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Paricalcitol attenuates indoxyl sulfate-induced apoptosis through the inhibition of MAPK, Akt, and NF-kB activation in HK-2 cells

Background/Aims Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods The fluorescent dye 2ʹ,7ʹ-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-κB (NF-κB) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-κB was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-κB p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-κB p65, and Akt in HK-2 cells. NF-κB promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-κB, and Akt signaling pathway in HK-2 cells.


INTRODUCTION
The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, accelerates the progression of chronic kidney disease and renal failure involving progressive tubulointerstitial fibrosis and glomerular sclerosis [1][2][3]. IS is an end metabolite of tryptophan in dietary proteins. Tryptophan is cleaved to indole by tryptophanase present in intestinal bacteria, such as Escherichia coli. The indole is transported to the liver through the portal vein, where it is oxidized to indoxyl by cytochrome p450 2E1 and sulfated by sulfotransferases 1A1, which is normally excreted in urine mainly via direct secretion by organic anion transporters 1, 3, and 4 in proximal tubular cells [2,[4][5][6]. However, under conditions of reduced renal function, IS is not excreted in the urine and accumulates in the serum due to its reduced renal clearance [7]. Accumulated IS is then incorporated into the basolateral membrane of renal proximal tubular cells by mediating organic anion transporter 1 and 3 [6,8].
IS stimulates renal expression of fibrotic genes such as transforming growth factor-β1 and a-smooth muscle actin, and inflammatory genes, including interleukin-6, interleukin-1β, tumor necrosis factor-a, and monocyte chemotactic protein-1 [9][10][11]. Importantly, IS induces intracellular production of reactive oxygen species (ROS) through nicotinamide adenine dinucleotide phosphate oxidase and extracellular O 2 − formation [12]. IS-induced ROS production activates nuclear factor-κB (NF-κB), c-Jun N-terminal domain kinase (JNK), signal transducer and activator of transcription 3, and p53, resulting in increased oxidative stress in tubular epithelia cells [13,14]. ROS production in renal tubular epithelia cells induces cell death via both apoptosis and necrosis dependent upon concentration and exposure time. High IS concentrations induce cell death in renal proximal tubular cells through the apoptosis pathway [15,16], and IS induces oxidative stress, promoting free-radical production and disturbing antioxidant enzymes, such as superoxide dismutase [10,17,18].
Paricalcitol is a synthetically manufactured analog of calcitriol, the biologically active form of vitamin D. Paricalcitol includes modifications to the D 2 side chain and the A (19-nor-1,25(OH) 2 D 2 ) ring, enabling binding to the vitamin D receptor [19] and resulting in attenuation of renal fibrosis [20,21]. The therapeutic effects of paricalcitol treatment may be attributed to its anti-inflammatory and anti-fibrotic effects in experimentally induced kidney diseases [22][23][24]. Additionally, decreased vitamin D concentration in renal-disease patients leads to increased cardiovascular mortality, as well as renal disease progression [25]. In the study, we determined the capability of paricalcitol to exert beneficial effects on IS-induced kidney tubule-cell injury, as well as the underlying molecular mechanisms associated with its anti-apoptotic effects.

MTT assay
Viability of HK-2 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HK-2 cells were sub-cultured in 96-well plates at an initial density of 5 × 10 4 cells/mL. Cells were incubathttps://doi.org/10.3904/kjim.2016.298 The Korean Journal of Internal Medicine Vol. 34, No. 1, January 2019 ed with fresh medium containing 0, 0.3, 0.5, 1, or 2 mM IS for 24 hours. At the end of the experimental period, MTT (Sigma-Aldrich) was added into each well to a final concentration of 0.5 mg/mL and subsequently incubated for 4 hours at 37°C. Supernatants were removed by aspiration, and dimethyl sulfoxide was added to solubilize the precipitated dyes. Absorbance was measured at 570 nm, and cell viability was expressed as the fraction of surviving cells relative to untreated controls.

Intracellular ROS levels
HK-2 cells were cultured in 48-well plates until reaching 60 % to 70 % confluence. Cells were incubated with fresh medium containing 0, 0.3, 0.5, 1, or 2 mM IS for 24 hours. At the end of the experimental period, cells were preloaded with 10 μM 2ʹ,-7ʹ-dichlorofluorescein diacetate (DCF-DA, Molecular Probes, Eugene, OR, USA) for 30 minutes at 37°C. Fluorescence intensity was analyzed using a fluorescence reader (Fluoroskan Ascent FL, Lab systems, Helsinki, Finland) with a 485-nm excitation and 538-nm emission filter. Additional HK-2 cells were cultured on a 6-well plate for DCF-DA staining. Cells were incubated with 1 mM IS for 24 hours and washed twice with Hank's balanced salt solution, then incubated with Hank's balanced salt solution (without phenol red) containing DCF-DA for 30 minutes at 37°C in the dark. Images were obtained with a fluorescence microscope (Nikon, Tokyo, Japan).

