TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease : Journal of the American Society of Nephrology

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TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease

Rayego-Mateos, Sandra1,2; Morgado-Pascual, Jose Luis1,3; Valdivielso, José Manuel2,3; Sanz, Ana Belén3,4; Bosch-Panadero, Enrique4; Rodrigues-Díez, Raúl R.1; Egido, Jesús5; Ortiz, Alberto3,4; González-Parra, Emilio4; Ruiz-Ortega, Marta1,3

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JASN 31(9):p 2026-2042, September 2020. | DOI: 10.1681/ASN.2019111206
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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.


PBMC Isolation

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.

Cultured Cells

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.

TWEAK Administration

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).

Protein Studies

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

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.

Coimmunoprecipitation Assays

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.

Statistical Analyses

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).

Figure 1.:
Paricalcitol (VDRA) inhibits noncanonical but not canonical activation of NF-κB, and decreases proinflammatory genes in PBMCs from patients with ESKD on hemodialysis. Activation of canonical and noncanonical NF-κB pathways in PBMCs was assessed by (A) RelA/p65 or (B) p52 and RelB DNA binding activity, respectively. (C) Gene expression was assessed by real-time PCR. Number of patients: five to eight per group. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus healthy control; # P<0.05 versus patients with ESKD not treated with paricalcitol.

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).

Figure 2.:
Paricalcitol decreased noncanonical NF-κB2 activation but not canonical NF-κB activation in PBMCs from healthy donors. Human PBMCs were pretreated with 12 µM paricalcitol for 24 hours, before stimulation with (A) 100 ng/ml TWEAK (TW) or (B) 200 µg/ml p-cresyl sulfate (p-CS) for 24 hours. (C) Activation of canonical NF-κB1 was assessed as phosphorylated p65 in cytosolic fractions and noncanonical activation of NF-κB2 as p52 levels. PBMCs from control individuals were pretreated with 12 μmol/L paricalcitol or vehicle for 24 hours, and then stimulated with TWEAK for 6 hours. Gene expression was assessed by real-time PCR. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus control; # P<0.05 versus TWEAK or p-CS–stimulated samples. Data are expressed as mean±SEM of four experiments for protein levels and five experiments for gene expression levels.

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.

Figure 3.:
Paricalcitol decreased noncanonical NF-κB2 activation but not canonical NF-κB activation induced by TWEAK in renal tubular epithelial cells. (A–D) Human tubular cells were pretreated with 12 µM paricalcitol for 48 hours before stimulation with 100 ng/ml TWEAK (TW) for 15 minutes (NF-κB1 pathway) or 6 hours (NF-κB2 pathway). (A and B) Activation of canonical NF-κB1 was assessed by IκBα or p65 phosphorylation levels in total protein extracts, and p65 levels in cytosolic or nuclear fractions. Activation of the noncanonical NF-κB2 pathway was assessed by (A and C) evaluating NIK and phosphorylated IKKα levels in total protein extracts and (A and D) the subcellular location of p100/p52 and Rel B subunits. GAPDH and histone H1 were used as total and nuclear protein loading controls, respectively. (A) Representative Western blots. (B–D) Quantification of protein levels expressed as mean±SEM of four experiments. *P<0.05 versus control; # P<0.05 versus TWEAK. Paricalcitol decreased proinflammatory genes in tubular cells. Cultured murine tubular epithelial cells were stimulated with 100 ng/ml TWEAK for 6 hours. Cells were preincubated for 48 hours with 12 μmol/L paricalcitol or 60 ng/ml SN52. (E and F) Gene expression was assessed by real-time PCR. Data expressed as mean±SEM of three to six experiments. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus control; # P<0.05 versus TWEAK-treated cells.

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).

