Research ArticleNLRP3 Deficiency Attenuates Renal Fibrosis andAmeliorates Mitochondrial Dysfunction in a Mouse UnilateralUreteral Obstruction Model of Chronic Kidney Disease

Honglei Guo1 Xiao Bi2 Ping Zhou3 Shijian Zhu2 and Wei Ding2

1Division of Nephrology The Fifth Peoplersquos Hospital of Shanghai Fudan University 128 Ruili Road Shanghai 200240 China2Division of Nephrology Shanghai Ninth Peoplersquos Hospital School of Medicine Shanghai Jiaotong University639 Zhizaoju Road Shanghai 200011 China3Department of Pediatrics The 2nd Hospital of Harbin Medical University Harbin 150086 China

Correspondence should be addressed to Wei Ding gump1015163com

Received 17 December 2016 Accepted 9 February 2017 Published 28 February 2017

Academic Editor Tania Silvia Frode

Copyright copy 2017 Honglei Guo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background andAimsThenucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) inflammasomehas beenimplicated in the pathogenesis of chronic kidney disease (CKD) however its exact role in glomerular injury and tubulointerstitialfibrosis is still undefined The present study was performed to identify the function of NLRP3 in modulating renal injury andfibrosis and the potential involvement of mitochondrial dysfunction in the murine unilateral ureteral obstruction (UUO) modelof CKD Methods Employing wild-type (WT) and NLRP3minusminus mice with or without UUO we evaluated renal structure tissueinjury and mitochondrial ultrastructure as well as expression of some vital molecules involved in the progression of fibrosisapoptosis inflammation and mitochondrial dysfunction Results The severe glomerular injury and tubulointerstitial fibrosisinduced in WT mice by UUO was markedly attenuated in NLRP3minusminus mice as evidenced by blockade of extracellular matrixdeposition decreased cell apoptosis and phenotypic alterations Moreover NLRP3 deletion reversed UUO-induced impairment ofmitochondrial morphology and function ConclusionsNLRP3 deletion ameliorates mitochondrial dysfunction and alleviates renalfibrosis in a murine UUOmodel of CKD

1 Introduction

Chronic kidney disease (CKD) is complicated progressivedisease that is becoming an increasingly important publichealth issue worldwide [1 2] A comprehensive understand-ing of the mechanisms underlying the progression of CKDis required Recent studies have shown a close relationshipbetween persistent inflammation and development of variousforms of CKD [3 4] indicating that this issue merits furtherinvestigation

Inflammation responses are induced by both infectionand tissue damage to restore homeostasis Pattern recognitionreceptors (PRRs) play a primary role in this process byrecognizing both intra- and extracellular danger signals [5]

Nucleotide-binding domain and leucine-rich repeat contain-ing PYD-3 (NLRP3) is one of four confirmed inflammasome-forming PRRs identified to date After formation and activa-tion the NLRP3 inflammasome associates with the adaptorprotein apoptosis speck-like protein containing a caspaserecruitment domain (ASC) via its pyrin domain ASC thenassociates with procaspase 1 in an interaction which inducesthe autocatalytic cleavage and activation of procaspase 1 Acti-vated caspase 1 cleaves pro-IL-1120573 and pro-IL-18 to generatethe mature secreted forms of these cytokines [6] Recentreports have demonstrated that the NLRP3 inflammasome isinvolved in the pathogenesis of CKD [7ndash10] Our previousstudy also showed that NLPR3 inflammasome activationcontributes to aldosterone-induced renal tubular injury [11]

Hindawi Publishing CorporationMediators of InflammationVolume 2017 Article ID 8316560 10 pageshttpdxdoiorg10115520178316560

2 Mediators of Inflammation

In addition to the role of inflammation mitochondrialdysfunction (MTD) is also implicated as an important fac-tor in the development of CKD [12ndash14] Mitochondria areimportant organelles involved in cellular energy productionand metabolism MTD is associated with increased oxidativestress and inflammation [15] and may also result in reducedATP production increased reactive oxygen species (ROS)generation and release of proapoptotic products such asmitochondrial DNA (mtDNA) and cytochrome 119888 Althoughour recent study demonstrated that inhibition of mitochon-drial complex I significantly attenuatedMTD and the inflam-masome response in aldosterone-infused rats [16] the roleof the NLRP3 inflammasome in modulating mitochondrialfunction in a murine unilateral ureteral obstruction (UUO)model of CKD disease is still unclear

In this study we investigated the contribution of NLRP3inflammasome activation to MTD and loss of kidney struc-ture and function in a murine UUOmodel of CKD

2 Materials and Methods

21 Materials Antibodies of the following specificities wereused in this study caspase 3 120572-SMA and E-cadherin(Cell Signaling Technology) IL-1120573 IL-18 collagen I andfibronectin (Abcam) caspase 1 and PGC-1a (Santa CruzBiotechnology) and 120573-actin (SigmandashAldrich)

22 Animal Studies All animal experiments were performedwith the approval of the Animal Care Committee at JiaoTong University C57BL6J (wild-type WT) mice were pur-chased from Shanghai SLAC Laboratory Animals (ShanghaiChina)NLRP3minusminusmice (C57BL6J genetic background)werepurchased from the Jackson Laboratory (Sacramento CA)Only age and sex-matched mice were used in experimentNLRP3 deficiency was genetically confirmed by RT-PCRscreening Male WT and NLRP3minusminus mice (aged 8ndash10 weeks119899 = 6 per group) underwent left ureteral ligation as describedpreviously [17] Complete ureteral obstruction was producedby double ligation with 4-0 silk threadThesemice comprisedthe WTUUO and NLRP3minusminusUUO groups respectivelyComparable groups of male mice underwent an identicalprocedure without ligation as the WTSham group andNLRP3minusminusSham groups (119899 = 6 per group) Mice were sacri-ficed on days 1 3 7 and 14 afterUUOAt the end of the experi-ment kidney samples were collected and immediately frozenin liquid nitrogen for storage at minus80∘C

23 Histological and IHC Studies Samples of kidney tissuewere sectioned (thickness 4 120583m) processed and stained withperiodic acid-Schiff reagent (PAS) or Massonrsquos trichromestain according to our previous report [18] After PASstaining we evaluated the severity of glomerular injury inexperimentalmice using lightmicroscopy in accordancewiththe following semiquantitative grades grade 0 normal grade1 segmental lesion lt 25 grade 2 25ndash50 grade 3 50ndash75 grade 4 75ndash100 At least 20 glomeruli were analyzedin each group The area of tubulointerstitial fibrosis was

evaluated in 10 random 400x magnification fields followingMasson trichrome staining

The sections were also stained with anti-collagen I(1 1000) or anti-fibronectin antibodies (1 1000) and theimmunohistochemistry was quantified as described in ourprevious study [19] In brief the areas of positive collagen Iand fibronectin staining were expressed as a percentage of thetotal area determined using an image analyzer (Winroof)

24 Western Blot Analysis Western blotting of renal tissuewas performed as previously described [20] After blottingthe membranes were incubated overnight with antibodiesagainst 120572-SMA E-cadherin cleaved caspase 3 caspase 1IL-18 and IL-1120573 (all diluted by Tris-buffered saline-Tween20 (TBST) containing 5 BSA) After washing with TBSTblots were incubated with secondary antibodies for 2 h Theimmune complexes were visualized with an enhanced chemi-luminescent system (Amersham Little Chalfont Bucks UK)and band intensity quantified using Quantity One (Bio-RadHercules CA USA)

25 Terminal Deoxyribonucleotide Transferase (TdT-) Medi-ated dUTP Nick-End Labeling (TUNEL) Assay Apoptosis inkidney tissue was detected using the TUNEL assay Brieflydeparaffinized kidney tissue section was treated in a perme-abilization solution (01 Triton X-100 in 01 sodium cit-rate) at 4∘C for 2min The tissue was then washed twice withPBS and incubated with 50120583L TUNEL reaction mixture for60min at 37∘C in the dark For quantification 20 fields wererandomly selected in each section and the number of TUNEL-positive cells was counted in a standard area (1mm2)

26 Electron Microscopy Essay To make a detailed analysisof the ultrastructural morphology of mitochondria kidneytissue samples were fixed with 25 glutaraldehyde at roomtemperature and then cut into 1mm3 pieces using a scalpelAccording to the method described in our previous report[16] we prepared ultrathin sections (60 nm) using a micro-tome The sections were then placed on copper grids andstained with uranyl acetate and lead citrate for evaluation byelectron microscopy

