Loss of sphingosine kinase 2 protects against cisplatin-induced kidney injury

Abstract

Cisplatin is an established chemotherapeutic drug for treatment of solid-organ cancers and is the primary drug used in the treatment of head and neck cancer; however, cisplatin-induced nephrotoxicity largely limits its clinical use. Inhibition of sphingosine kinase 2 (SphK2) has been demonstrated to alleviate various kidney diseases. Therefore, we hypothesized that inhibition of SphK2 could also protect against cisplatin-induced nephrotoxicity. Results from the present study showed that the SphK2 inhibitor ABC294640 or knockdown of SphK2 by siRNA blocked the cisplatin-induced increase of cellular injury markers (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and cleaved caspase-3) by Western blot analysis in HK-2 cells, a human renal tubular cell line. In addition, SphK2 inhibition blocked cisplatin-induced activation of NF-κB by Western blot analysis and immunostaining analysis. Furthermore, SphK2 inhibition suppressed cisplatin-induced increases of proinflammatory markers (NLR family pyrin domain containing 3, interleukin-1β, and interleukin-6). Genetic deletion of the SphK2 gene in mice further confirmed that inhibition of SphK2 protected against cisplatin-induced kidney damage in vivo. Compared with wild-type mice, SphK2 knockout mice exhibited less renal dysfunction and reduced promotion of kidney injury markers, inflammatory factors, tubular morphology damage, and fibrotic staining. At the same time, the SphK2 inhibitor ABC294640 failed to interfere with the activity of cisplatin or radiation in two cell culture models of head and neck cancer. It is concluded that inhibition of Sphk2 protects against cisplatin-induced kidney injury. SphK2 may be used as a potential therapeutic target for the prevention or treatment of cisplatin-induced kidney injury.

NEW & NOTEWORTHY The present study provides new findings that sphingosine kinase 2 (SphK2) is highly expressed in renal tubules, cisplatin treatment increases the expression of SphK2 in proximal tubular cells and kidneys, and inhibition of SphK2 alleviates cisplatin-induced kidney injury by suppressing the activation of NF-κB, production of inflammatory factors, and apoptosis. SphK2 may serve as a potential therapeutic target for the prevention or treatment of cisplatin-induced nephrotoxicity.

INTRODUCTION

Cisplatin is a widely used and effective drug for the treatment of solid-organ cancers, including head and neck cancer, lung cancer, ovarian cancer, and cervical cancer (1). However, nephrotoxicity of cisplatin largely limits its clinical use (2). About 20–40% of patients who received cisplatin developed kidney injury (3). The mechanisms of cisplatin-induced nephrotoxicity are complex and associated with various cellular processes, including inflammation, DNA damage, apoptosis, necrosis, oxidative stress, and mitochondrial dysfunction (4). Studies have showed that two membrane transporters, organic cation transporter 2 (OCT2) and Ctrl1, can actively transport cisplatin into cells (5). These transporters are primarily expressed in proximal tubular cells, which results in the accumulation of cisplatin in tubules and leads to tubular damage (6). Although several molecular pathways have been identified as potential therapeutic targets for renoprotection against cisplatin, no therapy is currently available, and nephrotoxicity is still the major obstacle in the clinical utilization of cisplatin (3, 7).

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite that plays an important in regulating various cellular processes, including cell migration, cell proliferation, vascular maturation, apoptosis, and inflammation (8). Studies have showed that S1P is involved in various kidney disease states. It has been reported that S1P induces the expression of fibrotic markers in kidney cells and that increased expression of S1P is found in diabetic kidneys and lupus nephritis (9, 10). Sphingosine kinase 2 (SphK2) is a rate-limiting enzyme that phosphorylates sphingosine into S1P. SphK2 is localized in the nucleus, mitochondria, and endoplasmic reticulum and is associated with DNA synthesis, histone acetylation, cellular arrest, and apoptosis (11). Studies have demonstrated that deletion of the SphK2 gene or inhibition of SphK2 alleviates the inflammatory response and reduces macrophage infiltration and renal fibrosis in a unilateral ureteral obstruction model (12, 13). Other studies have shown that deletion of SphK2 reduces the expression of proinflammatory factors and fibrotic markers and attenuates fibrosis in folic acid-induced kidney injury (14). Given the importance of SphK2 in the pathogenesis of kidney disease, the present work was designed to study the role of SphK2 in cisplatin-induced kidney injury based on the premise that inhibition of SphK2 could protect against cisplatin-induced nephrotoxicity.

