Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4+ T cell induced injury in human renal proximal tubular epithelial cells

Background The aim of this study was to investigate the protective effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on effector CD4+ T cells or on inflammatory cytokine-induced injury in human renal proximal tubular epithelial cells (HRPTEpiC). Methods First, we investigated the effect of 1,25(OH)2D3 on CD4+ T cell proliferation. Second, we examined the effect of 1,25(OH)2D3 on inflammatory cytokine secretion or fibrosis in HRPTEpiC induced by inflammatory cytokines or activated CD4+ T cells using ELISA and real-time PCR. Lastly, we compared urine inflammatory-cytokine (IL-6, IL-8) or KIM-1 levels in kidney transplant recipients low serum 25-hydroxyvitamin D (25(OH)D) group (< 20 ng/mL) (n = 40) and normal 25(OH)D group (n = 50). Results Pre-incubation with 1,25(OH)2D3 significantly reduced the percentages of Th1 and Th17 cells compared to that of Th0 condition (P < 0.05 for each). In contrast, 1,25(OH)2D3 increased the proportion of Th2 and Treg cells in a dose-dependent manner (P < 0.05 for each). Treatment of HRPTEpiC with inflammatory cytokines (TNF-α, IL-17, and TGF-β) or effector CD4+ T cells resulted in increased production of IL-6, IL-8, or KIM-1 from HRPTEpiC in a dose-dependent manner. However, treatment with 1,25(OH)2D3 significantly reduced the level of these cytokines (P < 0.05 for all). Western blot analysis demonstrated that the mTOR/STAT3/ERK pathway was downregulated by 1,25(OH)2D3 in HRPTEpiC. Furthermore, the concentrations of urine IL-6/creatinine (P < 0.05) and Kim-1/creatinine (P < 0.05) were higher in the low 25(OH)D group than in the normal 25(OH)D group in kidney transplant recipients. Conclusion The results of this study suggests that vitamin D may have a significant role in the regulation of inflammation in allograft tissue in kidney transplant recipients. Trial registration All participants provided written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (KC13TNMI0701).


Introduction
Recent studies have demonstrated the modulatory effects of vitamin D on various immune cells [1,2].Studies also demonstrate its significant association with immune disorders [3][4][5][6][7].Low serum levels of 25-hydroxyvitamin D (25(OH)D) are frequently associated with autoimmune diseases or graft versus host disease after hematopoietic stem cell transplantation.[3,8,9] Treatment with 1α,25 dihydroxyvitamin D3 (1,25(OH) 2 D3) was shown to have significant therapeutic effects on those disorders.[10,11] In kidney transplantation, 25(OH)D insufficiency was also associated with high incidence of acute rejection or the development of urinary tract infection.This may be owing to the modulatory effect of vitamin D on immune cells.[12][13][14] Previous studies have mainly focused on the effect of 1,25(OH) 2 D3 on the proliferation or activation of immune cells.However, the effect of 1,25(OH) 2 D3 on the target tissue or immune cell has not been extensively studied.[3][4][5][6][7] For example, the effect of 1,25(OH) 2 D3 on allograft tissue, which is the target of alloimmune effector CD4 + T cells or inflammatory cytokines in acute rejection, has not been fully investigated.The rejection process is not only the activation of alloimmune effector T cells, but also the injury to allograft kidney tissues.Therefore, the direct protective effect of 1,25(OH) 2 D3 on allograft tissue against immune cells or cytokines needs to be elucidated for establishing the preventive effect of 1,25(OH) 2 D3 on acute rejection.
Accordingly, we evaluated the effect of 1,25(OH) 2 D3 treatment on effector CD4 + T cell proliferation as well as on allograft tissue injury induced by T cells.To address this, we employed an in vitro experimental set up using human renal proximal tubular epithelial cell lines (HRPTEpiC) and human CD4 + T cells.Additionally, we examined the association between serum 25-hydroxyvitamin D (25(OH)D) levels and urine inflammatory cytokine levels or tubule injury markers in kidney transplant recipients.

