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Paricalcitol Inhibits Renal Inflammation by Promoting

Vitamin D Receptor–Mediated Sequestration of NF- BSignaling

Xiaoyue Tan, Xiaoyan Wen, and Youhua Liu

Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine,

Pittsburgh, Pennsylvania

ABSTRACTInflammation is a pathologic feature of a variety of chronic kidney diseases. Several lines of evidence

suggest a potential anti-inflammatory role for vitamin D in chronic kidney disease, but the underlying

mechanism remains unknown. Here, the effect of the synthetic vitamin D analogue paricalcitol on renalinflammation was investigated in a mouse model of obstructive nephropathy. Paricalcitol reduced

infiltration of T cells and macrophages in the obstructed kidney. This inhibition of inflammatory cell

infiltration was accompanied by a decreased expression of RANTES and TNF-. Induction of RANTES

was localized primarily to the tubular epithelium, underscoring a role for tubular cells in renal inflamma-

tion. In a human proximal tubular cell line (HKC-8), paricalcitol inhibited RANTES mRNA and protein

expression and abolished the ability of tubular cells to recruit lymphocytes and monocytes after TNF-

stimulation. Although RANTES induction depended on NF-B signaling, paricalcitol affected neither

TNF-–mediated IB phosphorylation and degradation nor p65 NF-B activation and nuclear translo-

cation. Instead, chromatin immunoprecipitation assay showed that paricalcitol abolished the binding of

p65 to its cognate cis-acting element in the RANTES promoter. The vitamin D receptor (VDR) and p65

formed a complex in tubular cells after paricalcitol treatment, which inhibited the ability of p65 to

trans-activate gene transcription. In vivo, paricalcitol did not block NF-B nuclear translocation afterobstructive injury but did increase the expression and nuclear distribution of VDR. These results suggest

that paricalcitol inhibits renal inflammatory infiltration and RANTES expression by promoting VDR-

mediated sequestration of NF-B signaling.

J Am Soc Nephrol 19: 1741–1752, 2008. doi: 10.1681/ASN.2007060666

Deficiency in vitamin D and its active metabolites isa common pathologic feature that occurs early in

the pathogenesis of chronic kidney disease (CKD).Numerous clinical studies have shown a high prev-

alence of calcitriol deficiency in CKD, even in pa-tients with reasonable GFR.1,2 In humans, active vi-

tamin D reduces proteinuria and all-causemortality in CKD, which is independent of serum

parathyroid hormone, phosphorus, and calciumlevels.3–5 Evidence is also mounting that vitamin D

analogue is renoprotective in different experimen-tal nephropathies.6 Although early studies largely

focused on primary glomerular diseases,7–9 activevitamin D was shown to display beneficial effects in

obstructive nephropathy,10

a model characterized

by inflammatory infiltration, tubular atrophy, andinterstitial fibrosis. The therapeutic effects of active

vitamin D in obstructive nephropathy are thoughtto be mediated by its ability to preserve tubular ep-

ithelial integrity via inhibiting epithelial-to-mesen-chymal transition (EMT)10; however, in view of its

Received June 13, 2007. Accepted April 8, 2008.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Youhua Liu, Department of Pathology, Uni-versity of Pittsburgh, S-405 Biomedical Science Tower, 200 Lo-throp Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax:412-648-1916; E-mail: [email protected]

Copyright 2008 by the American Society of Nephrology

BASIC RESEARCH www.jasn.org

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pleiotropic property, it is conceivable that vitamin D could

elicit its renoprotection by a multitude of actions.11,12 One of the potential mechanisms could be related to itsability to mod-

ulate renal inflammation after injury.Renal inflammation, characterized by the infiltration of in-

flammatory cells including T cells and macrophages to kidney parenchyma, is an imperative pathologic process in the evolu-

tion of CKD.13,14 Inflammatory infiltration contributes to theinitiation and progression of CKD in several ways. On the one

hand, inflammatory cells release proinflammatory, chemoat-

tractant cytokines (chemokines), thereby leading to the forma-tion of a vicious self-accumulation circle. On the other hand,

production and secretion of profibrotic cytokines by inflam-matory cells such as monocytes/macrophages and T cells create

a fibrogenic microenvironment, leading to generation of thematrix-producing effector cells through fibroblast activation

and tubular EMT. Not surprising, decline of renal function inpatients with CKD often correlates closely to the extent of in-

Figure 1. Paricalcitol reduces renal inflammatory T cell infiltration. (A through E) Immunohistochemical staining revealed an increasedinfiltration of CD3 T cells in the obstructed kidney at 7 d (B) and 14 d (D), respectively, after UUO, compared with sham control (A).Paricalcitol treatment at 0.3 g/kg body wt effectively inhibited the infiltration of T cells in the obstructed kidneys at 7 d (C) and 14 d(E) after UUO, respectively, as indicated. Bar 20 m. (F) Graphic presentation of quantitative data. Data are means SEM of fiveanimals per group. *P 0.01 versus sham control; †P 0.01 versus vehicle.

Figure 2. Paricalcitol inhibits renal inflammatory macrophage infiltration. (A through E) Immunohistochemical staining showed an increasedinfiltration of F4/80myeloidcellsincluding macrophages and renal dendritic cells in the obstructed kidney at 7 d (B) and 14 d (D), respectively, after UUO, compared with sham control (A). Paricalcitol at 0.3g/kg body wt inhibited the infiltration of the F4/80 cells in the obstructed kidneys at 7 d(C) and 14 d (E), respectively, after UUO as indicated. Bar 20 m. (F) Graphic presentation of quantitative data. Data are means SEM of fiveanimals per group. *P 0.01 versus sham control; †P 0.01 versus vehicle.