MATERIALS AND METHODS

Cell Culture, Treatment, and Transfection of SphK2 siRNA

Human renal tubular cell line HK-2 cells were cultured in DMEM with 10% FBS, streptomycin (100 µg/mL), and penicillin (100 IU/mL) at 37°C with 5% CO₂. Cells were grown in 6-cm dishes, cells were grown to ∼60% confluence, and then treated with 20 µM cisplatin (Sigma) for 24 h (15, 16) with or without 30-min pretreatment with 5 µM ABC294640 (SphK2 inhibitor, Echelon) (17, 18) or 1 µM (19) SLM6031434 (SphK2 inhibitor, Cayman) (20). The concentration of ABC294640 used in our study was determined based on our pilot experiments showing protection without cell damage on its own. This concentration was consistent with literature showing that 3 µM ABC294604 inhibited 80% of SphK2 activity in colorectal cancer cells (21) and that 5 µM ABC294604 totally inhibited SphK2 activity in bovine aortic vascular smooth muscle cells (22).

Transfection of SphK2 siRNA was performed as we have previously described (23). Briefly, HK-2 cells were seeded in six-well plates and incubated overnight until 80–90% confluence. Cells were then transfected with 100 nM SphK2 siRNA (siSphK2) or control siRNA (siCtrl) with Lipofectamine 3000 (Invitrogen) in reduced serum medium based on the manufacturer’s instructions. Control and cisplatin-treated groups were transfected with siCtrl to eliminate the effects of the transfection procedure. After 24 h of transfection, Opti-MEM medium was changed with complete culture medium for another 24 h, and 20 µM cisplatin was then added to the medium for 24 h. After the above treatments, cells were collected for further analysis. SphK2 knockdown efficiency was confirmed by Western blot analysis.

Experimental Animals

Experiments were performed in adult male SphK2 knockout (KO) mice (10–12 wk old, 25–30 g), which were purchased from Jax Lab and bred in the animal facility of our university. KO mice were generated on the C57BL/6 background, and C57BL/6 mice were used as the wild-type (WT) control. Mice were free to access food and water with a 12:12-h light-dark cycle. Mice were divided into the following four groups: WT + vehicle, SphK2 KO + vehicle, WT + cisplatin, and SphK2 KO + cisplatin. Cisplatin (10 mg/kg) was given weekly by intraperitoneal injection for 4 wk (24–26). Mice were then euthanized after blood and kidneys were collected for further study. All animal protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Immunohistochemistry of SphK2 in Mouse Kidney Tissue

The distribution of SphK2 in kidneys was assessed using immunohistochemistry (IHC). Kidneys from WT mice were collected and fixed with 4% formaldehyde, paraffin-embedded, and cut into 4-μm sections. IHC was done as we have previously described (27). The primary antibody against SphK2 was from Santacruz (P19, goat polyclonal, 1:50 dilution).

Western Blot Analysis

Western blot analysis was performed as we have previously described (28–30). Briefly, kidney samples were homogenized with lysis buffer, and cells were also lysed with lysis buffer on ice for 20 min. Supernatants were collected after centrifugation at 12,000 g and 4°C for 30 min and boiled in 4× loading buffer for 10 min at 100°C. Protein samples were separated in 10% or 12% SDS-PAGE at 120 V and transferred onto a PVDF membrane. Membranes were blocked with nonfat milk for 1 h and then incubated with primary antibodies of anti-NF-κB (ab16502, rabbit polyclonal, 1:1,000, Abcam), anti-neutrophil gelatinase-associated lipocalin (NGAL; ab63929, rabbit polyclonal, 1:1,000, Abcam), anti-α-smooth muscle actin (α-SMA; ab5694, rabbit polyclonal, 1:1,000, Abcam), anti-kidney injury molecule-1 (KIM-1; PA1-86790, rabbit polyclonal, 1:1,000, Invitrogen), anti-cleaved caspase-3 (D175, rabbit polyclonal, 1:1,000, Cell Signaling), anti-IL-1β (3A6, rabbit polyclonal, 1:1,000, Cell signaling), anti-IL-6 (D3K2N, rabbit polyclonal, 1:1,000, Cell Signaling), anti-SHPK2 (MBS2518663, rabbit polyclonal, 1:500, MyBioSource), anti-NLR family pyrin domain containing 3 (NLPR3; NBP2-12446, rabbit polyclonal, 1:1,000, Novus), or anti-OCT2 (A14061, rabbit polyclonal, 1:500, Abclonal) overnight at 4°C. After being washed with PBS with Tween 20, membranes were then incubated with horseradish peroxidase-labeled secondary antibody (1:5,000) and visualized by ECL. Anti-GAPDH (rabbit monoclonal, 1: 3,000, Cell Signaling Technology) was used as the internal control and detected by fluorescence-labeled secondary antibody ((IRDye 680RD, Li-Cor). Bands were detected using the Odyssey FC Imaging system (Li-Cor). Densitometric analysis of band intensities was performed using ImageJ (v. 1.44, National Institutes of Health). All ratios of band intensities over GAPDH were normalized to the mean value of the control group.

