LIGHT aggravates sepsis‐associated acute kidney injury via TLR4‐MyD88‐NF‐κB pathway

Abstract Sepsis‐associated acute kidney injury (SA‐AKI) is a common clinical critical care syndrome. It has received increasing attention due to its high morbidity and mortality; however, its pathophysiological mechanisms remain elusive. LIGHT, the 14th member of the tumour necrosis factor (TNF) superfamily and a bidirectional immunoregulatory molecule that regulates inflammation, plays a pivotal role in disease pathogenesis. In this study, mice with an intraperitoneal injection of LPS and HK‐2 cells challenged with LPS were employed as a model of SA‐AKI in vivo and in vitro, respectively. LIGHT deficiency notably attenuated kidney injury in pathological damage and renal function and markedly mitigated the inflammatory reaction by decreasing inflammatory mediator production and inflammatory cell infiltration in vivo. The TLR4‐Myd88‐NF‐κB signalling pathway in the kidney of LIGHT knockout mice was dramatically down‐regulated compared to the controls. Recombinant human LIGHT aggravated LPS‐treated HK‐2 cell injury by up‐regulating the expression of the TLR4‐Myd88‐NF‐κB signalling pathway and inflammation levels. TAK 242 (a selective TLR4 inhibitor) reduced this trend to some extent. In addition, blocking LIGHT with soluble receptor fusion proteins HVEM‐Fc or LTβR‐Fc in mice attenuated renal dysfunction and pathological damage in SA‐AKI. Our findings indicate that LIGHT aggravates inflammation and promotes kidney damage in LPS‐induced SA‐AKI via the TLR4‐Myd88‐NF‐κB signalling pathway, which provide potential strategies for the treatment of SA‐AKI.

has a convoluted and exclusive pathophysiology, which is not fully understood. Recent evidence shows that inflammation, oxidative stress, disturbances in coagulation and the adaptive response of renal tubular epithelial cells to injury may contribute to the development of SA-AKI. 1,2,[8][9][10][11] LIGHT (Homologous to Lymphotoxins, exhibits inducible expression and competes with HSV Glycoprotein D for HVEM, a receptor expressed by T lymphocytes), the 14th member of the TNF superfamily, has been identified as a novel immunoregulatory molecule. 12,13 LIGHT signals by combining its two receptors, the Herpes Virus Entry Mediator (HVEM) and the lymphotoxin β receptor (LTβR), may play a bidirectional regulatory role in inflammatory disorders. [14][15][16][17] By interacting with HVEM, LIGHT signalling can produce a co-stimulatory signal for the activation and proliferation of T cells and cause the production of cytokines and inflammatory factors to promote proinflammatory responses. 13,18 Meanwhile, the LIGHT-LTβR pathway may also increase the release of chemokines and adhesion molecules to induce immune cell recruitment to accelerate immunoreaction. 19 A range of studies have demonstrated that the LIGHT pathway plays an important role in the pathophysiology of several inflammatory diseases, including rheumatoid arthritis, IgA nephropathy and inflammatory bowel disease. [14][15][16][17] However, the effect of LIGHT on acute kidney injury has not yet been reported. Our study demonstrated that LIGHT deficiency significantly attenuated SA-AKI via the TLR4-MyD88-NF-κB pathway, suggesting that LIGHT may act as an innovative intervention target in the pathogenesis of SA-AKI.  20 Briefly, mice were administered a single intraperitoneal injection of LPS (20 mg/ kg, LPS diluted in 0.9% normal saline, n = 6). The control mice were injected with saline solution (n = 6). After 24 hours from the injection of LPS or 0.9%, the mice were killed and blood samples and kidney tissues were collected. The kidneys were snap-frozen in liquid nitrogen and stored at -80°C until total RNA or protein extraction.

| Mice and SA-AKI model
The kidneys were immediately embedded in 4% paraformaldehyde for haematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and immunofluorescence (IF). In addition, the dosage of the intraperitoneal injection was adjusted to 40 mg/kg for the survival study. 20 Briefly, the WT and LIGHT KO mice were we divided into four groups after LPS or 0.9% saline injection: WT + LPS group (n = 13), LIGHT KO + LPS group (n = 13), WT + Saline group (n = 11), and LIGHT KO + Saline group (n = 11). Mouse survival was monitored every 6 hours for a total of 4 days (96 hours).