Figure 4.:
Paricalcitol only inhibits TWEAK-induced noncanonical NF-κB activation in vivo. Mice were treated with paricalcitol 750 ng/kg per day starting 48 hours before 0.5 μg TWEAK (TW), and euthanized 24 hours after TWEAK administration. (A and B) In TWEAK-injected mice, increased (A) cytosolic phosphorylated IκBα levels and (B) nuclear p65 levels were found, but they were not modulated by paricalcitol treatment, suggesting that paricalcitol did not modulate NF-κB1 activation. GAPDH and Red Ponceau were used as loading controls. Data expressed as mean±SEM of five to eight mice per group. *P<0.05 versus control. (C) Nuclear location of p52 and RelB in renal tubules of TWEAK-injected mice assessed by immunohistochemistry. (D) Western blot assessment of NF-κB2 activation (p52 levels) in whole-kidney protein extracts. (E) In isolated renal nuclear protein extracts, p52 and RelB NF-κB2 DNA binding activity was measured by ELISA. All data expressed as mean±SEM of five to eight animals per group. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus control; # P<0.05 versus TWEAK.

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).

Figure 5.:
Paricalcitol inhibits NF-κB2 but not NF-κB1 activation in experimental UUO in mice. Mice were treated with paricalcitol 750 ng/kg per day, starting 24 hours before UUO, and studied 5 days after UUO. (A) Immunohistochemistry in paraffin-embedded kidney sections. Representative animal from each group (magnification 200×). (B) NF-κB2 activation was assessed as p52 levels in Western blots of total protein extracts. (C) In isolated renal nuclear proteins, p52 and RelB DNA binding activity was measured by ELISA. (D and E) Western blot assessment of nuclear levels of (D) p65 and (E) cytosolic IκBα phosphorylation were used to evaluate activation of the NF-κB1 pathway. In each mouse, obstructed (Ob) kidneys were compared with the corresponding contralateral nonobstructed (Nob) kidney. Data expressed as mean±SEM of four to eight animals per group. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus contralateral nonobstructed kidneys; # P<0.05 versus obstructed kidneys.

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).

Figure 6.:
Paricalcitol restored TRAF3 levels in PBMCs from patients with CKD, in experimental kidney injury, and in cultured cells. (A) TRAF3 protein levels in PBMCs from patients with ESKD treated or not with paricalcitol were determined by Western blot. Number of patients: five to eight per group. *P<0.05 versus control; # P<0.05 versus ESKD cells. (B) TRAF3 mRNA levels in PBMCs from patients with ESKD treated or not with paricalcitol were determined by real-time PCR. Number of patients: five to eight per group. *P<0.05 versus control. (C–E) Evaluation of TRAF3 levels in experimental models of renal damage, assessed by Western blot. Mice were treated with paricalcitol starting before induction of kidney injury. (C) TWEAK injection (0.5 μg/mouse), (D) 300 mg/kg FA, or (E) UUO. (F and G) Paricalcitol restored TRAF3 levels in cytokine-stimulated cells. (F) Human tubular cells or (G) PBMCs from healthy donors were pretreated with paricalcitol 48 hours before TWEAK stimulation (100 ng/ml) for 24 hours. TRAF3 protein levels by Western blot. Data expressed as mean±SEM of four to eight animals per group or of three in vitro experiments. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus control; # P<0.05 versus cells from patients with ESKD, injured-kidney cells, or TWEAK-treated cells. Nob, contralateral nonobstructed kidneys; Ob, obstructed kidneys.

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).

Figure 7.:
TRAF3 overexpression mimics the actions of paricalcitol. TRAF3 overexpression was achieved in cultured cells using activation TRAF3/CRISPR/Cas9 DNA plasmid. Cells were stimulated with recombinant human soluble TWEAK (100 ng/ml). In some experiments, cells were preincubated for 48 hours with 15 µmol/L paricalcitol before TWEAK stimulation. (A) TRAF3 mRNA levels are increased in cells transfected with CRISPR/Cas9 TRAF3 activation plasmid as evaluated by real-time PCR. (B) NF-κB2 pathway activation was assessed by Western blot of NF-κB2 p52 and the NF-κB2–regulated cytokine CCL-21A. (C and D) Gene expression of the proinflammatory factors (C)CCL-2, CCL-, and IL-6 or (D) CCL-21A and CCL-19 were evaluated by real-time PCR. Data expressed as mean±SEM of three to five independent experiments. Differences between intervention and control groups were assessed by Mann–Whitney test. *P<0.05 versus control; # P<0.05 versus TWEAK-treated cells.