Patient populations and study design
To investigate the protective effect of 1,25(OH) 2 D3 on HRPTEpiC from activated CD4 + T cells or inflammatory cytokines, we designed three separate experiments, two in vitro analyses and an ex vivo analysis.First, we investigated the suppressive effect of 1,25(OH) 2 D3 on CD4 + T cell proliferation.We enrolled six healthy volunteers for peripheral blood donation.The age was 31.8±5.4years and four were male and two were females.The serum 25(OH)D level at the time of blood donation was 30.3 ± 4.9 ng/mL.We investigated the effect of 1,25(OH) 2 D3 on CD4 + T cell proliferation by FACS analysis and ELISA.
Second, we investigated the effect of 1,25(OH) 2 D3 on inflammation or fibrosis induced by inflammatory cytokines or activated CD4 + T cells in HRPTEpiC, using ELISA and real-time PCR.Further, we used western blotting to analyze mTOR/STAT3 signaling as a potential mechanism by which 1,25(OH) 2 D3 exerts its effect on HRPTEpiC.
Third, we collected serum and urine samples from 90 kidney-transplant recipients with stable allograft function (Table 1).We measured serum 25

Coculture of Human Renal Proximal Tubular Epithelial Cell (HRPTEpiC) line and activated CD4+T cells
For co-culture experiments with CD4 + T cells, HRPTEpiC were seeded in 24-well plates at 2 × 10 4 cells/well with 1 mL of medium, and pre-incubated for 1 hour with 1,25(OH) 2 D3 (10 nM).CD4 + T cells (2 × 10 5 cells/well) with or without stimulation under Th0 polarizing conditions were added to the HRPTEpiC monolayers, and the culture plates were incubated for 48 hours.The culture supernatants were collected and stored at -80˚C until assayed.All cultures were set up in triplicate.

Enzyme-Linked Immunosorbent Assay (ELISA) for cytokine analysis
The levels of cytokines such as IL-6, IL-8, and Kim-1 in urine isolated from kidney transplant recipients or IFN-r, IL-17, IL-22, IL-23, IL-6 and IL-8 in the culture supernatants from PBMCs were measured by sandwich ELISA (R&D Systems) according to the manufacturer's instructions.Absorbance at 405 nm was measured using an ELISA microplate reader (Molecular Devices).

Expression of Kim-1 and FN-1 mRNA by real-time reverse transcription Polymerase Chain Reaction (real-time PCR)
mRNA was extracted from in vitro PBMCs samples using the TRIzol Reagent (Molecular Research Center, Inc., Cincinnati, OH), according to the manufacturer's instructions.cDNA was synthesized in a PerkinElmer Cetus DNA thermal cycler (PerkinElmer, Inc., Waltham, MA) using the SuperScript Reverse Transcription system (Takara, Shiga, Japan).A LightCycler 2.0 instrument (Roche Diagnostics; software version 4.0) was used for PCR amplification.All PCR reactions were performed using LightCycler FastStart DNA Master SYBR Green I (Takara), according to the manufacturer's instructions.S2 Table shows the sense and antisense primers used for each molecule (5 0 !3 0 ).

Statistical analysis
Statistical analysis was performed using SPSS software (version 16.0; SPSS Inc., Chicago, IL).Continuous variables were summarized as mean ± SD.Categorical variables were summarized as a percentage of the group total.A non-parametric, Wilcoxon signed-rank test was used to compare T cell suppression, cytokine production, and gene expression between the control and treatment groups.All data included in the ex vivo study were normally distributed.An independent t -test was used to compare urine cytokine levels between the low 25(OH)D and normal 25(OH)D groups.A p value of < 0.05 was considered statistically significant.