| Cell culture
The human kidney tubular epithelial cell line (HK-2 cell) was donated by Professor Yani He, who bought it from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM/ F12 supplemented with 10% foetal bovine serum (FBS) (Gibco, New Zealand) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) in a humidified atmosphere of 5% CO 2 and 95% O 2 at 37°C.

| Cell viability assay and treatment
The HK-2 cells were plated in 96-well plates at a density of 5 × 10 3 cells/mL per well for 24 hours. The cells were challenged with various concentrations of LPS (1,5,10,25,50 and 100 µg/mL) for 24 hours determine the optimal concentration of LPS for inducing cell injury.
Similarly, HK-2 cells were treated with recombinant human LIGHT (rhLIGHT) (0, 0.1, 0.25, 0.5, 1 and 5 µg/mL) with or without LPS for an additional 24 hours (data not shown) to determine the optimal concentration for aggravating or diminishing cell injury. Cell viability was determined using a Cell Counting Kit-8 (CCK-8) assay kit (Bioss, Beijing, China). The absorbance of the different groups was assayed at 450 nm using a spectrophotometer (Bio-Rad, USA). Then, the HK-2 cells were incubated with serum-free DMEM/F12 for 12 hours and divided into four groups before culturing for another 24 hours

Clinical Perspectives
• The role of LIGHT on SA-AKI has never been reported.
• The results allow us to further understand the effects of LIGHT in SA-AKI to develop novel therapies.

| Cell apoptosis assay
After treatment, the HK-2 cells were harvested. Apoptosis was assessed by flow cytometry following the manufacturer's instructions (Cwbiotech, China).

| Measurement of Scr and BUN levels in plasma
Blood samples were obtained from the periorbital sinus under anaesthesia. All mice were killed by cervical dislocation at 24 hours after LPS or 0.9% saline injection. The levels of serum creatinine (SCr) and blood urea nitrogen (BUN) were determined using a biochemical autoanalyser (Olympus AU5400; Olympus, Japan) to evaluate renal function, following the manufacturer's instructions.

| ELISA
The concentrations of TNF-α, IL-6 and IL-1β in serum were determined using ELISA kits (USCN Life Science Inc, China) following the manufacturer's instructions. The optical density (OD) was determined at 450 nm using a spectrophotometer (Bio-Rad, USA). The expression levels of TNF-α, IL-6 and IL-1β were calculated from a standard curve.

| Histological evaluation
After fixation in 4% paraformaldehyde overnight, the tissue from the right kidney was embedded in paraffin, sectioned and stained with H&E for observation under a light microscope (Olympus BX63; Olympus, Japan). Histological evaluation was performed by two experienced doctors in a double-blind manner. The 0-4 semi-quantitative scales were employed in the assessment, as previously described. 21,22 Ten random fields (400×) of cortical tissues in every mouse were counted and the percentage of injured area was determined. Tissue damage was scored according to the percentage of damaged tubules: 0, no damage; 1, <25%; 2, 25-50%; 3, 51-75%; 4, >75%. 21,22

| MPO assay
Renal tissue samples were mixed with buffer solution (1:19) to prepare 5% homogenate using a tissue homogenizer (Jingxin, Shanghai, China). To evaluate the accumulation of neutrophils, the MPO concentration was determined using commercial reagent kits (Jiancheng, Nanjing, China) following the manufacturer's instructions.

| RNA extraction and quantitative real-time PCR
Total RNA from kidney tissues and HK-2 cells was extracted using TRIzol reagent (TaKaRa Bio, Japan) according to the manufacturer's protocols. A NanoDrop spectrophotometer (ND-100; Thermo Scientific, USA) was used to assay the RNA ºconcentration. After reverse transcription, the target gene expression was determined by quantitative real-time PCR using SYBR Premix Ex Taq II kits (TaKaRa Bio, Japan) following the manufacturer's instructions in a PCR system (Mx3000; Stratagene, USA). As shown in Table 1, the primer sequences were synthesized by Invitrogen Co., Ltd. (Shanghai, China).
Target gene expression was standardized with GAPDH and calculated using the 2 −∆∆CT method.

