Tacrolimus ameliorates tubulointerstitial inflammation in diabetic nephropathy via inhibiting the NFATc1/TRPC6 pathway

Abstract Tubulointerstitial inflammation is crucial for the progression of diabetic nephropathy (DN), and tubular cells act as a driving force in the inflammatory cascade. Emerging data suggested that tacrolimus (TAC) ameliorates podocyte injury and macrophage infiltration in streptozotocin (STZ) mice. However, the effect of TAC on tubulointerstitial inflammation remains unknown. We found that albuminuria and tubulointerstitial damage improved in db/db mice treated with TAC. Macrophage infiltration and expression of IL‐6, TNF‐α, fibronectin, collagen 1 and cleaved caspase 3 were inhibited as well. In addition, the expression of nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) was up‐regulated in the kidneys of DN patients and correlated with tubular injury and inflammation. The expression of NFATc1 and TRPC6 also increased in the kidneys of db/db mice and HK‐2 cells with high glucose (HG), while TAC inhibited these effects. HG‐induced inflammatory markers and apoptosis were reversed by TAC and NFATc1 siRNA in HK‐2 cells, which was abolished by TRPC6 plasmid. Furthermore, HG‐induced TRPC6 expression was inhibited by NFATc1 siRNA, while NFATc1 nuclear translocation was inhibited by TAC, but was restored by TRPC6 plasmid in HK‐2 cells under HG conditions. These findings suggest that TAC ameliorates tubulointerstitial inflammation in DN through NFATc1/TRPC6 feedback loop.

a crucial role in the pathogenesis and progression of DN, which is characterized by increased macrophage infiltration and release of multiple inflammatory cytokines. [6][7][8] Moreover, it was demonstrated that renal tubular epithelial cells (TECs) are a key driving force in mediating macrophage recruitment and the subsequent local inflammatory cascade in the kidney under a hyperglycaemic state. [9][10][11] Thus, targeting TEC-mediated inflammation might be a novel therapeutic strategy to alleviate activation of macrophages and kidney inflammation, eventually delaying the progression of DN.
Tacrolimus (TAC, also known as FK506), a new immunosuppressive drug, has lower nephrotoxicity than cyclosporine, which has been widely used in post-kidney transplantation and immune-related chronic kidney disease by suppression of nuclear factor of activated T cell (NFAT) dephosphorylation and transcriptional activation of NFAT target genes. [12][13][14] Recently, TAC was attempted to be used in experimental DN patients and animal models. JIN H et al reported that TAC combined with a double dose of valsartan treatment ameliorated proteinuria and improved renal function by inhibiting high-sensitivity C-reactive protein and adiponectin levels in a small sample of DN patients. 15 Qi XM found that treatment with TAC mitigated collagen IV and TGF-β1 expression in streptozotocin (STZ)-induced diabetic rats. 16 Subsequent studies focused on the protective effect of TAC on podocytes and showed restored nephrin and podocin expression in early DN rats. 17 Regarding inflammation, it was shown that TAC reduced the infiltration of iNOS + /ED-1 + macrophages and NF-кB expression in STZ-induced diabetic rats. 18 However, the role of TAC in tubular-induced inflammation remains unknown.
The canonical transient receptor potential channel 6 (TRPC6) is a subtype of non-selective Ca 2+ -permeable cation channels. 19 It was shown that TRPC6 expression was up-regulated in the kidneys of STZ rats and Akita mice. 20,21 TRPC6 mediated podocyte calcium influx enhancement and renal damage in STZ rats. 20 Emerging evidence demonstrated TRPC6 contributes to inflammation through correlation with NFAT. It was shown that TAC ameliorates podocyte injury in type 2 DN rats by down-regulating TRPC6 and NFAT expression. 22 Angiotensin II (Ang Ⅱ) increased TRPC6 expression via an NFAT positive feedback pathway, 23 whereas the correlation between TRPC6 and NFAT in the inflammation of tubular cells under DN conditions is unclear.
In this study, we focused on the effect of TAC on tubulointerstitial inflammation and injury under a DN state in vitro and in vivo.
NFATc1/TRPC6 was investigated to explore the potential mechanism(s) of this process.