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flammation.15 Inhibition of renal inflammation by differentmaneuvers is therapeutically effective, resulting in an amelio-

ration of renal fibrotic lesions in various experimental animalmodels.16,17

Several lines of evidence have suggested a potential anti-inflammatory activity of vitamin D in CKD.6,18 In animalmod-

els of primary glomerular diseases, administration of vitaminD reduces glomerular infiltration of inflammatory cells.6,9

Consistently, a decreased inflammation is associated with

higher serum vitamin D level in patients with CKD.19 VitaminD may exert its immunomodulatory action through regulating

the activity of many types of immune cells such as macro-phages, dendritic cells, and T cells.20–22 Furthermore, evidence

also points to an inhibitory effect of vitamin D on the signalingof NF-B, a key transcription factor that is thought to mediate

acute and chronic inflammation by regulating the gene expres-sion of cytokines, chemokines, and adhesion molecules14;

however,it remains ambiguousas to the molecular mechanism

Figure 3. Paricalcitol inhibits RANTES/CCL5and TNF- but not MCP-1/CCL2 mRNA ex-pression in obstructive nephropathy. (A andB) RT-PCR results showed that paricalcitolsuppressed renal RANTES and TNF- mRNAexpression at 7 d (A) and 14 d (B), respec-tively, after UUO in a dosage-dependentmanner; however, no significant difference inMCP-1 mRNA abundance was observed after paricalcitol treatment, compared with vehiclecontrols. Numbers (1, 2, and 3) denote eachindividual animal in a given group. Dosagesof paricalcitol were 0.3 and 0.1 g/kg bodywt, respectively. (C and D) Renal mRNA levelsof RANTES, TNF-, and MCP-1 in variousgroups. Relative mRNA levels at 7 d (C) and14 d (D) after UUO were calculated and ex-pressed as fold induction over sham controls(value 1.0) after normalization with -actin.

Data are means SEM of five animals per group. *P 0.01 versus sham control; †P 0.01 versus vehicle.

Figure 4. Paricalcitol inhibits renal RANTES protein expression in the obstructed kidney after UUO. (A through E) Immunohistochemicalstaining demonstrated that RANTES protein was induced predominantly in renal tubular epithelium in the obstructed kidney at 7 d (B) and14 d (D) after UUO, compared with sham control (A). Tubular expression of RANTES was largely suppressed by paricalcitol treatment (0.3g/kgbody wt) at 7 d (C) and 14 d (E), respectively, after UUO. Bar 20 m. (F) Quantitative determination of RANTES protein expression in variousgroups as indicated. Data are means SEM of five animals per group. *P 0.05 versus sham control; †P 0.05 versus vehicle.

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by which vitamin D inhibits inflammation in the setting of

CKD.In this study, we demonstrated that paricalcitol, an active

vitamin D analogue, reduces inflammatory cell infiltration andRANTES, also known as CC-chemokine ligand 5 (CCL5), ex-

pression in vivo. We further showed that paricalcitol induced aphysical interaction of vitamin D receptor (VDR) and p65 NF-

B, resulting in sequestration of the ability of p65 to bind tocis-acting element, thereby causing the repression of NF-B–

mediated gene transcription. Our findings provide significantmechanistic insights into the understanding of the molecular

details underlying vitamin D inhibition of renal inflammation.

RESULTS

Paricalcitol Inhibits Renal Inflammatory InfiltrationTo assess the potential effect of paricalcitol on renal inflamma-tion, we first examined renal infiltration of the CD3T cells in

the obstructed kidney after unilateral ureteral obstruction(UUO). As shown in Figure 1, compared with sham controls,

obstructive injury caused significant T cell infiltration in thekidney at 7 d after UUO, as illustrated by immunohistochem-

ical staining for CD3 antigen. Prolonged, continuous obstruc-tion for 14 d markedly increased renal infiltration of the CD3

T cells in the obstructed kidney, when compared with 7 d afterUUO (Figure 1, D versus B); however, paricalcitol treatment

effectively inhibited the infiltration of T cells in the obstructed

kidney at different time points (Figure 1, C and E). A comput-er-aided quantitative analysis also demonstrated a dramatic

suppression of inflammatory T cell infiltration by paricalcitolat 7 and 14 d after UUO, respectively, in obstructive nephrop-

athy (Figure 1F).We also examined the effects of paricalcitol on monocyte/

macrophage infiltration in the obstructed kidney. As shown inFigure 2, UUO instigated significant infiltration of the F4/80

antigen–positive myeloid cells, including

macrophages and dendritic cells in renalinterstitium. Similarly, administration of

paricalcitol substantially reduced F4/80-

positive cell infiltration at different timepoints after UUO (Figure 2, C, E, and F).

Paricalcitol Inhibits Renal mRNAExpression of RANTES and TNF-We next examined the expression of

RANTES, an important proinflamma-tory chemokine that plays a crucial role

in inciting infiltration of T cells andother inflammatory cells such as mono-

cytes, in the obstructed kidney. Asshown in Figure 3, reverse transcriptase–

PCR (RT-PCR) results revealed that ap-proximately seven- and 11-fold induc-

tion of RANTES/CCL5 mRNA wasobserved in the obstructed kidney at 7 and 14 d, respectively,

when compared with sham controls. Interestingly, we foundthat paricalcitol markedly inhibited RANTES mRNA expres-

sion in different time points (Figure 3, A and B); however,paricalcitol seemed to have little effect on monocyte chemoat-

tractant protein-1 (MCP-1), also known as CCL2, mRNA ex-pression in the obstructed kidney (Figure 3).

We further investigated the expression of TNF-, a key in-flammatory cytokine that is produced primarily by the infil-

trated cells, in the obstructed kidney after UUO. As presentedin Figure 3, UUO dramatically induced renal TNF- mRNA

expression at 7 and 14 d after UUO, respectively. Similarly,paricalcitol inhibited TNF- expression in the obstructed kid-

ney in a dosage-dependent manner.

RANTES Protein Is Induced Specifically in RenalTubules after Obstructive InjuryWe also examined RANTES protein expression and its localiza-tion in the obstructed kidney by immunohistochemical staining.

