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Relationship of clusterin with renal inflammation and fibrosis after the recovery phase of ischemia-reperfusion… Guo, Jia; Guan, Qiunong; Liu, Xiuheng; Wang, Hao; Gleave, Martin E; Nguan, Christopher Y C; Du, Caigan Sep 20, 2016

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RESEARCH ARTICLE Open AccessRelationship of clusterin with renalinflammation and fibrosis after therecovery phase of ischemia-reperfusioninjuryJia Guo1,2, Qiunong Guan1, Xiuheng Liu2, Hao Wang3, Martin E. Gleave1,4, Christopher Y. C. Nguan1and Caigan Du1,5*AbstractBackground: Long-term outcomes after acute kidney injury (AKI) include incremental loss of function and progressiontowards chronic kidney disease (CKD); however, the pathogenesis of AKI to CKD remains largely unknown. Clusterin(CLU) is a chaperone-like protein that reduces ischemia-reperfusion injury (IRI) and enhances tissue repair after IRI in thekidney. This study investigated the role of CLU in the transition of IRI to renal fibrosis.Methods: IRI was induced in the left kidneys of wild type (WT) C57BL/6J (B6) versus CLU knockout (KO) B6 mice byclamping the renal pedicles for 28 min at the body temperature of 32 °C. Tissue damage was examined by histology,infiltrate phenotypes by flow cytometry analysis, and fibrosis-related gene expression by PCR array.Results: Reduction of kidney weight was induced by IRI, but was not affected by CLU KO. Both WT and KO kidneyshad similar function with minimal cellular infiltration and fibrosis at day 14 of reperfusion. After 30 days, KO kidneyshad greater loss in function than WT, indicated by the higher levels of both serum creatinine and BUN in KO mice, andexhibited more cellular infiltration (CD8 cells and macrophages), more tubular damage and more severe tissue fibrosis(glomerulopathy, interstitial fibrosis and vascular fibrosis). PCR array showed the association of CLU deficiency withup-regulation of CCL12, Col3a1, MMP9 and TIMP1 and down-regulation of EGF in these kidneys.Conclusion: Our data suggest that CLU deficiency worsens renal inflammation and tissue fibrosis after IRI in the kidney,which may be mediated through multiple pathways.Keywords: Clusterin, Kidney ischemia-reperfusion, Acute kidney injury, Chronic kidney disease, FibrosisBackgroundIschemic acute kidney injury (AKI) occurs when renalperfusion is decreased [1, 2], and it is the most commonform of clinical AKI, accounting for up to 70 % of allAKI cases [3–6]. With adequate therapy, quick recoveryof kidney function to baseline may occur and is consid-ered a hallmark of ischemic AKI [6, 7]. Many recentstudies have documented long-term sequelae followingAKI, including: increased risk of developing chronickidney disease (CKD) [8–12], worsening of preexistingCKD [8, 13], and progression to end-stage renal disease(ESRD) [8, 12, 14]. Increasing evidence in literature sug-gests that many pathological factors including micro-vascular rarefaction, hypoxia, inflammation and renaltubular injury play an important role in the progressionof CKD [15–20], but the precise pathophysiologic path-ways mediating the progressive insults associated withAKI leading to CKD are still not fully understood, andthus there are currently no viable therapeutic optionsavailable to prevent CKD progression after renal injury.Clusterin (CLU) is a 75–80 kDa disulfide-linked het-erodimeric glycoprotein that is encoded by a single genein both the human and mouse genomes [21, 22]. This* Correspondence: caigan@mail.ubc.ca1Department of Urologic Sciences, University of British Columbia, Vancouver,BC, Canada5Department of Urologic Sciences, The University of British Columbia,VGH-Jack Bell Research Centre, 2660 Oak St, Vancouver, BC V6H 3Z6, CanadaFull list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Guo et al. BMC Nephrology  (2016) 17:133 DOI 10.1186/s12882-016-0348-xprotein not only is a major glycoprotein in physiologicalfluids, such as plasma, milk, urine, cerebrospinal fluid,and semen [21], but is also upregulated in renal tissuesof both humans and experimental models by variousforms of cellular stress, such as unilateral ureteral ob-struction (UUO) [23], ischemia-reperfusion injury (IRI)[24–26], as well as in rejected renal allografts and thosenative kidneys with intrinsic renal disease [27–29]. How-ever, the biological importance of CLU for the kidney isnot fully understood. We and others have been investi-gating the biological impacts of CLU deficiency on adultkidneys using CLU knockout (KO) mice compared toCLU-expressing wild type (WT) control mice, and havedemonstrated: (i) CLU KO in aging mice results in de-veloping progressive glomerulopathy that is associatedwith glomerular antibody deposition [30]; (ii) CLU KOmice exhibit the higher levels of renal fibrosis in re-sponse to ureteral obstruction [31] or angiotensin IIstimulation [32]; and (iii) our group has shown that CLUKO results in more severe renal IRI [26], in an experi-mental model of ischemic AKI, and also impairs renalrepair after IRI [33]. However, the role of CLU in theprogression to CKD from AKI has not been investigatedyet. In the present study, the effects of CLU deficiencyon the transition of ischemic AKI to CKD were investi-gated using CLU KO mice in a model of renal IRI com-pared to WT