The effect of 1,25(OH) 2 D3 on Kidney Injury Marker (KIM-1) and fibronectin 1 in HRPTEpiC
We performed real-time PCR analyses to evaluate the effect of 1,25(OH) 2 D3 on KIM-1 mediated by IL-17 as well as TNF-α, and fibronectin 1 mediated by TGF-β as well as IL-17 in HRPTEpiC.HRPTEpiC were cultured with TNF-α and IL-17, and KIM-1 expression was measured after 24-hour incubation.

Protective effect of 1,25(OH) 2 D3 on activated CD4 + T cell-induced inflammation in HRPTEpiC
We co-cultured HRPTEpiC with CD4 + T cells with or without stimulation under Th0 conditions.Co-culturing HRPTEpiC with CD4 + T cells without stimulation did not increase the production of IL-6 or IL-8.However, co-culturing with activated CD4 + T cells under Th0 conditions significantly increased HRPTEpiC production of IL-6 and IL-8 compared to the unstimulated co-culture with CD4 + T cells (P < 0.05).However, addition of 1,25(OH) 2 D3 significantly decreased the HRPTEpiC production of these cytokines compared to that by activated CD4 + T cells ( Ã P < 0.05 vs. CD4 + T control, and † P < 0.05 vs. activated CD4 + T cells) (Fig 4A and 4B and S4 Table).