As shown in Figure 4A,no or littleRANTESproteinwas observedin normal, sham-operated kidney; however, RANTES protein ex-

pression was markedly induced in the obstructed kidney at 7 and14 d, respectively. RANTES protein was localized predominantly

in renal tubular epithelia (Figure 4, B and D). Consistent with themRNA data (Figure 3), paricalcitol treatment significantly re-duced RANTES protein expression in the obstructed kidney at

different time points (Figure 4, C and E).

Paricalcitol Inhibits Tubular RANTES Induction In VitroGiven that tubular epithelial cells arethe primary sitesof RANTES

production in vivo in obstructive nephropathy,we nextexaminedthe regulation of renal RANTES expression by paricalcitol using

an in vitro cell culture system. To this end, human proximal tubu-lar epithelial cells (HKC-8) were incubated with TNF- in the

presence or absence of paricalcitol, and RANTES expression wasexamined. As shown in Figure 5, A and B, Western blot analysis

Figure 5. Paricalcitol inhibits the TNF-–mediated RANTES expression in tubular epi-thelial cells in vitro. Human proximal tubular epithelial (HKC-8) cells were treated with 2ng/ml TNF- in the presence or absence of various concentrations of paricalcitol asindicated. (A and B) Representative Western blot (A) and graphic presentation (B) showedthat paricalcitol inhibited RANTES expression induced by TNF- in HKC-8 cells. (C, D, andE) RT-PCR showed that paricalcitol inhibited TNF-–induced RANTES mRNA expressionin HKC-8 cells. Data are means SEM of three experiments. *P 0.05 versus control;†P 0.05 versus TNF- treated alone.




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showed that proinflammatory cytokine TNF- dramatically in-duced RANTES expression in HKC-8 cells. Simultaneous incuba-

tion with paricalcitol, however, inhibited the TNF-–mediatedRANTES expression (Figure 5, A and B). Similarly, RT-PCR re-

sults demonstratedthat paricalcitolreduced RANTES mRNAex-

pression induced by TNF- in a dosage-dependent manner (Fig-ure5, C andD). Of note, paricalcitol did notaffect basalRANTES

expression under unstimulatedconditions in HKC-8 cells(Figure5E).

Paricalcitol Reduces the Recruitment of InflammatoryCells In Vitro

To evaluate the significance of tubular expression of RANTES

in recruiting inflammatory cells, we examined the chemoat-tractant ability of the supernatants of tubular cells by using

chemotaxis assay. To this end, HKC-8 cells were treated withTNF- in the absence or presence of paricalcitol for 24 h. The

supernatants of these cultures were used

to assess their ability to attract lympho-cyte or monocyte migration across the

Transwell filter. A shown in Figure 6,

conditioned medium derived fromTNF-–stimulated HKC-8 cells were

more potent in chemoattracting mousesplenocytes (Figure 6A) or THP-1 cells

(Figure 6C), compared with controls.These chemoattractant activities of the

conditioned medium were largely abol-ished when incubated with anti-RAN-

TES antibody (Figure 6, B and D), sug-gesting that RANTES is a key

chemotactic component secreted by tu-bular cells. Not surprising, treatment of

HKC-8 cells with paricalcitol signifi-cantly blocked the chemotaxis of

splenocytes and THP-1 cells in this assay (Figure 6, A and C).

NF- B Signaling Is Critical forMediating RANTES Induction In

Vitro

We next sought to investigate the mech-

anism underlying RANTES induction intubular epithelial cells. Because NF-B

signaling is known to be important inthe process of inflammation, we specu-

lated that RANTES expression may be

regulated by this signaling pathway. Totest this, we treated HKC-8 cells with

TNF- in the absence or presence of specific NF-B inhibitor (NF-B SN50),

a cell-permeable inhibitor peptide. Asshown in Figure 7A,inhibition of NF-B

signaling abolished RANTES expressioninduced by TNF- in tubular cells, indicating that an intact

NF-B signaling is required for RANTES induction. In recip-rocal experiments, ectopic expression of p65 NF-B sensitized

tubular epithelial cells to express RANTES in response to a low level of TNF- (0.2 ng/ml) stimulation (Figure 7B, lane 2 versus

lane 3). Interestingly, paricalcitol (107 M) also inhibited RAN-TES expression in the tubular cells overexpressing p65 NF-B

(Figure 7B, lane 3 versus lane 4).

Paricalcitol Does not Affect NF- B Early Activationand Its Nuclear TranslocationHaving established that NF-B signaling is important in me-diating RANTES induction (Figure 7), we reasoned whether

paricalcitol inhibits RANTES expression and renal inflamma-tion by modulating NF-B pathway. To testthis hypothesis, we

first examined the effect of paricalcitol on IB phosphoryla-tion and its subsequent degradation, as well as p65 NF-B

Figure 6. Paricalcitol reduces the chemoattraction of tubular epithelial cells to spleno-cytes and THP-1 cells in vitro. HKC-8 cells were treated without or with TNF- (2 ng/ml)in the absence or presence of various concentrations of paricalcitol for 24 h. The super-natants of these cultures were used in a chemotaxis assay to assess their ability to attractsplenocyte (A and B) or THP-1 cell (C and D) migration across the filter of Transwellchambers. Freshly isolated mouse splenocytes (A and B) and cultured THP-1 cells (C andD) were used. (A and C) Paricalcitol treatment reduced the ability of HKC-8 cell super-

natants to attract both splenocyte (A) and THP-1 cell (C) migration. (B and D) Neutraliza-tion of RANTES in HKC-8 cell supernatants with specific anti-RANTES antibody (2 g/ml)reduced splenocyte (B) and THP-1 cell (D) migration. The same amount of normal IgG wasused as controls. Data are expressed as the percentages of migrated cells in total cellsadded and presented as means SEM of three experiments. *P 0.05 versus control;†P 0.05 versus TNF- treated alone. (E) Representative pictures show the migratedTHP-1 cells in the bottom chambers of the Transwell plates in various groups as indicated.




