CKD is a devastating condition that affects 5%–7% of the population worldwide. The number of patients with CKD and ESKD) is increasing due to the growing incidence of diabetes mellitus type 2, hypertension, obesity, and aging.1 Patients with CKD frequently show vitamin D deficiency due to insufficient generation of the active metabolite 1,25-dihydroxyvitamin D by injured kidneys. CKD is associated with impaired immune system responses and chronic systemic inflammation, characterized by elevated circulating proinflammatory cytokines and activated circulating cells.2–345 Inflammation is thought to contribute to both CKD progression and cardiovascular mortality.1,6 Deficiency in vitamin D and its active metabolites has been associated with adverse outcomes in patients with ESKD and in the general population.7 However, despite evidence that vitamin D supplementation or vitamin D receptor (VDR) agonists (VDRAs), such as paricalcitol, may have antiproteinuric and anti-inflammatory properties in preclinical and clinical kidney disease, there is no agreement on the overall clinical effect.7–89101112131415161718192021 Thus, in clinical trials, vitamin D improved endothelial function in patients with CKD but did not preserve kidney function in patients with type 2 diabetes.22–2324 Paricalcitol had an erratic effect on albuminuria but did not alter left ventricular mass index.21,25,26 Systematic reviews and meta-analyses have also been inconsistent on the effect of vitamin D or its analogues on kidney or cardiovascular outcomes.27–28293031 These differences remain unexplained and may depend on specific patient characteristics such as the prior existence of vitamin D deficiency, dietary salt intake, or other features.32,33 An improved understanding of the underlying molecular mechanisms activated by paricalcitol may shed light on the contradictory clinical findings and facilitate the design of novel therapeutic strategies.
The NF-κB pathway is a key regulator of inflammation.34,35 The role of the canonical NF-κB1 pathway in kidney disease has been well characterized. The canonical pathway proceeds through phosphorylation of IκBα and p65, and the subsequent translocation of the active p65–NF-κB complex to the nucleus, where it binds to specific promoters and regulates gene transcription.34–353637 In particular, p65 phosphorylation on Ser536 has been linked to proinflammatory gene regulation.38 Activation of the noncanonical NF-κB2 pathway is regulated by NF-κB–inducing kinase (NIK), which collaborates with IκB kinase α (IKKα) to induce phosphorylation-dependent ubiquitination and processing of p100 NF-κB2 to p52 NF-κB2 and nuclear translocation of p52/RelB.34 In human and experimental kidney diseases, elevated renal NF-κB activity correlates with upregulation of proinflammatory parameters.34–353637,39,40 However, most studies focused on the canonical NF-κB1 pathway, and data about the noncanonical NF-κB2 pathway are scarce. TNF receptor–associated factors (TRAFs) are critical signaling adaptors downstream of proinflammatory receptors. Whereas TRAF2, TRAF5, and TRAF6 activate the canonical NF-κB1 signaling, TRAF3 inhibits the noncanonical NF-κB2 pathway by promoting NIK ubiquitin–dependent degradation.41 In this paper we described for the first time that circulating TRAF3 levels are decreased in CKD on hemodialysis and could be a biomarker of renal damage associated with the inflammatory state. Paricalcitol restored TRAF3 levels inhibiting the noncanonical NF-κB2 pathway and decreased renal inflammation.
Intracellular protein and mRNA levels were measured in eripheral blood mononuclear cells (PBMCs) from patients with ESKD on hemodialysis, treated or not with paricalcitol, as well as from healthy donors (used as controls). Inclusion criteria were as follows: age >45 years; male sex; informed consent; and absence of active inflammatory, infectious, or malignant diseases at the initiation or during the study. TheIIS-Fundación Jiménez Díaz (IIS-FJD) Ethics Committee approved this study. The primary aim was to assess the expression of inflammation-related genes in PBMCs. Patients were enrolled after providing written informed consent. PBMCs were separated from whole blood samples (30 ml into EDTA tubes) using Ficoll density gradient centrifugation. In total, 50×106 PBMCs were obtained from 30 ml of whole blood and 6×106 PBMCs were used to isolate protein, RNA, and nuclear/cytosolic protein levels using the NE-PER Reagent (Pierce) following the manufacturer’s instructions.
Human kidney proximal tubule epithelial cells (HK2 cell line, ATCC CRL-2190) were grown in RPMI 1640 with 10% FBS, 1% glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin-transferrin-selenite (ITS), and 36 ng/ml hydrocortisone in 5% carbon dioxide at 37°C. When cells reached 60%–70% confluence, they were serum depleted for 24 hours before the experiment.