| Western blot analysis
The total proteins of the kidney tissues and HK-2 cells were lysed and extracted using T-PER ™ Tissue Protein Extraction Reagent (Thermo Fisher, USA) following the manufacturer's instructions. The protein concentration was assessed using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China). 8%, 10% and 12% SDS-PAGE gels were prepared to separate proteins according to the molecular TA B L E 1 Sequences of the primers for real-time PCR

| Immunohistochemistry
After fixation in 4% paraformaldehyde overnight, the kidney tissue Lastly, the kidney sections were observed under a light microscope (Olympus BX63; Olympus, Japan) after DAB staining.

| In vivo LIGHT blocking experiments
WT mice were randomly divided into four groups (n

| Statistical analysis
All data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. GraphPad Prism 6.0 (La Jolla, California, USA) was used for statistical analysis. Student's t test was used to compare two groups, whereas the intergroup differences were analysed using one-way ANOVA with Dunnett's multiple comparisons tests. A P-value of 0.05 was considered statistically significant.

| Sepsis increased LIGHT expression in LPSinduced SA-AKI
A wealth of research has demonstrated that proximal tubular epithelial cells serve as a primary target of sepsis. 20 Figure 1E,F). These results demonstrate that LIGHT is closely related to LPS-induced SA-AKI, which forms the basis for our further study.

| LIGHT deficiency prolonged the survival of SA-AKI mice
To further elucidate the role of LIGHT in SA-AKI, LIGHT KO mice and WT mice were injected with LPS (40 mg/kg, i.p.) or saline, and the survival rate was monitored every 6 hours for a total of 96 hours. As shown in Figure 2A, at the end of the experiment, all mice in the WT group died (n = 13) and 9 mice in the LIGHT KO group died (n = 13), whereas none of the mice in the saline treatment group died (n = 11).
These findings suggest that LIGHT deficiency may play a protective role in a LPS-induced sepsis model.

| LIGHT deficiency protected against kidney injury in SA-AKI mice in vivo
To further explore the protective effect of LIGHT on SA-AKI, we in-

| LIGHT deficiency decreased inflammatory mediator production and inflammatory cell infiltration in SA-AKI in vivo
A wealth of research has corroborated that inflammatory cell infiltration and proinflammatory cytokines, including TNF-α, IL-6 and IL-1β, play a vital role in the pathological process of sepsis. 25 Here, ELISA revealed that LIGHT deficiency down-regulated LPS-induced TNF-α, IL-6 and IL-1β production in serum compared to WT mice

| Exogenous LIGHT protein promoted cell injury in LPS-treated HK-2 cells
To  Figure 5B). After the activation of TLRs, signals can be transmitted by two different signalling pathways: the MyD88-dependent pathway and the TRIFdependent pathway. 29 We also assessed the expression levels of TRIF mRNA, another downstream signal of TLR4, using quantitative RT-PCR. However, no significant differences were observed in the TRIF mRNA levels between the LIGHT KO mice and WT mice in the model groups ( Figure 5A). Intriguingly, MyD88 mRNA Therefore, these results demonstrate that LIGHT deficiency significantly down-regulates the expression of p-NF -κB in the model groups.
In accordance with the in vivo results, rhLIGHT directly promoted the expression of NF-κB in LPS-treated HK-2 cells, whereas TAK242 pretreatment inhibited the increased expression of p-NF-κB/P65 ( Figure 6A-E). In addition, immunofluorescence staining analysis revealed that rhLIGHT facilitated NF-κB/P65 translocation into the nucleus in HK-2 cells, a role that was suppressed by TAK242 in vitro ( Figure 6F). These results suggest that LIGHT deficiency mitigates LPSinduced inflammatory signals via the TLR4-MyD88-NF-κB pathway. These results further confirm that LIGHT aggravates SA-AKI.