27 Real-Time Reverse Transcription Polymerase Chain Reac-tion (RT-PCR) Total RNA was isolated from kidney tissueobtained from each group and reverse-transcribed to cDNART-PCR analysis was performed according to a previouslydescribed protocol [21] using the cDNA as a template andthe following primers mtDNA forward 51015840-TTTTATCTG-CATCTGAGTTTAATCCTGT-31015840 and reverse 51015840-CCACTT-CATCTTACCATTTATTATCGC-31015840 ATP synthase for-ward 51015840-TCCATCAAAAACATCCAGAAAA-31015840 and reverse51015840-GAGGAGTGAATAGCACCACAAA-31015840 ND1 forward51015840-ATCCTCCCAGGATTTGGAAT-31015840 and reverse 51015840-ACC-GGTAGGAATTGCGATAA-31015840 18S forward 51015840-TTCGGA-ACTGAGGCCATGATT-31015840 and reverse 51015840-TTTCGCTCT-GGTCCGTCTTG-31015840

28 Statistical Analyses Data are expressed as the mean plusmnstandard error of the mean (SEM) Comparisons between

Mediators of Inflammation 3

PAS staining

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

WTSham WTUUO

Masson staining

NLRP3minusminusSham NLRP3minusminusUUO

(b)

Figure 1 Demonstration of kidney histopathology at day 14 UUO Representative photomicrographs of periodic acid-Schiff-stained kidneysections ((a) magnification times400) and Masson trichrome-stained kidney sections ((b) magnification times400)

WTSham WTUUO

Collagen I staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

Fibronectin staining

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(b)

Figure 2 Immunohistochemical staining for deposition of collagen I ((a) magnification times400) and fibronection ((b) magnification times400)in kidney sections from different groups at day 14 after UUO

4 Mediators of Inflammation

WT WT

Sham UUO

NLRP3minusminus NLRP3minusminus

lowast

0

1

2

3

4G

lom

erul

ar in

jury

scor

e

(a)

WT WT

Sham UUO

NLRP3minusminus NLRP3minusminus

lowast

0

1

2

3

4

5

Tubu

loin

ters

titia

l fibr

osis

inde

x

(b)

WT WT

Sham UUO

NLRP3minusminusNLRP3minusminus

lowast

0

2

4

6

8

Qua

ntita

tion

of C

olla

gen

I (

)

(c)

WT WT

Sham UUO

NLRP3minusminusNLRP3minusminus

lowast

0

2

4

6

8

10

Qua

ntita

tion

of F

ibro

nect

ion

()

(d)

Figure 3 NLRP3 deletion attenuates UUO-induced glomerular injury and tubulointerstitial fibrosis as well as deposition of collagen I andfibronectin Glomerular injury score (a) and tubulointerstitial fibrosis index (b) are evaluated according to PAS staining andMasson stainingSemiquantitative analysis of collagen I (c) and fibronectin (d) positive areas is performed according to immunohistochemical staining Datarepresent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 WTSham group versusWTUUO group 119875 lt 005 NLRP3minusminusUUO group versusWTUUOgroup

groups were performed with one-way analysis of variance(ANOVA) followed by Dunnettrsquos multiple comparison testsor Studentrsquos 119905-test 119875 lt 005 was considered statisticallysignificant

3 Results

31 NLRP3 Deletion Attenuates UUO-Induced Renal InjuryRenal histopathology was examined following PAS stainingand Masson staining (Figures 1(a) and 1(b) resp) UUOcaused severe glomerular injury (306 plusmn 012) in WT mice(119875 lt 005 versus WTSham group) Compared with theWTUUO group the glomerular injury score was signifi-cantly decreased in the NLRP3minusminusUUO group (119875 lt 005Figures 1(a) and 3(a)) Similarly significant tubulointerstitialfibrosis was induced byUUO inWTmice and comparedwiththe WTUUO group the tubulointerstitial fibrosis index wassignificantly reduced in NLRP3minusminusUUO group (119875 lt 005Figures 1(b) and 3(b))

32 NLRP3 Deletion Attenuates Renal Collagen I Depositionand Fibrosis in UUO Mice Immunohistochemical analysisof collagen I and fibronectin in renal tissue sections (Figures2 3(c) and 3(d)) showed a significant increase in collagenI-positive and fibronectin-positive cells following UUO inWT mice Partial but significant reversal of these effects wasobserved in the NLRP3minusminusUUO group (119875 lt 005 Figures 23(c) and 3(d))

33 UUO-Induced Fibrosis-Related Phenotypic Alterationsand Apoptosis Are Reduced by NLRP3 Deletion 120572-SMA isregarded as a fibroblast marker and renal epithelial cellslose E-cadherin expression during renal fibrosis Thereforewe examined the expression of 120572-SMA and E-cadherin inrenal tissue (Figure 4) Expression of 120572-SMA protein wassignificantly increased in time-dependentmanner after UUOinduction inWTmice (119875 lt 005) and this increasewas signif-icantly decreased in the NLRP3minusminus mice at day 14 after UUO(119875 lt 005 versus WTU14 group) In contrast E-cadherin

Mediators of Inflammation 5

E-cadherin

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120572-SMA

120573-Actin

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

lowast

0

05

1

15

2

25

3

35

Rela

tive120572

-SM

A120573

-act

in le

vel

(b)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

0

02

04

06

08

1

12

Relat

ive E

-cad

herin

120573-a

ctin

leve

l

(c)

Figure 4 NLRP3 deletion decreases UUO-induced phenotypic alterations in UUO mice Expression of 120572-SMA and E-cadherin proteindetected in kidney samples from different groups (a) Semiquantitative analysis of 120572-SMA expression (b) and E-cadherin expression (c)normalized against 120573-actin Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

protein expression was significantly reduced in time-dependentmanner afterUUO inWTmice (119875 lt 005) Partialbut significant reversal of these effects was observed in theNLRP3minusminusUUO group (119875 lt 005 versus WTU14 group)

The effects of UUO on apoptosis were evaluated byTUNEL staining and detection of caspase 3 protein by West-ern blot analysis (Figure 5) TUNEL staining revealedmarked-ly more apoptotic cells in the WTUUO group comparedwith the WTSham group and this UUO-induced apoptosiswas evidently inhibited by NLRP3 deletion (Figures 5(a) and5(b))This was confirmed byWestern blot analysis of caspase3 Cleavage of caspase 3 to generate the activated proteinincreased significantly afterUUO inWTmice (119875 lt 005) thisincrease was significantly decreased in the NLRP3minusminus mice atday 14 after UUO (119875 lt 005 versus WTU14 group) (Figures5(c) and 5(d))

34 NLRP3 Deletion Ameliorates UUO-Induced Expres-sion of Inflammatory Cytokines The expression of NLRP3inflammasome-regulated inflammatory cytokines was eval-uated byWestern blot analysis (Figures 6(a) and 6(b)) At day14 after UUO significant increases in renal expression of cas-pase 1 (715-fold) IL-1120573 (298-fold) and IL-18 (742-fold) pro-teins were detected in WT mice (all 119875 lt 005 versus

WTSham group) and these effects were significantly inhib-ited in the NLRP3minusminus mice at day 14 after UUO (all 119875 lt 005versus WTU14 group)

35 NLRP3 Deletion Attenuates UUO-Induced MitochondrialDysfunction MTD is characterized by disordered intracellu-lar ATP synthesis mtDNA damage and excessive accumula-tion of reactive oxygen species in a process that is regulated bymitochondrial NADH dehydrogenase 1 (ND1) Observationof the ultrastructural morphology of renal cells of WT miceafter UUO revealed swollen mitochondria with disorganizedand fragmented cristae (Figure 7(a)) Moreover these effectswere accompanied by significantly decreased PGC-1a proteinmtDNA ATP synthase and ND1 at day 14 in the WTUUOgroup compared with the WTSham group (all 119875 lt 005Figures 7 and 8) The decreased levels of PGC-1a proteinmtDNA ATP synthase and ND1 induced by UUO in WTmice were significantly ameliorated in the NLRP3minusminus mice atday 14 after UUO (all 119875 lt 005 versus WTU14 group)

4 Discussion

During development of CKD loss of renal function is closelyrelated to renal fibrosis Although numerous studies suggest