| Animal experimental design
Twelve-week-old diabetic male db/db mice were used for the animal experiments, and week-matched db/m mice were used as a control. These mice were purchased from the Aier Matt Experimental Animal Company (Suzhou, China) and were randomly divided into four groups (n = 6 in each group): control (db/m), db/db, db/ db plus 0.5 mg/kg TAC (a gift from Huadong Pharmaceutical Co., Ltd, Hangzhou, China) and db/db plus 1.0 mg/kg TAC. They were housed in Second Xiangya Hospital of Central South University using a 12-hour/12-hour light/dark cycle. All mice were allowed free access to tap water and standard laboratory chow (Second Xiangya Hospital of Central South University). Chow contains ≥18% protein, ≤10% water, ≥4% fat, ≤5% fibre, ≤8% ash, 1.0%-1.8% calcium and 0.6%-1.2% calcium. TAC was dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na, Sigma-Aldrich, St. Louis, MO, USA) and was administered intragastrically to db/db mice. Untreated db/db and db/m mice received identical intragastric administration of the CMC-Na for 8 weeks. Administration routes and drug doses were selected based on previous studies. [16][17][18]22,24 Experimental mice were euthanized with an intraperitoneal injection of 50 mg/kg bodyweight sodium pentobarbital at 20 weeks, and samples of serum, urine and kidney tissues from each group were collected for further studies. The Animal Care and Use Committee of Second Xiangya Hospital of Central South University approved all animal procedures.

| Assessment of biological chemistry parameters and urine albuminuria excretion
The blood glucose levels were detected twice a week with the blood sample withdrawn from mouse tail vein using the blood glucose monitor and test strips (Boehringer Mannheim, Mannheim, Germany).
First, insert the blood glucose test strip into the blood glucose monitor. Then, fix the tail of the mouse and pierce the tail blood vessels of the mouse with a blood collection needle, and gently squeeze the centrifugal end with the hand. Last, the blood was transferred to test strips and the blood glucose value was recorded. Serum creatinine was assayed by standard automated enzymatic methods (Hitachi 912 automated analyzer, Hitachi, Mannheim, Germany).
Prior to killing, mice were placed in metabolic cages for collection of urine over 24-hour period of time. After centrifugation (4°C, 3000 g, 10 minutes), the supernatant from urine sample was frozen at −70°C for subsequent analysis. Urine albumin concentration was analysed using urine albumin ELISA kit (Bethyl Laboratories, Montgomery, TX, USA). Urinary albumin excretion (ACR) was calculated as the urine albumin/creatinine ratio.

| Morphological analysis and immunohistochemistry assay of kidneys in type 2 diabetic patients and experimental mice
Human kidney biopsy sections were obtained from DN patients (n = 10), and an equal number of patients with glomerular minor lesion (n = 10) were recruited for the study as controls as previously described. 25 According to the pathological classification of DN, 26 the DN patients observed in this study were class IIb (n = 4) and III (n = 6).
All of the DN patients use metformin combined with insulin to control blood glucose. Patients who used adrenal cortical hormones or immunosuppressive agents were excluded. All procedures followed were in accordance with the World Medical Association Declaration of Helsinki, and all subjects provided written informed consent. The human protocol was approved by the Ethics Committee of Second Xiangya Hospital, Central South University.
The threshold of images was adjusted to select the positive area (shown in red) and calculated the integral optical density (IOD) of each picture.

| Analysis of renal apoptosis
Terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL) staining was used to gauge apoptosis in the kidney sections of the mouse groups according to the manufacturer's instructions. 29

| Flow cytometry analysis of macrophage markers
Single renal cell suspensions obtained from db/db mice were blocked with antimouse CD45, CD11b and F4/80 in flow cytometry buffer (2% foetal bovine serum in phosphate buffer saline) for 30 minutes on ice in the dark, followed by flow cytometric analysis with FlowJo software (Tree Star Inc., Ashland, CA, USA). The data were analysed with FlowJo 10 software as previously described.

| Expression and translocation of NFATc1 and TRPC6 by immunofluorescence assay
HK-2 cells received the above treatments and were then detected by an immunofluorescence (IF) assay. Briefly, HK-2 cells were fixed, infiltrated, blocked and then incubated with anti-NFATc1 or anti-TRPC6 antibodies overnight at 4°C. The cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI), and images were obtained with confocal microscopy as described previously. 27 The mRNA levels of NFATc1 and TRPC6 were normalized to β-actin.
The primer sequences used for amplification are shown in Table 1.