controls.MethodsAnimalsBoth wild type (WT) C57BL/6 (B6) and CLU knockout(KO) mice in B6 background (B6-Clu−/−) were receivedfrom the breeding colonies in the animal facility at theJack Bell Research Centre (Vancouver, BC, Canada). Allthe animals (males, 12- weeks old) for the experimentswere cared in accordance with the Canadian Council onAnimal Care guideline under the protocols approvedby the Animal Use Subcommittee at the University ofBritish Columbia (Vancouver, BC, Canada).Renal IRIUnilateral renal IRI was induced in the left kidney ofWT versus CLU KO mice following a routine surgicalprocedure in the lab. In brief, mice were anesthetizedwith the combination of ketamine (100 mg/kg) and xyla-zine (10 mg/kg), and isoflurane as needed. The left kid-neys were exposed through a flank incision, followed bythe induction of ischemia in these kidneys through clamp-ing renal pedicles at the body temperature of 32 °C for28 min. After the clamps were released, reperfusion of thekidneys was confirmed visually. The non-ischemic rightkidneys in the same mice were kept as contralateral con-trols as well as for life support.Determination of renal functionThe function of the left kidneys after 14 or 30 days ofIRI was determined by using serum levels of both cre-atinine and blood urea nitrogen (BUN). In brief, thecontralateral/right kidney was removed on day 13 or 29after IRI, after 24 h of right kidney removal, mice wereeuthanized and serum samples were collected. Bothcreatinine and BUN in the serum samples were measuredin the Chemistry Laboratory at the Vancouver CoastalHealth Regional Laboratory Medicine (Vancouver, BC,Canada) by using the Dimension Vista® System with CRE2and BUN Flex® reagent cartridges (Siemens HealthcareDiagnostics Inc., Newark, DE, USA), respectively.Isolation of infiltrating leukocytes and splenocytesThe procedure of infiltrate isolation from the kidneys orsplenocytes from the spleens was described in our previousstudies [34, 35]. Mice were randomly selected from eachgroup for this experiment. In brief, after perfusion withphosphate buffered saline (PBS), kidney tissues wereminced and digested with collagenases D (WorthingtonBiochemicals, Lakewood, NJ, USA). The resultant cell sus-pensions were filtered through 30-μm nylon mesh, andleukocytes from the cell suspension were recovered frominterphase by a density centrifugation using 1.083 g/mlHistopaque (Sigma-aldrich Canada, Oakville, ON, Canada),and were finally suspended in PBS after washing with PBS.A single cell suspension of splenocytes was prepared bygently crushing the spleens of mice in PBS in a Cellstrainer (BD — Canada, Mississauga, ON, Canada),followed by removal of erythrocytes by a brief incuba-tion (~4 min) with lysis buffer (0.15 M NH4Cl, 1.0 mMKHCO3, 0.1 mM EDTA, pH 6.8). After washing withPBS, the splenocytes were finally suspended for exam-ination. The total number of isolated cells from eachkidney or spleen was counted using a TC10™ automatedcell counter (Bio-Rad Laboratories Canada, Missis-sauga, ON, Canada) with trypan blue stain.Flow cytometric analysisThe phenotypes (CD4+, CD8+, CD45R/B220+, pan NK+,CD11b+, and Mac3+ cells) of leukocyte population in cellsuspensions (kidney infiltrates and splenocytes) was de-termined by fluorescence-activated cell sorting (FACS)analysis using fluorescent-labeled rat monoclonal anti-bodies [anti-CD4 (clone YTS191-1), anti-CD8A (clone53-6-7), anti-CD45R/B220 (clone RA3-6B2), anti-panNK (clone DX5), anti-CD11b (clone M1-70-15), andanti-Mac3 (clone M3-84) antibodies] following manu-facture’s protocol (eBioscience, San Diego, CA, USA).Briefly, cells were probed with each fluorescence dye-conjugated antibody in the dark for 30 min at 4 °C. Afterwashing with PBS, the positive stain was counted byGuo et al. BMC Nephrology  (2016) 17:133 Page 2 of 15using a flow cytometry and further quantified usingFlowJo software (Tree Star Inc., Ashland, OR, USA).Determination of renal pathologies by histologyKidneys after IRI insult were randomly selected fromeach group for histological analyses. A coronal tissueslice through the mid-portion of each kidney was fixedin 10 % neutral buffered formalin, followed by embed-ded in paraffin wax. Sections were cut at 4-μm thick-ness and stained with hematoxylin and eosin (HE) forthe examination of cellular infiltration, periodic acid-Schiff (PAS) for tubular injury or Masson’s trichrome(MT) method for collagen fiber deposition. The sectionswere scanned with Leica SCN400 Slide scanner (LeicaMicrosystens Inc., Concord, ON, Canada). The patho-logical parameters of renal fibrosis after IRI were deter-mined in two separate sections of each kidney using theDigital Image Hub – A slidepath Software Solution (LeicaMicrosystems Inc.) in a blinded fashion as described pre-viously [36].The extent of mononuclear cell infiltration in renalcortex of a kidney was assessed in HE-stained sectionsusing a 0 to 4 scale, depending on the percentage ofcellular infiltrates-occupied area in each microscopicview: 0 (normal or no sign of infiltration), 1 (1–10 % ofthe area affected with cellular infiltration), 2 (11–25 %),3 (26–50 %), and 4 (>50 %). The average of at least 20randomly selected views represented the infiltration ineach kidney.The number of injured tubules, including cellular loss(atrophy), intratubular cast formation, tubular