Quantification of S1P by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Cells were collected on ice, snap frozen in liquid N₂, and then processed and analyzed by the VCU Lipidomics Core using liquid chromatography-electrospray ionization tandem mass spectrometry as previously described (31, 32).

Immunostaining and Fluorescent Microscopy in Cultured Cells

Cells were cultured on glass coverslips and, after the experiment was finished, fixed in 4% paraformaldehyde, incubated with anti-NF-κB p65 (ab16502, rabbit polyclonal, 1:200, Abcam) at 4°C overnight, incubated with fluorophore-conjugated secondary antibody for 1 h in the dark, mounted with DAPI, and sealed with a cover glass for 5 min. Pictures were then taken by fluorescence microscopy.

Histological Analysis

Periodic acid-Schiff staining (Sigma-Aldrich staining kit) was used to examine the tubular structures. Tubular injuries were scored by two independent and blinded examiners on a minimum of 10 cortical fields (27). Tubular injury is defined as cast formation, tubular dilation, tubular atrophy, and sloughing of tubular epithelial cells. The score was calculated based on the percentage of the injured area as follows: 0 = normal, 1 = <10%, 2 = 10–25%, 3 = 26–50%, 4 = 51–75%, and 5 = >75% (33). Masson trichrome staining (Polysciences) was used to examine extracellular matrix deposition. The percentage of the positive area in Masson trichrome staining was assessed by a computer program (Image-Pro Plus).

Renal Function

Blood samples were centrifuged at 1,500 g for 10 min to isolate the serum. According to the manufacturer’s instructions, serum creatinine was assessed by commercial kits (Fujifilm) and blood urea nitrogen (BUN) was assessed by a Urea Reagent Kit (Alfa Wassermann) using a Vet Axcel Chemistry Analyzer.

In Vitro Drug Sensitivity and Clonogenic Survival Assays

The sensitivity of head and neck cancer cell line HN30 and HN12 cells to cisplatin, the Sphk2 inhibitor ABC294640, and the combination of the two agents was determined by an MTS assay. In this assay, metabolically active cells catalyze the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Abcam) into a formazan product that is soluble in culture medium. Five thousand cells were seeded in 96-well plates and treated with concentrations of cisplatin ranging from 0 to 40 µM. After cisplatin removal, cells were treated with 2.5 and 5 µM ABC294640 for 24 h. After this, cells were treated with 10% MTS in culture media and incubated at 37°C for 4 h. The spectrophotometric absorbance of the samples was measured by a plate reader at a wavelength of 490 nm. For clonogenic survival assays, cells were seeded at a low density (5 × 10³ cells/10-cm dish) and exposed to 6-Gy radiation in the absence and presence of 5 µM ABC294640. Colony formation was monitored over time, and at the experiment end point (when the vehicle-treated condition formed distinct colonies with >50 cells), colonies were fixed with 100% methanol, air-dried, and stained with 0.05% crystal violet.

Statistics

Means ± SE was used to present data. One-way ANOVA followed by Tukey's test was used to compare among groups. Student’s t test was used to assess statistically significant differences between two groups. Statistical significance was defined as P < 0.05.

RESULTS

SphK2 Is Highly Expressed in Renal Proximal Tubules, Cisplatin Increased Expression of SphK2, and SphK2 siRNA Blocked the Cisplatin-Induced Increase of SphK2 in HK-2 Cells

SphK2 was found to be highly expressed in proximal tubules of the kidney by IHC (Fig. 1A). These results are consistent with the same expression pattern in human kidneys (12). Based on the fact that cisplatin primarily causes proximal tubule damage (33) and that SphK2 was highly expressed in proximal tubular cells, proximal tubular cells were used as the experimental model system in this project.

Figure. 1.

A: immunohistochemical analysis of sphingosine kinase 2 (SphK2) in the mouse kidney. Representative photomicrographs of kidney sections from three mice were analyzed. The negative control was performed in the absence of the primary anti-SphK2 antibody. B and D: effect of cisplatin (Cis) and SphK2 siRNA (siSphK2) on levels of SphK2 in HK-2 cells by Western blot analysis. C and E: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the control. siCtrl, control siRNA. *P < 0.05 vs. other groups. n = 3–4 batches of cells for each group.