Nuclear extracts preparation
For nuclear extracts, cells were lysed using NE-PER nuclear-extraction reagent (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer's protocol as described previously [23].

NF-κB-promotor activity
The transcriptional regulation of NF-κB was examined by transient transfection of a NF-κB-promoter luciferase-reporter construct (pGL3-NF-κB). HK-2 cells (5 × 10 5 ) were seeded and grown until they reached 60% to 70% confluence, followed by transfection of pGL3-NF-κB wild-type and pGL3-empty into the cells using FuGENE HD reagent (Promega, Durham, NC, USA) according to manufacturer protocol. The pRL-null plasmid encoding Renilla luciferase was included in all samples to monitor transfection efficiency. At 24 hours post-transfection, the levels of firefly and Renilla luciferase activity were measured sequentially from a single sample using the Dual-Glo Luciferase Assay system (Promega). Firefly luciferase activity was normalized to Renilla activity and the relative amount of luciferase activity in the untreated cells.

Flow cytometry
An Annexin V FLUOS staining kit (Sigma-Aldrich) was used to measure the level of Annexin V binding according to manufacturer instructions. Briefly, after treatment with 0, 0.5, or 1 mM IS for 24 hours, HK-2 cells were harvested and washed twice with pre-cooled phosphate-buffered saline and resuspended in a binding buffer containing Annexin V. After incubation in the dark for 15 minutes, cells were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA). Several controls were used to optimize the instrument settings and determine the gating for the Windows-based platform.

Statistical analysis
Results are presented as the mean ± standard error of the mean of three individual experiments. Differences were analyzed by analysis of variance with post hoc comparison. Statistical significance of differences was accepted at the level of p < 0.05.

ROS production and cytotoxicity
IS treatment (0, 0.3, 0.5, 1, and 2 mM) for 24 hours decreased HK-2 cell viability in a concentration-dependent manner as determined by MTT assay (Fig. 1A). IS-induced decrease in cell viability, which was attenuated by paricalcitol pretreatment (Fig. 1B). ROS formation was detected using the ROS-sensitive fluorescent dye DCF-DA in HK-2 cells. The level of intracellular ROS increased progressively after incubation with 0 to 2 mM IS for 24 hours, whereas pretreatment with paricalcitol attenuated IS-induced ROS production (Fig. 1C-1G).

Cell apoptosis
The expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 was measured by immunoblotting. Incubation with 1 mM IS for 3, 6, 12, or 24 hours increased the ratio of Bax/Bcl-2 expression ( Fig. 2A). Additionally, ISinduced increase in the Bax/Bcl-2 ratio in HK-2 cells, which was attenuated by pretreatment with 5 ng/mL paricalcitol for 1 hour (Fig. 2B). Furthermore, HK-2 cells treated with IS exhibited increased Annexin V positive staining, which was attenuated by pretreatment with 5 ng/mL paricalcitol ( Fig. 2C and 2D).

MAPK and Akt expression
The expression of p-Erk, p-Jnk, and p-p38 was increased after incubation of HK-2 cells with 1 mM IS for 0.5, 1, 2, or 4 hours. Akt expression was also increased in HK-2 cells incubated with 1 mM IS for 2, 4, 8, and 12 hours. MAPK phosphorylation was peaked at 2 hours after IS treatment (Fig. 3A), whereas p-Akt levels peaked at 8 hours after IS treatment (Fig. 3B). In contrast, total Erk, Jnk, p38, and Akt expression was not affected by IS treatment. To investigate the effects of paricalcitol pretreatment on phosphorylation of MAPK and Akt, HK-2 cells were co-treated with IS and paricalcitol. IS-induced increases in MAPK phosphorylation and p-Akt levels in HK-2 cells, which was attenuated by pretreatment with 5 ng/mL paricalcitol for 1 hour (Fig. 4).