| D ISCUSS I ON
As a bidirectional immunoregulatory molecule, LIGHT has been reported to be involved in the pathogenesis of a variety of inflammatory and autoimmune diseases. 14-16 However, the effect and Additionally, LIGHT and its receptors HVEM and LTβR are constitutively expressed in kidney tissues. 24 In line with the literature, we found that LIGHT, HVEM and LTβR were expressed in kidney tissues.
Intriguingly, in this study, we found for the first time that LIGHT was highly co-localized with CK-18, a marker of renal tubular epithelial cells. Given that renal tubular epithelial cells are the primary target of AKI, 23 we speculated that the LIGHT pathway was closely associated with the pathogenesis of SA-AKI. Previous research from our group has confirmed that LIGHT-HVEM/LTβR is involved in IFNγmediated MIN6 cell injury. 26 Consistent with these findings, we further found that LIGHT blocking with soluble receptor fusion proteins HVEM-Fc or LTβR-Fc attenuated renal dysfunction and pathological injury in SA-AKI. To the best of our knowledge, this study is the first to demonstrate that both receptors play an instrumental role in kidney disease, whereas previous studies have focused on the effect of LTβR activation. 33 In addition, these data raised the fundamental question: by what mechanism does LIGHT aggravate LPS-induced SA-AKI? We speculated that there were two possible mechanisms. Firstly, LIGHT might indirectly mediate renal damage to aggravate SA-AKI by promoting inflammatory responses. As a co-stimulatory molecule, a myriad of research has demonstrated that LIGHT aggravates inflammatory responses and inflammatory-related diseases. [14][15][16] In line with these data, our study demonstrated that LIGHT deficiency led to a remarkable decrease in inflammatory mediator production and inflammatory cell infiltration in mice with SA-AKI. Secondly, LIGHT might directly mediate renal damage to aggravate SA-AKI. Renal tubular epithelial cells are a primary target of kidney injury and the progression of kidney disease. 23 In the present study, LIGHT was found to be highly co-localized with CK-18, a marker of renal tubular epithelial cells, and the ultrastructural observation revealed the significant autophagy of tubular epithelial cells (data not shown). Moreover, exogenous LIGHT promoted LPS-treated HK-2 cell injury. These findings suggest that these mechanisms may collectively promote the pathogenesis of SA-AKI to some extent.
TLR4 plays a pivotal role in the pathogenesis of LPS-induced SA-AKI. 34 Consistent with previous studies, 27,28,34 we found that the expression of TLR4 and the downstream signalling molecules was significantly up-regulated in the SA-AKI model in vivo and in vitro.
After the activation of TLRs, signals can be transmitted by two different signalling pathways: the MyD88-dependent pathway and the TRIF-dependent pathway. 29 In addition, LPS induces the activation of the TLR4-MyD88-dependent signalling pathway, which contributes to the nuclear translocation and phosphorylation of NF-κB. 35 Our study showed that LIGHT deficiency significantly down-regulated the levels of TLR4-MyD88-NF-κB. TAK242, a selective TLR4 inhibitor, attenuated exogenous LIGHT-induced HK-2 cell injury and down-regulated the expression of the TLR4-MyD88-NF-κB pathway. These results suggest that the downstream signal of TLR4 may be activated through MyD88. In addition, we found that blocking LIGHT with an LTβR-Fc or HVEM-Fc fusion protein attenuated SA-AKI. In contrast to our results, a previous study reported that LTβR activation induces TLR4 tolerance by combining with LTα1β2 ligand in vivo. 36 Although seemingly contradictory, the findings of the previous study support our results: LPS increased LTβR activation, which can only bind with the LTα1β2 ligand if the LIGHT gene is knocked out. Therefore, LIGHT KO mice showed milder tissue injury and renal function and prolonged survival after LPS administration.
Additionally, it has been previously demonstrated that LIGHT-LTβR induced the activation of the NF-κB pathway in a non-canonical F I G U R E 8 Schematic representation of the mechanism for LIGHT aggravating SA-AKI manner. 37 Undoubtedly, LTβR costimulation synergistically improved the late NF-κB reaction to TLR4 NF-κB target gene-expressions. 38 However, recent studies have indicated that the classical NF-κB activity has the ability to suppress non-canonical NF-κB signalling. 39 Therefore, LIGHT-induced TLR signalling was the main pathway to activate NF-κB to mediate LPS-induced SA-AKI ( Figure 8).
Taken together, our results demonstrate that LIGHT mediates SA-AKI by promoting the TLR4-MyD88-NFκB signalling pathway.
Our results provide the basis for a novel therapeutic strategy for the treatment of sepsis-associated AKI in humans.

ACK N OWLED G EM ENTS
We would like to thank Prof.

CO N FLI C T O F I NTE R E S T
All the authors declare no competing interests.