Tacrolimus alleviates LPS‐induced AKI by inhibiting TLR4/MyD88/NF‐κB signalling in mice

Abstract Lipopolysaccharide (LPS)‐induced sepsis‐associated acute kidney injury (SA‐AKI) is a model of clinical serious care syndrome, with high morbidity and mortality. Tacrolimus (TAC), a novel immunosuppressant that inhibits inflammatory response, plays a pivotal role in kidney diseases. In this study, LPS treated mice and cultured podocytes were used as the models of SA‐AKI in vivo and in vitro, respectively. Medium‐ and high‐dose TAC administration significantly attenuated renal function and renal pathological manifestations at 12, 24 and 48 h after LPS treatment in mice. Moreover, the Toll‐like receptor 4 (TLR4)/myeloid differential protein‐88 (MyD88)/nuclear factor‐kappa (NF‐κB) signalling pathway was also dramatically inhibited by medium‐ and high‐dose TAC administration at 12, 24 and 48 h of LPS treatment mice. In addition, TAC reversed LPS‐induced podocyte cytoskeletal injury and podocyte migratory capability. Our findings indicate that TAC has protective effects against LPS‐induced AKI by inhibiting TLR4/MyD88/NF‐κB signalling pathway and podocyte dysfunction, providing another potential therapeutic effects for the LPS‐induced SA‐AKI.

SA-AKI. 9,10 In addition, inhibition of TLR4 signalling protects against SA-AKI development in mice. 11,12 Thus, agents that are capable of suppressing TLR4 signalling might be effective on treating SA-AKI. Tacrolimus (TAC) is an effective immunosuppressant. It acts to bind to FKBP12 and in turn prevents calcium-induced calcineurin activation, which inhibits the induction of inflammatory cytokines. 13,14 TAC is exploited in patients with transplant rejection and autoimmune diseases, including systemic lupus erythematosus and autoimmune hepatitis. [15][16][17] Meanwhile, studies have demonstrated that TAC can alleviate proteinuria, podocyte injury and further renal dysfunction. [18][19][20] However, whether TAC is effective for SA-AKI has not been explored. In this study, we demonstrate that TAC is capable of alleviating LPS-induced SA-AKI through inhibiting the TLR4/MyD88/NF-κB signalling pathway and podocyte dysfunction.

| Reagents
The reagents used in current study include: LPS (Sigma, Chemical

| Animal model of LPS nephrosis
Sixty male C57BL/6 mice (Laboratory Animals Center of Xuzhou Medical College, Xuzhou, China) were housed in cages at 24 to 26°C with alternating 12 h light/dark cycles, and had free access to regular food and water. Mice were divided into five groups: normal control (NC) group (n = 12), LPS model group (n = 12) and low-(n = 12), medium-(n = 12) and high-dose (n = 12) TAC treatment groups. Except for NC group, mice were injected intraperitoneally with 200 μg LPS, which dissolved into 200 μL sterile saline. NC group received the same volume of sterile saline. After LPS injection, the mice were injected with TAC (low dose of 1 mg/kg, medium dose of 2 mg/kg and high dose of 4 mg/kg per 24 h); treatment continued throughout the experiment. The study was approved by the Committee on Ethical Use of Animals of Xuzhou Medical College. At 12,24 and 48 h after LPS treatment, mice were sacrificed under anaesthesia with 1% pentobarbital. We collected the kidney tissues, which were processed for standard histological examination, including fixation in 10% formalin, dehydration in graded alcohol solution, embedding in paraffin, etc. The renal cortex tissue was also used for immunoblotting assay after being lysed with RIPA buffer as described below. At 12,24 and 48 h after LPS treatment, blood samples were collected for biochemical analysis. All samples were kept at -80°C, blood urea nitrogen (BUN) and creatinine levels were measured using Nanjing Jiancheng Urea Assay kit and Creatinine (Cr) Assay kit (sarcosine oxidase).

| Histological examination
Two-micrometre-thick sections were cut and stained with haematoxylin and eosin and periodic acid-Schiff reagent, respectively, following the methods described. 7 All slides were imaged by an Olympus microscope and the images were evaluated by the same pathologist.

| Western blot
The renal tissues were dissolved in RIPA lysis buffer (Beyotime Biotechnology) supplemented with protease inhibitors and Cell Signaling Technology) and anti-beta-actin (1:1000; Beyotime Biotechnology). After washing with TBST, the membranes were then incubated with the second antibody for 1 h at room temperature. Finally, the target proteins were detected by highly sensitive ECL reagent (Bio-Rad Laboratories), and exposed in the BIO-RAD Chemical Imaging System.