6 Mediators of Inflammation

WTSham WTUUO

TUNEL staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

WTWT

Sham UUONLRP3minusminusNLRP3minusminus

lowast

0

3

6

9

12

15

Apop

tosis

cells

sco

pe

(b)

cleaved Caspase 3

Caspase 3

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120573-Actin

(c)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowastlowast

lowast

lowast

0

2

4

6

8

10

Rela

tive c

leav

ed ca

spas

e 3120573

-act

in le

vel

(d)

Figure 5 NLRP3 deletion ameliorates UUO-induced cell apoptosis in UUOmice Representative images of TUNEL staining are shown ((a)magnification times200) Bar graph indicates the mean number of TUNEL-positive tubular cells per field (b) Expression of caspase 3 proteinwas detected in kidney samples (c) Semiquantitative analysis of cleaved caspase 3 normalized against 120573-actin (d) Data represent the meanplusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

that persistent inflammation contributes to chronic kidneyinjury the underlying mechanism of inflammation-inducedCKD is still unknown The results of the present studyindicate that activation of the NLRP3 inflammasome acceler-ates renal fibrosis through mitochondrial dysfunction in themurine UUO model of CKD Thus the NLPR3 inflamma-some is implicated as a potential target in the prevention ofrenal fibrosis progression

Inflammation is part of the normal response to tissuedamage Despite its original purpose of restoring homeosta-sis overt or chronic inflammation can lead to secondary

tissue damage and organ dysfunction [22] Activation of theinflammasome and subsequent release of proinflammatorycytokines have been shown to be involved in many diseaseconditions [23] NLRP3 expression was found to be increasedin renal biopsy samples from patients with various renal dis-eases with increased levels correlating with decreased renalfunction [7] Patients with CKD also exhibited elevated levelsof inflammasome-regulated cytokines such as IL-18 [24]

The UUO model of progressive CKD is often used toinvestigate the mechanisms of renal fibrosis Vilaysane etal reported less renal inflammation and fibrosis at 14 days

Mediators of Inflammation 7

Pro-IL-18IL-18

Procaspase 1Caspase 1

Sham U14ShamU14U7U3U1WT NLRP3minusminus

120573-Actin

Pro-IL-1120573IL-1120573

(a)

Sham U14ShamU14U7U3U1WT

Caspase 1IL-18

NLRP3minusminus

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

IL-1120573

05

101520253035

Relat

ive p

rote

in120573

-act

in le

vel

(b)

Figure 6 NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines Expression of caspase 1IL-1120573 and IL-18 in kidney samples was detected (a) Semiquantitative analysis of caspase 1 IL-1120573 and IL-18 normalized against 120573-actin (b)Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versusWTU14 groupU1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

Sham U14ShamU14

PGC-1a

WT NLRP3minusminus

lowast

120573-actin

0

03

06

09

12

Relat

ive P

GC-

1a le

vel

(b)

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive m

tDN

A1

8srR

NA

leve

l

(c)

Figure 7 NLRP3 deletion reverses UUO-induced mitochondrial dysfunction Representative electron microscopy photomicrographs ofultrastructural morphology of mitochondria (a) (magnification times12000) Arrow indicates swollen mitochondria Expression of PGC-1a inkidney sample was detected and semiquantitative analysis of PGC-1a normalized against 120573-actin (b) Semiquantitative analysis of mtDNAexpression normalized against 18S performed by real-time PCR (c) Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 13 7 and 14 after UUO

after UUO in the absence of NLRP3 compared with thelevels detected in its presence which was associated withthe activation of the inflammasome-dependent pathway ofNLRP3 [7] Furthermore Bani-Hani et al showed that IL-18

neutralization prevents renal injury and fibrosis after UUO inmice [25] In the present study we found that activation of theNLRP3 inflammasome and its downstream cytokines wereincreased in a time-dependent manner after UUO in WT

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

Mediators of Inflammation 3

PAS staining

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

WTSham WTUUO

Masson staining

NLRP3minusminusSham NLRP3minusminusUUO

(b)

Figure 1 Demonstration of kidney histopathology at day 14 UUO Representative photomicrographs of periodic acid-Schiff-stained kidneysections ((a) magnification times400) and Masson trichrome-stained kidney sections ((b) magnification times400)

WTSham WTUUO

Collagen I staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

Fibronectin staining

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(b)

Figure 2 Immunohistochemical staining for deposition of collagen I ((a) magnification times400) and fibronection ((b) magnification times400)in kidney sections from different groups at day 14 after UUO

4 Mediators of Inflammation

WT WT

Sham UUO

NLRP3minusminus NLRP3minusminus

lowast

0

1

2

3

4G

lom

erul

ar in

jury

scor

e

(a)

WT WT

Sham UUO

NLRP3minusminus NLRP3minusminus

lowast

0

1

2

3

4

5

Tubu

loin

ters

titia

l fibr

osis

inde

x

(b)

WT WT

Sham UUO

NLRP3minusminusNLRP3minusminus

lowast

0

2

4

6

8

Qua

ntita

tion

of C

olla

gen

I (

)

(c)

WT WT

Sham UUO

NLRP3minusminusNLRP3minusminus

lowast

0

2

4

6

8

10

Qua

ntita

tion

of F

ibro

nect

ion

()

(d)

Figure 3 NLRP3 deletion attenuates UUO-induced glomerular injury and tubulointerstitial fibrosis as well as deposition of collagen I andfibronectin Glomerular injury score (a) and tubulointerstitial fibrosis index (b) are evaluated according to PAS staining andMasson stainingSemiquantitative analysis of collagen I (c) and fibronectin (d) positive areas is performed according to immunohistochemical staining Datarepresent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 WTSham group versusWTUUO group 119875 lt 005 NLRP3minusminusUUO group versusWTUUOgroup

groups were performed with one-way analysis of variance(ANOVA) followed by Dunnettrsquos multiple comparison testsor Studentrsquos 119905-test 119875 lt 005 was considered statisticallysignificant

3 Results

31 NLRP3 Deletion Attenuates UUO-Induced Renal InjuryRenal histopathology was examined following PAS stainingand Masson staining (Figures 1(a) and 1(b) resp) UUOcaused severe glomerular injury (306 plusmn 012) in WT mice(119875 lt 005 versus WTSham group) Compared with theWTUUO group the glomerular injury score was signifi-cantly decreased in the NLRP3minusminusUUO group (119875 lt 005Figures 1(a) and 3(a)) Similarly significant tubulointerstitialfibrosis was induced byUUO inWTmice and comparedwiththe WTUUO group the tubulointerstitial fibrosis index wassignificantly reduced in NLRP3minusminusUUO group (119875 lt 005Figures 1(b) and 3(b))

32 NLRP3 Deletion Attenuates Renal Collagen I Depositionand Fibrosis in UUO Mice Immunohistochemical analysisof collagen I and fibronectin in renal tissue sections (Figures2 3(c) and 3(d)) showed a significant increase in collagenI-positive and fibronectin-positive cells following UUO inWT mice Partial but significant reversal of these effects wasobserved in the NLRP3minusminusUUO group (119875 lt 005 Figures 23(c) and 3(d))

33 UUO-Induced Fibrosis-Related Phenotypic Alterationsand Apoptosis Are Reduced by NLRP3 Deletion 120572-SMA isregarded as a fibroblast marker and renal epithelial cellslose E-cadherin expression during renal fibrosis Thereforewe examined the expression of 120572-SMA and E-cadherin inrenal tissue (Figure 4) Expression of 120572-SMA protein wassignificantly increased in time-dependentmanner after UUOinduction inWTmice (119875 lt 005) and this increasewas signif-icantly decreased in the NLRP3minusminus mice at day 14 after UUO(119875 lt 005 versus WTU14 group) In contrast E-cadherin

Mediators of Inflammation 5

E-cadherin

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120572-SMA

120573-Actin

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

lowast

0

05

1

15

2

25

3

35

Rela

tive120572

-SM

A120573

-act

in le

vel

(b)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

0

02

04

06

08

1

12

Relat

ive E

-cad

herin

120573-a

ctin

leve

l

(c)

Figure 4 NLRP3 deletion decreases UUO-induced phenotypic alterations in UUO mice Expression of 120572-SMA and E-cadherin proteindetected in kidney samples from different groups (a) Semiquantitative analysis of 120572-SMA expression (b) and E-cadherin expression (c)normalized against 120573-actin Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

protein expression was significantly reduced in time-dependentmanner afterUUO inWTmice (119875 lt 005) Partialbut significant reversal of these effects was observed in theNLRP3minusminusUUO group (119875 lt 005 versus WTU14 group)