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).

Figure 8.:
Modulation of TRAF3 ubiquitination and cIAP1-TRAF3 complex formation was involved in paricalcitol restoration of TRAF3 levels. (A) In PBMCs from patients with ESKD on hemodialysis treated or not with paricalcitol (VDRA), TRAF3 was immunoprecipitated (I.P.) by an anti-TRAF3 antibody followed by SDS-PAGE and Immunoblotting (I.B.) using an anti-ubiquitin antibody. Representative experiment. TRAF3 antibody was used as loading control. cIAP1 levels were also evaluated. Number of patients five to eight per group. (B) TWEAK-induced TRAF3 ubiquitination was prevented by paricalcitol in HK2 cells stimulated with TWEAK and treated or not with paricalcitol. Figures show a representative I.P. experiment out of three performed.* IgG heavy chain.

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 (

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.

Published online ahead of print. Publication date available at

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.

Supplemental Material

This article contains the following supplemental material online at

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.


1. Ortiz A, Fernandez-Fernandez B: Humble kidneys predict mighty heart troubles. Lancet Diabetes Endocrinol 3: 489–491, 2015 26028595
2. Niewczas MA, Pavkov ME, Skupien J, Smiles A, Md Dom ZI, Wilson JM, et al.: A signature of circulating inflammatory proteins and development of end-stage renal disease in diabetes. Nat Med 25: 805–813, 2019 31011203
3. Heine GH, Ortiz A, Massy ZA, Lindholm B, Wiecek A, Martínez-Castelao A, et al.; European Renal and Cardiovascular Medicine (EURECA-m) working group of the European Renal Association-European Dialysis and Transplant Association (ERA-EDTA): Monocyte subpopulations and cardiovascular risk in chronic kidney disease. Nat Rev Nephrol 8: 362–369, 2012 22410492
4. Zhang J, Hua G, Zhang X, Tong R, Du X, Li Z: Regulatory T cells/T-helper cell 17 functional imbalance in uraemic patients on maintenance haemodialysis: A pivotal link between microinflammation and adverse cardiovascular events. Nephrology (Carlton) 15: 33–41, 2010 20377769
5. Betriu A, Martinez-Alonso M, Arcidiacono MV, Cannata-Andia J, Pascual J, Valdivielso JM, et al.; Investigators from the NEFRONA Study: Prevalence of subclinical atheromatosis and associated risk factors in chronic kidney disease: the NEFRONA study. Nephrol Dial Transplant 29: 1415–1422, 2014 24586070
6. Vanholder R, Fouque D, Glorieux G, Heine GH, Kanbay M, Mallamaci F, et al.; European Renal Association European Dialysis; Transplant Association (ERA-EDTA) European Renal; Cardiovascular Medicine (EURECA-m) working group: Clinical management of the uraemic syndrome in chronic kidney disease. Lancet Diabetes Endocrinol 4: 360–373, 2016 published correction appears in Lancet Diabetes Endocrinol 4: e4, 2016
7. Gonzalez-Parra E, Rojas-Rivera J, Tuñón J, Praga M, Ortiz A, Egido J: Vitamin D receptor activation and cardiovascular disease. Nephrol Dial Transplant 27[Suppl 4]: iv17-iv21, 2012 23258805
8. Agarwal R, Acharya M, Tian J, Hippensteel RL, Melnick JZ, Qiu P, et al.: Antiproteinuric effect of oral paricalcitol in chronic kidney disease. Kidney Int 68: 2823–2828, 2005 16316359
9. Liu L-J, Lv JC, Shi SF, Chen YQ, Zhang H, Wang HY: Oral calcitriol for reduction of proteinuria in patients with IgA nephropathy: a randomized controlled trial. Am J Kidney Dis 59: 67–74, 2012 22019331