The influence of cisplatin on the expression of SphK2 was examined in HK-2 cells. The effective knockdown of SphK2 by siRNA was first validated, as shown by the significant reduction of SphK2 levels in cells treated with siSphK2 compared with cells treated with siCtrl (Fig. 1, B and C). The results shown in Fig. 1, D and E, demonstrate that cisplatin significantly increased the expression of SphK2 in HK-2 cells, suggesting that cisplatin-induced toxicity is associated with activation of the SphK2 pathway. In addition, compared with the siCtrl + cisplatin group, siSphK2 + cisplatin significantly reduced the expression of SphK2, indicating inhibition of cisplatin-induced SphK2 levels by siRNA and further confirming that siRNA effectively silenced the expression of SphK2 (Fig. 1, D and E).

SphK2 Inhibitor ABC294640 Inhibited the Cisplatin-Induced Increase of S1P Levels in HK-2 Cells

As the results in Fig. 1 showed that cisplatin increased the expression of SphK2, we further assessed the S1P levels. The results showed that cisplatin significantly increased S1P levels. However, compared with the cisplatin-treated group, cells treated with cisplatin + SphK2 inhibitor ABC294640 showed significantly lower S1P levels. These results further indicate that cisplatin-induced toxicity is associated with activation of the SphK2 pathway and that SphK2 inhibitor sufficiently blocked the increase of S1P levels (Fig. 2).

Figure 2.

Effect of the sphingosine kinase 2 (SphK2) inhibitor ABC294640 (A) on cisplatin (Cis)-induced sphingosine 1-phosphate (S1P) levels. A: representative chromatograph of the S1P standard and internal control (Ctrl). B: S1P levels in cells with different treatments. *P < 0.05 vs. other groups. n = 4. A, ABC294640.

ABC294640, SLM6031434, or SphK2 siRNA Alleviated Cisplatin-Induced HK-2 Cell Injury

NGAL and KIM-1 are well-established kidney injury markers (34, 35), whereas cleaved caspase-3 is a cell injury and apoptosis marker (36). Cisplatin significantly increased the expression of NGAL, KIM-1, and cleaved caspase-3 in HK-2 cells. Compared with cisplatin treatment alone, pretreatment with the SphK2 inhibitor ABC294640 prevented the elevation of injury markers induced by cisplatin (Fig. 3, A and B). A more recent SphK2 inhibitor, SLM6031434 (20), showed the same effects on cisplatin-induced expression of NGAL, KIM-1, and cleaved caspase-3 (Fig. 3, C and D). Considering the possibility that pharmacological inhibitor might have off-target effects, SphK2 siRNA was used to silence SphK2. Silence of SphK2 also alleviated cisplatin-induced HK-2 cell injury (Fig. 3, E and F). These results indicate that the protective effect of SphK2 inhibitor is unlikely to be a consequence of off-target effects.

Figure 3.

Effect of sphingosine kinase 2 (SphK2) inhibitor or SphK2 siRNA (siSphK2) on cisplatin (Cis)-induced levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and cleaved caspase-3 in HK-2 cells by Western blot analysis. A and B: results from cells treated with control (Ctrl) or the SphK2 inhibitor ABC294640. C and D: results from cells treated with control or SphK2 inhibitor SLM6031434 (SLM). E and F: results from cells treated with Ctrl siRNA (siCtrl) or siSphK2. Summarized band intensity ratios to GAPDH were normalized to the value in the control. *P < 0.05 vs. other groups. n = 4. A, ABC294640.

ABC294640 Inhibited NF-κB Activation in Cisplatin-Treated HK-2 Cells

Several studies have shown that cisplatin can activate NF-κB (37, 38), a major transcription factor that regulates many inflammatory cytokines (39, 40). Western blot analysis showed that cisplatin increased the expression of NF-κB p65 in HK-2 cells, an effect that was reversed by SphK2 inhibitor (Fig. 4, A and B). Translocation of NF-κB p65 from the cytoplasm to the nucleus is also an important component in activation of the NF-κB pathway (40). Immunostaining showed that cisplatin induced NF-κB p65 translocation from the cytoplasmic to nuclear compartments, a transition that was blocked by ABC294640 (Fig. 4C). These results indicate that inhibition of NF-κB p65 translocation contributes to protection from cisplatin-induced HK-2 cell injury by SphK2 inhibition.

Figure 4.

Effect of sphingosine kinase 2 (SphK2) inhibitor on cisplatin (Cis)-induced changes in levels of NF-κB p65 by Western blot analysis and staining patterns of NF-κB p65 by immunostaining. A: representative gel documents depicting protein levels of NF-κB p65. B: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the control (Ctrl). C: representative images of immunostaining of NF-κB p65 (green) and nuclear marker (blue). *P < 0.05 vs. other groups. n = 4. A, ABC294640.