The effects of UUO on apoptosis were evaluated byTUNEL staining and detection of caspase 3 protein by West-ern blot analysis (Figure 5) TUNEL staining revealedmarked-ly more apoptotic cells in the WTUUO group comparedwith the WTSham group and this UUO-induced apoptosiswas evidently inhibited by NLRP3 deletion (Figures 5(a) and5(b))This was confirmed byWestern blot analysis of caspase3 Cleavage of caspase 3 to generate the activated proteinincreased significantly afterUUO inWTmice (119875 lt 005) thisincrease was significantly decreased in the NLRP3minusminus mice atday 14 after UUO (119875 lt 005 versus WTU14 group) (Figures5(c) and 5(d))

34 NLRP3 Deletion Ameliorates UUO-Induced Expres-sion of Inflammatory Cytokines The expression of NLRP3inflammasome-regulated inflammatory cytokines was eval-uated byWestern blot analysis (Figures 6(a) and 6(b)) At day14 after UUO significant increases in renal expression of cas-pase 1 (715-fold) IL-1120573 (298-fold) and IL-18 (742-fold) pro-teins were detected in WT mice (all 119875 lt 005 versus

WTSham group) and these effects were significantly inhib-ited in the NLRP3minusminus mice at day 14 after UUO (all 119875 lt 005versus WTU14 group)

35 NLRP3 Deletion Attenuates UUO-Induced MitochondrialDysfunction MTD is characterized by disordered intracellu-lar ATP synthesis mtDNA damage and excessive accumula-tion of reactive oxygen species in a process that is regulated bymitochondrial NADH dehydrogenase 1 (ND1) Observationof the ultrastructural morphology of renal cells of WT miceafter UUO revealed swollen mitochondria with disorganizedand fragmented cristae (Figure 7(a)) Moreover these effectswere accompanied by significantly decreased PGC-1a proteinmtDNA ATP synthase and ND1 at day 14 in the WTUUOgroup compared with the WTSham group (all 119875 lt 005Figures 7 and 8) The decreased levels of PGC-1a proteinmtDNA ATP synthase and ND1 induced by UUO in WTmice were significantly ameliorated in the NLRP3minusminus mice atday 14 after UUO (all 119875 lt 005 versus WTU14 group)

4 Discussion

During development of CKD loss of renal function is closelyrelated to renal fibrosis Although numerous studies suggest

6 Mediators of Inflammation

WTSham WTUUO

TUNEL staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

WTWT

Sham UUONLRP3minusminusNLRP3minusminus

lowast

0

3

6

9

12

15

Apop

tosis

cells

sco

pe

(b)

cleaved Caspase 3

Caspase 3

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120573-Actin

(c)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowastlowast

lowast

lowast

0

2

4

6

8

10

Rela

tive c

leav

ed ca

spas

e 3120573

-act

in le

vel

(d)

Figure 5 NLRP3 deletion ameliorates UUO-induced cell apoptosis in UUOmice Representative images of TUNEL staining are shown ((a)magnification times200) Bar graph indicates the mean number of TUNEL-positive tubular cells per field (b) Expression of caspase 3 proteinwas detected in kidney samples (c) Semiquantitative analysis of cleaved caspase 3 normalized against 120573-actin (d) Data represent the meanplusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

that persistent inflammation contributes to chronic kidneyinjury the underlying mechanism of inflammation-inducedCKD is still unknown The results of the present studyindicate that activation of the NLRP3 inflammasome acceler-ates renal fibrosis through mitochondrial dysfunction in themurine UUO model of CKD Thus the NLPR3 inflamma-some is implicated as a potential target in the prevention ofrenal fibrosis progression

Inflammation is part of the normal response to tissuedamage Despite its original purpose of restoring homeosta-sis overt or chronic inflammation can lead to secondary

tissue damage and organ dysfunction [22] Activation of theinflammasome and subsequent release of proinflammatorycytokines have been shown to be involved in many diseaseconditions [23] NLRP3 expression was found to be increasedin renal biopsy samples from patients with various renal dis-eases with increased levels correlating with decreased renalfunction [7] Patients with CKD also exhibited elevated levelsof inflammasome-regulated cytokines such as IL-18 [24]

The UUO model of progressive CKD is often used toinvestigate the mechanisms of renal fibrosis Vilaysane etal reported less renal inflammation and fibrosis at 14 days

Mediators of Inflammation 7

Pro-IL-18IL-18

Procaspase 1Caspase 1

Sham U14ShamU14U7U3U1WT NLRP3minusminus

120573-Actin

Pro-IL-1120573IL-1120573

(a)

Sham U14ShamU14U7U3U1WT

Caspase 1IL-18

NLRP3minusminus

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

IL-1120573

05

101520253035

Relat

ive p

rote

in120573

-act

in le

vel

(b)

Figure 6 NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines Expression of caspase 1IL-1120573 and IL-18 in kidney samples was detected (a) Semiquantitative analysis of caspase 1 IL-1120573 and IL-18 normalized against 120573-actin (b)Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versusWTU14 groupU1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

Sham U14ShamU14

PGC-1a

WT NLRP3minusminus

lowast

120573-actin

0

03

06

09

12

Relat

ive P

GC-

1a le

vel

(b)

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive m

tDN

A1

8srR

NA

leve

l

(c)

Figure 7 NLRP3 deletion reverses UUO-induced mitochondrial dysfunction Representative electron microscopy photomicrographs ofultrastructural morphology of mitochondria (a) (magnification times12000) Arrow indicates swollen mitochondria Expression of PGC-1a inkidney sample was detected and semiquantitative analysis of PGC-1a normalized against 120573-actin (b) Semiquantitative analysis of mtDNAexpression normalized against 18S performed by real-time PCR (c) Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 13 7 and 14 after UUO

after UUO in the absence of NLRP3 compared with thelevels detected in its presence which was associated withthe activation of the inflammasome-dependent pathway ofNLRP3 [7] Furthermore Bani-Hani et al showed that IL-18

neutralization prevents renal injury and fibrosis after UUO inmice [25] In the present study we found that activation of theNLRP3 inflammasome and its downstream cytokines wereincreased in a time-dependent manner after UUO in WT

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

4 Mediators of Inflammation

WT WT

Sham UUO

NLRP3minusminus NLRP3minusminus

lowast

0

1

2

3

4G

lom

erul

ar in

jury

scor

e

(a)

WT WT

Sham UUO

NLRP3minusminus NLRP3minusminus

lowast

0

1

2

3

4

5

Tubu

loin

ters

titia

l fibr

osis

inde

x

(b)

WT WT

Sham UUO

NLRP3minusminusNLRP3minusminus

lowast

0

2

4

6

8

Qua

ntita

tion

of C

olla

gen

I (

)

(c)

WT WT

Sham UUO

NLRP3minusminusNLRP3minusminus

lowast

0

2

4

6

8

10

Qua

ntita

tion

of F

ibro

nect

ion

()

(d)

Figure 3 NLRP3 deletion attenuates UUO-induced glomerular injury and tubulointerstitial fibrosis as well as deposition of collagen I andfibronectin Glomerular injury score (a) and tubulointerstitial fibrosis index (b) are evaluated according to PAS staining andMasson stainingSemiquantitative analysis of collagen I (c) and fibronectin (d) positive areas is performed according to immunohistochemical staining Datarepresent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 WTSham group versusWTUUO group 119875 lt 005 NLRP3minusminusUUO group versusWTUUOgroup

groups were performed with one-way analysis of variance(ANOVA) followed by Dunnettrsquos multiple comparison testsor Studentrsquos 119905-test 119875 lt 005 was considered statisticallysignificant

3 Results

31 NLRP3 Deletion Attenuates UUO-Induced Renal InjuryRenal histopathology was examined following PAS stainingand Masson staining (Figures 1(a) and 1(b) resp) UUOcaused severe glomerular injury (306 plusmn 012) in WT mice(119875 lt 005 versus WTSham group) Compared with theWTUUO group the glomerular injury score was signifi-cantly decreased in the NLRP3minusminusUUO group (119875 lt 005Figures 1(a) and 3(a)) Similarly significant tubulointerstitialfibrosis was induced byUUO inWTmice and comparedwiththe WTUUO group the tubulointerstitial fibrosis index wassignificantly reduced in NLRP3minusminusUUO group (119875 lt 005Figures 1(b) and 3(b))