10. Cheng J, Zhang W, Zhang X, Li X, Chen J: Efficacy and safety of paricalcitol therapy for chronic kidney disease: a meta-analysis. Clin J Am Soc Nephrol 7: 391–400, 2012 published correction appears in Clin J Am Soc Nephrol 7: 1053, 2012
11. Zhang Z, Sun L, Wang Y, Ning G, Minto AW, Kong J, et al.: Renoprotective role of the vitamin D receptor in diabetic nephropathy. Kidney Int 73: 163–171, 2008 17928826
12. Branisteanu DD, Leenaerts P, van Damme B, Bouillon R: Partial prevention of active Heymann nephritis by 1 alpha, 25 dihydroxyvitamin D3. Clin Exp Immunol 94: 412–417, 1993 8252801
13. Wang Y, Zhou J, Minto AW, Hack BK, Alexander JJ, Haas M, et al.: Altered vitamin D metabolism in type II diabetic mouse glomeruli may provide protection from diabetic nephropathy. Kidney Int 70: 882–891, 2006 16820793
14. Sanchez-Niño MD, Sanz AB, Carrasco S, Saleem MA, Mathieson PW, Valdivielso JM, et al.: Globotriaosylsphingosine actions on human glomerular podocytes: implications for Fabry nephropathy. Nephrol Dial Transplant 26: 1797–1802, 2011 20504837
15. Freundlich M, Quiroz Y, Zhang Z, Zhang Y, Bravo Y, Weisinger JR, et al.: Suppression of renin-angiotensin gene expression in the kidney by paricalcitol. Kidney Int 74: 1394–1402, 2008 18813285
16. Piecha G, Kokeny G, Nakagawa K, Koleganova N, Geldyyev A, Berger I, et al.: Calcimimetic R-568 or calcitriol: equally beneficial on progression of renal damage in subtotally nephrectomized rats. Am J Physiol Renal Physiol 294: F748–F757, 2008 18199601
17. Matsui I, Hamano T, Tomida K, Inoue K, Takabatake Y, Nagasawa Y, et al.: Active vitamin D and its analogue, 22-oxacalcitriol, ameliorate puromycin aminonucleoside-induced nephrosis in rats. Nephrol Dial Transplant 24: 2354–2361, 2009 19297354
18. Teng M, Wolf M, Lowrie E, Ofsthun N, Lazarus JM, Thadhani R: Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy. N Engl J Med 349: 446–456, 2003 12890843
19. Alborzi P, Patel NA, Peterson C, Bills JE, Bekele DM, Bunaye Z, et al.: Paricalcitol reduces albuminuria and inflammation in chronic kidney disease: a randomized double-blind pilot trial. Hypertens 52: 249–255, 2008
20. Fishbane S, Chittineni H, Packman M, Dutka P, Ali N, Durie N: Oral paricalcitol in the treatment of patients with CKD and proteinuria: a randomized trial. Am J Kidney Dis 54: 647–652, 2009 19596163
21. de Zeeuw D, Agarwal R, Amdahl M, Audhya P, Coyne D, Garimella T, et al.: Selective vitamin D receptor activation with paricalcitol for reduction of albuminuria in patients with type 2 diabetes (VITAL study): a randomised controlled trial. Lancet 376: 1543–1551, 2010 21055801
22. de Boer IH, Zelnick LR, Ruzinski J, Friedenberg G, Duszlak J, Bubes VY, et al.: Effect of vitamin D and omega-3 fatty acid supplementation on kidney function in patients with type 2 diabetes: a randomized clinical trial. JAMA 322: 1899, 2019 31703120
23. Levin A, Tang M, Perry T, Zalunardo N, Beaulieu M, Dubland JA, et al.: Randomized controlled trial for the effect of vitamin D supplementation on vascular stiffness in CKD. Clin J Am Soc Nephrol 12: 1447–1460, 2017 28550081
24. Kumar V, Yadav AK, Lal A, Kumar V, Singhal M, Billot L, et al.: A randomized trial of vitamin D supplementation on vascular function in CKD. J Am Soc Nephrol 28: 3100–3108, 2017 28667080