cell flat-tening or vacuolation, in each microscopic view in renalcortex of a kidney was counted in PAS-stained section,and the average number of at least 20 randomly selectedviews represented the injured tubules in each kidney.The percentage of damaged tubule (combined bothnecrosis and vacuolization) in total of tubules wascounted in each view, randomly selected in the region ofrenal cortex under 400× magnification (high-poweredfield – hpf), and was averaged at least of 20 nonoverlap-ping fields for each kidney.The severity of renal fibrosis was semi-quantitativelydetermined by the degree of tubulointerstitial fibrosis inrenal cortex of the kidney, the percentage of affectedglomeruli (glomerulopathy) and of interlobular arteriolesor arteries in Masson’s trichrome-stained sections. Thetubulointerstitial fibrosis was determined using a 0 to 4scale based on the severity of tubular dilation or the per-centage of the area stained positively with collagen fi-bers: 0 (normal interstitium with the absence of collagenfiber stain), 1 (minimal collagen fiber stain or less than5 % of affected area), 2 (mild or 6–25 %), 3 (moderate or26–50 %) and 4 (severe, or affecting >50 % of thecortical area). The percentage of affected glomeruli(glomerulopathy), including glomerulosclerosis (focaland segmental sclerosis) and glomerular hypertrophy,was determined in a range of 180 to 250 glomeruli ofeach kidney. And the percentage of affected interlobulararterioles or arteries, determined by the presence of fi-brous intima, was counted in a range of 10 to 20 inter-lobular arteries or arterioles of each kidney.Immunohistochemical analysisThe kidneys were harvested from mice after perfusionwith PBS, followed by formalin fixation, paraffin embed-ding and section as described above. The expression ofalpha-smooth muscle actin (α-SMA) protein in kidneysections was assessed by a standard immunohistochemi-cal method. Briefly, after deparaffin and rehydrationbuffered-formalin-fixed sections were treated with 3 %H2O2 in Tris buffer saline (TBS) (pH 7.4) for 30 min atroom temperature to quench endogenous peroxidase,followed by permeabilization with 0.2 % Triton X-100 for10 min. After being washed with TBS containing 0.1 %Tween 20 (TBS-T) and blocked with 5 % normal rabbitserum, the sections were incubated with 1:50 dilution ofprimary mouse monoclonal anti-α-SMA (clone 1A4,Sigma-Aldrich Canada, Oakville, ON, Canada) overnightat 4 °C. The immune complexes of α-SMA and the anti-body on the tissue section were detected using VectorM.O.M. Immunodetection kit following manufacturer’sprotocol (Vector Labs, Burlington, ON, Canada), and werevisualized using a 3,3′-diaminobenzidine (DAB) peroxid-ase substrate. The control negative staining included thesections incubated with normal mouse IgG instead ofmouse anti-α-SMA antibody as the primary antibody.Nuclei are counterstained with hematoxlin.Fibrosis PCR arrayThe expression of 84 fibrosis-associated genes was quanti-tatively examined in WT kidney tissues compared to CLUKO controls using Mouse Fibrosis RT2 Profiler™ PCRArrays kits following manufacturer’s instruction (SABios-ciences – QIAGEN Inc., Valencia, CA, USA). In brief, fol-lowing perfusion with PBS, one part of kidney tissue wassnap-frozen in liquid nitrogen and stored at −80 °C priorto RNA extraction. Four kidney samples from each groupwere randomly selected for the determination of fibrosis-related gene expression profile. The total RNA from kid-ney tissues was extracted and purified using the RNeasyMicroarray Tissue Mini kit (QIAGEN Canada, Toronto,ON, Canada), and converted to cDNA using RT2 FirstStrand Kit (QIAGEN Canada). The expression of selectedgenes was amplified by using real-time Mouse FibrosisRT2 Profiler™ PCR Arrays. Data were analyzed usingWeb-based PCR Array Data Analysis Software (www.SABiosciences.com/pcrarraydataanalysis.php.).Guo et al. BMC Nephrology  (2016) 17:133 Page 3 of 15Statistical analysisData were collected from individual experiment or mousein each study, and were presented as mean ± standard der-ivation (SD) for each group in the text. The difference be-tween groups was analyzed using t-tests or analysis ofvariance (ANOVA) as appropriate with Prism® software(GraphPad Software, Inc., La Jolla, CA, USA). A p valueof ≤0.05 was considered significant.ResultsSignificant reduction of kidney weight is induced by IRI,but not affected by CLU deficiencyIt has been demonstrated that IRI initiates progressiverenal atrophy, indicated by the reduction in renal weight,volume and cortical thickness accompanying tubular celldeath (both apoptosis and necrosis) and interstitial infil-tration [37–39]. CLU plays an anti-apoptotic or prosurvi-val role in the kidney against IRI [26, 40]. Surprisingly, thepresent study showed that the renal atrophy indicated bythe loss of renal mass was not statistically different be-tween WT and KO groups (Fig. 1). The kidney weight inWT mice at age of 12 weeks old was 192 ± 20.31 mg, andwas not different from 179 ± 17.91 mg in age-matched KOmice (P = 0.3741, two-tailed t-test, n = 4). As shown inFig. 