32 NLRP3 Deletion Attenuates Renal Collagen I Depositionand Fibrosis in UUO Mice Immunohistochemical analysisof collagen I and fibronectin in renal tissue sections (Figures2 3(c) and 3(d)) showed a significant increase in collagenI-positive and fibronectin-positive cells following UUO inWT mice Partial but significant reversal of these effects wasobserved in the NLRP3minusminusUUO group (119875 lt 005 Figures 23(c) and 3(d))

33 UUO-Induced Fibrosis-Related Phenotypic Alterationsand Apoptosis Are Reduced by NLRP3 Deletion 120572-SMA isregarded as a fibroblast marker and renal epithelial cellslose E-cadherin expression during renal fibrosis Thereforewe examined the expression of 120572-SMA and E-cadherin inrenal tissue (Figure 4) Expression of 120572-SMA protein wassignificantly increased in time-dependentmanner after UUOinduction inWTmice (119875 lt 005) and this increasewas signif-icantly decreased in the NLRP3minusminus mice at day 14 after UUO(119875 lt 005 versus WTU14 group) In contrast E-cadherin

Mediators of Inflammation 5

E-cadherin

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120572-SMA

120573-Actin

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

lowast

0

05

1

15

2

25

3

35

Rela

tive120572

-SM

A120573

-act

in le

vel

(b)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

0

02

04

06

08

1

12

Relat

ive E

-cad

herin

120573-a

ctin

leve

l

(c)

Figure 4 NLRP3 deletion decreases UUO-induced phenotypic alterations in UUO mice Expression of 120572-SMA and E-cadherin proteindetected in kidney samples from different groups (a) Semiquantitative analysis of 120572-SMA expression (b) and E-cadherin expression (c)normalized against 120573-actin Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

protein expression was significantly reduced in time-dependentmanner afterUUO inWTmice (119875 lt 005) Partialbut significant reversal of these effects was observed in theNLRP3minusminusUUO group (119875 lt 005 versus WTU14 group)

The effects of UUO on apoptosis were evaluated byTUNEL staining and detection of caspase 3 protein by West-ern blot analysis (Figure 5) TUNEL staining revealedmarked-ly more apoptotic cells in the WTUUO group comparedwith the WTSham group and this UUO-induced apoptosiswas evidently inhibited by NLRP3 deletion (Figures 5(a) and5(b))This was confirmed byWestern blot analysis of caspase3 Cleavage of caspase 3 to generate the activated proteinincreased significantly afterUUO inWTmice (119875 lt 005) thisincrease was significantly decreased in the NLRP3minusminus mice atday 14 after UUO (119875 lt 005 versus WTU14 group) (Figures5(c) and 5(d))

34 NLRP3 Deletion Ameliorates UUO-Induced Expres-sion of Inflammatory Cytokines The expression of NLRP3inflammasome-regulated inflammatory cytokines was eval-uated byWestern blot analysis (Figures 6(a) and 6(b)) At day14 after UUO significant increases in renal expression of cas-pase 1 (715-fold) IL-1120573 (298-fold) and IL-18 (742-fold) pro-teins were detected in WT mice (all 119875 lt 005 versus

WTSham group) and these effects were significantly inhib-ited in the NLRP3minusminus mice at day 14 after UUO (all 119875 lt 005versus WTU14 group)

35 NLRP3 Deletion Attenuates UUO-Induced MitochondrialDysfunction MTD is characterized by disordered intracellu-lar ATP synthesis mtDNA damage and excessive accumula-tion of reactive oxygen species in a process that is regulated bymitochondrial NADH dehydrogenase 1 (ND1) Observationof the ultrastructural morphology of renal cells of WT miceafter UUO revealed swollen mitochondria with disorganizedand fragmented cristae (Figure 7(a)) Moreover these effectswere accompanied by significantly decreased PGC-1a proteinmtDNA ATP synthase and ND1 at day 14 in the WTUUOgroup compared with the WTSham group (all 119875 lt 005Figures 7 and 8) The decreased levels of PGC-1a proteinmtDNA ATP synthase and ND1 induced by UUO in WTmice were significantly ameliorated in the NLRP3minusminus mice atday 14 after UUO (all 119875 lt 005 versus WTU14 group)

4 Discussion

During development of CKD loss of renal function is closelyrelated to renal fibrosis Although numerous studies suggest

6 Mediators of Inflammation

WTSham WTUUO

TUNEL staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

WTWT

Sham UUONLRP3minusminusNLRP3minusminus

lowast

0

3

6

9

12

15

Apop

tosis

cells

sco

pe

(b)

cleaved Caspase 3

Caspase 3

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120573-Actin

(c)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowastlowast

lowast

lowast

0

2

4

6

8

10

Rela

tive c

leav

ed ca

spas

e 3120573

-act

in le

vel

(d)

Figure 5 NLRP3 deletion ameliorates UUO-induced cell apoptosis in UUOmice Representative images of TUNEL staining are shown ((a)magnification times200) Bar graph indicates the mean number of TUNEL-positive tubular cells per field (b) Expression of caspase 3 proteinwas detected in kidney samples (c) Semiquantitative analysis of cleaved caspase 3 normalized against 120573-actin (d) Data represent the meanplusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

that persistent inflammation contributes to chronic kidneyinjury the underlying mechanism of inflammation-inducedCKD is still unknown The results of the present studyindicate that activation of the NLRP3 inflammasome acceler-ates renal fibrosis through mitochondrial dysfunction in themurine UUO model of CKD Thus the NLPR3 inflamma-some is implicated as a potential target in the prevention ofrenal fibrosis progression

Inflammation is part of the normal response to tissuedamage Despite its original purpose of restoring homeosta-sis overt or chronic inflammation can lead to secondary

tissue damage and organ dysfunction [22] Activation of theinflammasome and subsequent release of proinflammatorycytokines have been shown to be involved in many diseaseconditions [23] NLRP3 expression was found to be increasedin renal biopsy samples from patients with various renal dis-eases with increased levels correlating with decreased renalfunction [7] Patients with CKD also exhibited elevated levelsof inflammasome-regulated cytokines such as IL-18 [24]

The UUO model of progressive CKD is often used toinvestigate the mechanisms of renal fibrosis Vilaysane etal reported less renal inflammation and fibrosis at 14 days

Mediators of Inflammation 7

Pro-IL-18IL-18

Procaspase 1Caspase 1

Sham U14ShamU14U7U3U1WT NLRP3minusminus

120573-Actin

Pro-IL-1120573IL-1120573

(a)

Sham U14ShamU14U7U3U1WT

Caspase 1IL-18

NLRP3minusminus

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

IL-1120573

05

101520253035

Relat

ive p

rote

in120573

-act

in le

vel

(b)

Figure 6 NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines Expression of caspase 1IL-1120573 and IL-18 in kidney samples was detected (a) Semiquantitative analysis of caspase 1 IL-1120573 and IL-18 normalized against 120573-actin (b)Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versusWTU14 groupU1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

Sham U14ShamU14

PGC-1a

WT NLRP3minusminus

lowast

120573-actin

0

03

06

09

12

Relat

ive P

GC-

1a le

vel

(b)

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive m

tDN

A1

8srR

NA

leve

l

(c)

Figure 7 NLRP3 deletion reverses UUO-induced mitochondrial dysfunction Representative electron microscopy photomicrographs ofultrastructural morphology of mitochondria (a) (magnification times12000) Arrow indicates swollen mitochondria Expression of PGC-1a inkidney sample was detected and semiquantitative analysis of PGC-1a normalized against 120573-actin (b) Semiquantitative analysis of mtDNAexpression normalized against 18S performed by real-time PCR (c) Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 13 7 and 14 after UUO

after UUO in the absence of NLRP3 compared with thelevels detected in its presence which was associated withthe activation of the inflammasome-dependent pathway ofNLRP3 [7] Furthermore Bani-Hani et al showed that IL-18

neutralization prevents renal injury and fibrosis after UUO inmice [25] In the present study we found that activation of theNLRP3 inflammasome and its downstream cytokines wereincreased in a time-dependent manner after UUO in WT

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

Mediators of Inflammation 5

E-cadherin

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120572-SMA

120573-Actin

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

lowast

0

05

1

15

2

25

3

35

Rela

tive120572

-SM

A120573

-act

in le

vel

(b)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast lowast

lowast

0

02

04

06

08

1

12

Relat

ive E

-cad

herin

120573-a

ctin

leve

l

(c)