Murine proximal tubular epithelial cells (MCT cell line) were originally obtained from Dr. Eric Neilson (Vanderbilt University) and used for gene expression studies. These cells were grown in RPMI 1640 with 10% ineFBS, 1% glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in 5% carbon dioxide at 37°C. When cells reached 60%–70% confluence, they were maintained in RPMI with 1% FBS for 24 hours.
Cells were stimulated with 100 ng/ml recombinant human soluble TNF-like weak inducer of apoptosis (TWEAK; Millipore) or 200 μg/ml p-cresyl sulfate (APExBIO). Concentrations of TWEAK and p-cresyl sulfatep-CS) were based on previously published dose-response experiments.42,43 In some experiments, cells were preincubated for 48 hours with 12 µmol/L paricalcitol (Abbott) before stimulation. DMSO, used as a solvent in some cases, had no effect on cell viability or on gene expression levels (not shown).
Experimental Animal Models
All procedures on animals were performed according to the recommendations issued by the European Community and the protocol was approved by IIS-FJD Animal Research Ethical Committee and by the Community of Madrid All animal work took place in the Laboratory of Molecular and Cellular Biology in Renal and Vascular Pathology of the IIS-FJD at the Autonoma University of Madrid.
At the time of euthanasia, animals were anesthetized with 5 mg/kg xylazine (Bayer AG) and 35 mg/kg ketamine (Pfizer), and kidneys were perfused in situ with cold saline before removal. Then, kidney portions were fixed in buffered formalin for immunohistochemistry studies or immediately frozen in liquid nitrogen for gene and protein studies.
C57BL/6 female mice (9–12 weeks, weight 20 g, seven to eight animals per group) received a single intraperitoneal (i.p.) injection of 0.5 μg TWEAK dissolved in saline and were euthanized 24 hours later, as previously described.42 Controls were injected with the saline vehicle (n=8–10 mice per group). TWEAK endotoxin levels were <0.1 ng/mg, confirmed by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) (not shown). Some animals received paricalcitol (750 ng/kg per day, i.p.; Abbot) or the NF-κB2 inhibitor SN52 (0.7 mg/mouse per day), starting 48 hours before TWEAK administration. Animals were euthanized 24 hours later. The dose of paricalcitol was chosen based on prior experience in mild inflammatory kidney conditions.
Unilateral Ureteral Obstruction
Unilateral ureteral obstruction (UUO) is used as a model of accelerated CKD. It was established in male C57BL/6 mice, under isoflurane-induced anesthesia. The left ureter was ligated with silk (5/0) at two locations and cut between ligatures to prevent urinary tract infection (obstructed kidney).44,45 Two groups were studied: the untreated group or the group treated with paricalcitol (750 ng/kg per day), with n=6–8 mice per group. Studies compared both kidneys (contralateral versus obstructed) in each mouse.
Folic Acid Nephropathy
Folic acid (FA) nephropathy is a classic model of kidney tubulointerstitial injury, inflammation, and AKI that has been reported in humans. Mice received a single i.p. injection of FA (300 mg/kg; Sigma) in 0.3 mol/L sodium bicarbonate or vehicle, and were euthanized 48 hours later. Two groups were studied: the untreated group or the group treated with paricalcitol (25 μg/kg per day), starting 24 hours before FA, n=7–8 per group. The dose of paricalcitol was chosen based on dose-finding preliminary experiments.
Renal Histology and Immunohistochemistry
Paraffin-embedded kidney sections (3 μm) were stained using conventional methods. Antigen retrieval was performed by PT Link system (Dako Diagnostics) with sodium citrate buffer (10 mmol/L) adjusted to pH 6–9, depending on the immunohistochemical marker, followed by immunohistochemical staining in a Dako Autostainer. The steps were as follows: (1) endogenous peroxidase blockade; (2) incubation with primary antibodies anti-CD3 (1:300; Dako) or anti-F4/80 (1:5000; Serotec), anti-p52 NF-κB2 (1:50; Cell Signaling), anti-RelB (1:50; Santa Cruz Biotechnology), and anti-CCL-21 (1:90; Santa Cruz Biotechnology); (3) washing; and (4) DUOFLEX Doublestain EnVision treatment, using 3,3′-diaminobenzidine as chromogen. For F4/80 staining, a rabbit anti-rat linker was used before EnVision. Sections were counterstained with Carazzi hematoxylin. The intensity of the reactive mark was obtained using Image-Pro Plus software. For each sample (processed by duplicate in a blinded manner), the average value was obtained from the analysis of four fields (20× objective) as density/mm2 or percentage stained area versus total analyzed area. Data are expressed as fold increase over control mice, as mean±SEM of 8–10 animals per group. Negative controls include nonspecific Ig and no primary antibody (not shown).