25. Thadhani R, Appelbaum E, Pritchett Y, Chang Y, Wenger J, Tamez H, et al.: Vitamin D therapy and cardiac structure and function in patients with chronic kidney disease: the PRIMO randomized controlled trial. JAMA 307: 674–684, 2012 22337679
26. Parvanova A, Trillini M, Podestà MA, Iliev IP, Ruggiero B, Abbate M, et al.; PROCEED Study Organization and the Scientific Writing Academy (SWA) 2016: Moderate salt restriction with or without paricalcitol in type 2 diabetes and losartan-resistant macroalbuminuria (PROCEED): a randomised, double-blind, placebo-controlled, crossover trial. Lancet Diabetes Endocrinol 6: 27–40, 2018 29104158
27. de Borst MH, Hajhosseiny R, Tamez H, Wenger J, Thadhani R, Goldsmith DJ: Active vitamin D treatment for reduction of residual proteinuria: a systematic review. J Am Soc Nephrol 24: 1863–1871, 2013 23929770
28. Hu X, Shang J, Yuan W, Zhang S, Jiang Y, Zhao B, et al.: Effects of paricalcitol on cardiovascular outcomes and renal function in patients with chronic kidney disease: a meta-analysis. Herz 43: 518–528, 2018 28835982
29. Lundwall K, Jacobson SH, Jörneskog G, Spaak J: Treating endothelial dysfunction with vitamin D in chronic kidney disease: a meta-analysis. BMC Nephrol 19: 247, 2018 30253741
30. Beveridge LA, Khan F, Struthers AD, Armitage J, Barchetta I, Bressendorff I, et al.: Effect of vitamin D supplementation on markers of vascular function: a systematic review and individual participant meta-analysis. J Am Heart Assoc 7: e008273, 2018 29848497
31. Barbarawi M, Kheiri B, Zayed Y, Barbarawi O, Dhillon H, Swaid B, et al.: Vitamin D supplementation and cardiovascular disease risks in more than 83 000 individuals in 21 randomized clinical trials: a meta-analysis. JAMA Cardiol 4: 765–776, 2019 31215980
32. Fernandez-Fernandez B, Ortiz A: Paricalcitol and albuminuria: tread carefully. Lancet Diabetes Endocrinol 6: 3–5, 2018 29104157
33. Ortiz A, Sanchez Niño MD, Rojas J, Egido J: Paricalcitol for reduction of albuminuria in diabetes. Lancet 377: 635–636, author reply 636–637, 201121334525
34. Sanz AB, Sanchez-Niño MD, Ramos AM, Moreno JA, Santamaria B, Ruiz-Ortega M, et al.: NF-kappaB in renal inflammation. J Am Soc Nephrol 21: 1254–1262, 2010 20651166
35. Rangan G, Wang Y, Harris D: NF-kappaB signalling in chronic kidney disease. Front Biosci (Landmark Ed) 14: 3496–3522, 2009
36. Baud L, Fouqueray B, Bellocq A, Haymann J-P, Peltier J: [Inflammation, prelude to renal sclerosis: The importance of NF-kappa B]. J Soc Biol 196: 269–273, 2002 12645294
37. Guijarro C, Egido J: Transcription factor-kappa B (NF-kappa B) and renal disease. Kidney Int 59: 415–424, 2001 11168923
38. Cui R, Tieu B, Recinos A, Tilton RG, Brasier AR: RhoA mediates angiotensin II-induced phospho-Ser536 nuclear factor kappaB/RelA subunit exchange on the interleukin-6 promoter in VSMCs. Circ Res 99: 723–730, 2006 16960103
39. Mezzano S, Aros C, Droguett A, Burgos ME, Ardiles L, Flores C, et al.: NF-kappaB activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol Dial Transplant 19: 2505–2512, 2004 15280531
40. Ruiz-Ortega M, Ruperez M, Lorenzo O, Esteban V, Blanco J, Mezzano S, et al.: Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney. Kidney Int Suppl 62: S12–S22, 2002 12410849
41. Sun S-C: The noncanonical NF-κB pathway. Immunol Rev 246: 125–140, 2012 22435551
42. Sanz AB, Justo P, Sanchez-Niño MD, Blanco-Colio LM, Winkles JA, Kreztler M, et al.: The cytokine TWEAK modulates renal tubulointerstitial inflammation. J Am Soc Nephrol 19: 695–703, 2008 18235096