1, after IRI, the left kidneys in CLU KO mice were194.5 ± 0.71 mg on day 3 (n = 4), 145.0 ± 21.21 mg onday 7 (n = 4), 136.0 ± 65.04 mg on day 14 (n = 5) and88.13 ± 34.46 mg on day 30 (n = 15) (P = 0.0073, one-way ANOVA), while the weight of their contralateralcontrols was not significantly changed, and it was163.5 ± 0.71 mg on day 3 (n = 4), 161.0 ± 15.56 mg onday 7 (n = 4), 180.0 ± 44.72 mg on day 14 (n = 5) and198.67 ± 12.46 mg on day 30 (n = 15) (P = 0.0553, one-way ANOVA). Similar results were seen in WT mice, inwhich IRI induced kidney weight loss from 206.5 ±9.19 mg on day 3 (n = 4) to 107.15 ± 62.11 mg on day30 (n = 22) (P = 0.0060, one-way ANOVA) (Fig. 1). Moreimportantly, statistical analysis revealed that the atrophicdegree or the weight loss of the left kidneys after IRI be-tween CLU KO and WT mice was not significantly differ-ent (P = 0.3542, two-way ANOVA), demonstrating theoccurrence of severe atrophy in the kidney after IRI, butat the same time not significantly affected by a deficiencyin CLU expression.In CLU KO mice, kidney recovery from IRI occurs after14 daysOur previous study demonstrated there were no signs oftissue repair in the kidneys of CLU KO mice until day 7after IRI, whereas WT controls showed significant im-provement [33]. After induction of IRI in the left kidneysunder the same conditions as performed in the previousstudy, kidney atrophy was no different between WT andKO group on day 14 [CLU KO: 136.0 ± 65.04 mg (n = 5)vs WT: 138.36 ± 34.73 mg (n = 11) (P = 0.9885, two-tailedt-test)]. Similarly, the function of these kidneys was notdifferent, indicated by the fact that after removal ofcontralateral kidneys the serum levels of creatinine orBUN in KO mice were 0.44 ± 0.08 mg/dL (n = 8) or 41.17± 10.42 mg/dL (n = 8), which were not statistically differ-ent from those (creatinine: 0.45 ± 0.11 mg/dL; BUN: 47.19± 15.44 mg/dL, n = 8) in WT mice (creatinine: P = 0.8345;BUN: P = 0.3764, two-tailed t-test) (Fig. 2). Histologicalanalysis revealed the absence of notable inflammatoryinfiltrates as well as the remarkable repair of tubular/nephron in CLU KO or WT kidneys after 14 days of IRI(Fig. 3). Under the microscopic views of both HE- andMT-stained sections, the kidneys from either KO or WTmice exhibited relatively intact tissues (tubular epitheliumand interstitium) with the minimal levels of cellular infil-tration (KO: 0.5–1; WT: 0.5) and interstitial fibrosis (KO:0.5–1; WT: 0.5–2), which were mostly seen in the perivas-cular areas (Fig. 3). We also noticed that 2 out of 9 WTkidneys with ≥1.5 of fibrosis score showed some tubulardilatation and degeneration (reduced HE-stain of thecytoplasm) in outer area of renal cortex (Additional file 1:Figure S1).CLU deficiency decreases the function of the kidneyswith atrophyTo investigate the role of CLU in the long-term effectsof IRI on the kidney, the function of the kidneys with at-rophy was examined in CLU KO mice (n = 21) comparedwith WT controls (n = 22) on day 30 after IRI. Becausethe serum volume of some mice was not sufficient forboth measurements, the sample sizes for BUN wereFig. 1 No difference in the progression of kidney atrophy betweenCLU KO mice and WT controls after IRI. Renal IRI in left kidneys ofCLU KO versus WT mice was induced by clamping renal pedicles for28 min at the body temperature of 32 °C, and the weight of the leftkidney and contralateral right kidney from each mouse was recordedat different time points after reperfusion. Data are presented in thefigure as mean ± standard error of the mean (SEM) of each group.KO-IRI: the left kidneys of CLU KO mice (n = 4–15) after IRI (p = 0.0073,one-way ANOVA); KO-Contralateral: the right/contralateral kidneys ofCLU KO mice (p = 0.0553, one-way ANOVA); WT-IRI: the left kidneysof WT mice (n = 4–22) after IRI (p = 0.0060, one-way ANOVA); andWT-Contralateral: the right/contralateral kidneys of CLU KO mice(p = 0.5140, one-way ANOVA). Kidney atrophy in KO versus WTmice, p = 0.3542 (two-way ANOVA)Guo et al. BMC Nephrology  (2016) 17:133 Page 4 of 15smaller (WT: n = 11; KO: n = 16). As shown in Fig. 4,the serum levels of both creatinine and BUN werehigher in KO group than those in WT control, andwere indicated by 4.1 ± 1.55 mg/dL of the creatinine inKO group (n = 21) compared to 2.63 ± 2 mg/dL in WTgroup (n = 22) (P = 0.0104, two-tailed t-test), and108.71 ± 29.72 mg/dL of the BUN in KO group (n = 16)compared to 82.28 ± 29.34 mg/dL in WT group (n = 11)(P = 0.0312, two-tailed t-test). These data suggested thatalthough the severity of renal atrophy was not affectedby the deficiency in CLU expression (Fig. 1), the loss ofkidney function was worse in CLU null kidneys with at-rophy than that in WT controls after IRI.CLU deficiency increases cellular infiltration (CD8+ T cellsand macrophages) in the kidneys with atrophyIt has been documented previously that increased cellu-lar infiltration is a long-term consequence of renalischemia [41, 42], but was not significantly seen in thekidneys after 14 days of IRI in this study (Fig. 3). Tounderstand the role of CLU expression in this pathologicchange of the kidneys, the infiltrates of the kidneys withatrophy in the CLU KO group were compared to thosein WT group after 30 days of IRI. The samples fromeach group were randomly selected for this study. Asshown in Fig. 5, there was severe cellular infiltration in theleft kidneys with atrophy in both groups, but not incontralateral kidneys. A semi-quantitative scoring showedthat the severity of renal infiltration in KO group (3.28 ±0.18) was higher than that in WT