FTY720 attenuates tubulointerstitial inflammation and fibrosis in subtotally nephrectomized rats

Abstract

Tubulointerstitial fibrosis is a common pathway that leads to kidney failure, and persistent tubulointerstitial inflammation is a key event in the development of tubulointerstitial fibrosis. The new immunosuppressive drug FTY720 modifies lymphocyte migration into injured tissues by sequestering lymphocytes within secondary lymphoid organs. However, its therapeutic effect on tubulointerstitial inflammation and fibrosis had not been well understood. This study was designed to explore the effect of FTY720 on tubulointerstitial inflammation and fibrosis in subtotally nephrectomized (SNX) rats. In total, 24 male Sprague–Dawley rats were used. Seven days after 5/6 nephrectomy, rats were randomized to FTY720 (1 mg/kg/d) and placebo-treated groups. Sham-operated rats served as controls. FTY720 significantly attenuated the rise in proteinuria, serum creatinine, urea nitrogen and N-acetyl-β-D-glucosaminidase activity in SNX rats, and reduced the count of peripheral white blood cells and lymphocytes in SNX rats. Morphological analysis revealed that there was severe tubulointerstitial inflammation and fibrosis in SNX group and much more tubulointerstitial infiltrating inflammatory cells with high expression of CD3, CD4, CD8, CD20, CD68, CD163 and CCR-7 in SNX group, as compared with the controls, but the lesions were attenuated significantly by treatment with FTY720. Furthermore, the expressions of proinflammatory molecules (IL-6, TNF-α and MCP-1), profibrotic molecule (TGF-β1) and production of extracellular matrix proteins such as fibronectin and types I and III collagens were upregulated in SNX rats. FTY720 administration significantly reduced these abnormalities. In summary, FTY720 exerts therapeutic effects on tubulointerstitial fibrosis in SNX rats by inhibiting the tubulointerstitial inflammatory response.

• Chronic kidney disease
• FTY720
• inflammation
• interstitial fibrosis
• lymphocyte

Introduction

Renal fibrosis is the principal process underlying the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD).1 It involves glomerulosclerosis, tubulointerstitial fibrosis and vascular sclerosis. Tubulointerstitial fibrosis (TIF) has been regarded as the most consistent predictor of an irreversible loss of renal function and progression to ESRD.2 Renal interstitial inflammation is closely related to the progressive renal fibrosis. Inflammatory infiltrates in chronic renal disease are composed of heterogeneous cell types, such as macrophages and lymphocytes.3 Although some cells are derived from the proliferation of resident macrophages, the majority enter the kidney from the circulation. Macrophage infiltration is driven by the local expression of molecules, such as monocyte chemoattractant protein-1 (MCP-1).4 Infiltrating cells release proinflammatory cytokines, such as TNF-α, IL-6 and IL-1β, and profibrotic molecules such as TGF-β1.5 These factors in turn activate interstitial resident fibroblasts to produce extracellular matrix proteins, such as fibronectin and types I and III collagens.6

Halting the process of renal inflammation and fibrosis remains a huge challenge for nephrologists. FTY720 {fingolimod; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3- propanediol hydrochloride} is a sphingosine-1-phosphate (S1P) analog originally developed as a novel immunosuppressant and has currently been tested in phase III studies for the treatment of renal transplantation.7 The mechanism of immunosuppressive action of FTY720 has not been fully elucidated, although a characteristic depletion of circulating lymphocytes in the peripheral blood and a reduction of lymphocyte infiltration into injured tissues by accelerating lymphocyte homing into peripheral lymph nodes has been observed in some experimental models.8 More interestingly, previous studies have shown that FTY720 attenuates renal injury in many experimental models, such as anti-thy1-induced chronic glomerulosclerosis,9 renal ischemia reperfusion injury,10 subtotal nephrectomy,11 experimental hypertensive nephropathy12 and antiglomerular basement membrane glomerulonephritis.13 Tubulointerstitial inflammation and fibrosis are common pathways of CKD leading to ESRD, regardless of the underlying cause. There are insufficient reports on the role of FTY720 in tubulointerstitial inflammation and fibrosis. Therefore, a more detailed insight into the potential pathomechanism is of interest to investigators. This study was designed to explore the effect of FTY720 on tubulointerstitial inflammation and fibrosis in 5/6 subtotal nephrectomized rats.