43. Poveda J, Sanchez-Niño MD, Glorieux G, Sanz AB, Egido J, Vanholder R, et al.: p-cresyl sulphate has pro-inflammatory and cytotoxic actions on human proximal tubular epithelial cells. Nephrol Dial Transplant 29: 56–64, 2014 24166466
44. Ucero AC, Benito-Martin A, Izquierdo MC, Sanchez-Niño MD, Sanz AB, Ramos AM, et al.: Unilateral ureteral obstruction: beyond obstruction. Int Urol Nephrol 46: 765–776, 2014 24072452
45. Rodrigues-Díez R, Rodrigues-Díez RR, Rayego-Mateos S, Suarez-Alvarez B, Lavoz C, Stark Aroeira L, et al.: The C-terminal module IV of connective tissue growth factor is a novel immune modulator of the Th17 response. Lab Invest 93: 812–824, 2013 23648563
46. Rossi M, Campbell K, Johnson D, Stanton T, Pascoe E, Hawley C, et al.: Uraemic toxins and cardiovascular disease across the chronic kidney disease spectrum: an observational study. Nutr Metab Cardiovasc Dis 24: 1035–1042, 2014 24880738
47. Starkey JM, Haidacher SJ, LeJeune WS, Zhang X, Tieu BC, Choudhary S, et al.: Diabetes-induced activation of canonical and noncanonical nuclear factor-kappaB pathways in renal cortex. Diabetes 55: 1252–1259, 2006 16644679
48. Ortiz A, Husi H, Gonzalez-Lafuente L, Valiño-Rivas L, Fresno M, Sanz AB, et al.: Mitogen-activated protein kinase 14 promotes AKI. J Am Soc Nephrol 28: 823–836, 2017 27620989
49. Tan X, Wen X, Liu Y: Paricalcitol inhibits renal inflammation by promoting vitamin D receptor-mediated sequestration of NF-kappaB signaling. J Am Soc Nephrol 19: 1741–1752, 2008 18525004
50. Yao Z, Xing L, Boyce BF: NF-kappaB p100 limits TNF-induced bone resorption in mice by a TRAF3-dependent mechanism. J Clin Invest 119: 3024–3034, 2009 19770515
51. Zarnegar B, Yamazaki S, He JQ, Cheng G: Control of canonical NF-kappaB activation through the NIK-IKK complex pathway. Proc Natl Acad Sci U S A 105: 3503–3508, 2008 18292232
52. Liao G, Zhang M, Harhaj EW, Sun S-C: Regulation of the NF-kappaB-inducing kinase by tumor necrosis factor receptor-associated factor 3-induced degradation. J Biol Chem 279: 26243–26250, 2004 15084608
53. Hu J, Zhu XH, Zhang XJ, Wang PX, Zhang R, Zhang P, et al.: Targeting TRAF3 signaling protects against hepatic ischemia/reperfusions injury. J Hepatol 64: 146–159, 2016 26334576
54. Häcker H, Tseng P-H, Karin M: Expanding TRAF function: TRAF3 as a tri-faced immune regulator. Nat Rev Immunol 11: 457–468, 2011 21660053
55. Yang X-D, Sun S-C: Targeting signaling factors for degradation, an emerging mechanism for TRAF functions. Immunol Rev 266: 56–71, 2015 26085207
56. Hu H, Brittain GC, Chang JH, Puebla-Osorio N, Jin J, Zal A, et al.: OTUD7B controls non-canonical NF-κB activation through deubiquitination of TRAF3. Nature 494: 371–374, 2013 23334419
57. Vallabhapurapu S, Matsuzawa A, Zhang W, Tseng PH, Keats JJ, Wang H, et al.: Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling. Nat Immunol 9: 1364–1370, 2008 18997792
58. Hostager BS, Haxhinasto SA, Rowland SL, Bishop GA: Tumor necrosis factor receptor-associated factor 2 (TRAF2)-deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling. J Biol Chem 278: 45382–45390, 2003 12958312
59. Gardam S, Turner VM, Anderton H, Limaye S, Basten A, Koentgen F, et al.: Deletion of cIAP1 and cIAP2 in murine B lymphocytes constitutively activates cell survival pathways and inactivates the germinal center response. Blood 117: 4041–4051, 2011 21300983