| Protein extraction and Western blot analysis
Protein extraction from mouse kidney tissues and HK-2 cells was performed with radio immunoprecipitation assay (RIPA) lysis buffer. Nucleoprotein and plasma protein extraction was performed using the nuclear and cytoplasmic protein extraction kits

| Statistical analysis
All statistical analyses were performed using SPSS 20.0 software.
Values are presented as the mean ± SD and were assessed by Student's t test or one-way analysis of variance. Correlation analysis was performed using Pearson's correlation analysis. P < .05 indicated a significant difference.

| Protective effect of TAC on renal function and morphological changes in the kidneys of db/db mice
The bodyweight and serum creatinine levels notably increased in db/db mice compared with those of db/m mice, while no significant difference was observed with TAC treatment ( Figure 1A,C).
The blood glucose level was slightly higher in 0.5 mg/kg TAC treatment group as compared to db/db mice. There was no aggravated effect on blood glucose level of db/db mice as the dose of TAC treated from 0.5 to 1.0 mg/kg ( Figure 1B). The level of ACR was significantly increased in db/db mice, whereas it was down-regulated by TAC treatment, especially in high dose TAC treatment group ( Figure 1D). In addition, HE, PAS and Masson's staining showed that morphological changes in the kidney were also attenuated with TAC treatment, which was characterized by amelioration of glomerular hypertrophy, mesangial matrix proliferation, tubular atrophy and tubulointerstitial fibrosis ( Figure 1E).
Subsequent semiquantification analysis and glomerular and tubulointerstitial scores verified these results ( Figure 1F,G).
Transmission electron microscopy (TEM) showed basement membrane thickening, podocyte foot process fusion and mitochondrial fragmentation in tubular cells. Encouragingly, TAC alleviated these pathological changes in a dose-dependent manner ( Figure 1H,I). Similarly, TAC also reduced the expression of cleaved caspase 3 (C-CAS3) in the kidneys of db/db mice ( Figure 2E), which was verified by densitometry analysis ( Figure 2G).

| Macrophage infiltration and the level of inflammatory cytokines in db/db mice were ameliorated by TAC
In addition to the effect of TAC on tubulointerstitial injury, changes in inflammation after treatment were also investigated. As shown in Figure 3A, the intensity of IL-6 and TNF-α expression was sig-

| Overexpression of NFATc1 and TRPC6 in the kidney tissues of db/db mice was inhibited by TAC treatment
Immunostaining showed that the intensity of NFATc1 and TRPC6 was low in the kidneys of db/m mice, whereas expression was ob-

| NFATc1 and TRPC6 expression in kidney sections positively correlated with inflammation and tubular injury in type 2 DN patients
The basal clinical data of the DN patients and controls in this study were shown in Table 2. HE, PAS and Masson's staining of the renal sections of DN patients showed mesangial matrix proliferation, tubular atrophy and interstitial fibrosis ( Figure 5A). In addition, the IFTA and glomerular damage scores of patients with DN were higher than those of control subjects ( Figure 5C1,C2).

IHC staining revealed increased expression of NFATc1 and TRPC6
as well as IL-6 in DN patients compared with that of controls ( Figure 5B,

| TAC alleviated inflammation and apoptosis in HK-2 cells under hyperglycaemia conditions
As shown in Figure 6A, Western blotting showed a significant time- showed up-regulated IL-6 and C-CAS3 in HK-2 cells exposed to HG, which was reversed by TAC treatment ( Figure 7A, A2 and A3), indicating that TAC exerts a beneficial role in inflammation, production of extracellular matrix and apoptosis of HK-2 cells under hyperglycaemia.

| TAC inhibited HG-induced high expression of NFATc1 and TRPC6 in HK-2 cells
It was shown that an increased expression of TRPC6 in HK-2 cells was induced by HG in a time-dependent manner ( Figure 6A,A1).
Western blotting of nuclear protein extracts revealed a significant

| NFATc1/TRPC6 mediated the effect of TAC on inflammation and apoptosis of HK-2 cells under HG conditions
As shown in Figure 7A,