Proteins were obtained from treated cells or mouse kidneys using lysis buffer (50 mmol/L Tris-hydrochloride, 150 mol/L sodium chloride, 2mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% IGEPAL, 10 μl/ml proteinase inhibitor cocktail, 0.2 mmol/L PMSF, and 0.2 mmol/L orthovanadate). To determine protein content, the bicinchoninic acid method was used.
For Western blot, cell (25 μg/lane) and kidney (100–150 μg/lane) protein extracts were separated on 6%–12% polyacrylamide-SDS gels under reducing conditions. Samples were then transferred onto nitrocellulose membranes (Bio-Rad), blocked with Tris-buffered saline/5% defatted milk/0.05% Tween 20, and incubated overnight at 4°C with the following antibodies (dilution): anti–NF-κB2 p52 (1:500), phosphorylated-p65 NF-κB (1:500), and anti–p65 NF-κB (1:500; Cell Signaling); and anti–CCL-21A (sc-25445, 1:500), anti–RelB (sc-226, 1:500), anti–p-IKKα (sc-101706, 1:500), anti–p-IκBα (sc-8404, 1:500), anti–IκBα (sc-371, 1:500), anti–TRAF3 (sc-6933, 1:250), anti–cIAP1 (sc-271419, 1:500), anti–NIK (sc-7211, 1:500 Santa Cruz Biotechnology). Membranes were subsequently incubated with peroxidase-conjugated IgG secondary antibody and developed using an ECL Chemiluminescence Kit (Amersham). Loading controls included anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10000; Chemicon), anti–α tubulin (1:5000; Sigma-Aldrich), or anti–histone H1 (sc-8030, 1:250; Santa Cruz Biotechnology) for nuclear proteins or total protein levels in phosphorylation studies. Autoradiographs were scanned using the Gel Doc EZ imager and analyzed with the Image Lab 3.0 software (Bio-Rad).
ELISA was used to evaluate levels of the chemokines CCL-2 and CCL-5 (eBioscience). In renal samples, total protein content was determined by the bicinchoninic acid method, and equal amounts of protein were analyzed. Data are expressed as n-fold increase over the mean of control levels.
Gene Expression Studies
Total RNA was isolated from cells and mouse kidney samples with Trizol (Invitrogen). The cDNA was synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems) using 2 μg total RNA primed with random hexamer primers. Multiplex real-time PCR was performed using the following Applied Biosystems expression assays for murine and human samples: ccl-2 Mm00441242_m1, ccl-5 Mm_ 01302428_m1, il-6 Mm_00446190_m1, ccl-19 Mm_00839967_g1, ccl-21a Mm_036466971_gH, TRAF3 Hs_00936781_m1, CCL-2 Hs00234140_m1, CCL-5 Hs00982282_m1, IL-6 Hs00174131_m1, CCL-19 Hs_00171149_m1, and CCL-21A Hs_00989654_m1. Data were normalized to 18S; 4210893E (VIC) and gapdh Mm99999915_g1. The mRNA copy numbers were calculated for each sample by the instrument software using Ct value. Results are expressed in copy numbers, calculated relative to unstimulated cells or control mice, after normalization against 18S or gapdh. Spike-in was not used as an internal control.
ELISA-Based NF-κB2 and RelB Assay
Nuclear and cytoplasmic fractions were separated from renal tissues using the NE-PER Reagent following the manufacturer’s instructions. In renal nuclear extracts, NF-κB2 and RelB DNA binding activity was measured as binding to an oligonucleotide containing the NF-κB consensus site using a TransAM NF-κB Family kit (Active Motif) with an antibody that only recognizes active NF-κB2 p52 and RelB.
Activation of Gene Expression by Magnetofection
Gene overexpression was achieved in cultured cells using the activation TRAF3 CRISPR/Cas9 DNA plasmid (Santa Cruz Biotechnology). Subconfluent cells were transfected with magnetofection (OZ biosciences) mixture (1 µl PolyMag CRISPR magnetofection reagent plus 1 µg DNA plasmid) for 30 minutes above the magnetic plate according to the manufacturer’s instructions. Then, cells were incubated with 10% heat-inactivated FBS for 24 hours and incubated in serum-free medium for 24 hours before experiments. Cells were stimulated with recombinant human soluble TWEAK (Millipore). In some experiments, cells were preincubated for 48 hours with 12 µmol/L paricalcitol (Abbott) before stimulation. Activation of the NF-κB2 pathway was assessed by gene expression and Western blot analysis using 1:200 anti-p52/NF-κB2 and 1:200 anti–CCL-21A. Anti-TRAF3 (1:250) was used for overexpression specificity and efficiency assessment, and anti-GAPDH (1:10,000) as loading control.
Gene silencing in cultured cells was performed using a predesigned small interfering RNA (siRNA) corresponding to membrane associated, rapid response steroid-binding (MARRS; Ambion). Subconfluent cells were transfected for 24 hours with 25 nmol/L siRNA using 50 nmol/L Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Cells were then incubated with 10% heat-inactivated FBS for 24 hours, followed by 24 hours in serum-free medium.