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phosphorylation and activation. When HKC-8 cells were incu-

bated with proinflammatory TNF-, IB and p65 NF-Bwere rapidly phosphorylated starting at the time point as early

as 5 min (Figure 8, A and B); however, treatment of HKC-8

cells with paricalcitol did not significantly affect the kineticsand magnitude of IB and p65 NF-B phosphorylation.

Likewise, paricalcitol also did not influence IB degradationtriggered by TNF- in HKC-8 cells (Figure 8B). We next as-

sessed the p65 NF-B nuclear translocation after various treat-ments. Immunofluorescence staining demonstrated that upon

stimulation with TNF-, p65 NF-B rapidly translocated intothe nuclei in HKC-8 cells, and paricalcitol did not affect the

nuclear translocation of p65 NF-B (Figure 8C). These resultssuggest that paricalcitol inhibits tubular RANTES expression

by a mechanism independentof NF-B early activation and itsnuclear translocation.

Paricalcitol Abolishes NF- B Binding to RANTES

Promoter by Facilitating VDR/p65 InteractionThe inability of paricalcitol to affect NF-B activation prompted

us to examine whether it modulates the signaling events after p65nuclear translocation. To test this, we first investigated the possi-

bility that paricalcitol might negatively influence the in vivo bind-ing of p65 NF-B to RANTES promoter region. As shown in

Figure 9, A and B, TNF- induced p65 binding to its cognatecis-acting NF-B element in human RANTES promoter, as re-

vealed by chromatin immunoprecipitation (ChIP) assay; how-ever, treatment of HKC-8 cells with paricalcitol abolished the

binding of p65 to RANTES promoter induced by TNF-. To as-sess the consequence of this paricalcitol-mediated inhibition of

p65/DNA binding, we examined the effect of paricalcitol on NF-B–mediated gene expressionby using a luciferase reporter assay.

As demonstrated in Figure 9C, paricalcitol effectively repressed

Figure 7. NF-B signaling is critical for mediating RANTES in-duction in tubular epithelial cells. (A) Inhibition of NF-B signalingabrogated RANTES induction by TNF- in tubular epithelial cells.HKC-8 cells were treated with 2 ng/ml TNF- in the absence or presence of NF-B inhibitor (NF-B SN50). Cell lysates wereimmunoblotted with antibodies against RANTES and actin, re-spectively. (B) Ectopic expression of p65 NF-B sensitized tubular epithelial cells to express RANTES in response to a low level of TNF- stimulation. HKC-8 cells were transfected with either p65NF-B expression vector or pcDNA3 empty vector as indicated,followed by incubation with a low level of TNF- (0.2 ng/ml) in thepresence or absence of paricalcitol (107 M). Western blotshowed that ectopic expression of p65 NF-B rendered HKC-8cells more sensitive to the stimulation with low concentration of TNF- to express RANTES, whereas this effect was largely

blocked by paricalcitol.

Figure 8. Paricalcitol does not affect NF-B activation and its nuclear translocation induced by TNF-. HKC-8 cells were pretreatedwith or without 107 M paricalcitol for 0.5 h, followed by incubation with 2 ng/ml TNF- for various periods as indicated. (A) Westernblot showed that paricalcitol did not significantly affect p65 NF-B phosphorylation and activation induced by TNF-. (B) Paricalcitolalso did not significantly alter IB phosphorylation and subsequent degradation induced by TNF-. Cell lysates were immunoblottedwith specific antibodies against phospho- and total p65 NF-B and against phospho- and total IB, respectively. The low band in thedoublet (B, lane 1) likely represents the unphosphorylated IB. (C) Immunofluorescence staining demonstrated that paricalcitolpretreatment did not affect p65 NF-B to undergo nuclear translocation upon TNF- stimulation in HKC-8 cells.




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the NF-B–mediated reporter gene expression after TNF- stim-ulation.These results indicatethatparicalcitol can specifically tar-

get a key eventin NF-B signaling.We next investigated the potential mechanism underlying

the paricalcitol inhibition of p65 binding to RANTES pro-moter. By co-immunoprecipitation, we found that paricalcitol

facilitated the interaction between VDR and p65 NF-B inHKC-8 cells that overexpressed p65. As shown in Figure 9, D

and E, after stimulation by TNF- and paricalcitol, increasedp65 was detectable in the immunocomplexes precipitated by

anti-VDR antibody. Similarly, in HKC-8 cells without ectopicexpression of exogenous p65, paricalcitol induced a physical

interaction between VDR and endogenous p65 NF-B (Figure9F).Thissuggests thatan increasedVDR/p65 complex formation

after paricalcitol treatment would lead to a reduced availability of free p65, thereby sequestering its ability to bind to cis-acting ele-ment, causing a repression of p65-mediated gene transcription.

Paricalcitol Does not Affect NF- B NuclearTranslocation but Restores VDR Expression In VivoWe further investigated how paricalcitol inhibits NF-B sig-

naling and renal inflammation in obstructive nephropathy in

vivo. As showed in Figure 10A, UUO markedly induced p65

phosphorylation and activation, as revealed by Western blotanalysis of whole-kidney lysates using phospho-specific anti-

p65 antibody; however, paricalcitol did not inhibit p65 phos-phorylation in the obstructed kidney at 7 d after UUO (Figure

10A). Immunofluorescence staining demonstrated that p65NF-B was localized largely in the cytoplasm of the tubular

cells in normal kidney (Figure 10C); however, obstructive injury apparently induced its nuclear translocation. Similar to an

in vitro situation, administration of paricalcitol did not affectthe activation and nuclear translocation of p65 in obstructed

kidney (Figure 10C).We also examined the expression and localization of VDR

after different treatments. Consistent with a previous report,10

obstructive injury caused a suppression of VDR, and parical-

citol largely restored VDR expression after UUO (Figure 10B).Interestingly, immunostaining exhibited that a large propor-

tion of VDR was localized in the nuclei in normal kidney (Fig-ure 10C), suggesting an active role of the vitamin D system in

the maintenance of normal tubular integrity. After UUO, theoverall level of VDR was reduced and little nuclear VDR wasobserved; however, paricalcitol not only restored VDR protein

level but also induced VDR to redistribute completely into thenuclei. It is plausible, therefore, that the nuclear VDR after

paricalcitol treatment would bind to the activated p65 in thenuclei and sequester its gene-transactivating ability.