Discussion
In this study, we intended to find the direct protective effect of 1,25(OH) 2 D3 on HRPTEpiC against effector T cells or cytokines.We observed that pre-treatment with 1,25(OH) 2 D3 effectively regulated not only the proliferation of effector T cells but also suppressed injury induced by activated T cells or inflammatory cytokines, which may represent allograft rejection.Tubular epithelial cells are a key feature of acute graft rejection as well as of any inflammatory reaction in transplanted kidneys [18][19][20].
In this regard, we performed in vitro analyses to investigate the effect of 1,25(OH) 2 D3 on the proliferation of effector CD4 + T cells as well as the direct protective effect on HRPTEpiC from effector T cell-induced injury.Our in vitro study using human PBMCs pre-incubated with 1,25 (OH) 2 D3 demonstrated significant reduction in the percentage of Th1 and Th17 cells compared to that of Th0 condition (P < 0.05 for each).In contrast, 1,25(OH) 2 D3 increased the proportion of Th2 and Treg cells in a dose-dependent manner (P < 0.05 for each).These observations correlate with those from previous studies showing that 1,25(OH) 2 D3 suppresses T cell proliferation, [21] and results in a shift from a Th1 to a Th2 phenotype [22,23].1,25(OH) 2 D3 has also been shown to affect T cell maturation with a skewing away from the inflammatory Th17 phenotype, [24,25] as well as to facilitate the induction of T regulatory cells [25][26][27][28].Further, we evaluated the direct protective effect of 1,25(OH) 2 D3 on activated T cellinduced target organ injury, which may be similar to allograft rejection.
Most of kidney diseases is associated with acute tubular cell injury and dysfunction [29,30].Renal tubular epithelial cells play a crucial role in renal function.Renal tubular epithelial cells can produce inflammatory mediators such as cytokines (IL-1, TNF-α, IL-17) and chemokines and actively participate in acute inflammatory processes by affecting and directing leukocyte chemotaxis via the production of IL-8 [19,[31][32][33][34][35].
Based on the above background, we designed two separate experiments using HRPTEpiC: first, pre-treatment with inflammatory cytokines; second, co-culture with activated T cells.In response to the treatment of HRPTEpiC with inflammatory cytokines such as TNF-α, IL-17 induced the production of IL-6, IL-8, and KIM-1, whereas TGF-β increased the expression of fibronectin-1 in a dose-dependent manner.However, treatment with 1,25(OH) 2 D3 significantly reduced the levels of these cytokines or molecules (P < 0.05 for all).In the second experiment, we investigated whether 1,25(OH) 2 D3 protects HRPTEpiC against activated CD4 + T cellinduced inflammation.Our results showed that activated CD4 + T cell-induced inflammation also induced IL-6 and IL-8 production from HRPTEpiC.Similarly, treatment with 1,25 (OH) 2 D3 significantly reduced the levels of these cytokines (P < 0.05 for all).
Next, we investigated the molecular signaling pathway involved in the suppressive effects of 1,25(OH) 2 D3 on HRPTEpiC.We focused on the mTOR/Akt pathway, which plays an essential role in the regulation of kidney inflammation and fibrosis.[36][37][38][39][40].We also investigated the activity of Akt and p70S6k, which are located either upstream or downstream of the mTOR/ STAT3 pathway, and are significantly associated with the activity of mTOR [41,42].Activation of mTOR most prominently results in the phosphorylation of two downstream targets, ribosomal S6 Kinase (S6K) and eukaryotic translation-initiation factor 4E-binding protein (4E-BP), which stimulate ribosome biogenesis and translation in order to increase cell mass [43,44].
Our results showed that 1,25(OH) Finally, we investigated the association between serum 25(OH)D levels and urine IL-6, IL-8, KIM-1, and creatinine levels in kidney transplant recipients with stable allograft function.In the clinical setting, the measurement of urinary IL-6, IL-8, and KIM-1 levels is thought to be a potential biomarker of the localization and severity of inflammation within the renal tubular system [45,46].In this ex vivo study, we used serum 25(OH)D levels instead of 1,25(OH) 2 D3 levels because it shows some advantages.First, 25(OH)D is easy to measure and hence is widely used for the diagnosis of vitamin D deficiency in the clinical practice.Second, it better represents the immune status of patients and also, its level is not affected by renal function [47,48].In addition, 25(OH)D level shows very significant correlation with 1,25(OH)2D3 level [49].As a result, an inverse relationship between serum 25(OH)D and urine IL-6/creatinine (p < 0.05) as well as between serum 25(OH)D and KIM-1/creatinine (p < 0.05) in kidney transplant recipients without rejection or urinary tract infection were detected.These findings provide evidence of the advantages of sustaining normal vitamin D level in kidney transplant recipients.However, this study has some limitations.First, the study only evaluated T cell mediated rejection in renal tubular epithelial cell, which accounts for only a small portion of allograft rejection.Additional in vitro studies using vascular endothelial cells, which are also important targets of alloimmune cells or alloantibodies, may be required to fully represent the entire spectrum of allograft rejection in clinical practice.Second, this study did not show the direct clinical benefit of normal 25(OH)D levels and low inflammatory cytokine expression in urine from kidney transplant recipients.However, several previous studies have already demonstrated the clinical significance of normal 25(OH)D levels for the prevention of allograft rejection or urinary tract infection.Therefore, we thought that the observations from this study might explain one of the mechanisms through which vitamin D exerts its beneficial effect.[12,13,50] In conclusion, 1,25(OH) 2 D3 treatment showed significant suppressive effect on renal tubular inflammation induced by activated T cells or inflammatory cytokines via regulation of the mTOR/STAT3 pathway.In our ex vivo study, serum 25(OH)D showed significant association with urine inflammatory cytokine levels, which is consistent with the findings of our in vitro study.
The results of this study suggests that vitamin D may have an significant role in the regulation of inflammation in allograft tissue in kidney transplant recipients.
-hydroxyvitamin D (25(OH)D) levels and divided them into normal 25(OH)D group (25(OH)D !20 ng/mL) and low 25(OH)D group (25(OH)D < 20 ng/mL).We also measured urine IL-6, IL-8, KIM-1, and creatinine levels in each subject.We compared urine IL-6/creatinine and urine IL-8/creatinine and KIM-1/ creatinine between normal 25(OH)D group and low 25(OH)D group.All participants provided written informed consent in accordance with the Declaration of Helsinki.This study was approved by the Institutional Review Board of Seoul St. Mary's Hospital (KC13TNMI0701).

Table 1 . Baseline characteristics of the patients cohort to investigate the association between 25(OH)2D and urine cytokine level. Normal 25(OH)D (n = 50) Low 25
(OH)D (n = 40) P