Figure 4 NLRP3 deletion decreases UUO-induced phenotypic alterations in UUO mice Expression of 120572-SMA and E-cadherin proteindetected in kidney samples from different groups (a) Semiquantitative analysis of 120572-SMA expression (b) and E-cadherin expression (c)normalized against 120573-actin Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

protein expression was significantly reduced in time-dependentmanner afterUUO inWTmice (119875 lt 005) Partialbut significant reversal of these effects was observed in theNLRP3minusminusUUO group (119875 lt 005 versus WTU14 group)

The effects of UUO on apoptosis were evaluated byTUNEL staining and detection of caspase 3 protein by West-ern blot analysis (Figure 5) TUNEL staining revealedmarked-ly more apoptotic cells in the WTUUO group comparedwith the WTSham group and this UUO-induced apoptosiswas evidently inhibited by NLRP3 deletion (Figures 5(a) and5(b))This was confirmed byWestern blot analysis of caspase3 Cleavage of caspase 3 to generate the activated proteinincreased significantly afterUUO inWTmice (119875 lt 005) thisincrease was significantly decreased in the NLRP3minusminus mice atday 14 after UUO (119875 lt 005 versus WTU14 group) (Figures5(c) and 5(d))

34 NLRP3 Deletion Ameliorates UUO-Induced Expres-sion of Inflammatory Cytokines The expression of NLRP3inflammasome-regulated inflammatory cytokines was eval-uated byWestern blot analysis (Figures 6(a) and 6(b)) At day14 after UUO significant increases in renal expression of cas-pase 1 (715-fold) IL-1120573 (298-fold) and IL-18 (742-fold) pro-teins were detected in WT mice (all 119875 lt 005 versus

WTSham group) and these effects were significantly inhib-ited in the NLRP3minusminus mice at day 14 after UUO (all 119875 lt 005versus WTU14 group)

35 NLRP3 Deletion Attenuates UUO-Induced MitochondrialDysfunction MTD is characterized by disordered intracellu-lar ATP synthesis mtDNA damage and excessive accumula-tion of reactive oxygen species in a process that is regulated bymitochondrial NADH dehydrogenase 1 (ND1) Observationof the ultrastructural morphology of renal cells of WT miceafter UUO revealed swollen mitochondria with disorganizedand fragmented cristae (Figure 7(a)) Moreover these effectswere accompanied by significantly decreased PGC-1a proteinmtDNA ATP synthase and ND1 at day 14 in the WTUUOgroup compared with the WTSham group (all 119875 lt 005Figures 7 and 8) The decreased levels of PGC-1a proteinmtDNA ATP synthase and ND1 induced by UUO in WTmice were significantly ameliorated in the NLRP3minusminus mice atday 14 after UUO (all 119875 lt 005 versus WTU14 group)

4 Discussion

During development of CKD loss of renal function is closelyrelated to renal fibrosis Although numerous studies suggest

6 Mediators of Inflammation

WTSham WTUUO

TUNEL staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

WTWT

Sham UUONLRP3minusminusNLRP3minusminus

lowast

0

3

6

9

12

15

Apop

tosis

cells

sco

pe

(b)

cleaved Caspase 3

Caspase 3

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120573-Actin

(c)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowastlowast

lowast

lowast

0

2

4

6

8

10

Rela

tive c

leav

ed ca

spas

e 3120573

-act

in le

vel

(d)

Figure 5 NLRP3 deletion ameliorates UUO-induced cell apoptosis in UUOmice Representative images of TUNEL staining are shown ((a)magnification times200) Bar graph indicates the mean number of TUNEL-positive tubular cells per field (b) Expression of caspase 3 proteinwas detected in kidney samples (c) Semiquantitative analysis of cleaved caspase 3 normalized against 120573-actin (d) Data represent the meanplusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

that persistent inflammation contributes to chronic kidneyinjury the underlying mechanism of inflammation-inducedCKD is still unknown The results of the present studyindicate that activation of the NLRP3 inflammasome acceler-ates renal fibrosis through mitochondrial dysfunction in themurine UUO model of CKD Thus the NLPR3 inflamma-some is implicated as a potential target in the prevention ofrenal fibrosis progression

Inflammation is part of the normal response to tissuedamage Despite its original purpose of restoring homeosta-sis overt or chronic inflammation can lead to secondary

tissue damage and organ dysfunction [22] Activation of theinflammasome and subsequent release of proinflammatorycytokines have been shown to be involved in many diseaseconditions [23] NLRP3 expression was found to be increasedin renal biopsy samples from patients with various renal dis-eases with increased levels correlating with decreased renalfunction [7] Patients with CKD also exhibited elevated levelsof inflammasome-regulated cytokines such as IL-18 [24]

The UUO model of progressive CKD is often used toinvestigate the mechanisms of renal fibrosis Vilaysane etal reported less renal inflammation and fibrosis at 14 days

Mediators of Inflammation 7

Pro-IL-18IL-18

Procaspase 1Caspase 1

Sham U14ShamU14U7U3U1WT NLRP3minusminus

120573-Actin

Pro-IL-1120573IL-1120573

(a)

Sham U14ShamU14U7U3U1WT

Caspase 1IL-18

NLRP3minusminus

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

IL-1120573

05

101520253035

Relat

ive p

rote

in120573

-act

in le

vel

(b)

Figure 6 NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines Expression of caspase 1IL-1120573 and IL-18 in kidney samples was detected (a) Semiquantitative analysis of caspase 1 IL-1120573 and IL-18 normalized against 120573-actin (b)Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versusWTU14 groupU1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

Sham U14ShamU14

PGC-1a

WT NLRP3minusminus

lowast

120573-actin

0

03

06

09

12

Relat

ive P

GC-

1a le

vel

(b)

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive m

tDN

A1

8srR

NA

leve

l

(c)

Figure 7 NLRP3 deletion reverses UUO-induced mitochondrial dysfunction Representative electron microscopy photomicrographs ofultrastructural morphology of mitochondria (a) (magnification times12000) Arrow indicates swollen mitochondria Expression of PGC-1a inkidney sample was detected and semiquantitative analysis of PGC-1a normalized against 120573-actin (b) Semiquantitative analysis of mtDNAexpression normalized against 18S performed by real-time PCR (c) Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 13 7 and 14 after UUO

after UUO in the absence of NLRP3 compared with thelevels detected in its presence which was associated withthe activation of the inflammasome-dependent pathway ofNLRP3 [7] Furthermore Bani-Hani et al showed that IL-18

neutralization prevents renal injury and fibrosis after UUO inmice [25] In the present study we found that activation of theNLRP3 inflammasome and its downstream cytokines wereincreased in a time-dependent manner after UUO in WT

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

6 Mediators of Inflammation

WTSham WTUUO

TUNEL staining

NLRP3minusminusSham NLRP3minusminusUUO

(a)

WTWT

Sham UUONLRP3minusminusNLRP3minusminus

lowast

0

3

6

9

12

15

Apop

tosis

cells

sco

pe

(b)

cleaved Caspase 3

Caspase 3

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

120573-Actin

(c)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowastlowast

lowast

lowast

0

2

4

6

8

10

Rela

tive c

leav

ed ca

spas

e 3120573

-act

in le

vel

(d)

Figure 5 NLRP3 deletion ameliorates UUO-induced cell apoptosis in UUOmice Representative images of TUNEL staining are shown ((a)magnification times200) Bar graph indicates the mean number of TUNEL-positive tubular cells per field (b) Expression of caspase 3 proteinwas detected in kidney samples (c) Semiquantitative analysis of cleaved caspase 3 normalized against 120573-actin (d) Data represent the meanplusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

that persistent inflammation contributes to chronic kidneyinjury the underlying mechanism of inflammation-inducedCKD is still unknown The results of the present studyindicate that activation of the NLRP3 inflammasome acceler-ates renal fibrosis through mitochondrial dysfunction in themurine UUO model of CKD Thus the NLPR3 inflamma-some is implicated as a potential target in the prevention ofrenal fibrosis progression

Inflammation is part of the normal response to tissuedamage Despite its original purpose of restoring homeosta-sis overt or chronic inflammation can lead to secondary

tissue damage and organ dysfunction [22] Activation of theinflammasome and subsequent release of proinflammatorycytokines have been shown to be involved in many diseaseconditions [23] NLRP3 expression was found to be increasedin renal biopsy samples from patients with various renal dis-eases with increased levels correlating with decreased renalfunction [7] Patients with CKD also exhibited elevated levelsof inflammasome-regulated cytokines such as IL-18 [24]