DISCUSSION

The results presented in this study demonstrate that paricalci-tol, an active vitamin D analogue, displays a potent anti-in-

Figure 9. Paricalcitol induces a physical as-sociation between VDR and p65 NF-B, in-hibits its binding to RANTES promoter, andrepresses NF-B–mediated gene transcrip-tion. (A) Partial sequence of human RANTESgene promoter region. Highlighted is theNF-B element in this region. (B) ChIP assaydemonstrated that paricalcitol abrogated thebinding of p65 to its cognate cis -acting ele-ment in the RANTES promoter. HKC-8 cellswere treated with TNF- and paricalcitol asindicated. (C) Luciferase reporter assay dem-onstrated that paricalcitol repressed the NF-B–mediated gene transcription in responseto TNF- treatment. Relative luciferase activ-ity (with control group 1.0) is presented.Data are means SEM from three experi-ments. *P 0.01 versus control; †P 0.01versus TNF- treatment group. (D and E)Co-immunoprecipitation revealed that pari-

calcitol induced an increased binding of VDRto p65 NF-B. HKC-8 cells were transfectedwith p65 NF-B expression vector, followedby treatment without or with TNF-, parical-citol, or both as indicated. Cell lysates were then immunoprecipitated with anti-VDR antibody, followed by immunoblotting withanti-p65. Whole-cell lysates were also immunoblotted with anti-p65 for normalizing p65 abundance. Data from three independentexperiments are shown (E). *P 0.05 versus control; †P 0.05 versus TNF- or paricalcitol treated alone. (F) Paricalcitol also induceda physical interaction between VDR and endogenous p65 NF-B. HKC-8 cells were treated without or with TNF-, paricalcitol, or bothand then assessed for endogenous p65/VDR complex formation by co-immunoprecipitation.




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flammatory activity by effectively inhibiting T lymphocyte andmacrophage infiltration and proinflammatory cytokines

RANTES and TNF- expression in a mouse model of obstruc-tive nephropathy. Mechanistically, paricalcitol specifically tar-

get NF-B, a principal signaling transducer that is involved inmediating proinflammatory responses in virtually all circum-

stances. Paricalcitol induces VDRbinding to the p65subunit of NF-B and prevents it from interacting with cis-acting DNA

element, thereby sequestering its ability to transactivate thetranscription of its targeted genes. Because chronic inflamma-

tion is a critical process that contributes to the pathogenesis of CKD in many ways,23,24 inhibition of inflammation could bean important mechanism by which vitamin D exerts its bene-

ficial activity in ameliorating renal interstitial fibrosis after ob-structive injury; therefore, our findings shed new light on the

understanding of the therapeutic effects of active vitamin D inCKD.

Although an anti-inflammatory effect of vitamin D is welldocumented, previous studies largely emphasized its role in

modulating immune cell activity 22; however, paricalcitol in-hibits RANTES expression that is localized almost exclusively

in renal tubules after injury, suggesting that tubular epithelialcells are likely the primary target of vitamin D in eliciting its

anti-inflammatory action. Tubular epithelial cells, the largestcell population in kidney parenchyma, are not a bystander in

the development of renal inflammation. Instead, increasingevidence indicates that they contribute directly and actively to

renal inflammatory response after injury. Tubular productionof RANTES inevitably builds up the chemokine gradient,

which serve as chemotactic signals to attract the migrating leu-kocytes to move along the gradient and eventually reach the

tubulointerstitial site of inflammation.25 In this regard,tubularexpression and secretion of RANTES is a critical step that sets

in motion toward the peritubular infiltration of inflammatory cells. As shown by chemotaxis assay, RANTES is a major che-motactic component in the supernatants of tubular cells after

TNF- stimulation, which plays an important role in attract-ing both splenocyte (rich in lymphocytes) and THP-1 cell

(monocytic cell line) migration (Figure 6); therefore, by inhib-iting RANTES expression in tubular cells, paricalcitol directly

targets a key event in the circuit of inflammatory responses inobstructive nephropathy.

The influx of inflammatory cells from the peripheral circu-lation to the injured sites is a complex process in which che-

mokines play a fundamental role. Chemokines not only act asthe directional signals to sort and guide effector leukocyte mi-

Figure 10. Paricalcitol does not block NF-B activation and its nuclear translocation but restores VDR expression and enhances itsnuclear translocation in the obstructed kidney after UUO in vivo. (A) Western blot demonstrated that UUO induced p65 NF-Bphosphorylation and paricalcitol treatment did not block p65 NF-B activation in vivo. Kidney lysates were immunoblotted withanti–phospho-specific p65 NF-B antibody. (B) Western blot showed that VDR expression was reduced significantly in the obstructedkidney and paricalcitol treatment largely restored VDR expression in vivo. Numbers (1, 2, and 3) denote each individual animal in a givengroup. (C) Immunofluorescence staining also demonstrated that paricalcitol restored VDR expression but did not block p65 NF- Bnuclear translocation in the obstructed kidney in vivo. Kidney sections were immunostained for total p65 (red) and the nuclei (green;top). (Bottom) Staining for VDR (red). Arrowheads indicate the nuclear staining of VDR and p65 NF-B. Bar 20 m.