Materials and methods

Animal experiments

In total, 24 male Sprague–Dawley rats (Shanghai Super-B&K Laboratory Animal Corp. Ltd, Shanghai, China) with an initial weight of 200–220 g were housed in pairs in cages at a constant room temperature (22 °C) and humidity (55%) under a 12:12 h light–dark cycle. Chronic renal failure was initiated by a one-step subtotally nephrectomy as described previously.14 In brief, the right kidney was removed and a weight-controlled 2/3 left-side nephrectomy was performed (according to the weight of the left kidney). Sham operation was done by laparotomy and bilateral decapsulation of the kidneys. The rats had free access to tap water. The experiments were performed according to the Chinese law on animal protection.

After 7 d of surgery, the animals were divided into the following groups: sham + vehicle (Sham, n = 8), 5/6 subtotal nephrectomy + vehicle (SNX, n = 8) and SNX + FTY720 (FTY720, n = 8, FTY720 was purchased from Selleck Chemicals (Houston, TX). Treatment with FTY720 was started at the first week after complete surgery and the drug was administered by gavage in a dose of 1 mg/kg/d in sterile water.9,11,15 Systolic blood pressure was measured on Weeks 0, 2, 4, 6, 8, 10 and 12 after the subtotal nephrectomy in conscious animals by tail-cuff plethysmography and calculated as an average of three separate measurements at each session. Rats were killed on the 12th week after surgery. Before killed, rats were housed in metabolic cages for 24-h urine collection. Blood samples were collected by cardiac punctureand examined for white blood cell counts, blood urea nitrogen (BUN) and serum creatinine (Scr). Urine samples were examined for 24-h proteinuria and N-acetyl-β-D-glucosaminidase activity (NAG). White blood cell counts, BUN and Scr were measured at the Clinical Biochemistry Department of Zhong Da Hospital by automatic analyzers (Hitachi, Tokyo, Japan). NAG activity was determined from urine sample using an assay kit (Jiancheng, Nanjing, China).16

Tissue preparation

Rats were perfused with a saline solution containing 0.5 g/L of procaine hydrochloride at a pressure of 120–140 mm Hg by the retrograde insertion of a cannula into the abdominal aorta before sacrifice. After perfusion, the renal tissue was weighed, and several midcoronal sections were fixed in 10% buffered formalin and embedded in paraffin for histological investigation and immunohistochemical analysis. Pieces of kidneys were snap-frozen and stored at −80 °C for protein extraction.

Morphological analysis of the kidney

For the quantitative evaluation of a number of interstitial inflammatory cells, periodic acid-Schiff (PAS) staining sections were analyzed using a grid containing 100 fields. The interstitial cells per grid were counted in 20 nonoverlapping tubulointerstitial areas.11 For the evaluation of the percentage of tubulointerstitial fibrosis, Masson-stained sections were analyzed, and the severity of tubulointerstitial fibrosis was estimated.17

Immunohistochemistry

The following antibodies were used for immunohistochemical staining: polyclonal goat anti-rat CD3 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), polyclonal rabbit anti-rat CD4 (Santa Cruz Biotechnology Inc.), polyclonal rabbit anti-rat CD8 (Santa Cruz Biotechnology Inc.), polyclonal goat anti-rat CD20 antibody (Santa Cruz Biotechnology Inc.), monoclonal mouse anti-rat ED-1 (CD68) antibody (Santa Cruz Biotechnology Inc.), CCR7 (Epitomics, CA), monoclonal mouse anti-rat ED-2 (CD163) (Santa Cruz Biotechnology Inc.), polyclonal goat anti-rat IL-6 (Santa Cruz Biotechnology Inc.), polyclonal goat anti-rat TNF-α (Santa Cruz Biotechnology Inc.), polyclonal rabbit anti-rat MCP-1 (Santa Cruz Biotechnology Inc.), polyclonal rabbit anti-rat TGF-β1 (Santa Cruz Biotechnology Inc.), polyclonal rabbit anti-rat collagen I (Santa Cruz Biotechnology Inc.), polyclonal rabbit anti-rat collagen III (Santa Cruz Biotechnology Inc.) and polyclonal rabbit anti-rat fibronectin (Santa Cruz Biotechnology Inc.).

The sections were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase in methanol for 30 min at room temperature. The sections were microwaved for 5 min to retrieve the antigen by the antigen unmasking solution. After incubation with 5% skimmed milk for 1 h at room temperature, the sections were incubated with the primary antibodies overnight at 4 °C. Then, the tissues were washed several times in phosphate buffer saline (PBS) and incubated for 45 min with the secondary antibody; subsequently, the tissues were visualized with a Diaminobenzidine (DAB) kit, examined under a microscope and photographed.