Cells growing in tissue culture dish plates were lysed in 300–500 µl Triton–NP-40 lysis buffer (50 mmol/L Tris-hydrochloride pH 8, 150 mmol/L sodium chloride, 1 mmol/L PMSF, 1% NP-40/IGEPAL, and a phosphatase-inhibitor cocktail [Set II; Calbiochem]), scraped off the dish, and incubated for 1 hour at 4°C with shaking. Cell lysates were precleared by incubating with 10 µl protein A agarose bead slurries (0.5 ml agarose/2 ml PBS) for 30 minutes at 4°C and then centrifuged three times for 5 minutes at 2500 rpm to wash supernatants. Precleared lysates were incubated with 2.5–5 µg mouse monoclonal anti-TRAF3 antibody (sc-6933; Santa Cruz Biotechnology) overnight at 4°C. The immune complexes were captured by the addition of protein A/G PLUS-agarose (20 μl) bead slurries for 1 hour at 4°C. The agarose beads were collected by centrifugation, washed three times with lysis buffer, resuspended in 2× Laemmli sample buffer, boiled for 5 minutes, and subjected to SDS-PAGE. Then Western blot was performed as described above, using 1:250 anti-ubiquitin Lys48 specific (05-1307; Millipore) or 1:250 anti-TRAF3 (sc-6933) antibodies.
All results are expressed as mean±SEM. Differences between intervention groups and controls were assessed by Mann–Whitney test. P<0.05 was considered significant. Statistical analysis was conducted using the SPSS statistical software (version 11.0).
Paricalcitol Inhibits NF-κB2 Activation without Modulating NF-κB1 Pathway in PBMCs from Patients with CKD
We used PBMCs to study paricalcitol anti-inflammatory mechanisms PBMCs. In PBMCs from patients with ESKD, both the canonical and noncanonical NF-κB pathways were activated, as demonstrated by increased p65/NF-κB1 and RelB or p52/NF-κB2 DNA binding activity (Figure 1, A and B). Interestingly, in PBMCs from patients with ESKD who were treated with paricalcitol, p65/NF-κB1 activation was similar to patients with ESKD who were not treated with paricalcitol, where the activation of the NF-κB2 pathway was inhibited (Figure 1, A and B). In PBMCs from patients with ESKD, proinflammatory gene expression levels were higher than in controls, but were significantly lower in patients with ESKD treated with paricalcitol (Figure 1C).
Paricalcitol Inhibits NF-κB2 Activation in Cytokine-Stimulated Cultured Cells
TWEAK is one of the few cytokines that activates both NF-κB pathways.34 In PBMCs from healthy donors, paricalcitol inhibited TWEAK-induced NF-κB2 activation without modulating the canonical NF-κB1 pathway (Figure 2A). Similar effects were observed in cells stimulated with p-cresyl sulfate, a representative uremic toxin that is not adequately cleared by dialysis (Figure 2B).43,46 Additionally, paricalcitol inhibited TWEAK-induced upregulation of several proinflammatory genes, such as ccl-2, ccl-5, and il-6, as well as the specific NF-κB2–regulated genes ccl-21a and ccl-19(Figure 2C).
Next, we performed studies in cultured human tubular epithelial HK2 cells to represent renal cells expressing components of the noncanonical NF-κB2 pathway that promote kidney injury.47,48 In cultured tubular cells, TWEAK increased p65 and IκBα phosphorylation and downregulated cytosolic IκBα levels (Figure 3, A and B). Preincubation with paricalcitol did not modulate TWEAK-induced NF-κB1 activation (Figure 3, A and B) but inhibited TWEAK-induced IKKα phosphorylation, the increase in NIK, and nuclear p52 and RelB (Figure 3, A, C and D). Paricalcitol also inhibited TWEAK-induced cytokine gene expression (Figure 3, E and F). Moreover, NF-κB2 inhibition with SN52 prevented the TWEAK-induced upregulation of proinflammatory genes (Figure 3, E and F), mimicking the effects of paricalcitol.
Paricalcitol Inhibits NF-κB2 Activation In Vivo
We next explored the effect of paricalcitol on NF-κB2 activation in three different preclinical models: TWEAK-induced renal inflammation, UUO, and FA-induced AKI.