group (2.05 ± 0.22) (KOvs. WT, P < 0.0001, two-tailed t-test, n = 8) (Fig. 5b).The phenotypes of infiltrates (CD4+, CD8+, B220+, panNK+ CD11b+ and Mac3+) in the kidneys with atrophywere further examined using FACS analysis. Again, thesamples for this study were randomly selected from eachgroup, but were not the same as those for histologicalanalysis in Fig. 5. As indicated in Table 1, similar to thehigher infiltration scores in KO group by histologicalanalysis (Fig. 5b), the total number of isolated infiltratesfrom CLU null kidneys was more than that in WTcontrols although the difference was not statistically sig-nificant in this limited number of samples (P = 0.0814,one-tailed t-test, n = 4). Statistically, only the percentagesof both CD8+ and Mac3+ cells in KO group were sig-nificantly higher than those in WT group (CD8+ cells,P = 0.0377; Mac3+ cells, P = 0.0387, one-tailed t-test, n = 4),while the rest of phenotypes (CD4 T cells, B cell, NK cells,dendritic cells) were the same (Table 1). These data also in-dicated that the total numbers of infiltrating CD8 T cellsand macrophages were more significantly higher in KOkidneys than those in WT controls. In addition, the per-centage of each phenotype of leukocytes in the spleen fromthe same animal was also determined and used as a controlfor determining the specificity of kidney infiltrates. Asshown in Table 2, there was no difference in the total num-bers of splenocytes and the percentages of every phenotypebetween KO and WT groups. Taken together, these datasuggested that CLU deficiency was associated with morecellular infiltrates, especially CD8+ (CD8 T cells) andMac3+ cells (macrophages), in the kidneys with atrophy.CLU deficiency increases the damage of renal tubularintegrity in the kidney with atrophyOur previous studies have demonstrated that CLU defi-ciency worsens renal injury after IRI and impairs its re-pair during the early phase of IRI [26, 33]. However, itsimpact on the maintenance of the nephron of the kidneywith atrophy after IRI was not known. After 30 days ofIRI, histological analysis revealed that there were fewerintact tubules in the kidneys with atrophy in the CLUKO group compared with those in the WT group [24.52± 6.3 damaged tubules per hpf vs 10.52 ± 3.03 respect-ively (P < 0.0001, two-tailed t-test, n = 8)] (Fig. 6). Thesedata suggested that CLU deficiency resulted in more se-vere renal tubular damage over the long term after IRI.Fig. 2 No difference in renal function between CLU KO and WT mice on day 14 after IRI. On day 13 after IRI, contralateral kidneys were removed.After 24 h of surgery, serum from each mouse was collected, and the levels of both serum creatinine (Scr) and blood urea nitrogen (BUN) weremeasured as biomarkers of renal function. Data are presented as mean ± standard derivation (SD) of each group. a Scr in CLU KO group (n = 8)compared to WT control (n = 8), p = 0.8345 (two-tailed t-test). b BUN in CLU KO group (n = 8) compared to WT control (n = 8), P = 0.3764(two-tailed t-test)Guo et al. BMC Nephrology  (2016) 17:133 Page 5 of 15Fig. 3 (See legend on next page.)Guo et al. BMC Nephrology  (2016) 17:133 Page 6 of 15CLU deficiency worsens tissue fibrosis in the kidneywith atrophyIn addition to cellular infiltration (Fig. 5), progression offibrosis is recognized as one of long-term consequencesof IRI in the kidney [43, 44]. To evaluate the effect ofCLU deficiency on IRI-initiated renal fibrosis, the degreeof tubulointerstitial fibrosis in the cortex, the percentageof affected glomeruli (glomerulopathy) and of interlobu-lar arterioles or arteries of the kidneys in CLU KO micewere compared to those in WT controls. As shown inFig. 7, abnormal collagen accumulation (blue stain) wasseen in the tubulointerstitium, glomeruli, interlobular ar-terial wall and perivascular spaces of both CLU KO andWT kidneys. Semi-quantitative scoring showed that therenal fibrosis in KO group was significantly worse in allthree areas (interstitial fibrosis, glomerulopathy, and vas-cular fibrosis) as compared to that in WT group. Theinterstitial fibrosis score of CLU null kidneys was 3.65 ±0.15 and statistically higher than 2.92 ± 0.2 of WT controls(P < 0.0001, two-tailed t-test, n = 8) (Fig. 7b). Similar re-sults were seen in the assessment of both glomerulopathy(P = 0.0300, one-tailed t-test, n = 8) and vascular fibrosis(P = 0.0144, two-tailed t-test, n = 8) in these two groups(Fig. 7c and d).The α-SMA-expressing myofibroblasts plays an import-ant role in the progression of renal fibrosis in differentconditions including AKI [45, 46]. To further confirm theresults from MT stain, the myofibroblast population, iden-tified by α-SMA expression, was examined in CLU nullkidneys compared to WT controls by using immunohisto-chemical stain. As shown in Fig. 8, two types of renal cellswere found α-SMA positivity: infiltrates that were mainlylocalized in the interstitial and perivascular areas, and in-jured tubular epithelial cells. Furthermore, there weremore α-SMA positive cells, especially tubular epithelialcells, in the sections of CLU null kidneys than in WT con-trols, which were consistent with the more collagen accu-mulation seen by MT-stained sections. Taken together,these data suggested that CLU deficiency worsened IRI-initiated fibrosis in the kidneys.CLU deficiency associates