60. Mizobuchi M, Morrissey J, Finch JL, Martin DR, Liapis H, Akizawa T, et al.: Combination therapy with an angiotensin-converting enzyme inhibitor and a vitamin D analog suppresses the progression of renal insufficiency in uremic rats. J Am Soc Nephrol 18: 1796–1806, 2007 17513326
61. Kuhlmann A, Haas CS, Gross ML, Reulbach U, Holzinger M, Schwarz U, et al.: 1,25-Dihydroxyvitamin D3 decreases podocyte loss and podocyte hypertrophy in the subtotally nephrectomized rat. Am J Physiol Renal Physiol 286: F526–F533, 2004 14600034
62. Hirata M, Makibayashi K, Katsumata K, Kusano K, Watanabe T, Fukushima N, et al.: 22-Oxacalcitriol prevents progressive glomerulosclerosis without adversely affecting calcium and phosphorus metabolism in subtotally nephrectomized rats. Nephrol Dial Transplant 17: 2132–2137, 2002 12454223
63. Panichi V, Migliori M, Taccola D, Filippi C, De Nisco L, Giovannini L, et al.: Effects of 1,25(OH)2D3 in experimental mesangial proliferative nephritis in rats. Kidney Int 60: 87–95, 2001 11422740
64. Tan X, Li Y, Liu Y: Paricalcitol attenuates renal interstitial fibrosis in obstructive nephropathy. J Am Soc Nephrol 17: 3382–3393, 2006 17082242
65. Wiley SR, Winkles JA: TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TweakR/Fn14 receptor. Cytokine Growth Factor Rev 14: 241–249, 2003 12787562
66. Winkles JA: The TWEAK-fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting. Nat Rev Drug Discov 7: 411–425, 2008 18404150
67. Brown SAN, Ghosh A, Winkles JA: Full-length, membrane-anchored TWEAK can function as a juxtacrine signaling molecule and activate the NF-kappaB pathway. J Biol Chem 285: 17432–17441, 2010 20385556
68. Ruiz-Ortega M, Ortiz A, Ramos AM: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and kidney disease. Curr Opin Nephrol Hypertens 23: 93–100, 2014 24257157
69. Sanz AB, Izquierdo MC, Sanchez-Niño MD, Ucero AC, Egido J, Ruiz-Ortega M, et al.: TWEAK and the progression of renal disease: clinical translation. Nephrol Dial Transplant 29[Suppl 1]: i54–i62, 2014
70. Valiño-Rivas L, Vaquero JJ, Sucunza D, Gutierrez S, Sanz AB, Fresno M, et al.: NIK as a druggable mediator of tissue injury. Trends Mol Med 25: 341–360, 2019 30926358
71. Zhao Y, Banerjee S, LeJeune WS, Choudhary S, Tilton RG: NF-κB-inducing kinase increases renal tubule epithelial inflammation associated with diabetes. Exp Diabetes Res 2011: 192564, 2011 21869881
72. Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071, 1997 9091804
73. Tomita N, Morishita R, Lan HY, Yamamoto K, Hashizume M, Notake M, et al.: In vivo administration of a nuclear transcription factor-kappaB decoy suppresses experimental crescentic glomerulonephritis. J Am Soc Nephrol 11: 1244–1252, 2000 10864580
74. Loverre A, Ditonno P, Crovace A, Gesualdo L, Ranieri E, Pontrelli P, et al.: Ischemia-reperfusion induces glomerular and tubular activation of proinflammatory and antiapoptotic pathways: differential modulation by rapamycin. J Am Soc Nephrol 15: 2675–2686, 2004 15466272
75. Feng B, Chen G, Zheng X, Sun H, Zhang X, Zhang ZX, et al.: Small interfering RNA targeting RelB protects against renal ischemia-reperfusion injury. Transplantation 87: 1283–1289, 2009 19424026