ABC294640 Inhibited the Cisplatin-Induced Proinflammatory Response in HK-2 Cells

NLRP3, IL-1β, and IL-6 are inflammatory markers that are associated with cisplatin-induced kidney injury (41) and are molecules that can be regulated by NF-κB (42–44). As shown in Fig. 5, cisplatin significantly increased the expression of NLRP3, IL-1β, and IL-6; ABC294640 inhibited increases of NLRP3, IL-1β, and IL-6 in cisplatin-treated HK-2 cells (Fig. 5).

Figure 5.

Effect of sphingosine kinase 2 (SphK2) inhibitor ABC294640 on cisplatin (Cis)-induced changes in levels of NLR family pyrin domain containing 3 (NLRP3), IL-6, and IL-1β in HK-2 cells by Western blot analysis. A: representative gel documents depicting protein levels of NLRP3, IL-6, and IL-1β. B: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the control (Ctrl). *P < 0.05 vs. other groups. n = 4. A, ABC294640.

Cisplatin Significantly Increased the Expression of SphK2 in Mouse Kidneys; SphK2 Deficiency Improved Cisplatin-Induced Kidney Dysfunction

Assessment of the expression of SphK2 in kidneys from mice indicated that cisplatin significantly increased the expression of SphK2, which was consistent with results from our experiments with HK-2 cells, indicating that activation of the SphK2 pathway participates in cisplatin-induced kidney damage (Fig. 6, A and B).

Figure 6.

Effect of cisplatin (Cis) on levels of sphingosine kinase 2 (SphK2) in mouse kidneys as well as serum creatinine and blood urea nitrogen (BUN). A: representative Western blot gel documents depicting protein levels of SphK2. B: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the control. C: serum creatinine levels. D: BUN levels. *P < 0.05 vs. other groups. n = 4–5. KO, knockout; Veh, vehicle; WT, wild type.

Serum creatinine and BUN are widely recognized as important and clinically used biomarkers for the assessment of renal function. The results showed that cisplatin treatment significantly increased serum creatinine and BUN levels in WT mice. However, compared with WT + cisplatin mice, SphK2 KO + cisplatin mice exhibited significantly reduced elevations of serum creatinine and BUN (Fig. 6, C and D). These results demonstrated that deletion of SphK2 improved cisplatin-induced kidney dysfunction.

SphK2 Deficiency Inhibited Cisplatin-Induced Injury and Renal Fibrosis in Mouse Kidneys

NGAL and KIM-1 as kidney injury markers and cleaved caspase-3 as an apoptotic marker were monitored in both WT and SphK2 KO mice exposed to cisplatin. Cisplatin significantly increased levels of NGAL, KIM-1, and cleaved caspase-3 in WT + cisplatin mice; in contrast, SphK2 KO + cisplatin mice exhibited only minor elevations of NGAL, KIM-1, and cleaved caspase-3 (Fig. 7). These results further demonstrated that deletion of SphK2 was protective against cisplatin-induced kidney injury and apoptosis.

Figure 7.

Effect of sphingosine kinase 2 (SphK2) deficiency on cisplatin (Cis)-induced levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), α-smooth muscle actin (α-SMA), and cleaved caspase-3 in kidney tissues by Western blot analysis. A: representative gel documents depicting protein levels. B: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the wild type (WT) + vehicle (Veh) group. *P < 0.05 vs. all other groups. n = 4–5. KO, knockout.

Previous literature has demonstrated that cisplatin also causes renal fibrosis (45). To assess whether deletion of the SphK2 gene could protect against renal fibrosis induced by cisplatin, α-SMA, as one of the most important markers, in renal fibrosis was assessed by Western blot analysis (46). The results showed that cisplatin significantly increased the expression of α-SMA; however, compared with WT + cisplatin mice, SphK2 KO + cisplatin mice exhibited a minor elevation of α-SMA (Fig. 7), indicating that deletion of SphK2 was protective against cisplatin-induced renal fibrosis.

SphK2 Deficiency Inhibited Cisplatin-Induced Activation of NF-κB and Increase of Inflammatory Factors in the Kidneys

The results showed that cisplatin significantly increased the level of NF-κB, NLRP3, and IL-1β in WT + cisplatin mice; however, compared with WT + cisplatin mice, SphK2 KO + cisplatin mice exhibited significantly reduced elevations of NF-κB, NLRP3, and IL-1β (Fig. 8). These results demonstrated that deletion of SphK2 inhibited cisplatin-induced activation of the NF-κB-mediated inflammatory response in the kidneys.

Figure 8.

Effect of sphingosine kinase 2 (SphK2) deficiency on cisplatin (Cis)-induced levels of NF-κB p65, NLR family pyrin domain containing 3 (NLRP3), and IL-1β in kidney tissues by Western blot analysis. A: representative gel documents depicting protein levels. B: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the wild type (WT) + vehicle (Veh) group. *P < 0.05 vs. all other groups. n = 4–5. KO, knockout.