with down-regulation of EGFand up-regulation of CCL12 and MMP9To further understand the molecular pathways by whichCLU deficiency worsens IRI-induced fibrosis in the kid-ney, the expression of 84 fibrosis-related genes in CLUnull kidneys compared to WT controls, also randomlyselected from each group (n = 4), was examined usingPCR array (Additional file 2: Table S1). Surprisingly, theexpression of well-characterized pro-fibrogenic factors,such as transforming growth factor (TGF)-β1 and con-nective tissue growth factor (CTGF), was not correlatedwith an increase of fibrosis in CLU KO kidneys. As indi-cated in Table 3, by using a cutoff line (>2-fold change,P < 0.05), the expression levels of five genes in CLU nullkidneys compared to WT controls (n = 4) were signifi-cantly changed after 30 days of IRI, which were the down-regulation of EGF as well as up-regulation of CCL12,(See figure on previous page.)Fig. 3 No difference in tissue architecture of the kidneys between CLU KO and WT mice on day 14 after IRI. The sections of the left kidneys ofWT versus CLU KO mice, harvested on day 14 after reperfusion, were stained with hematoxylin and eosin (HE) or Masson’s trichrome (MT). Dataare presented as a typical microscopic image of renal cortex. KO: CLU null kidney sections; WT: WT kidney sections. a Typical microscopic imagesof HE-stained sections showing intact tubules with minimal cellular infiltration in the perivascular space. b Typical microscopic images of MT-stainedsections showing red cytoplasm and mild blue collagen in the perivascular space. Black arrows: glomeruli; IA: interlobular artery; and IV: interlobularvein. c The infiltration was semi-quantitatively scored in two separate sections of each injured left kidney, and was presented in average per view.Data are presented as mean ± SD of each group. KO group vs. WT control: P = 0.1510 (two-tailed t-test, n = 9). d The interstitial fibrosis wassemi-quantitatively scored in two separate sections of each injured left kidney, and was presented in average per view. Data are presentedas mean ± SD of each group. KO group vs. WT control: P = 0.1284 (two-tailed t-test, n = 9)Fig. 4 An association of CLU deficiency with worse kidney function after 30 days of IRI. On day 29 after IRI, contralateral kidneys were removed.After 24 h of surgery, the serum levels of both serum creatinine (Scr) and blood urea nitrogen (BUN) were measured. Data are presented asmean ± SEM of each group. a Scr in CLU KO group (n = 21) compared to WT control (n = 22), p = 0.00104 (two-tailed t-test). b BUN in CLU KOgroup (n = 16) compared to WT control (n = 11), P = 0.0312 (two-tailed t-test)Guo et al. BMC Nephrology  (2016) 17:133 Page 7 of 15Fig. 5 An association of CLU deficiency with worse cellular infiltration after 30 days of IRI. As described in Fig. 4, the contralateral right kidneyswere harvested on day 29, and the injured left kidney on day 30 after IRI. The sections (two per each kidney) of both kidneys (the left and rightfrom KO or WT) were stained with HE for the examination of the content of mononuclear infiltrates, labeled by dark/black nuclear staining.a A typical microscopic image of renal cortex in each group (KO: CLU null kidneys; WT: WT kidney sections). Left column: tubule damage andcellular infiltration of the injured left kidneys; right column: normal tissue architecture of the contralateral right kidneys. G: glomerulus; PT:proximal convoluted tubule; DT: distal convoluted tubule; IA: interlobular artery; and IV: interlobular vein. b The infiltration was semi-quantitativelyscored in at least 20 randomly selected views in two separate sections of each injured left kidney, and was presented in average per view. Data arepresented as mean ± standard derivation (SD) of each group. KO group vs. WT control: P < 0.0001 (two-tailed t-test, n = 8)Table 1 The percentage of each phenotype of infiltrating immune cells in the kidneysWT (n = 4) KO (n = 4) p value (WT vs. KO)Total isolated cells (× 106) 20.39 ± 4.31 25.57 ± 4.78 0.0814CD4 (%) 1.37 ± 0.91 2.38 ± 1.21 0.1547CD8 (%) 3.51 ± 2.48 5.79 ± 3.76 0.0377CD45R/B220 (%) 0.83 ± 0.62 1.08 ± 0.42 0.2853Mac-3 (%) 4.22 ± 4.72 8.56 ± 5.21 0.0387CD11b (%) 6.83 ± 1.43 8.02 ± 1.68 0.2195pan-NK (%) 1.65 ± 0.68 2.4 ± 0.97 0.1384Four kidneys/animals were randomly selected from each group for FACS analysis of infiltrating T cells (CD4+ and CD8+ cells), B cells (CD45R/B220+ cells),macrophages (Mac-3+ cells), dendritic cells (CD11b+ cells) and natural killer (NK) cells (pan-NK+ cells) on day 30 after IRI. The data were presented as mean ± SD,and were compared using one-tailed t-testGuo et al. BMC Nephrology  (2016) 17:133 Page 8 of 15Col3a1, MMP9 and TIMP1. Also, it was noticed that asignificant increase (1.3–1.7 fold change, p < 0.05) wasseen in the expression of AKT1, IL-1α, ITGB1, MMP14,PDGFA and TGIF1 (Additional file 2: Table S1). All thesedata indicated that these genes/pathways might mainlymediate the biological impacts of CLU deficiency on theinitiation and progression of renal fibrosis and chronic in-flammation after ischemic AKI.DiscussionRecent literature suggests that the full recovery of renalfunction after AKI is suboptimal because the long-term ef-fects of AKI are associated with development of CKD,worsening of preexisting CKD and progression to ESRD[8, 47, 