| RT-qPCR
The renal tissues were dissolved in RNAiso Plus Reagent (TaKaRa), and the RNA was extracted. Then, cDNA was synthesized accord- and 30 s at 72°C. After the last cycle, incubation at 72°C for 10 min was followed. These experiments were repeated three times independently. All data were normalized by beta-action levels.

| Podocyte culture and treatment
The immortalized mouse podocyte cell line was obtained from the group. Except for NC group, the cells were exposed to LPS (25 μg/ ml) for 6 h and then incubated in medium containing 0.1% DMSO.
In the low-, medium-and high-dose TAC group, cells were treated with TAC (low dose of 0.5 μg/ml, medium dose of 1μg/mL and high dose of 2 μg/ml); treatment continued throughout the experiment.
NC group cells were grown in the control medium. Cells were collected after TAC treatment for 6, 12 and 24 h. All experiments were repeated at least three times for each indicated condition.

| Phalloidin staining for F-actin
Mouse podocytes growing on type I collagen-coated (Sigma-Aldrich) glass slides were pretreated with different conditions and then fixed in 4% paraformaldehyde for 10 min followed by being permeabilized in 0.2% Triton ×-100 for 10 min. Phalloidin (Sigma-Aldrich) was used to stain F-actin in the podocytes, and the resulting images were examined by an inverted fluorescent microscope (Olympus).

| Wound healing assays
Mouse podocyte pretreated with different conditions was allowed to grow to confluence on type I collagen-coated (Sigma-Aldrich) culture dishes. Cell monolayers were washed and scratch wounds were applied using a 200 μl pipet tip. Podocytes were imaged using microscope at time 0 immediately after wound creation. Cells were then returned to non-permissive growth conditions for 24 h before final imaging of wound healing.

| Transwell migration assay
Transwell cell culture inserts (Corning) were coated with type I collagen (Sigma-Aldrich), rinsed once with PBS and placed in RPMI medium in the lower compartment. About 1 × 10 4 /ml podocytes from each group were seeded in the inserts and then allowed to migrate for 24 h at 37°C. Non-migratory cells were removed from the upper surface of the membrane and the migrated cells were fixed with 4% paraformaldehyde and stained with haematoxylin.
The number of migrating cells was counted using phase contrast microscopy (Leica).

| Statistical analysis
Statistical analyses were performed with SPSS 16.0 software (version 16.0, SPSS Inc.). The results were expressed as the mean ± SD.
Statistical analysis of the differences between two groups of treatment was performed using variance analysis combined with the rank sum test. p < 0.05 was considered statistically significant.

| Renal function of the mice treated with LPS and TAC
To analyse kidney function, BUN and serum creatinine were determined. As shown in Figure 1, LPS induced significant increases in both BUN and serum creatinine levels, and the increases were significantly attenuated by medium-and high-dose TAC treatment.

| Pathological Findings in mice treated with LPS and TAC
The light microscopy with haematoxylin and eosin staining revealed that LPS treatment caused inflammatory cells infiltration, glomerular capillary congestion, mesangial cells proliferation, mesangial matrix expansion, focal segmental glomerulosclerosis, capsule adhesion, glomerular basement membrane abnormality and the pathological changes were reversed by medium-and high-dose TAC treatment ( Figure 2).

| TLR4, MyD88 and p65 expression examination by IHC
As shown in Figure 3, IHC studies revealed that LPS treatment upregulated the expression of TLR4, MyD88 and p65 in glomeruli of the mice, and treatment with the medium-dose and high-dose TAC prevented the upregulation of TLR4, MyD88 and p65 expression.

| qRT-PCR analysis of TLR4, p65 and MyD88
According to the qRT-PCR results, LPS significantly upregulated the mRNA levels of TLR4, MyD88 and p65 in kidney tissues, and the mRNA levels upregulation was attenuated by the medium-and highdose TAC treatments, and the medium-and high-dose TAC groups did not exhibit any difference in preventing upregulation of these proteins ( Figure 5).

| The wound healing and transwell experiments
The wound healing and transwell experiments both showed that the migration of podocytes in the LPS group and low-dose TAC group was faster than that of the normal group. However, podocyte migration in the medium-and high-dose TAC groups was much slower than that of the LPS group (Figure 7), indicating that TAC inhibited LPS-induced increase in podocyte migratory capability.

| DISCUSS ION
SA-AKI has shown high hospital mortality in patients, and LPS is one of the most common causes of this disease. [3][4][5] Therefore, there is an urgent need for appropriate therapeutic drugs to halt the progres- In conclusion, this is the first report demonstrating that TAC has protective effects on LPS-induced AKI by inhibiting TLR4/MyD88/ NF-κB signalling pathway and preventing podocyte dysfunction.
Therefore, TAC may be a potential therapeutic drug for treating LPSinduced SA-AKI in patients.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

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