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gration but also are instrumental in modulating immune cell

activation.13,26 Among many chemokines, RANTES/CCL5 isone of the best characterized.25 Increased RANTES expression

has been documented in a variety of kidney disorders ranging

from acute kidney injury and renal transplant rejection tochronic renal fibrosis.23,27,28 RANTES is a broad chemoattrac-

tant that is capable of recruiting many types of immune cells,including T lymphocytes, monocytes/macrophages, and natu-

ral killer cells, and promoting the chemotaxis of these cellsalong its gradient25,29; therefore, RANTES induction in tubular

cells after obstructive injury not only is important for T cellinfiltration but also is critical for the influx of monocytes/mac-

rophages.29 Of note, although the importance of macrophagesin renal inflammationis widely recognized, the role of T cells in

the tubulointerstitial inflammation and injury that followsUUO is poorly understood. Our study clearly implicates T cells

as a major constituent of the infiltrated inflammatory cells inthis model (Figure 1), suggesting a potential role of T cells in

obstructive nephropathy. It is worthwhile to point out thatinhibition of RANTES expression by paricalcitol in tubular

cells is compatible with several previous observations per-formed in cultured human dermal fibroblasts and vascular en-

dothelial cells,30,31 in which active vitamin D also suppressesthis chemokine expression; however, MCP-1/CCL2 mRNA is

not significantly inhibited by paricalcitol (Figure 3), suggestingthat its effect on chemokine expression seems to be gene spe-

cific. Interestingly, the expression of TNF-, another proin-flammatory cytokine that is produced predominantly by the

infiltrated cells, is markedly induced in the obstructed kidney.TNF-, in turn, stimulates tubular epithelial cells to produce

RANTES in vitro. This would create a vicious circuit betweentubular RANTES induction and inflammatory infiltration,

leading to a sustained, chronic inflammation, a characteristicfeature of CKD. That administration of paricalcitol inhibits

both RANTES and TNF- expression in vivo underscores itsability to break up such a vicious cycle, resulting in effective

inhibition of renal inflammation in this model. Notably, be-cause TNF- is mainly produced by infiltrating leukocytes, a

reduction of TNF- expression in vivo probably reflects a de-creased infiltration of leukocytes after paricalcitol treatment.

This study has unraveled the molecular details in whichparicalcitol inhibits RANTES expression, thereby providing

significant mechanistic insights into how vitamin D inhibitsrenal inflammation. RANTES expression in tubular epithelialcells is controlled primarily by NF-B, a “master” transcrip-

tion factor that is silenced by its specific inhibitor, IB, in thecytoplasm in normal restingstate.Upon activation, IB under-

goes phosphorylation, ubiquitination, and subsequent degra-dation.14,32 Thus, NF-B can be released and translocated into

the nucleus, where it dictates the transcription of its targetedgenes. Presumably, the activity of NF-B can be regulated ei-

ther positively or negatively at multiple sites along this signal-ing pathway; however, paricalcitol treatment influences nei-

ther NF-B phosphorylation nor its nuclear translocation(Figures 8 and 10), suggesting that any opposing effect of pari-

calcitol on NF-B signaling is operated at the postnuclear

translocation stage. Of note, several reports suggested that vi-tamin D inhibits NF-B signaling by increasing IB and re-

ducing p65 NF-B nuclear translocation in mesangial cells,

mouse embryonic fibroblasts, and pancreatic islet cells.33–35

The reason behind this discrepancy remains unknown, but it

could be related to the cell-type specificity. In this study, wedemonstrated that paricalcitol induced a complex formation

between VDR and p65 NF-B, consistent with previous re-ports in fibroblasts and osteoblastic cells.34,36 It should be

noted that in the p65-overexpressing tubular cells, addition of TNF- (or paricalcitol) alone slightly induced the p65/VDR

interactions (Figure 9). This could be attributable to the pres-ence of a trivial amount of vitamin D in the cultured condi-

tions, which promoted the p65/VDR interaction when the cellsoverproduce p65. Neither TNF- nor paricalcitol alone in-

duced endogenous p65/VDR interaction (Figure 9F), whenthese cells did not overexpress p65. The physical interaction

between VDR and p65 evidently prevents activated NF-Bfrom binding to the DNAelementin the promoter of RANTES

gene (Figure 9B). These findings underscore that paricalcitoltargets specifically and directly a key nuclear event in NF-B

signal circuit, resulting in an effective sequestration of thisproinflammatory signaling.

The data presented here strongly suggest that paricalcitolinhibits renal inflammation primarily by triggering a direct,

VDR-mediated sequestration of NF-B signaling; however, al-though the in vitro data are clear, it remains to be determined

whether this mechanism is operative in vivo and whether theVDR ligation is necessary for the paricalcitol effect. In addi-

tion, we cannot exclude the possibility that paricalcitol may regulate RANTES and TNF- expression by a NF-B–inde-

pendent mechanism. Furthermore, paricalcitol may exert itsanti-inflammatory actions by other indirect routes as well,

given that vitamin D has pleiotropic effects.37,38 Along this line,Li et al.39 reported that vitamin D is a negative regulator of

renin gene expression, and, therefore, vitamin D analoguecould inhibit renin expression, resulting in a reduced activa-

tion of the renin-angiotensin system in diseased kidney. Be-cause angiotensin II is a known proinflammatory stimulus, it is

conceivable that paricalcitol could inhibit renal inflammationby targeting the renin-angiotensin system in CKD. In addition,

vitamin D is shown to induce the expression of hepatocytegrowth factor,40 a cytokine that has potent anti-inflammatory and antifibrotic properties.41–43

In summary, we have shown in this study that paricalcitolattenuates renal infiltration of inflammatory cells and inhibits

tubular RANTES expression in obstructive nephropathy. Thisanti-inflammatory effect of paricalcitol seems to be mediated

by its ability to induce the VDR-mediated sequestration of NF-B signaling; therefore, in addition to preserving tubular

epithelial integrity by blocking EMT, as previously reported,10

active vitamin D also exerts its beneficial activities in CKD by

inhibiting renal inflammation through directly disruptingNF-B signaling.