48]. Thus, understanding of the pathways mediatingthe long-term pathologic effects of AKI may eventuallylead to develop a strategy for reducing or preventing theincidence of CKD or ESRD. Our previous studies havedemonstrated that CLU deficiency worsens IRI and delaysinjury repair in adult kidneys [22, 26, 33]; however, the roleof CLU deficiency in the long-term effects of ischemicAKI has not been investigated. The present study demon-strates that although CLU expression does not preventrenal atrophy after IRI, CLU deficiency is associated withthe loss of renal function, more damage in the renal tu-bules and an increase in both cellular infiltrates (particu-larly CD8+ T cells and Mac3+ macrophages) and tissuefibrosis. All these negative impacts of CLU deficiency onTable 2 The percentage of each phenotype of immune cells in the spleensWT (n = 4) KO (n = 4) p value (WT vs. KO)Total isolated splenocytes (× 106) 369.36 ± 177.5 373.5 ± 169.31 0.4871CD4 (%) 15.85 ± 1.34 16.43 ± 1.92 0.3095CD8 (%) 6.67 ± 1.56 8.13 ± 1.8 0.1839CD45R/B220 (%) 59.63 ± 6.38 56.03 ± 6.37 0.2275Mac-3 (%) 1.84 ± 0.42 1.37 ± 0.41 0.1620CD11b (%) 5.67 ± 1.48 6.27 ± 1.38 0.2460pan-NK (pan) (%) 3.41 ± 0.32 3.19 ± 0.33 0.1182The spleens were collected from the same animals as described in Table 1, and the phenotypes of their splenocytes were determined using the FACS analysis,showing T cells (CD4+ and CD8+ cells), B cells (CD45R/B220+ cells), macrophages (Mac-3+ cells), dendritic cells (CD11b+ cells) and natural killer (NK) cells(pan-NK+ cells). The data were presented as mean ± SD, and were compared using one-tailed t-testFig. 6 An association of CLU deficiency with more renal tubular injury after 30 days of IRI. The sections of CLU null and WT kidneys, harvestedafter 30 days of IRI, were stained with periodic acid-Schiff (PAS), and the number of injured tubules, including cellular loss (atrophy), intratubularcast formation, tubular cell flattening and vacuolation, in renal cortex was counted in each microscopic view (200x magnification). a Typical microscopicviews of kidney sections in each group (KO: CLU KO kidneys; WT: WT kidneys). G: glomerulus; *: damaged tubules. b The number of injured tubules wascounted in at least 20 randomly selected views in two separate sections of each kidney and was presented in average per view. Data are presented asmean ± SD of each group. KO group vs. WT control: P< 0.0001 (two-tailed t-test, n= 8)Guo et al. BMC Nephrology  (2016) 17:133 Page 9 of 15the kidney after IRI may be mainly mediated by a cascadenetwork of down-regulation of EGF and up-regulation ofCCL12, Col3a1, MMP9 and TIMP1.During the early or initial phase of AKI, sub-lethal in-jury of both tubular epithelial and endothelial/vascularcells is seen along with acute inflammation, followed bycellular repair and organ integrity re-establishmentuntil functional recovery [2, 15, 49]. However, evenafter kidney function returns to baseline, patients arestill at increased risk for the development of CKD after2.5–3.4 years [47, 50]. The AKI-to-CKD transition isconfirmed in rodent models of ischemic AKI and 3–4weeks after IRI, the kidneys exhibit remarkable accu-mulation of tissue fibrosis [51–54]. Currently, thepathophysiology of AKI to CKD or AKI-initiated renalfibrosis is not completely clear, especially in humans,Fig. 7 An association of CLU deficiency with more renal fibrosis (tubulointerstitial fibrosis, glomerulopathy and vascular fibrosis) after 30 days ofIRI. The sections of CLU KO and WT kidneys, harvested after 30 days of IRI, were stained with MT. a Typical microscopic views of kidney sectionsin each group (KO: CLU KO kidneys; WT: WT kidneys), showing blue stain of collagen accumulation in the tubulointerstitial area, inside the glomerulusand interlobular arterial wall, and in the perivascular space. G: glomerulus; PT: proximal convoluted tubule; IA: interlobular artery; IF: interstitial fibrosis.b The extent of tubulointerstitial fibrosis was semi-quantitatively scored in at least 20 randomly selected views in two separate sections of each kidneyand was presented in average per view. Data are presented as mean ± SD of each group. KO group vs. WT control: P < 0.0001 (two-tailed t-test, n = 8).c The percentage of affected glomeruli (glomerulopathy), including glomerulosclerosis (focal and seqmental sclerosis) and glomerular hypertrophy,was counted in two separate sections of each kidney, and the range of 180 to 250 glomeruli of each kidney was examined. Data are presented asmean ± SD of each group. KO group vs. WT control: P = 0.0300 (one-tailed t-test, n = 8). d The percentage of affected interlobular arterioles orarteries, determined by the presence of the fibrous intima, was counted in two separate sections of each kidney, and the range of 10 to 20interlobular arteries or arterioles of each kidney was examined. Data are presented as mean ± SD of each group. KO group vs. WT control:P = 0.0144 (two-tailed t-test, n = 8)Guo et al. BMC Nephrology  (2016) 17:133 Page 10 of 15but experimental studies have identified several mecha-nisms, such as activation of memory or effector T cellsand M2 macrophages [17, 54, 55], infiltration of bonemarrow-derived myofibroblasts [51, 53], endothelial injurywith vascular rarefaction and hypoxia [16, 17, 56, 57], andepigenetic changes and G2/M cell