For the evaluation of the numbers of the T lymphocytes (CD3), T helper cells (CD4), cytotoxic T cells (CD8), B lymphocytes (CD20), macrophages (ED-1/CD68) and macrophage subtypes (CCR7/M1, CD163/M2), interstitial cross-sections were analyzed using a grid containing 100 fields (Leica). In each kidney, at least 30 nonoverlapping cortical areas from two different sections were evaluated.11 For the immunoperoxidase stains to detect TGF-β1, 30 interstitial areas were graded by a qualitative score of 0–3 according to the intensity of the immunostaining (0 = no staining, 1 = low intensity, 2 = medium intensity and 3 = high intensity). Expression for collagens I, III and fibronectin in tubulointerstitium was analyzed by randomly selecting at least 15 cortical sections/animal using ×200 magnification. Matrix protein expression is indicated by the percentage of the positive-staining area per tubulointerstitium.18

Western blot analysis

Proteins from the kidney cortex extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, MA). Nonspecific antibody binding was blocked by a pre-incubation of the membrane in 1× Tris-buffered saline containing 5% skim milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with the primary antibodies against IL-6, TNF-α and MCP-1, followed by incubation with the appropriate peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, the signals were detected using an enhanced chemiluminescence advanced system (GE Healthcare, Chalfont St. Giles, UK).

Statistical analysis

The results are presented as the mean ± SD. Comparisons among several groups were determined with one-way analysis of variance (ANOVA) using SPSS18.0. If the ANOVA indicated an overall significant difference (p < 0.05), Student–Newman–Keuls (SNK) test was used to determine the significance of a difference in the means between any two groups. A level of p < 0.05 was considered significant.

Results

Effects of FTY720 on physiological and biochemical parameters in subtotal nephrectomized rats

After the subtotal nephrectomy, blood pressure increased significantly in the SNX group compared with the sham group (p < 0.01). FTY720 did not show significant effect on blood pressure in the SNX group compared with the vehicle-treated SNX rats (p > 0.05, Figure 1).

Figure 1. Effects of FTY720 on systolic blood pressure in the subtotally nephrectomized rats. After the subtotal nephrectomy, blood pressure rose significantly in the SNX group compared with the sham group. Treatment with FTY720 did not attenuate these abnormalities. n = 8, *p < 0.01 versus sham.

The SNX group exhibited proteinuria, increased plasma urea and creatinine and NAG compared to the sham group (p < 0.01). Treatment with FTY720 significantly attenuated these abnormalities (p < 0.01, Table 1).

Table 1. Effect of FTY720 on basic data in 5/6 nephrectomized rats.

Group	n	Proteinuria(mg/24 h)	NAG(U/L)	SCR(µmol/L)	BUN(mmol/L)
Sham + vehicle	8	6.29 ± 1.68	6.46 ± 3.50	42.20 ± 6.81	5.98 ± 0.45
SNX + vehicle	8	98.12 ± 12.55*	61.07 ± 7.72*	122.50 ± 15.78*	13.43 ± 0.61*
SNX + FTY720	8	50.45 ± 9.21#	41.93 ± 2.05#	61.90 ± 9.46#	10.33 ± 1.59#

Notes: Results are expressed as mean ± SD.

*p < 0.01 versus sham. #p < 0.01 versus SNX.

Effects of FTY720 on the peripheral white blood cell count.

In SNX rats, the numbers of circulating leukocytes and lymphocytes were similar to those in the sham animals (p > 0.05). However, the counts of peripheral lymphocytes and leukocytes were significantly reduced by treatment with FTY720 (p < 0.01, Figure 2). In SNX rats, the numbers of circulating monocytes were similar to those in the sham animals (p > 0.05), FTY720 did not reduce the number of peripheral monocytes (p > 0.05)

Figure 2. Effects of FTY720 on the number of peripheral white blood cells, lymphocytes and monocytes. The whole white blood cell count (A), blood lymphocyte count (B) and blood monocyte count (C) 12 weeks after the 5/6 nephrectomy. FTY720 significantly reduced the number of peripheral whole white blood cells and lymphocyte, but FTY720 did not reduce the number of peripheral monocytes. #p < 0.01 versus SNX.