SphK2 Deficiency Attenuated Cisplatin-Induced Tubular Damage and Collagen Deposition in the Kidneys

To assess whether SphK2 deficiency was protective against morphological damage induced by cisplatin in renal tubules, periodic acid-Schiff staining was used to assess morphological injury to kidney tubules. Compared with the WT + vehicle group, WT + cisplatin mice showed severe tubular damage, including tubular dilation, tubular atrophy, cast formation, and sloughing of tubular epithelial cells. In conformation with the biomarker results shown in Figs. 7 and 8, tubular damage was significantly attenuated in SphK2 KO mice (Fig. 9).

Figure 9.

Effect of sphingosine kinase 2 (SphK2) deficiency on cisplatin (Cis)-induced morphology changes by periodic acid-Schiff (PAS) staining and collagen deposition by Masson trichrome staining in kidney tissue slides. A: representative photomicrographs showing tubular structures. B: representative photomicrographs showing the staining area. C: semiquantitation of tubular damage. D: summarized positive area. *P < 0.05 vs. all other groups. n = 4–5. KO, knockout; Veh, vehicle; WT, wild type.

Masson trichrome staining was used to stain collagen. The results showed that cisplatin led to collagen deposition in kidney tissue; however, SphK2 KO + cisplatin mice had much less collagen deposition than WT + cisplatin mice (Fig. 9). These results further confirmed that deletion of SphK2 attenuated cisplatin-induced renal fibrosis in vivo.

SphK2 Deficiency Did Not Alter OCT2 Levels in the Kidneys

Cisplatin reduced levels of OCT2 in the kidneys (Fig. 10), which was consistent with the literature (47, 48). However, there was no difference in OCT2 levels between WT and KO mice (Fig. 10), suggesting that SphK2 inhibition did not change levels of OCT2 in the kidneys.

Figure 10.

Effect of sphingosine kinase 2 (SphK2) deficiency and cisplatin (Cis) on levels of organic cation transporter 2 (OCT2) in kidney tissues by Western blot analysis. A: representative gel documents depicting protein levels. B: summarized data displaying the band intensity ratio to GAPDH normalized to the value in the wild type (WT) + vehicle (Veh) group. *P < 0.05 vs. other groups. n = 4. KO, knockout.

ABC294640 Did Not Interfere With the Response of Tumor Cells to Cisplatin or Radiation

To evaluate whether inhibition of SphK2 would potentially obstruct the antitumor effect of cisplatin, two different head and neck cancer models, HN30 and HN12 cells with WT and nonfunctional p53, respectively, were treated with ABC294640 in combination with cisplatin. Cell viability assays, as shown in Fig. 11A, demonstrated that the SphK2 inhibitor had no influence on cisplatin effectiveness in combination therapy, suggesting that SphK2 inhibition does not interfere with the antitumor effect of cisplatin.

Figure 11.

Effect of the sphingosine kinase 2 (SphK2) inhibitor ABC294640 on cisplatin (Cis)- or radiation-induced toxicity in head and neck cancer HN30 and HN12 cells. A: MTS assay showing cell viability in response to cisplatin with or without SphK2 inhibitor. B: clonogenic survival assays showing the colony formation of cells exposed to 6-Gy radiation alone or in combination with Sphk2 inhibitor. n = 3. A, ABC294640; Ctrl, control.

Because radiation is a standard procedure combined with cisplatin in head and neck cancer management, we also evaluated whether inhibition of SphK2 would interfere with radiation therapy when combating cisplatin nephrotoxicity. Figure 11B shows that the SphK2 inhibitor had no influence on the effectiveness of radiation in these tumor cells.

DISCUSSION

The present study demonstrates that SphK2 is highly expressed in renal proximal tubules and that its expression is increased by cisplatin. In our in vitro experiments, both the SphK2 inhibitor and siRNA targeting SphK2 attenuated cisplatin-induced cellular injury, as evidenced by suppression of increases in KIM-1, NGAL, and cleaved caspase-3 by cisplatin. In addition, inhibition of SphK2 was found to block cisplatin-induced activation of NF-κB and attenuate cisplatin-induced increases in inflammatory factors. In our in vivo experiments, genetic deletion of SphK2 diminished cisplatin-induced kidney dysfunction, kidney injury, tubular damage, and renal fibrosis as well as activation of NF-κB-associated inflammatory responses. These results demonstrate that inhibition of SphK2 can protect against cisplatin-induced kidney injury.