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CONCISE METHODS

AnimalsMale CD-1 mice that weighed approximately 18 to 22 g were pur-

chased from Harlan Sprague-Dawley (Indianapolis, IN). UUO was

performed using an approved protocol by the Institutional Animal

Care and Use Committee at the University of Pittsburgh, as describedpreviously.10,44 Sham-operated mice were used as normal controls.

Paricalcitol (provided by Abbott laboratories, Abbott Park, IL) was

administeredby daily subcutaneous injectionat thedosages of 0.1and

0.3 g/kg body wt, respectively. Mice that underwent UUO and re-

ceived an injection of the same volume of vehicle (ethanol) were used

as vehicle controls. Groups of mice (n 5) were killed at 7 and 14 d

after UUO, respectively, and the kidneys were removed for various

analyses.

Cell Culture and Cytokine TreatmentHuman proximal tubular epithelial cells (HKC, clone-8) were pro-

vided by Dr. L. Racusen (Johns Hopkins University, Baltimore, MD).Cell culture andcytokinetreatments were carried outaccording to the

procedures described previously.44 Briefly, HKC-8 cells were seeded

in complete medium that contained 10% FBS at approximately 70%

confluence. After an overnight incubation, cells were serum-starved

in serum-free medium for 24 h before addition of cytokines. Recom-

binant human TNF- was purchased from R&D Systems (Minneap-

olis, MN). HKC-8 cells were incubatedwith various concentrations of

paricalcitol in the absence or presence of TNF- (2 ng/ml) for 24 h,

unless otherwise indicated. For some experiments, such as assessing

NF-B activation, HKC-8 cells were pretreated with paricalcitol for

0.5 h, followed by incubation with TNF- for various periods as in-

dicated. For blocking NF-B signaling, HKC-8 cells were pretreatedwith a cell-permeable inhibitorpeptide NF-B SN50 (Calbiochem,La

Jolla, CA; 20 M) for 1 h and then incubated with TNF-. In some

experiments, HKC-8 cells were transfected with either p65 NF-B

expression vector (pNF-B/p65; provided by Dr. J.A. Schmid, Medi-

cal University Vienna, Vienna, Austria) or empty pcDNA3 control

vectorfor 48 h andthen treated with lowconcentration of TNF- (0.2

ng/ml). Whole-cell lysates or conditioned media were prepared and

then subjected to various analyses.

Western Blot AnalysisThe preparation of whole-cell lysates and kidney tissue homogenates

and Western blot analysis of protein expression were carried out by using routine procedures as described previously.10 The primary an-

tibodies were obtained from following sources: Anti-RANTES (sc-

1410) and anti-VDR (sc-1008;Santa Cruz Biotechnology, SantaCruz,

CA); anti-p65 NF-B, anti–phospho-p65 NF-B (Ser536), anti-

IB, and anti–phospho-IB (Ser32; Cell Signaling Technology,

Danvers, MA); anti–glyceraldehyde-3-phosphate dehydrogenase

(Ambion, Austin, TX).

Immunohistochemical and ImmunofluorescenceStainingImmunohistochemical staining of kidney sections was performed by

an established protocol.10 In brief, paraffin-embedded sections were

stained with anti-CD3 (sc-20047; Santa Cruz Biotechnology), anti-

F4/80 (14-4801-82; eBioscience, San Diego, CA), and anti-RANTES

(500-P118; PeproTech, Rocky Hill, NJ) antibodies using the Vector

M.O.M. immunodetection kit, according to the protocol specified by

the manufacturer (Vector Laboratories, Burlingame, CA). Indirect

immunofluorescence staining was carried out according to the pro-

cedures described previously.45 Briefly, cells or kidney cryosectionswere incubated with the specific primary anti-p65 and anti-VDR an-

tibodies, followed by staining with cyanine Cy3–conjugated second-

ary antibody (Jackson ImmunoResearch Laboratories, West Grove,

PA). Some slides were double stained with sytox green (Invitrogen,

Carlsbad, CA)to visualize the nuclei. Slides were viewed with a Nikon

Eclipse E600 microscope equipped with a digital camera (Melville,

NY) or Leica TCS-SL confocal microscope (Microsystems, Heidel-

berg, Germany).Nonimmune normalcontrol IgGwas used to replace

the primary antibody as negative control, and no staining occurred.

CD3, F4/80,and RANTES staining were semiquantified by a comput-

er-aided morphometric analysis (MetaMorph; Universal Imaging

Co., Downingtown, PA). Briefly, a grid containing 117 (13

9) sam-pling pointswas superimposed on images of cortical high-power field

(400). The number of grid points overlying positive area (except

tubular lumen and glomeruli) was counted and expressed as a per-

centage of all sampling points, as described previously.10 For each

kidney, 10 randomly selected, nonoverlapping fieldswere analyzed in

a blinded manner.