TWEAK administration in mice causes an acute renal inflammatory response and activates both canonical and noncanonical NF-κB pathways.34 In TWEAK-injected mice, paricalcitol significantly decreased the number of infiltrating monocytes/macrophages and T lymphocytes (Supplemental Figure 1, A and B) and upregulated proinflammatory genes, such as ccl-2, ccl-5, and il-6 (Supplemental Figure 1, C and D). In kidneys from TWEAK-injected mice, IκBα phosphorylation and nuclear p65 NF-κB levels were higher than in controls, but were not modified by paricalcitol. However, paricalcitol inhibited TWEAK-induced activation of kidney NF-κB2, as demonstrated by reduced nuclear p52 and RelB accumulation in tubular cells. Moreover, paricalcitol inhibited TWEAK-induced p52 and RelB DNA binding activity (Figure 4).
TWEAK via the NF-κB2 pathway drives the synthesis of specific chemokines, such as CCL-21A and CCL-19 in tubular cells.34 In TWEAK-injected mice, paricalcitol reduced renal ccl-21a and ccl-19 gene expression and CCL-21A protein levels (Supplemental Figures 1E and 2A). Interestingly, CCL-21A was located in renal tubules in TWEAK-injected mice (Supplemental Figure 2, C and D), that is, in the same renal structures where active NF-κB2 components RelB and p52 were located (Figure 4C).
In TWEAK-injected mice, inhibition of the NF-κB2 pathway with SN52, a synthetic peptide that blocks the nuclear translocation of the p52/NF-κB2 heterodimer (Supplemental Figure 3), significantly decreased the inflammatory cell infiltration (Supplemental Figure 1), thus displaying a clear-cut anti-inflammatory effect. Interestingly, in TWEAK-injected mice, SN52 prevented the upregulation of specific NF-κB2 target genes, but also NF-κB1–regulated genes (il-6, ccl-2, and ccl-5) (Supplemental Figure 1).
In UUO, paricalcitol was previously shown to decrease kidney inflammatory cell infiltration and expression of ccl-2 and ccl-5, without blocking nuclear p65/NF-κB1 translocation.49 Therefore, we investigated whether paricalcitol modulated the NF-κB2 pathway in UUO. Paricalcitol prevented the increased RelB levels and nuclear localization observed in obstructed kidneys (Figure 5A). Moreover, it blocked p100/p52 processing as well as p52 nuclear translocation and DNA binding activity (Figure 5, A–C). By contrast, activation of NF-κB1, evaluated by p65 nuclear translocation and IκBα phosphorylation, was not modified in paricalcitol-treated obstructed kidneys (Figure 5, D and E), confirming prior reports.49 Importantly, in obstructed kidneys, paricalcitol prevented inflammatory cell infiltration and the increased expression of proinflammatory factors (such as il-6, ccl-2, and ccl-5) and specific NF-κB2–regulated chemokines (Supplemental Figure 4).
Next, we explored paricalcitol effects in FA-induced renal damage in mice. Paricalcitol only blocked NF-κB2 activation (as assessed by RelB and p52 nuclear translocation, p52 protein levels, DNA binding activity of RelB, and NF-κB2–dependent cytokines such as ccl-21a), whereas NF-κB1 activation was not modified. Paricalcitol also prevented the decrease in renal function as assessed by BUN levels (Supplemental Figure 5).
Paricalcitol Increases TRAF3 Levels
TRAF3 plays an essential role in regulating the activation of the noncanonical NF-κB2 signaling pathway by the TNF receptor superfamily.41,50,51
First, we investigated TRAF3 levels in patients with ESKD and their modulation by paricalcitol. In PBMCs from patients with ESKD, TRAF3 protein was downregulated compared with healthy controls, without a decrease in TRAF3 mRNA levels (Figure 6, A and B), suggesting that TRAF3 protein levels were regulated post-translationally. Moreover, in patients with ESKD treated with paricalcitol, PBMC TRAF3 levels were similar to the range of healthy controls, without differences in TRAF3 mRNA levels (Figure 6, A and B), suggesting that paricalcitol prevents TRAF3 protein degradation. Next, we evaluated TRAF3 levels in experimental kidney injury. Kidney TRAF3 levels were downregulated in the models of TWEAK administration, FA, and UUO, and this effect was prevented by paricalcitol (Figure 6, C–E). In cultured tubular cells and in PBMCs from healthy donors, TWEAK downregulated TRAF3 levels and this was again prevented by paricalcitol (Figure 6, F and G).
Mechanisms of TRAF3 Modulation by Paricalcitol
In cultured cells, TRAF3 overexpression mimicked paricalcitol actions. In HK2 cells, transfection with a CRISPR/Cas9 TRAF3 activation plasmid blocked TWEAK-induced p52 activation, and the increased expression of proinflammatory genes such as CCL-2, CCL-5, and IL-6, as well as NF-κB2–regulated genes (Figure 7).