NF-κB expression
We then examined changes in NF-κB p65-subunit levels in nuclear extracts of HK-2 cells incubated with 1 mM IS. The expression of p65 subunits began to increase after a 1 hour incubation with IS, whereas total cytoplas- mic IκBa expression began to decrease at 1 hour, continued to decrease at 2 hours, then remained stationary for at least 4 hours (Fig. 5A).
The effects of specific chemical inhibitors, such as those toward Erk (PD98059), Jnk (SP600125), and p38 (SB203580), are shown in Fig. 5B. Treatment of HK-2 cells with PD98059, SP600125, or SB203580 attenuated IS-induced levels of nuclear NF-κB p65 subunit. As shown in Fig. 5C, Bay 11-7082 (NF-κB inhibitor) and Ly294 (Akt inhibitor) noticeably reduced levels of nuclear NF-κB p65 subunit. These findings suggested that MAPK and Akt activation are involved in IS-induced NF-κB nuclear activation. The IS-induced activation of NF-κB p65 subunit in nuclear extracts of HK-2 cells was also attenuated by pretreatment with 5 ng/mL paricalcitol for 1 hour (Fig. 5D).

DISCUSSION
IS induces nephrotoxic kidney injury by increased production of ROS and induction of inflammatory and fibrotic gene expression [26,27]. In this study, we showed that treatment of proximal tubular HK-2 cells with IS enhanced ROS production and led to dose-dependent cell death. Accordingly, IS treatment led to phosphorylation of MAPK and Akt and subsequent initiation of their signaling pathway, resulting in NF-κB nuclear translocation. However, following paricalcitol treatment, we observed reduced MAPK and Akt phosphorylation and decreased NF-κB activation.
Although some studies demonstrated that altered IS levels promote antioxidant functions under normal physiological conditions, including those catalyzed by superoxide dismutase [28][29][30], high concentration of IS induced ROS production. Miyamoto et al. [31] reported that high serum IS levels could function as a promoter of pro-oxidant activity due to enhanced oxidative stress, while it might also function as a promoter of antioxidant activity under normal physiological conditions. Our study showed that high IS concentrations induced ROS production and possibly initiated apoptosis in HK-2 cells. We also observed that IS-induced oxidative stress enhanced the phosphorylation of Erk1/2-, Jnk-, p38-, and Akt-signaling pathways. Other studies reported a role for p38-and MAPK-signaling pathways in apoptosis, as well as ROS-induced apoptosis in kidney epithelial cells [32,33]. Here, we showed that paricalcitol pretreatment attenuated IS-induced increases in p-Erk1/2, p-Jnk, p-p38, and p-Akt levels. Furthermore, treatment with the inhibitors PD98059, SB203580, SP600125, and Ly294002 attenuated nuclear levels of NF-κB p65 sub-unit. These findings suggest the involvement of MAPKand Akt-signal pathway in NF-κB translocation in HK-2 cells. Activation of NF-κB pathways promote initiation of cell death signal and pro-inflammatory responses [34].
Here, we observed that IS treatment increased nuclear translocation of the NF-κB p65 subunit and increased NF-κB-promoter activity in HK-2 cells, whereas paricalcitol pretreatment attenuated this activity. Additionally, paricalcitol treatment rescued IS-treated apoptotic HK-2 cells from NF-κB-dependent cell death through inactivation of the MAPK and Akt pathways. Apoptosis describes programmed cell death initiated by specific signaling pathways. ROS is capable of damaging kidney tubular epithelial cells and promoting formation of kidney disease through the activation of apoptosis pathways, such as the mitochondria-mediated intrinsic cell-death pathway. Mitochondria-mediated apoptosis is regulated by Bcl-2 family proteins, which can be classified into two groups: pro-apoptotic  proteins; such as Bax, Bad, and Bid; and anti-apoptotic proteins, such as Bcl-2 and Bcl-X L . Bax resides in the cytosol under normal circumstances; however, ROS-mediated Bax activation initiates apoptosis by altering mitochondrial membrane permeability [35].
Our results indicated that treatment of HK-2 cells with IS significantly upregulated Bax expression and decreased Bcl-2 expression. We also showed that IS-treated HK-2 cells exhibited significant progressive increases in Annexin V positive staining, indicating induction of apoptosis. We were subsequently able to modulate Bax and Bcl-2 expression by pretreatment with paricalcitol. These findings suggest that pretreatment of HK-2 cells with paricalcitol contributes to the inhibition of IS-induced apoptosis, and paricalcitol prevents initiation of mitochondria-mediated apoptosis triggered by IS through regulation of Bcl-2 and Bax.
In summary, IS induces apoptosis in renal tubular epithelial cells via excessive intracellular ROS and subsequent activation of the NF-κB-signaling pathway through activation of the MAPK and Akt pathways. Paricalcitol treatment attenuates IS-induced apoptosis by regulating MAPK, Akt, and NF-κB signaling pathway in HK-2 cells.