Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4

Abstract

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Figure 1.

Dying renal cells release histones into extracellular compartments. (A) Necrosis was induced in primary tubular epithelial cells as described in the Concise Methods section. Histone H4 was detected in necrotic supernatants by using anti-H4 antibody. Recombinant histone H4 was loaded as a positive control. (B) Renal endothelial cell proliferation was determined in a period of 24 hours by bioluminescence assay, as described in the Concise Methods. Data represent mean OD ± SEM of three experiments measured at a wavelength of 492 nm. (C and D) Renal endothelial cells were stimulated with CpG, 6 µg/ml; camptothecin (CPT), 10 µM; or histones. Doses are given in µg/ml. Results show flow cytometry from floating cells in culture supernatants. Data represent the mean of total positive cell numbers of positive cells of three independent experiments. PI, propidium iodine.

Figure 2.

In vivo microscopy of cremaster muscles. In vivo microscopy was performed on cremaster muscle postcapillary venules as described in the Concise Methods section. Six mice in each group were treated with intrascrotal injections of vehicle or total histones as indicated. Leukocyte rolling (A), firm adhesion (B), and transendothelial migration (C) were determined 6 hours after injection. Histone+APC means that histone had been pre-incubated with activated protein C (APC) before injection. Data are means ± SEM. #P<0.05 versus control, *P<0.05 versus histones. (D) Representative images illustrate the increase in leukocyte adhesion and transendothelial migration after histone challenge (right) versus control (left). (E) Microvascular FITC-dextran leakage was determined 6 hours after injection. Data are means ± SEM. #P<0.05 versus control, *P<0.05 versus histones. (F) Representative images illustrate the increase in vascular dextran permeability after histone challenge (right) versus control (left).

Figure 3.

Histone injection into the renal artery. (A) Twelve hours after intraperitoneal LPS injection (1 mg/kg body weight), the abdominal aorta and the left renal artery was prepared and a microcannula was placed into the left renal artery (left) for histone injection. The puncture site was mounted with glue before closure of the wound (right). (B) Representative images of periodic acid-Schiff (PAS) stainings and for neutrophils are shown at a magnification of ×50. The quantitative analysis of tissue injury and neutrophil numbers per high-power field are shown on the right. (C) Dilated peritubular capillaries and interstitial edema are illustrated by transmission electron microscopy. Endothelial cells with condensed nuclear chromatin seem to undergo apoptosis. Original magnification ×7500. (D) Total kidney mRNA levels of TNF-α, IL-6, and inducible nitric oxide synthetase (iNOS) were determined in LPS-primed and histone-injected left kidneys. Histone preincubation (before injection) with recombinant activated protein C (APC) reduced intrarenal cytokine expression. (E) Renal mRNA levels of TLR2 and TLR4 with and without LPS priming. 18s rRNA levels were used as internal control. Data represent means ± SEM from nine mice. ^†P<0.05, **P<0.01, ***P<0.001 versus LPS (B and C) or saline control (D).

Figure 4.

Neutralizing histone H4 protects from septic AKI. (A) Serum creatinine was determined 12 hours after intraperitoneal LPS injection. Two hours before, groups of mice had received anti-histone H4 or control IgG. Data represent means ± SEM. ***P<0.001. (B) Renal tissue from mice of both groups was stained with periodic acid-Schiff (PAS) or for neutrophils. Representative images are shown at magnifications as indicated. Data represent means ± SEM from six mice of each group. hpf, high-power field.

Figure 5.

Neutralizing histone H4 improves postischemic renal pathologic findings. Renal tissue was obtained 24 hours after bilateral renal ischemia/reperfusion from anti-H4 IgG–and control IgG–treated mice and stained with periodic acid-Schiff (PAS), lectin (primal tubular cells), Tamm-Horsfall protein (THP) (distal tubular cells), and TUNEL (apoptotic cells) as indicated. Representative images are shown at a magnification of ×400. The quantitative analysis of tissue injury or TUNEL-positive cells is shown next to the respective staining procedure. Data represent means ± SEM from six mice of each group. **P<0.01, ***P<0.001.