Next, we investigated upstream mechanisms involved in TRAF3 degradation triggered by paricalcitol. A previous study demonstrated that TNF superfamily receptor–induced activation of NF-κB2 signaling is coupled with TRAF3 degradation and concomitant accumulation of NIK.52 The E3 ubiquitin ligases cIAPs promote TRAF3 K48-linked ubiquitination, thus tagging it for proteasomal degradation.53–545556575859 In PBMCs from patients with ESKD, TRAF3 immunoprecipitation studies showed increased TRAF3 ubiquitination in the K48-linked chains, leading to the formation of cIAP1-TRAF3 complexes (Figure 8A). In patients treated with paricalcitol, TRAF3 ubiquitination and cIAP1-TRAF3 interaction were significantly lowered to values in the range of healthy controls (Figure 8A). Moreover, cIAP1 levels were higher in whole protein extracts of PBMCs from patients with ESKD than from healthy controls, whereas in patients with ESKD treated with paricalcitol they were close to control values (Figure 8A). Similar results were observed in cell culture experiments. In HK2 cells, TWEAK stimulation induced TRAF3 ubiquitination and CIAP1-TRAF3 complex formation and this was prevented by paricalcitol (Figure 8B).
Paricalcitol Decreases NF-κB2 Activation Independently of VDR and MARRS
To evaluate the role of VDR in paricalcitol actions, we used VDR knockout mice. Surprisingly, paricalcitol decreased TWEAK-induced renal inflammation and prevented TRAF3 downregulation and NF-κB2–dependent gene upregulation, as seen by the increase in ccl-21a expression in VDR knockout mice (Supplemental Figure 6), suggesting a VDR-independent anti-inflammatory effect of paricalcitol.
MARRS has been suggested as a VDRA receptor. To test whether MARRS may mediate paricalcitol effects on NF-κB2 activation, MARRS was blocked by gene silencing. HK2 cells were transfected with a MARRS siRNA or a control siRNA and then pretreated or not with paricalcitol before stimulation with TWEAK. In MARRS silenced cells, paricalcitol diminished TWEAK-induced NF-κB2 activation, suggesting a MARRS-independent effect of paricalcitol (Supplemental Figure 7).
Several lines of evidence suggest an anti-inflammatory activity of vitamin D and VDRAs in CKD.60 However, clinical outcomes have been inconsistent, suggesting an incomplete understanding of the pathways involved. This work, studies in vitro, in experimental renal damage, and in patients with ESKD on hemodialysis demonstrate a novel mechanism implicated in the anti-inflammatory actions of paricalcitol that involves TRAF3 modulation and subsequent inhibition of the noncanonical NF-κB2 pathway.
Vitamin D or VDRAs reduce renal inflammatory cell infiltration in experimental renal damage.14,60–616263 In the UUO model, paricalcitol was previously shown to decrease interstitial fibrosis64 and renal inflammation, without blocking nuclear p65/NF-κB1 translocation.49 We have now observed that paricalcitol inhibited activation of the NF-κB2 pathway without interfering with NF-κB1 activation, both in injured murine kidneys and in cultured renal cells. TWEAK is one of the few cytokines that activates both NF-κB pathways. Experimental studies, using pharmacologic or genetic approaches, have clearly demonstrated that TWEAK activates its receptor Fn14 to induce renal inflammation,65–66676869 suggesting this cytokine is relevant in renal diseases. In cultured tubular epithelial cells and in the kidneys in vivo, paricalcitol inhibited the successive steps of noncanonical NF-κB activation: p-IKKα; NIK, NF-κB2/p52, and RelB protein expression, and ccl-21a and ccl-19 gene expression, suggesting anti-inflammatory effects mediated by the modulation of NF-κB2–controlled genes. Prior in vitro studies support our findings. In human proximal tubular cells, paricalcitol did not affect TNF-α–mediated IκBα phosphorylation and degradation, nor p65 activation and its nuclear translocation.49 Despite this, we have found that paricalcitol downregulated many canonical NF-κB1 genes, such as ccl-2 and ccl-5, in experimental renal damage and in cytokine-stimulated cells. Recent studies suggest a crosstalk between both NF-κB pathways in which the recruitment of the noncanonical pathway, such as NIK, boosts the consequences of NF-κB activation.48,49,70 Moreover, paricalcitol diminished TWEAK-induced CCL-21A upregulation and blocked NF-κB2 activation in tubular epithelial cells in vivo, suggesting these cells are likely the primary target of vitamin D anti-inflammatory actions. Accordingly, in cultured human tubular epithelial cells, NIK overexpression increased nuclear RelB/p52 levels and DNA binding activity and expression of proinflammatory cytokines, including CCL-2,CCL-5, IL-6 and IL-8.71