FTY720 attenuates the tubulointerstitial histological changes in subtotal nephrectomized rats

Compared to the controls (Figure 3A and D), the SNX kidneys showed severe tubular dilatation, tubular atrophy, widened interstitial space and a greater number of inflammatory cells (Figure 3B and E), which were ameliorated by treatment with FTY720 (Figure 3C and F). The administration of FTY720 significantly inhibited the infiltration of tubulointerstitial inflammatory cells compared to that in SNX rats (p < 0.01, Figure 3G). The tubulointerstitial deposition of collagen detected by Masson staining was significantly increased in the SNX rats than that in the sham-operated controls, which was significantly attenuated when treated with FTY720. (p < 0.01, Figure 3H).

Figure 3. FTY720 attenuates the tubulointerstitial histological changes in the subtotally nephrectomized rats. Representative examples of PAS stains (A–C) and Masson stains (D, E) showing the tubulointerstitial histological changes (original magnification: ×200). Semiquantitative analyses of the tubulointerstitial inflammatory cells (G) and tubulointerstitial fibrosis (H). The tubulointerstitial fibrotic area and the number of inflammatory cells was markedly increased in the SNX animals compared with sham and was lower in SNX+FTY720 group; n = 8, *p < 0.01 versus sham, #p < 0.01 versus SNX.

Effects of FTY720 on tubulointerstitial infiltrating inflammatory cells

In the sham-operated rats, the T lymphocytes (CD3-positive), the subtypes of T helper cells (CD4-positive) and cytotoxic T cells (CD8-positive) were almost undetectable. However, in the SNX group, a markedly increased level of interstitial infiltration by different T cells was detected compared to the sham animals (p < 0.01). The interstitial infiltration by different T lymphocyte subtypes was significantly attenuated by treatment with FTY720 (p < 0.01, Figure 4A–C).

Figure 4. Effects of FTY720 on tubulointerstitial staining of T cells (CD3), T cell subtypes (CD4, CD8), B cells (CD20), macrophages (CD68) and macrophage subtypes (CCR7/M1, CD163/M2). FTY720 treatment significantly reduced the number of the interstitial infiltration of CD3-positive (A), CD4-positive (B), CD8-positive T cells (C), CD20-positive B cells (D), CD68-positive (E), CCR7-positive (F) and CD163-positive macrophages (G) in the SNX rats. Original magnification: ×400. Data are presented as the mean ± SD, n = 8, *p < 0.01 versus sham, #p < 0.01 versus SNX.

Interstitial B lymphocytes (CD20-positive) were significantly elevated in the SNX group compared with the sham group. Treatment with FTY720 significantly reduced the number of interstitial B lymphocytes in the SNX rats (p < 0.01, Figure 4D).

The number of interstitial monocytes/macrophages (ED-1/CD68-positive) and their different phenotypes, M1 (CCR7) and M2 (CD163), were significantly increased in the SNX group compared to the sham group (p < 0.01). Treatment with FTY720 markedly reduced the interstitial positive cell counts of monocytes/macrophages and their different phenotypes (M1 and M2) in the SNX rats (p < 0.01, Figure 4E–G).

Effects of FTY720 on the expression of proinflammatory and profibrotic molecules

To determine whether FTY720 could reduce the expression of proinflammatory molecules, we analyzed the expression of IL-6, TNF-α and MCP-1 by western blot analysis (Figure 5A). The protein expression of some proinflammatory molecules, such as IL-6, TNF-α and MCP-1 were increased in the SNX group, while FTY720 administration significantly attenuated these abnormalities (p < 0.01, Figure 5B–D). The immunostaining for TGF-β1 in the tubulointerstitial space was significantly enhanced in the rats with SNX compared to the sham group, while treatment with FTY720 significantly attenuated the expression (p < 0.01, Figure 6D).

Figure 5. Effects of FTY720 on the expression of proinflammatory molecules. Representative western blotting analysis of IL-6, TNF-α and MCP-1 are shown for the sham, SNX and SNX+FTY720 groups (A). Densitometric results showed that FTY720 treatment prevented the upregulation of IL-6 (B), TNF-α (C) and MCP-1 (D). β-Actin was used as an internal control. Data are presented as the mean ± SD, n = 3, *p < 0.01 versus sham, #p < 0.01 versus SNX.

Figure 6. Effects of FTY720 on the expression of profibrotic molecules (TGF-β1). Representative tubulointerstitial immunostaining of TGF-β1 are shown for the sham (A), untreated SNX (B) and SNX+FTY720 groups (C). FTY720 treatment prevented the upregulation of TGF-β1 in the tubulointerstitial area (D). Original magnification: ×200. Data are presented as the mean ± SD, n = 8, *p < 0.01 versus sham, #p < 0.01 versus SNX.