RT-PCRFordetermination of RANTESand TNF-mRNA expression, a semi-

quantitative RT-PCR was used. Total RNA was prepared from kidney

tissue and cultured HKC-8 cells. After reverse transcription of the

RNA, cDNA was used as a template in PCR reactions using gene-specific primer pairs. Approximately 20 to 25 cycles for amplification

in the linear range were used. After quantification of band intensities

by use of densitometry, the relative steady-state level of mRNA was

calculated afternormalizationto -actin.The sequencesof theprimer

sets were as follows: RANTES(human),5-ACC CTG CTG CTT TGC

CTA C (sense), and5-GGT TCA CGC CAT TCT CCT G (antisense);

RANTES (mouse), 5-GTG CCC ACG TCA AGG AGT AT (sense)

and 5-GGG AAG CGT ATA CAG GGT CA (antisense); TNF-, 5-

CTG GGA CAG TGA CCT GGA CT-3 (sense) and 5-GCA CCT

CAG GGA AGA GTC TG-3 (antisense); MCP-1, 5-CCC ACT CAC

CTG CTG CTA C-3(sense) and 5-TTC TTG GGG TCA GCA CAG

A-3 (antisense). The sequences of -actin primer set were described

previously.46

Chemotaxis AssayCell chemotaxis assay was performed using a 24-well Transwell plate

as described previously.29 Human monocytic cell line (THP-1; Amer-

ican Type Culture Collection, Manassas, VA) was maintained in

RPMI-1640 medium supplemented with 10% FCS. Splenocytes,

which contain lymphocytes, as well as macrophages, natural killer

cells, and dendritic cells, were freshly prepared from mouse spleen

using an established procedure as described previously.47 Briefly,

spleens were removed from CD1 mice and a single-cell suspension in

Hank’s buffer prepared using 40-m cell strainers. Red blood cells




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were lysed using standard lysis buffer (0.15 M NH4Cl, 10 mM

KHCO3, and 0.1mM EDTA [pH7.4]). Splenocytes were then washed

and used in chemotaxis assays. Cultured THP-1 cells and mouse

splenocytes (5105 in 100l) were added onto theupperchamber of

the Transwell insert (5-m polycarbonate filter; Corning, Corning,

NY). HKC-8 cell–conditionedmedia(0.5ml) were added in thelower

chamber of the Transwell. For the neutralizing experiments, anti-RANTES antibody (5 g/ml; Santa Cruz Biotechnology) or control

goat IgG was added to the conditioned media in the lower chamber.

After incubation at 37°C for 4 h, the number of cells migrated to the

lower chamber of the Transwell was counted. Data are expressed as

percentage of the migrated cells in total number of input cells.

ChIP AssayChIP assay was performed to analyze in vivo interactions of NF-B

and its cognate cis-acting element in RANTES promoter. This assay

was carried out essentially according to the protocols specified by the

manufacturer (ChIP assay kit; Upstate, Charlottesville, VA). Briefly,

HKC-8 cells after various treatments as indicated were cross-linkedwith 1% formaldehyde and then resuspended in SDS lysis buffer con-

taining protease inhibitors. The chromatin solution was sonicated,

and the supernatant was diluted 10-fold. An aliquot of total diluted

lysate was used for total genomic DNA as input DNA control. The

anti-p65 NF-B antibody was added and incubated at 4°C overnight,

followed by incubation with protein A–agarose for 1 h. The precipi-

tates were washed, andchromatin complexeswere eluted. After rever-

sal of the cross-linking at 65°C for 4 h, the DNA was purified, and

ChIP samples were used as a template for PCR using the primer sets

for human RANTES promoter regions (from208 to10) contain-

ing NF-B response element.48 The sequences of primers used for

ChIP assay were as follows: Forward, 5-TTGGTGCTTGGTCAAA-

GAGG-3; and reverse, 5-CCCTTTATAGGGCCAGTTGA-3.

Transient Transfection and Luciferase Reporter AssaysThe effect of paricalcitol on NF-B–mediated gene transcription was

assessed by using the PathDetect NF-B-Luc cis-reporter system

(Stratagene, La Jolla, CA), in which NF-B response element was

linked to the firefly luciferase gene. HKC-8 cells were co-transfected

by using Lipofectamine 2000 reagent (Invitrogen) with pNF-B-Luc

plasmid (1g). An internal control reporter plasmid (0.05g) Renilla

reniformis luciferase driven under thymidine kinase (TK) promoter

(pRL-TK; Promega, Madison, WI) was also co-transfected for nor-

malizing the transfection efficiency. The transfected cells were incu-bated in serum-free medium without or with TNF- (2 ng/ml) in the

absence or presence of paricalcitol (107 M) as indicated. Luciferase

assay was performed using the Dual Luciferase Assay System kit ac-

cording to the manufacturer’s protocols (Promega). Relative lucif-

erase activity (arbitrary unit) was reported as fold induction over the

controls after normalization for transfection efficiency.

ImmunoprecipitationImmunoprecipitation was carried out by using an established

method.49 Briefly, HKC-8 cells were transfected with p65 NF-B ex-

pression vector (pNF-B/p65) by using lipofectamine 2000 reagent

(Invitrogen) and then incubated with or without 107 M paricalcitol

in the absence or presence of 2 ng/ml TNF- for 1 h. In the experi-

ments for assessing endogenous p65/VDR interaction, HKC-8 cells

were directly treated with paricalcitol, TNF-, or both and then sub-

jected to co-immunoprecipitation. Cells were lysed on ice in 1 ml of

nondenaturing lysis buffer that contained 1% Triton X-100, 0.01 M

Tris-HCl (pH 8.0), 0.14 M NaCl, 0.025% NaN3, 1% protease inhibi-

tors cocktail, and 1% phosphatase inhibitors cocktails I and II(Sigma). After preclearing with normal IgG, cell lysates (0.5 mg of

protein) were incubated overnight at 4°C with 4 g of anti-VDR

(Santa Cruz Biotechnology), followed by precipitation with 30 l of

protein A/G Plus-Agarose for 1 h at 4°C. The precipitated complexes

were separated on SDS–polyacrylamide gels and immunoblottedwith

anti-p65 NF-B antibody.

Statistical AnalysisStatistical analysis of the data was carried out using SigmaStat soft-

ware (Jandel Scientific, San Rafael, CA). Comparison between groups

was made using one-way ANOVA followed by Student-Newman-

Keuls test. P

0.05 was considered significant.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants

DK061408, DK064005, and DK071040 and a Grant-in-Aid from Ab-

bott Laboratories. X.T. was supported by a postdoctoral fellowship

from the American Heart Association Great Rivers Affiliate.

DISCLOSURESNone.

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40. paricalcitol inhibits renal inflammation by promoting

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