Blockade of the canonical NF-κB activation, using different inhibitors, such as parthenolide or SN50 peptides, inhibited experimental inflammation, including kidney damage.37,72,73 There is recent evidence of noncanonical NF-κB2 activation in human and experimental kidney disease, including increased IKKα phosphorylation, and NIK and RelB levels, as well as protection from AKI by RelB downregulation or NIK deficiency.47,48,74,75 Additionally, NIK or RelB silencing protected cultured tubular cells from cell death or inflammatory responses to advanced glycation end products or inflammatory cytokines.48,53,75,76 However, there are no studies specifically linking NF-κB2 to renal inflammation. Now, we have found renal activation of the NF-κB2 pathway in several models of renal damage, which is associated with kidney infiltration by inflammatory cells. SN52, a specific blocker p52/NF-κB2 nuclear translocation, inhibited TWEAK-induced renal inflammation and upregulation of proinflammatory genes both in vitro and in vivo. Interestingly, SN52 inhibited genes not regulated by NF-κB1, such as ccl-21a and ccl-19, supporting the existence of a crosstalk between NF-κB pathways.41
The mechanisms by which TNF superfamily receptors such as Fn14 activate the NF-κB2 pathway remain controversial. NIK is continuously synthesized and degraded in resting cells but becomes stabilized and accumulates in response to TNF receptor superfamily members.78 TRAF3 is a key regulator of NIK ubiquitin-dependent degradation and protein levels by forming a complex with the E3 ubiquitin ligase cIAP (cIAP1 or cIAP2), in which TRAF3 serves as the NIK-binding adapter.41,77,79,80 Activation of TNF superfamily receptors induces TRAF3 degradation, thereby promoting NIK accumulation and NF-κB2 p100 processing to p52.41,52,55,81,82 Indeed, TRAF3 knockout cells exhibit elevated NIK expression and constitutive NF-κB2 p100 processing to p52. More importantly, deletion of the NF-κB2 p100 gene rescued the TRAF3 null phenotype,51 showing that TRAF3 regulates NF-κB2 p100 processing. We found that TRAF3 overexpression blocked TWEAK/Fn14-induced NF-κB2 p100 processing to p52 and upregulation of NF-κB2 target cytokines, suggesting a link between TRAF3 and the NF-κB2 pathway. Thus, TRAF3 overexpression in vitro mimics the effects of paricalcitol, suggesting a role for TRAF3 modulation in the anti-inflammatory action of paricalcitol.
TRAF2/3 and cIAP1/2 form a cytoplasmic ubiquitin complex that induces Lys-48–linked ubiquitination and degradation of TRAF3.82 In PBMCs from patients with ESKD, cIAP1-TRAF3 complex formation and Lys-48–linked ubiquitination were increased in association to NF-κB2 activation and a proinflammatory phenotype. Importantly, paricalcitol reduced TRAF3 K48-linked chain ubiquitination and prevented cIAP1-TRAF3 complex formation in all of the experimental systems studied.
Paricalcitol and calcitriol bind to and activate the VDR to regulate gene expression.83 As an example, paricalcitol promotes VDR/p65 complex formation to inhibit p65 transactivation of CCL-5/RANTES gene transcription. However, this mechanism did not occur for CCL-2/MCP1.49 To evaluate the role of VDR in the action of paricalcitol, we studied VDR knockout mice.84–8586 Surprisingly, paricalcitol decreased TWEAK-induced renal inflammation and normalized TRAF3 levels in VDR knockout mice. This indicates that paricalcitol has VDR-independent anti-inflammatory effects. The VDR-independent effects of VDRAs have been known for some time. Calcitriol also exerts fast nontranscriptional responses involving an increase in intracellular calcium and activation of intracellular kinases.87,88 A novel calcitriol receptor, MARRS, was proposed to mediate nongenomic actions of calcitriol in chick intestinal basolateral membranes.89 However, in MARRS-silenced cells, paricalcitol still prevented TWEAK-induced NF-κB2/p52 activation, indicating that paricalcitol modulates NF-κB2 activation in a VDR- and MARRS-independent manner.
As indicated in the introduction, interventional studies on vitamin D or VDRAs in patients with CKD have been inconclusive, with more recent meta-analyses being disappointing.27–28293031 In some instances, potential confounders have been identified, such as salt intake or baseline vitamin D status.32,33 Some of these confounders, such as salt intake, are known to modulate macrophage activation, but whether they modulate TRAF3 is currently unknown.90,91 Interestingly, sodium retention has been linked to modulation of a TRAF3-interacting protein (TRAF3 interacting protein 2, TRAF3IP2), resulting in cardiovascular injury.92 The present findings of an anti-inflammatory action of the VDRA paricalcitol mediated by the modulation of TRAF3 levels and NF-κB2 activation open new avenues to further optimize therapeutic approaches and clinical trial design. Thus, paricalcitol analogues selected for TRAF3 modulation and baseline risk stratification by TRAF3 expression in patient PBMCs represent novel strategies for improving drug selection and clinical trial design.