Effects of FTY720 on production of extracellular matrix proteins

The protein expression of extracellular matrix proteins showed a similar trend as histological fibrosis (Figure 7A–C). Tubulointerstitial protein deposition of collagens I, III and fibronectin were increased in the SNX group compared to the sham group, while FTY720 administration significantly attenuated these abnormalities (p < 0.01, Figure 7D–F).

Figure 7. Effects of FTY720 on protein expression of collagens I, III and fibronectin. Representative tubulointerstitial immunostaining of collagens I (A), III (B) and fibronectin (C) are shown for the sham (a), untreated SNX (b) and FTY720-treated SNX groups (c). Original magnification: ×200. the positive-staining areas of collagens I, III and fibronectin are larger in the SNX rats than in the sham; FTY720 administration significantly reduced these abnormalities (D, E, F). Data are presented as the mean ± SD, n = 8, *p < 0.01 versus sham, #p < 0.01 versus SNX.

Discussion

Tubulointerstitial fibrosis is a common consequence of a diverse range of kidney diseases leading to end-stage renal failure.2 As recently reviewed,3,19 chronic inflammation is one of the key risk factors contributing to tubulointerstitial fibrosis. Therefore, it is hypothesized that inhibiting renal interstitial inflammation is a pivotal strategy to halt the progression of interstitial fibrosis and thus prevent ESRD. In this study, we demonstrated that FTY720 attenuated the infiltration of inflammatory cells into the tubulointerstitial area, inhibited the expression of proinflammatory and profibrotic molecules and blocked production of extracellular matrix proteins in SNX rats. Our results provide novel insight into the use of FTY720 for treating chronic kidney disease by inhibiting tubulointerstitial inflammation and fibrosis.

It is well recognized that inflammation plays a crucial role in the initiation and progression of tubulointerstitial fibrosis after an injury. The classic view on the connection between inflammation and fibrosis is that they are mediated in a paracrine fashion, whereby inflammatory cells secrete proinflammatory and profibrotic cytokines that act on resident fibroblasts and tubular cells to promote fibrogenesis.5 Merely reducing the accumulation of extracellularmatrix (ECM) might not result in an improvement or stabilization of renal function.20 Advanced tubulointerstitial fibrosis represents a vicious cycle of inflammation, tissue damage and fibrosis.21 Furthermore, most therapeutic approaches to renal fibrosis are not specific to fibrosis but rather affect generic processes that act on multiple levels. Therefore, controlling renal inflammation may be the best way to target renal fibrosis and to maintain renal function.

However, the role of inflammation in the progression of fibrosis is more complicated than previously thought. Inflammatory cells exhibit heterogeneous phenotypes and have incredible functional plasticity in the development of kidney diseases.22 It remains largely unclear why and how inflammatory cell subtypes or phenotypes influence the rather unspecific inflammation that accompanies progressive renal diseases. Recently, it was found that lymphocytes, specifically CD4+ T cells, play a critical role in renal fibrosis following a ureteric obstruction.23 In addition, macrophages can switch phenotypes in response to the ever-changing microenvironment and alter their effect from proinflammatory to profibrotic.22 These new data demonstrate the plasticity of inflammatory cell function and call for conceptual and strategic innovations to develop effective cellular-based anti-inflammatory therapies for the treatment of CKD patients. Most immunosuppressive drugs currently used for autoimmune diseases therapy interfere with discrete events during T and B cell activation. The sphingosine-1-phosphate receptor agonist FTY720 could downregulate autoimmune inflammatory responses by modifying the migration of lymphocytes into injured tissues. It is the primary intention of this study to explore the effect of FTY720 on the tubulointerstitial inflammatory response in SNX rats. In this study, we found that FTY720 attenuated the number of lymphocyte cells infiltrating the tubulointerstitial area of SNX animals. The marked reduction of lymphocyte infiltration into the tubulointerstitial area of FTY720-treated rats may be explained by S1P receptor internalization and a downregulation by FTY720.