The results from the present study showed that cisplatin upregulated expression of SphK2, which indicated that SphK2 may participate in the development of cisplatin-induced kidney injury. These results are consistent with previous reports showing that expression of SphK2 was upregulated in transforming growth factor-β-induced fibrosis and unilateral ureteral obstruction models (12, 49). Cisplatin-induced kidney injury was successfully established in the cell and animal models used in the present study, as evidenced by the increase in KIM-1 and NGAL levels. These results were consistent with previous studies by Chen et al. (50) and Florova et al. (51). Interestingly, inhibition of SphK2 significantly suppressed the increased levels of NGAL and KIM-1 in cisplatin-induced kidney injury. Our results were consistent with most of the current literature, where inhibition of SphK2 alleviated kidney damage in different models. However, one study did show that an absence of SphK2 enhanced kidney damage in ischemia-reperfusion injury (52). The discrepant role of SphK2 in kidney injury might be associated with different disease models.

NF-κB is considered an important response factor at the early stage in treatment with cisplatin and exerts important effects in stimulating the production of proinflammatory factors (53). Inhibition of the activation of NF-κB has been demonstrated to protect against cisplatin-induced kidney injury (37, 38). The present study showed that cisplatin activated NF-κB, and, interestingly, SphK2 inhibitor suppressed activation of NF-κB, which indicates that blockade of the activation of NF-κB contributes to the protective mechanisms of SphK2 inhibition against cisplatin-induced kidney injury. These results from the present study were consistent with previous studies showing that inhibition of SphK2 blunted NF-κB activation (54–56) and improved drug resistance in cancer therapeutics (55). In addition, the present study showed that cisplatin increased S1P levels and that inhibition of SphK2 blocked the increase of S1P induced by cisplatin. S1P has been previously demonstrated to activate NF-κB, and inhibition of SphK2 blocked NF-κB activation and improved antineoplastic resistance through the SphK2/S1P pathway (57). Another study showed that cytosolic delivery of S1P led to NF-κB activation and induced production of cytokines (58).

In addition, activation of NF-κB upregulates proinflammatory factors, IL-1β, IL-6, and NLRP3, and then causes the inflammatory response, leading to cellular death and fibrosis (42–44, 59, 60). Furthermore, increases in IL-1β and IL-6 are associated with different kidney injuries, including cisplatin-induced kidney injury (61–63). Consistently, the present study showed that inhibition of SphK2 blocked activation of IL-1β and IL-6, which was similar to previous observations in a different kidney injury model showing that deletion of SphK2 protected against unilateral ureteral obstruction-induced kidney damage and was associated with suppression of inflammatory cytokines, including IL-1β and IL-6 (12). NLRP3 is another downstream effector of NF-κB (60) and also an important regulator of IL-1β in inflammatory kidney damages (64). Interestingly, cisplatin-induced activation of NLRP3, which has been shown to mediate cisplatin-induced kidney injury (45), was also blocked by SphK2 inhibition in the present study. Taken together, the present study suggests that upregulation of SphK2 is responsible for the activation of NF-κB-mediated inflammatory signaling, which represents a novel mechanism in cisplatin-induced nephrotoxicity, and that the underlying mechanism of renoprotection produced by SphK2 inhibition is through inhibition of NF-κB-mediated production of inflammatory factors. However, the detailed interaction between SphK2 and NF-κB signaling pathways requires future investigation.

Apoptosis of tubular cells is regarded as one of the major mechanisms contributing to cisplatin-induced kidney injury (65). Our results showed that inhibition of SphK2 reduced cisplatin-induced apoptosis in HK-2 cells, which was similar to a previous report showing that silence of SphK2 reduced serum deprivation-induced apoptosis (66). Although inhibition of SphK2 was also found to induce apoptosis, these effects are generally observed with relatively large doses of the inhibitors and in tumor cells; consequently, we speculate that this discrepancy may be due to cell type, disease model, and doses (67, 68).

Finally, our in vivo experiments also showed increased levels of SphK2 in the kidneys promoted by cisplatin treatment. Critically, mice with a deficiency of SphK2 demonstrated reduced elevation in markers of renal dysfunction and kidney damage as well as inhibited activation of NF-κB-associated inflammatory responses, further suggesting that inhibition of SphK2 protects against cisplatin-induced nephrotoxicity. OCT2 has been shown to contribute to the uptake of cisplatin and cisplatin nephrotoxicity (69–71). However, our results did not show a difference in OCT2 levels between WT and KO mice, suggesting that the renal protection by SphK2 inhibition may not be via actions on OCT2.