Currently, there is no drug in clinical use targeting NF-κB2. However, information from patients deficient in NFKB2 is in line with our observations because it is associated with immune deficiency, implying a role for NF-κB2 in inflammation and immune defenses (https://omim.org/entry/615577).
In conclusion, our data clearly demonstrate that, in patients with ESKD, paricalcitol decreases inflammatory factors in PBMCs cells by preserving TRAF3 expression and preventing activation of the noncanonical NF-κB2 pathway, thus identifying TRAF3/NF-κB2 as an important target for VDRAs. Paricalcitol upregulation of TRAF3 was independent from the presence of VDR and MARRS, thus facilitating the design of novel molecules with enhanced anti-inflammatory activity that may be used for kidney protection. Further studies should characterize the effect of earlier CKD stages on TRAF3 levels and their modulation by paricalcitol or other VDRAs.
J. Egido, J.L. Morgado-Pascual, A. Ortiz, E. Gonzalez-Parra, S. Rayego-Mateos, and M. Ruiz-Ortega have a patent entitled “In Vitro Method for Detecting Renal Disease” (European patent application number, EP19382470.3; patent holder Autonomous University of Madrid and IIS-FJD) pending. All remaining authors have nothing to disclose.
This work was supported by Instituto de Salud Carlos III (ISCIII; Carlos III Health Institute), Centro de Investigación Biomédica en Red Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM; Biomedical Research Networking Center in Diabetes and Associated Metabolic Disorder), Ministerio de Economía y Competitividad, and European Union European Regional Development Fund grants PI17/00119, PI19/00588, PI19/00815, and PI17/01495. This work was also supported by Red de Investigación Renal (REDinREN) grant RD16/0009; Sociedad Española de Nefrologia; NOVELREN-CM: Enfermedad renal crónica: nuevas Estrategias para la prevención, Diagnóstico y tratamiento grants B2017/BMD-3751, B2017/BMD-3686, and CIFRA2-CM; and the European Union’s Horizon 2020 research and innovation program for the IMPROVE-PD (“Identification and Management of Patients at Risk–Outcome and Vascular Events in Peritoneal Dialysis”) project under the Marie Skłodowska-Curie grant agreement number 812699. A.B. Sanz was supported by an ISCIII Miguel Servet grant. S. Rayego-Mateos’s salary was supported by the Ministerio de Economía, Industria y Competitividad, Gobierno de España “Juan de la Cierva de Formacion” training program, grant FJCI-2016-29050.
We want to thank Susana Carrasco at the IIS-FJD for help with immunohistochemical procedures and María Soledad Sanchez Fernández for help with the acquisition of healthy donor samples.
Dr. Sandra Rayego-Mateos contributed to the design of the experiments, data acquisition, analysis, interpretation of the data, and drafted the manuscript; Dr. José Luis Morgado-Pascual contributed to data acquisition, mouse model development, and data analysis; Dr. José Manuel Valdivielso and Dr. Ana Belén Sanz contributed data acquisition, critical review, and financial support; Dr. Enrique Bosch-Panadero and Dr. Raul R. Rodrigues-Díez contributed to data acquisition and critical review; Dr. Alberto Ortiz, Dr. Jesús Egido, and Dr. Emilio González-Parra contributed to the critical review of the manuscript and financial support; and Dr. Marta Ruiz-Ortega contributed to the design of the experiments, analysis and interpretation of the data, financial support, and drafted the manuscript. All authors have approved the final version of this manuscript for publication.
Dr. Alberto Ortiz reports personal fees from Amicus, Amgen, AstraZeneca, Fresenius Medical Care, Genzyme, Kyowa-Kirin, Menarini, Mundipharma, Otsuka, and Shire, as well as grants from Genzyme, outside the submitted work.
This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019111206/-/DCSupplemental.
Supplemental Figure 1. Paricalcitol or NF-κB2 blockade decreased TWEAK-induced renal inflammation.
Supplemental Figure 2. Paricalcitol inhibits TWEAK-induced upregulation of specific NF-κB2 targets in the kidney.
Supplemental Figure 3. SN52 peptide blocks NF-κB2 activation and NF-κB2 nuclear translocation in a experimental TWEAK-induced renal damage.
Supplemental Figure 4. Paricalcitol decreases renal inflammation in experimental Unilateral Ureteral Obstruction (UUO) in mice.
Supplemental Figure 5. Paricalcitol inhibits NF-κB2, but not NF-κB1 activation in folic acid-induced renal injury.
Supplemental Figure 6. Paricalcitol reduces TWEAK-induced renal inflammation in VDR KO mice.
Supplemental Figure 7. Paricalcitol inhibits TWEAK-induced upregulation of specific NF-κB2 targets in MARRS gene silenced cells.
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