Figure 6.

Assessment of postischemic renal inflammation. (A) Total RNA was extracted from kidneys from anti-H4 IgG– and control IgG–treated mice. KIM-1 and cytokine mRNA expression levels were determined by real-time PCR and expressed as mean of the ratio 18S rRNA ± SEM; ^‡P<0.05 versus control. (B) Renal sections were stained for neutrophils, and representative images are shown at a magnification of ×400. The quantitative analysis of interstitial neutrophils is shown next to the respective staining procedure. Data represent means ± SEM from six mice of each group. **P<0.01.

Figure 7.

Histones activate TNF and IL-6 cytokine production. (A and B) TNF and IL-6 ELISA of supernatants from mouse BMDCs stimulated for 6 hours with total histones (30 µg/ml) and individual histones (30 µg/ml) or LPS (1 µg/ml). (C) BMDCs were stimulated with H4 at the indicated time points. Cell lysates were immunoblotted and probed for the indicated proteins. (D) BMDCs were stimulated for 6 hours with H4 and LPS in the presence or absence of polymyxin B treatment. Supernatants were analyzed for TNF and IL-6 by ELISA. (E) BMDCs were stimulated for 6 hours with H4 or pretreated H4 with deoxyribonuclease, ribonuclease, and proteinase K. Supernatants were analyzed for TNF and IL-6 by ELISA. (F) BMDCs were stimulated for 6 hours with H4 in the presence or absence of anti-H4 antibodies (H4 Ab) or control IgG antibodies. Supernatants were analyzed for TNF and IL-6 by ELISA. In A, B, and D–F, the data represent the mean ± SD of three independent experiments. ^¶P<0.05 by t test. Data shown in part C were repeated two times. Related data are presented in Supplemental Figure 1. ND, not detected.

Figure 8.

Histone H4 activates TLR2/TLR4-MyD88 to induce cytokines. ELISA for TNF and IL-6 in supernatants from wild-type (WT) and different gene-deficient BMDCs stimulated with H4 (30 µg/ml), LPS (1 µg/ml), Pam3Cys (1 µg/ml), and Poly I:C RNA (5 µg/ml) for 6 hours as indicated. A–F illustrate dendritic cell stimulation with histones and various other TLR agonists in wild-type or gene-deficient mice as indicated. The data represent the mean ± SD of three independent experiments. #Not detected. See also Supplemental Figures 2 and 3. ND, not detected.

Figure 9.

Histone H4 directly interacts with TLR2 and TLR4/MD2. (A and C) Binding of NT-647 fluorescence-labeled H4 to recombinant human TLR2 (A) and TLR4/MD2 complex (C). To determine the affinity of binding, a titration series of TLR2 (1000–1.95 nM) and TLR4/MD2 proteins (350–0.68 nM) was performed while fluorescence-labeled H4 was kept at a constant concentration of 5 nM. The change in the thermophoretic signal of H4 suggested a Kd of 4.2±1.7 nM for TLR2 and 6.0±3.7 nM for TLR4/MD2. Kd was calculated from three independent thermophoresis measurements using NanoTemper software. The fluorescence was measured before laser heating (F initial) and after 30 seconds of laser on time (F hot). The normalized fluorescence F norm = F hot/F initial reflects the concentration ratio of labeled molecules. F norm is plotted directly and multiplied by a factor of 10, yielding the relative change in fluorescence per mill (F_Norm [‰]). (B and D) NT-647 fluorescence-labeled albumin (5 nM) tested for its binding to TLR2 (B) and TLR4/MD2 (D) was used as negative control. (E) Mice were injected intravenously with histone H4 (20 mg/kg). After 6 hours, IL-6 and TNF cytokines were measured in plasma by ELISA. The data represent mean ± SD from four mice in each group. **P<0.01 by t test. #Not detected. KO, knockout; WT, wild-type.