Although chronic kidney disease is characterized by widespread kidney lymphocyte infiltration, the role of lymphocytes in the progression of tubulointerstitial injury is not clearly defined. It remains largely unclear why and how different kinds of lymphocytes and subtypes of T lymphocytes influence the unspecific inflammation that occurs with progressive renal diseases. Our experiments showed the numbers of the subtypes of T helper cells (CD4) and cytotoxic T cells (CD8) were significantly increased in the SNX group compared to the controls and FTY720 treatment led to a significant reduction. Previously, the relative percentage of B lymphocytes in the renal tubulointerstitium in chronic kidney disease was considered low.24,25 In our study, there was a marked staining for CD-20-positive B cells in the SNX rats compared to the sham-operated controls. FTY720 treatment significantly reduced the infiltration of tubulointerstitial CD-20-positive B cells in the SNX rats.

In this study, we demonstrated that FTY720 treatment could induce a marked lymphopenia, while without any effect on monocyte count. In addition, we also found a reduction of macrophages infiltration in the tubulointerstitium in FTY720-treated SNX rats. Previous studies have indicated that MCP-1 serves as a chemotactic signal to attract macrophages to the injured site in the kidney.26,27 Recruitment of lymphocytes to the kidney could induce the production of MCP-1 by renal cells.28 It is therefore we assumed that the reduction of infiltration of macrophage in the tubular interstitium by FTY720 treatment may relate to the reduced production of MCP-1 in the kidney.

The contribution of macrophages to renal injury is well documented.27 Recent reports indicate that the activation status of infiltrating macrophages is a major determinant of injury.29 When monocytes from circulating blood are recruited to the injured site, they differentiate into two broad but distinct subsets of macrophages that are categorized as either classically activated (M1) or alternatively activated (M2).30 In general, M1 macrophages display a typical proinflammatory phenotype, produce a variety of chemokines and therefore have pathogenic functions that lead to tissue damage. M2 macrophage subpopulations resolve inflammation and repair injury. However, insufficient vascular and epithelial healing, abundant growth factor secretion promotes profibrotic M2 subpopulations that accelerate fibrogenesis.22 It remains largely unclear how macrophage phenotypes determine the progression or resolution of renal inflammation and fibrosis during the different phases of renal injury. CCR7 is a surface marker indicative of an M1 phenotype, and CD163 is a surface marker representative of an M2 phenotype.31 In this study, we found that both M1 and M2 macrophage subsets were markedly increased in the SNX rats compared to the sham group. Treatment with FTY720 significantly inhibited the infiltration of M1 and M2 macrophages, further suggesting that FTY720 might exert its antifibrotic effect by inhibiting the inflammatory process that occurs in injured renal tissue.

A series of proinflammatory and profibrotic molecules secreted by lymphocytes and macrophages directly contribute to renal injury.4,5 There is a complicated interaction between lymphocytes and macrophages in a mutual and complex manner. Previous work has shown a positive correlation between renal injury and the over expression of TNF-α, IL-6 and MCP-1.4 TGF-β1 is a central mediator of fibrosis that works primarily by inducing ECM production and the proliferation of myofibroblasts and fibroblasts.5 Our study showed that FTY720 treatment significantly attenuated the expression of proinflammatory molecules (TNF-α, IL-6 and MCP-1) and profibrotic molecules (TGF-β1) in the SNX group, further suggesting the potential for this treatment to be an anti-inflammatory agent in CKD treatment.

Our results showed that the counts of peripheral lymphocytes and leukocytes were significantly reduced in the SNF group. It naturally causes some concern about its potential side-effects. In an initial phase II study in multiple sclerosis, Kappos et al.32 found that giving fingolimod at dosage of 1.5 or 5 mg daily, no severe infectious complications occurred in the placebo-controlled core phase (6 months). Furthermore, treatment with FTY720 did not promote spontaneous bacterial infections after experimental stroke in mice.33 It is noted that fingolimod-mediated lymphopenia is primarily due to the retention of lymphocytes in lymphatic tissue and is fully reversible in most patients. FTY720 does not seem to repress the function of lymphocytes in the context of the primary immune response.34 However, pharmacovigilance should be practiced when administering FTY720 clinically.

In conclusion, our study demonstrates that FTY720 ameliorates the tubulointerstitial injury in 5/6 nephrectomized rats by inhibiting the infiltration of tubulointerstitial inflammatory cells, the expression of proinflammatory molecules and tubulointerstitial fibrosis. These findings provide novel insights into this newly developed drug with regard to the prevention of the progression of chronic kidney disease.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study was financially supported by the National Natural Scientific Fund (no. 81130010), the National Key Basic Research Project (no. 2012CB517700) and the Jiangsu Natural Scientific Fund (no. BK2011061) with Professor Bi-Cheng Liu as the PI.