The UUO model of progressive CKD is often used toinvestigate the mechanisms of renal fibrosis Vilaysane etal reported less renal inflammation and fibrosis at 14 days

Mediators of Inflammation 7

Pro-IL-18IL-18

Procaspase 1Caspase 1

Sham U14ShamU14U7U3U1WT NLRP3minusminus

120573-Actin

Pro-IL-1120573IL-1120573

(a)

Sham U14ShamU14U7U3U1WT

Caspase 1IL-18

NLRP3minusminus

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

IL-1120573

05

101520253035

Relat

ive p

rote

in120573

-act

in le

vel

(b)

Figure 6 NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines Expression of caspase 1IL-1120573 and IL-18 in kidney samples was detected (a) Semiquantitative analysis of caspase 1 IL-1120573 and IL-18 normalized against 120573-actin (b)Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versusWTU14 groupU1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

Sham U14ShamU14

PGC-1a

WT NLRP3minusminus

lowast

120573-actin

0

03

06

09

12

Relat

ive P

GC-

1a le

vel

(b)

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive m

tDN

A1

8srR

NA

leve

l

(c)

Figure 7 NLRP3 deletion reverses UUO-induced mitochondrial dysfunction Representative electron microscopy photomicrographs ofultrastructural morphology of mitochondria (a) (magnification times12000) Arrow indicates swollen mitochondria Expression of PGC-1a inkidney sample was detected and semiquantitative analysis of PGC-1a normalized against 120573-actin (b) Semiquantitative analysis of mtDNAexpression normalized against 18S performed by real-time PCR (c) Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 13 7 and 14 after UUO

after UUO in the absence of NLRP3 compared with thelevels detected in its presence which was associated withthe activation of the inflammasome-dependent pathway ofNLRP3 [7] Furthermore Bani-Hani et al showed that IL-18

neutralization prevents renal injury and fibrosis after UUO inmice [25] In the present study we found that activation of theNLRP3 inflammasome and its downstream cytokines wereincreased in a time-dependent manner after UUO in WT

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

Mediators of Inflammation 7

Pro-IL-18IL-18

Procaspase 1Caspase 1

Sham U14ShamU14U7U3U1WT NLRP3minusminus

120573-Actin

Pro-IL-1120573IL-1120573

(a)

Sham U14ShamU14U7U3U1WT

Caspase 1IL-18

NLRP3minusminus

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

IL-1120573

05

101520253035

Relat

ive p

rote

in120573

-act

in le

vel

(b)

Figure 6 NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines Expression of caspase 1IL-1120573 and IL-18 in kidney samples was detected (a) Semiquantitative analysis of caspase 1 IL-1120573 and IL-18 normalized against 120573-actin (b)Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versusWTU14 groupU1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 after UUO

WTSham WTUUONLRP3minusminusSham NLRP3minusminusUUO

(a)

Sham U14ShamU14

PGC-1a

WT NLRP3minusminus

lowast

120573-actin

0

03

06

09

12

Relat

ive P

GC-

1a le

vel

(b)

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive m

tDN

A1

8srR

NA

leve

l

(c)

Figure 7 NLRP3 deletion reverses UUO-induced mitochondrial dysfunction Representative electron microscopy photomicrographs ofultrastructural morphology of mitochondria (a) (magnification times12000) Arrow indicates swollen mitochondria Expression of PGC-1a inkidney sample was detected and semiquantitative analysis of PGC-1a normalized against 120573-actin (b) Semiquantitative analysis of mtDNAexpression normalized against 18S performed by real-time PCR (c) Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared withWTSham group 119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 13 7 and 14 after UUO

after UUO in the absence of NLRP3 compared with thelevels detected in its presence which was associated withthe activation of the inflammasome-dependent pathway ofNLRP3 [7] Furthermore Bani-Hani et al showed that IL-18

neutralization prevents renal injury and fibrosis after UUO inmice [25] In the present study we found that activation of theNLRP3 inflammasome and its downstream cytokines wereincreased in a time-dependent manner after UUO in WT

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

8 Mediators of Inflammation

Sham U14ShamU14U7U3U1WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12Re

lativ

e ATP

synt

hase

18s

leve

l

(a)

Sham U14ShamU14U7U3U1

WT NLRP3minusminus

lowast

lowast

0

02

04

06

08

1

12

Relat

ive N

D1

18s l

evel

(b)

Figure 8 NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction Semiquantitative analysis of ATP synthase (a) and ND1 (b)normalized against 18S performed by real-time PCR Data represent the mean plusmn SEM (119899 = 6) lowast119875 lt 005 compared with WTSham group119875 lt 005 NLRP3minusminusU14 group versus WTU14 group U1 U3 U7 and U14 indicate that mice were sacrificed on days 1 3 7 and 14 afterUUO

mice Moreover NLRP3 deletion significantly amelioratedUUO-induced glomerular injury and tubulointerstitial fibro-sis through decreased accumulation of extracellular matrixcomponents such as collagen I and fibronectin as well asinhibition of apoptosis and phenotypic transition of renalintrinsic cells In addition our results implicate the releaseof mature IL-1120573 and IL-18 due to NLRP3 inflammasomeactivation in glomerular injury and tubulointerstitial fibrosisTaken together these findings suggested an important rolefor canonical NLRP3 inflammasome activation (the NLRP3inflammasome-caspase 1-IL-1120573IL-18 axis) in renal fibrosis inthe murine UUOmodel of CKD

MTD is closely associated with cell apoptosis and tissueinjury Recent studies have suggested that MTD is a keyevent in the pathogenesis of CKD [26] In this study we havedemonstrated increasingly severe MTD in the UUO modelmanifested swollen mitochondria together with decreasedmtDNA ATP synthase and ND1 MtDNA which is distinctfrom nuclear DNA encodes mitochondrial proteins that areessential for oxidative phosphorylation as well as productionof uridine and pyruvate [27] ND1 is one subunit of mito-chondrial complex I the loss of which leads to disruptionof complex I assembly followed by mitochondrial metabolicdysfunction [28] ATP synthase is vital for ATP productionwith loss of this enzyme resulting causing a serious deficiencyin the production of energy [29] Thus the reductions inmtDNA ATP synthase and ND1 are implicated in MTDobserved in the UUOmodel may correlate with the observedincreases in glomerular injury and tubulointerstitial fibrosis

In many reports MTD is regarded as important con-tribution to activation of NLRP3 inflammasome in kidneydamage [30] Moreover mitochondrial ROS inhibitors mito-TEMPO and rotenone significantly inhibited Nlrp3 inflam-masome activation [11 16] However other studies havesuggested that the NLRP3 inflammasome mediates renalinjury via impaired mitochondrial function [31 32] whichimplies the existence of positive feedback regulation betweenactivation of NLRP3 inflammasome and mitochondrial dys-function It has been noted that a number of proinflam-matory cytokines including TNF-120572 and IL-1120573 may induce

mitochondrial injury by decreasing the activity of complex Iproduction of ATP and mitochondrial transcription whichis related to the induction of apoptosis These cytokines alsoinduce marked accumulation of ROS [33] In addition theNLRP3 inflammasome has been shown to activate caspase1 via increased mitochondrial membrane permeability andcytosolic mtDNA release [34] In this study NLRP3 deletionsignificantly preserved kidney function via attenuating mito-chondrial dysfunction evidenced by improved swollen mito-chondria ameliorated mtDNA ATP synthase and ND1 at 14days after UUOThese data presented suggest that inflamma-some activation in the UUOmodel occurs upstream of mito-chondrial dysfunction which indicates the potential role ofthe NLRP3 inflammasome in the induction of mitochondrialdysfunction in the UUOmodel

In summary our study demonstrates mitochondrial dys-function and the involvement of NLRP3 inflammasomeactivation and subsequent proinflammatory cytokine pro-duction in glomerular injury and tubulointerstitial fibrosisin the UUO model Moreover NLRP3 deletion significantlydecreased proinflammatory cytokine release reversed mito-chondrial dysfunction and preserved kidney ultrastructureThese results support the concept that activation of NLRP3inflammasome induces mitochondrial dysfunction and sub-sequent renal fibrosis in themurineUUOmodel of CKDOurcharacterization of theNLRP3 inflammasomemitochondrialdysfunction axis may offer new insights into the preventionof renal fibrosis in CKD