76. Benedetti G, Fokkelman M, Yan K, Fredriksson L, Herpers B, Meerman J, et al.: The nuclear factor κB family member RelB facilitates apoptosis of renal epithelial cells caused by cisplatin/tumor necrosis factor α synergy by suppressing an epithelial to mesenchymal transition-like phenotypic switch. Mol Pharmacol 84: 128–138, 2013 23625948
77. Dejardin E, Droin NM, Delhase M, Haas E, Cao Y, Makris C, et al.: The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-kappaB pathways. Immunity 17: 525–535, 2002 12387745
78. Cuarental L, Sucunza-Sáenz D, Valiño-Rivas L, Fernandez-Fernandez B, Sanz AB, Ortiz A, et al.: MAP3K kinases and kidney injury. Nefrologia 39: 568–580, 2019 31196660
79. Vucic D: The role of ubiquitination in TWEAK-stimulated signaling. Front Immunol 4: 472, 2013 24391645
80. Boutaffala L, Bertrand MJ, Remouchamps C, Seleznik G, Reisinger F, Janas M, et al.: NIK promotes tissue destruction independently of the alternative NF-κB pathway through TNFR1/RIP1-induced apoptosis. Cell Death Differ 22: 2020–2033, 2015 26045047
81. He JQ, Zarnegar B, Oganesyan G, Saha SK, Yamazaki S, Doyle SE, et al.: Rescue of TRAF3-null mice by p100 NF-kappa B deficiency. J Exp Med 203: 2413–2418, 2006 17015635
82. Gardam S, Sierro F, Basten A, Mackay F, Brink R: TRAF2 and TRAF3 signal adapters act cooperatively to control the maturation and survival signals delivered to B cells by the BAFF receptor. Immunity 28: 391–401, 2008 18313334
83. Valdivielso JM: The physiology of vitamin D receptor activation. In: Peritoneal Dialysis - from Basic Concepts to Clinical Excellence, edited by Ronco C, Crepaldi C, Cruz DN, , Basel, Switzerland, Karger Medical and Scientific Publishers, 2009, pp 206–212
84. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP: 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 110: 229–238, 2002 12122115
85. Kato S, Takeyama K, Kitanaka S, Murayama A, Sekine K, Yoshizawa T: In vivo function of VDR in gene expression-VDR knock-out mice. J Steroid Biochem Mol Biol 69: 247–251, 1999 10418998
86. Xiang W, Kong J, Chen S, Cao LP, Qiao G, Zheng W, et al.: Cardiac hypertrophy in vitamin D receptor knockout mice: role of the systemic and cardiac renin-angiotensin systems. Am J Physiol Endocrinol Metab 288: E125–E132, 2005 15367398
87. de Boland AR, Nemere I: Rapid actions of vitamin D compounds. J Cell Biochem 49: 32–36, 1992 1644851
88. Marcinkowska E, Wiedłocha A, Radzikowski C: 1,25-Dihydroxyvitamin D3 induced activation and subsequent nuclear translocation of MAPK is upstream regulated by PKC in HL-60 cells. Biochem Biophys Res Commun 241: 419–426, 1997 9425286
89. Nemere I, Safford SE, Rohe B, DeSouza MM, Farach-Carson MC: Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS) binding protein. J Steroid Biochem Mol Biol 89–90: 281–285, 2004 15225786
90. Jantsch J, Schatz V, Friedrich D, Schröder A, Kopp C, Siegert I, et al.: Cutaneous Na+ storage strengthens the antimicrobial barrier function of the skin and boosts macrophage-driven host defense. Cell Metab 21: 493–501, 2015 25738463
91. Zhang W-C, Zheng XJ, Du LJ, Sun JY, Shen ZX, Shi C, et al.: High salt primes a specific activation state of macrophages, M(Na). Cell Res 25: 893–910, 2015 26206316
92. Sakamuri SSVP, Valente AJ, Siddesha JM, Delafontaine P, Siebenlist U, Gardner JD, et al.: TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis. Mol Cell Endocrinol 429: 84–92, 2016 27040306

vitamin D receptor; paricalcitol; chronic kidney disease; inflammation; NF-κB; TRAF3

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