage-labeled fibroblasts differentiate into diverse fibroblasts with distinct phenotypes.
(A–I) Immunofluorescence (IF) of enhanced cyan fluorescent protein–positive (ECFP+) cells in kidneys subjected to ischemic reperfusion injury (IRI) in aged P0-Cre/R26ECFP mice. ECFP was visualized with an anti-GFP antibody. IF of GFP and (A and D) PDGFRβ, (B and E) p75 neurotrophin receptor (p75NTR), (C and F) retinaldehyde dehydrogenase 2 (RALDH2), (G) CXCL13, (H) CCL19, and (I) CD21. Magnified views of the outlined regions in (A–C) are shown in (D–F), respectively. Arrows in (A–C) indicate tertiary lymphoid tissue (TLT) localization. Scale bars: (A–C) 50 μm, (D–I) 10 μm.
Figure 5
Figure 5. Superaged mice spontaneously develop renal tertiary lymphoid tissues (TLTs) without injury.
(A–I) Histological analysis of a 23-month-old mouse kidney. (A) Periodic acid-methenamine silver (PAM) staining showing a TLT containing PAM-positive basement membranes of nephron segments. (B and C) PAS staining showed signs of injury (yellow arrows) in tubules adjacent to TLTs. A magnified view of the outlined region in B is shown in C. (D–I) Immunofluorescence analysis of (D) lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), α-smooth muscle actin (αSMA), and CD45; (E) CD3ε and B220; (F) Ki67; (G) p75 neurotrophin receptor (p75NTR); (H) CXCL13 and αSMA; and (I) CCL19 and αSMA. White arrows indicate the localization of TLTs. Scale bars: (A, B, D, F, G, and I) 100 μm, (C, E, and H) 50 μm. (J) The expression of Cxcl13, Ccl19, Ifng, and Tnfa mRNA in mouse kidneys at various ages (n = 4 per group). The expression levels were normalized to those of Gapdh and expressed relative to those of young (2-month-old) mouse kidneys. In J, the box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values. *P < 0.001, **P < 0.01, #P < 0.05 versus values at 2 months of age (2-tailed Student’s t test).
Figure 6
Figure 6. Targeting tertiary lymphoid tissue (TLT) formation has the potential to ameliorate renal fibrosis and inflammation.
(A–C) Analysis of the kidneys from aged CXCL13-deficient mice (KO) and their littermates (WT) 45 days after ischemic reperfusion injury (IRI) (n = 6 per group). (A) Periodic acid-Schiff (PAS) staining, (B) TLT sizes, and (C) Ccl19 and Ifng mRNA levels in IRI kidneys. (D–H) GK1.5 treatment study (n = 5 or 6 per group). (D) Experimental protocol, (E) PAS staining, (F) TLT sizes, (G) fibrosis scores, (H) Cxcl13, Ccl19, Ifng, Tnfa, Col1a1 (type 1 collagen α-1 subunit), and Fn1 (fibronectin) mRNA levels in the IRI kidneys of GK1.5-treated mice (GK1.5) and isotype antibody–treated mice (Isotype control, Con). (I–M) Dexamethasone (Dex) treatment study (n = 5 per group). (I) Experimental protocol, (J) PAS staining, (K) TLT sizes, (L) fibrosis scores, and (M) Cxcl13, Ccl19, Ifng, Tnfa, Col1a1, and Fn1 mRNA levels in the IRI kidneys of Dex-treated (DEX) and vehicle-treated mice (Control, Con). Mice were subjected to 37-minute IRI (A–C) or 30-minute IRI (D–M). The expression levels were normalized to those of Gapdh and expressed relative to those of aged (12-month-old) mouse kidney. The box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values. *P < 0.001, **P < 0.01, #P < 0.05 versus control (2-tailed Student’s t test). n.s, not significant. Scale bars: (A, E, and J) 100 μm.
Figure 7
Figure 7. Tertiary lymphoid tissues (TLTs) form in aged human kidneys.
(A–L) Histological analyses of aged human kidney samples. (A and B) Periodic acid-Schiff (PAS) staining and immunofluorescence analysis of (C) CD20 and CD3ε; (E) Ki67 and CD3ε/CD20 (arrowheads indicate double-positive cells); (G) CXCL13 and CD21; (J) CXCL13 and CD45; (K) CD21, α-smooth muscle actin (αSMA), and CD45; and (L) p75 neurotrophin receptor (p75NTR) and CD21, and immunohistochemical analysis of (D) peripheral lymph node addressin (PNAd); (F) CD21 (arrowheads indicate the localization of CD21+ follicular dendritic cell [FDC] networks); (H) CD20, activation-induced cytidine deaminase (AID), CD21, and retinaldehyde dehydrogenase 2 (RALDH2) (serial sections); and (I) RALDH2 (arrowheads indicate the localization of B cell follicles). The outlined region in (H) is magnified in (I). Arrows indicate TLT localization. Scale bars: (A, D, H, K, and L) 50 μm, (B, C, and F) 100 μm, (E, G, I, and J) 10 μm. (M) Quantitative analysis of TLT frequencies in human samples (n = 56; young = 13, aged = 43). #P < 0.05 (Pearson’s χ2 test).
Figure 8
Figure 8. A model for renal tertiary lymphoid tissue (TLT) formation.
Heterogeneous fibroblasts play crucial roles in steering the development and maturation of renal TLTs in mice and humans. (A) Mouse: In the early stage, fibroblasts around arteries receive retinoic acid (RA) from retinaldehyde dehydrogenase 2+ (RALDH2+) fibroblasts in a paracrine manner and dedifferentiate into p75 neurotrophin receptor+ (p75NTR+) fibroblasts, some of which acquire the ability to produce CXCL13/CCL19 (Stage 1). As the TLT grows and expands, the number of RA-producing fibroblasts around it gradually decreases (Stage 2). In this stage, CD21+/p75NTR– follicular dendritic cells (FDCs) emerge as a part of a stromal network, and peripheral lymph node addressin+ (PNAd+) high endothelial venules (HEVs) develop within the TLT. Human: Although the major components in human renal TLTs are quite similar to those in mouse TLTs, p75NTR colocalizes with CD21, and RALDH+ stromal cells surrounded these FDC networks. (B) A model of stromal cell maturation in the aged injured kidney. Upon kidney injury, resident fibroblasts differentiate into RALDH+ fibroblasts, which promote the transdifferentiation of other fibroblasts into p75NTR+ fibroblasts with 3 phenotypes. These p75NTR+ fibroblasts act in concert to form TLTs. In the later phase of kidney injury, some of these fibroblasts lose their p75NTR expression and mature into CD21+/CXCL13+/p75NTR– FDCs.
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