It is worth noting that inhibition of SphK2 has been shown to have efficacy against tumor growth and survival (21, 72–74). Previous studies have demonstrated that SphK2 is upregulated in cancer cells from patients and in cancer cell lines (21, 72, 73), which is associated with chemoresistance in cancer cells (75). Furthermore, inhibition of SphK2 has been reported to enhance antitumor effects of cisplatin (21). Silence of SphK2 in combination with gefitinib has been reported to increase tumor cell apoptosis (73). SphK2 inhibition has been reported to have a synergistic effect with sorafenib in the suppression of proliferation of cancer cells (67). Therefore, inhibition of SphK2 would unlikely interfere with the antitumor effect of cisplatin. Because radiation is a standard procedure combined with cisplatin in head and neck cancer management, we also evaluated whether inhibition of SphK2 would interfere with radiation therapy when combating cisplatin nephrotoxicity. In our hands, no sensitization to the antitumor effects of cisplatin or radiation was evident in combination with SphK2 inhibition. Nevertheless, the SphK2 inhibitor had no influence on cisplatin or radiation effectiveness against head and neck tumor cell growth in the present study. This indicates that SphK2 inhibition could potentially be used to suppress cisplatin toxicity without interfering with chemoradiation.

The potential mechanism for the absence of interference with the antitumor effect of cisplatin by SphK2 inhibition is probably also associated with blockade of NF-κB activation, which may be accountable for the potential antitumor actions of SphK2 inhibition. Cisplatin-produced activation of NF-κB has been shown to contribute to both chemotoxicity and chemoresistance in cisplatin therapy (76–79). Therefore, by blocking NF-κB activation, SphK2 inhibition would unlikely compromise the antitumor effect of cisplatin and, in contrast, may sensitize the antitumor effect of cisplatin. Indeed, the literature shows that SphK2 inhibition has demonstrated antitumor activity as well as sensitizes tumor cells to several chemotherapeutic agents (75), including cisplatin (21). The present study did not show the enhancement of the antitumor effect of SphK2 inhibitor in combination with cisplatin. This discrepancy might be due to the different sensitivity of cancer types to SphK2 inhibition.

We recognize that there are limitations in the present study. We showed that inhibition of SphK2 protected against cisplatin-induced damages. However, the underlying mechanisms by which SphK2 inhibition provided the pretension remain to be clarified. For example, cisplatin uptake and renal content of cisplatin are known to contribute to cisplatin-induced damage; whether SphK2 inhibition causes changes in the uptake and concentration of cisplatin in the kidneys requires future investigations. In addition, we showed blockade of NF-κB activation by SphK2 inhibition; how SphK2 signaling interacts with the NF-κB pathway remains unclear. Furthermore, how cisplatin activates SphK2 is still unknown. Moreover, inhibition of SphK2 would result in both a decrease of the downstream product, S1P, as well as increase of the upstream substrates, sphingosine, and ceramides. Both S1P and ceramides are bioactive sphingolipids. The present study could not distinguish whether the protective effects of SphK2 inhibition were a consequence of the reduced S1P, increased ceramides, or both. Nevertheless, the present study reveals the important role of the sphingolipid pathway in cisplatin-induced nephrotoxicity, which provides clues for further investigations in this regard and may lead to discoveries of more novel insights into the pathophysiology of cisplatin-induced nephrotoxicity.

In conclusion, the results from the present study demonstrate that inhibition or deletion of SphK2 protected against cisplatin-induced nephrotoxicity. Potential mechanisms involve inhibition of NF-κB activation, inflammatory factor production, and apoptosis.

Perspectives and Significance

The present study demonstrated that cisplatin treatment activated the SphK2 signaling pathway, which consequently augmented NF-κB-mediated production of inflammatory factors to produce kidney damage. Thus, activation of SphK2 represents a novel mechanism in cisplatin-induced nephrotoxicity, and SphK2 inhibition may be used as a potential therapeutic strategy for the prevention or treatment of cisplatin-induced nephrotoxicity. Noteworthily, the SphK2 inhibitor ABC294640 has been shown to be well tolerated in cancer clinical trials (80) although it has not yet been tested in cisplatin-induced nephrotoxicity; thus, findings from the present study bear the potential for clinical translation.

GRANTS

This work was supported by National Institutes of Health Grants HL145163, DK107991, DK054927, CA206028, CA239706, and CA260819.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.X., P-L.L., D.A.G., and N.L. conceived and designed research; D.X., G.H., C.C., F.A., and W.W. performed experiments; D.X., G.H., C.C., F.A., W.W., and N.L. analyzed data; D.X., G.H., C.C., W.W., P-L.L., D.A.G., and N.L. interpreted results of experiments; D.X. and C.C. prepared figures; D.X. drafted manuscript; D.X., D.A.G., and N.L. edited and revised manuscript; D.X., P-L.L., D.A.G., and N.L. approved final version of manuscript.