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Honglei Guo and Xiao Bi contributed equally

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (81300590)

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

Mediators of Inflammation 9

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] T J Hoerger S A Simpson B O Yarnoff et al ldquoThe futureburden of CKD in the United States a simulation model for theCDC CKD initiativerdquo American Journal of Kidney Diseases vol65 no 3 pp 403ndash411 2015

[3] N Ledo Y-A Ko A-S D Park et al ldquoFunctional genomicannotation of genetic risk loci highlights inflammation andepithelial biology networks in CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 3 pp 692ndash714 2015

[4] D Impellizzeri E Esposito J Attley and S Cuzzocrea ldquoTar-geting inflammation new therapeutic approaches in chronickidney disease (CKD)rdquo Pharmacological Research vol 81 pp91ndash102 2014

[5] D L Mattson ldquoInfiltrating immune cells in the kidney in salt-sensitive hypertension and renal injuryrdquo American Journal ofPhysiology - Renal Physiology vol 307 no 5 pp F499ndashF5082014

[6] C M Artlett and J D Thacker ldquoMolecular activation of theNLRP3 inflammasome in fibrosis common threads linkingdivergent fibrogenic diseasesrdquo Antioxidants and Redox Signal-ing vol 22 no 13 pp 1162ndash1175 2015

[7] A Vilaysane J Chun M E Seamone et al ldquoThe NLRP3inflammasome promotes renal inflammation and contributes toCKDrdquo Journal of the American Society of Nephrology vol 21 no10 pp 1732ndash1744 2010

[8] W P Pulskens L M Butter G J Teske et al ldquoNlrp3 preventsearly renal interstitial edema and vascular permeability inunilateral ureteral obstructionrdquo PLoS ONE vol 9 no 1 ArticleID e85775 2014

[9] K Chen J Zhang W Zhang et al ldquoATP-P2X4 signalingmediates NLRP3 inflammasome activation a novel pathway ofdiabetic nephropathyrdquo International Journal of Biochemistry andCell Biology vol 45 no 5 pp 932ndash943 2013

[10] S Granata V Masola E Zoratti et al ldquoNLRP3 inflammasomeactivation in dialyzed chronic kidney disease patientsrdquo PLoSONE vol 10 no 3 Article ID e0122272 2015

[11] WDingHGuo C Xu BWangM Zhang and FDing ldquoMito-chondrial reactive oxygen species-mediated NLRP3 inflam-masome activation contributes to aldosterone-induced renaltubular cells injuryrdquo Oncotarget vol 7 no 14 pp 17479ndash174912016

[12] M Tamaki K Miyashita S Wakino M Mitsuishi K HayashiandH Itoh ldquoChronic kidney disease reducesmusclemitochon-dria and exercise endurance and its exacerbation by dietary pro-tein through inactivation of pyruvate dehydrogenaserdquo KidneyInternational vol 85 no 6 pp 1330ndash1339 2014

[13] J L Gamboa F T Billings M T Bojanowski et al ldquoMitochon-drial dysfunction and oxidative stress in patients with chronickidney diseaserdquo Physiological Reports vol 4 no 9 Article IDe12780 2016

[14] S Granata G Zaza S Simone et al ldquoMitochondrial dysreg-ulation and oxidative stress in patients with chronic kidneydiseaserdquo BMC Genomics vol 10 article 388 2009

[15] T Ide H Tsutsui S Hayashidani et al ldquoMitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarctionrdquo Circulation Researchvol 88 no 5 pp 529ndash535 2001

[16] W Ding C Xu B Wang and M Zhang ldquoRotenone attenuatesrenal injury in aldosterone-infused rats by inhibiting oxidativestress mitochondrial dysfunction and inflammasome activa-tionrdquoMedical Science Monitor vol 21 pp 3136ndash3143 2015

[17] R L Chevalier M S Forbes and B A Thornhill ldquoUreteralobstruction as a model of renal interstitial fibrosis and obstruc-tive nephropathyrdquo Kidney International vol 75 no 11 pp 1145ndash1152 2009

[18] W Ding BWang M Zhang and Y Gu ldquoInvolvement of endo-plasmic reticulum stress in uremic cardiomyopathy protectiveeffects of tauroursodeoxycholic acidrdquo Cellular Physiology andBiochemistry vol 38 no 1 pp 141ndash152 2016

[19] B Wang W Ding M Zhang H Li and Y Gu ldquoRapamycinattenuates aldosterone-induced tubulointerstitial inflammationand fibrosisrdquo Cellular Physiology and Biochemistry vol 35 no1 pp 116ndash125 2015

[20] C Xu W Ding L Yang M Yang M Zhang and Y GuldquoContributions of endoplasmic reticulum stress and reactiveoxygen species to renal injury in aldosteronesalt-induced ratsrdquoNephron - Experimental Nephrology vol 126 no 1 pp 25ndash322014

[21] W Ding BWang M Zhang and Y Gu ldquoTempol a superoxidedismutase-mimetic drug ameliorates progression of renal dis-ease in CKD micerdquo Cellular Physiology and Biochemistry vol36 no 6 pp 2170ndash2182 2015

[22] M Lech R Grobmayr M Ryu et al ldquoMacrophage phenotypecontrols long-term AKI outcomes-kidney regeneration versusatrophyrdquo Journal of the American Society of Nephrology vol 25no 2 pp 292ndash304 2014

[23] H-J Anders and D AMuruve ldquoThe inflammasomes in kidneydiseaserdquo Journal of the American Society of Nephrology vol 22no 6 pp 1007ndash1018 2011

[24] G Lonnemann D Novick M Rubinstein and C A DinarelloldquoInterleukin-18 interleukin-18 binding protein and impairedproduction of interferon-g in chronic renal failurerdquo ClinicalNephrology vol 60 no 5 pp 327ndash334 2003

[25] A H Bani-Hani J A Leslie H Asanuma et al ldquoIL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosisrdquo Kidney Interna-tional vol 76 no 5 pp 500ndash511 2009

[26] J-F Chen H Liu H-F Ni et al ldquoImproved mitochondrialfunction underlies the protective effect of pirfenidone againsttubulointerstitial fibrosis in 56 nephrectomized ratsrdquo PLoSONE vol 8 no 12 Article ID e83593 2013

[27] M Pinto and C T Moraes ldquoMechanisms linking mtDNAdamage and agingrdquo Free Radical Biology and Medicine vol 85pp 250ndash258 2015

[28] S C Lim J Hroudova N J VanBergenM I G Lopez SanchezI A Trounce and M McKenzie ldquoLoss of mitochondrialDNA-encoded protein ND1 results in disruption of complex Ibiogenesis during early stages of assemblyrdquoThe FASEB Journalvol 30 no 6 pp 2236ndash2248 2016

[29] J Houstek S Kmoch and J Zeman ldquoTMEM70 proteinmdashanovel ancillary factor of mammalian ATP synthaserdquo Biochimicaet Biophysica Acta vol 1787 no 5 pp 529ndash532 2009

[30] Y Zhuang M Yasinta C Hu et al ldquoMitochondrial dysfunctionconfers albumin-inducedNLRP3 inflammasome activation andrenal tubular injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 8 pp F857ndashF866 2015

[31] Y Zhuang G Ding M Zhao et al ldquoNLRP3 inflamma-some mediates albumin-induced renal tubular injury through

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011

10 Mediators of Inflammation

impaired mitochondrial functionrdquo The Journal of BiologicalChemistry vol 289 no 36 pp 25101ndash25111 2014

[32] W Gong S Mao J Yu et al ldquoNLRP3 deletion protects againstrenal fibrosis and attenuates mitochondrial abnormality inmousewith 56 nephrectomyrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 310 no 10 pp F1081ndashF1088 2016

[33] R Zell P Geck K Werdan and P Boekstegers ldquoTNF-120572 andIL-1120572 inhibit both pyruvate dehydrogenase activity and mito-chondrial function in cardiomyocytes evidence for primaryimpairment of mitochondrial functionrdquoMolecular and CellularBiochemistry vol 177 no 1-2 pp 61ndash67 1997

[34] K Nakahira J A Haspel V A K Rathinam et al ldquoAutophagyproteins regulate innate immune responses by inhibiting therelease of mitochondrial DNA mediated by the NALP3 inflam-masomerdquo Nature Immunology vol 12 no 3 pp 222ndash230 2011