| D ISCUSS I ON
The present study delineated that administration of TAC exerted a beneficial role in tubulointerstitial injury, especially in inflammation of db/db mice, which was characterized by suppressed tubular atrophy and tubulointerstitial fibrosis, reduced macrophage infiltration and inflammatory cytokine (IL-6, TNF-a) expression. Moreover, in vitro experiment also demonstrated that TAC ameliorated inflammation, the production of extracellular matrix and apoptosis of HK-2 cells exposed to exogenous HG. It has been demonstrated that inflammation is crucial for the pathogenesis and progression of DN. 6,35,36 Renal pathological analysis revealed that macrophages are the most abundant immune cell in DN patients and animal models, which correlates with tubular atrophy and tubulointerstitial fibrosis. [37][38][39] Macrophage depletion in diabetic mice ameliorated albuminuria and morphological changes in DN. 40 Notably, accumulating data suggested that tubular cells act as a driving force rather than as 'victims' in the inflammation in DN. It was found that MCP-1 increased in HK-2 cells exposed to HG, which enhanced the migration ability of co-cultured macrophage. However, TAC, a calcineurin inhibitor, could reduce the phosphorylation activity of calcineurin by binding to FK506 binding protein (FKBP12), 43 and preventing the dephosphorylation and nuclear translocation of NFAT, 12 and has been widely used in immune-associated kidney diseases. 14,44,45 Emerging data demonstrated that TAC exerts a beneficial effect on proteinuria and fibrosis, 15,16 especially against podocyte injury in DN. 17,22 It was found that TAC reduces calpain-mediated NFAT activation, which reduces tubular cell death and the transcription of pro-inflammatory cytokine genes in ischaemia-reperfusion injury (IRI) mice. 46 However, few studies have reported the effect of TAC on tubulointerstitial inflammation in DN.
In the present study, it was shown that TAC not only reduced proteinuria, and improved tubulointerstitial pathological changes, but  also markedly ameliorated macrophage infiltration and pro-inflammatory cytokine expression in db/db mice (Figures 1 and 3). Moreover, we observed that TAC has no obvious influence on blood glucose level of db/db mice, although a little higher in 0.5mg/kg TAC treatment ( Figure 1). The result was in accordance with previous literatures in STZ-induced diabetic rats. [16][17][18]22 Thus, we suppose that there was no and knockout of TRPC6 reduced proteinuria and tubular injury in Akita mice. 21 In this study, TRPC6 expression was obviously up-regulated in the kidneys of DN patients, which positively correlated with tubulointerstitial damage score and IL-6 expression ( Figure 5). In addition, TRPC6 expression increased in the kidney tissue of db/db mice and in HK-2 cells incubated with HG, and these effects were reversed by TAC treatment (Figures 4, 6 and 7). However, the beneficial effect of TAC on tubular inflammation and apoptosis was partially neutralized by the TRPC6 overexpression plasmid (Figure 7), indicating TRPC6 contributes to the effect of TAC on the inflammation of tubular cells in DN.
Interestingly, it was shown that there was a tight correlation between NFAT and TRPC6.
TRPC6 is regulated by NFAT in cardiomyocytes and vice versa. 49 Moreover, TRPC6 expression increased through positive feedback with NFAT in podocytes incubated with Ang II. 23 Precisely, the promoter of the TRPC6 gene contains 2 conserved NFAT sites; therefore, upon NFAT binds to these domains, it could initiate the transcription of TRPC6. 49 On the other hand, TRPC6, in turn, promotes the nuclear translocation of NFAT via calcium-dependent activation of calcineurin, which enhances the dephosphorylation of NFAT. 23,50 In the present study, we found that HG-induced TRPC6 expression was inhibited by In conclusion, this study delineated that TAC has a potential effect on the amelioration of tubuloinflammation and fibrosis, partially through NFATc1/TRPC6 (Figure 8), which provides more evidence for the renoprotective effects of TAC under hyperglycaemia.

ACK N OWLED G EM ENTS
The authors are grateful to the time and effort from all who contributed to this study.

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data included in this study are available from the corresponding author upon reasonable request.

R E FE R E N C E S F I G U R E 8
Diagram of the potential mechanism by which tacrolimus (TAC) protects against tubulointerstitial inflammation and fibrosis in diabetic nephropathy (DN). Nuclear factor of activated T cell 1 (NFATc1) is dephosphorylated and translocates to the nucleus in tubular cells in hyperglycaemia conditions. NFAT targets downstream genes, such as transient receptor potential channel 6 (TRPC6), and causes macrophage aggregation and inflammatory cytokine release, eventually leading to tubulointerstitial inflammation and fibrosis. Encouragingly, treatment with TAC significantly inhibits NFATc1 dephosphorylation and nuclear translocation and reduces the transcriptional activation of TRPC6, which ameliorates macrophage infiltration and inflammatory cytokine expression. On the other hand, TRPC6 also induces NFATc1 dephosphorylation and nuclear translocation. The positive feedback loop between NFATc1 and TRPC6 contributes to tubulointerstitial inflammation and injury in DN with TAC treatment