cycle arrest in epithelialcells [17, 58]. All of these components (pro-inflammatoryimmune cells, myofibroblasts, vascular rarefaction/hyp-oxia, and tubular epithelial cells) may play different rolesduring the transition from AKI to CKD in a cascademanner, but how they interact with each other remainsfor further investigation. Recently, Tanaka et al. [16] haveproposed a central role of hypoxia in the AKI to CKDtransition, and have hypothesized that after functional re-covery from the early phase of AKI, the expression of vas-cular factors, such as vascular endothelial growth factor(VEGF) in tubular cells, is not yet restored, which resultsin capillary rarefaction leading to renal hypoxia, and con-sequently activation of inflammatory reactions, inducesmyofibroblast migration and/or differentiation and dam-ages the regenerated tubules. All of these events togetherlead to tubulointerstitial fibrosis [16]. The primary role ofhypoxia is supported by the fact that the density of bloodvessels decreases by almost 45 % after 4 weeks of ischemicAKI [56], and treatment with VEGF preserves microvascu-lar density and prevents renal fibrosis [59]. In this study,however, by PCR array analysis the expression of VEGFand EDN 1 (endothelin 1) was not different between CLUKO and WT kidneys (Additional file 2: Table S1), suggest-ing that the more severe fibrosis in CLU null kidneys maynot be related to the hypoxia/vascular rarefaction, or thehypoxia is not a major factor for the CLU deficiency-related renal fibrosis after IRI.It has been demonstrated that IRI initiates progressiverenal weight or volume loss accompanying with tubularcell death (both apoptosis and necrosis) and interstitialinfiltration [37–39], and our group and others usingWT mice compared with CLU negative controls (CLUKO mice) have demonstrated that CLU is an anti-apoptotic or a prosurvival molecule in the kidneyagainst IRI [22, 26, 33, 40]. In the present study of thetransition of IRI to renal fibrosis, the data show thatFig. 8 An association of CLU deficiency with more positivity in α-SMA stain. The expression of α-SMA (a myofibroblast marker) in the sections ofCLU null and WT kidneys, harvested after 30 days of IRI, was examined by using a routine immunohistochemical method. Data were a typicalmicroscopic view of the renal cortex in each group (KO: CLU KO kidneys; WT: WT kidneys), showing dark brown stain of α-SMA-expressing cells inthe tubulointerstitial area and tubular epithelium, inside the glomerulus and interlobular arterial wall, and in the perivascular space. G: glomerulus;PT: proximal convoluted tubule; IA: interlobular artery; and IV: interlobular vein. Black arrows: infiltrating α-SMA+ cells; red arrows: tubular α-SMA+ cellsTable 3 The change (>2-fold change, p < 0.05) of fibrosis-related genes in CLU KO kidneys compared to WT controls after 30 days of IRIGene (protein) Fold change in KOkidneys vs. WT controlsp value(n = 4)Main functionsEgf (Epidermal growth factor) −2.7208 0.0341 The growth of epidermal and epithelial cells, and some of fibroblastsccl12 (Chemokine (C-C Motif) ligand 12) 2.1439 0.0130 The recruitment of monocytes, lymphocytes and fibrocytescol3a1 (Collagen, Type III, alpha 1) 2.0162 0.0457 Fibril formingmmp9 (Matrix metallopeptidase 9) 2.573 0.0132 The degradation of the extracellular matrixtimp1 (Tissue inhibitor of metalloproteinase 1) 2.0831 0.0260 Inhibition of the matrix metalloproteinasesFour renal tissues (cortex) were randomly selected from each group for PCR array analysis of gene expression using mouse fibrosis PCR array (CatalogNo. PAMM-120Z). Negative in fold change: down-regulated; positive in fold change: up-regulatedGuo et al. BMC Nephrology  (2016) 17:133 Page 11 of 15CLU deficiency does not affect the kidney weight or at-rophy (Fig. 1), but it is associated with a decrease in thenumber of intact tubules (Fig. 6), and at the same timewith an increase in the cellular infiltrates in the intersti-tial spaces (CD8 T cells and macrophages) and α-SMA+myofibroblasts (Figs. 5, 7 and 8), suggesting that thesurvival and/or proliferation of different kidney cells,particularly tubular epithelial cells versus (myo) fibro-blasts, may be regulated by CLU differently. Also, weshowed that up-regulation of MMP9, TIMP1 and per-haps the membrane-type MMP14 was found in CLUKO kidneys after IRI. In general, MMP9 directly de-grades extracellular matrix and is produced by a rangeof immune cells (e.g. lymphocytes and macrophages),and other cells including fibroblasts and endothelialcells following the exposure to pro-inflammatory cyto-kines (e.g. TNF-α, IFN-γ and IL-1β) [60], which is inagreement with the increase in cellular infiltrates, par-ticularly CD8+ T cells and macrophages, and perhapsIL-1α within the CLU KO kidneys. More interestingly,MMP9 produced by renal tubular cells, macrophagesand myofibroblasts contributes to renal fibrogenesis viaosteopontin cleavage, which in turn recruits macrophageand induces renal tubular epithelial-mesenchymal transi-tion (EMT) [61, 62], suggesting that MMP9 but not TGF-β is a major fibrotic factor for fibrosis in CLU KOkidneys. The major biological function of TIMPs in-cluding TIMP1 is to inhibit MMPs by forming a 1:1enzyme-inhibitor complex, but the role of TIMP1 inkidney fibrosis has not been well investigated as oftoday. TIMP1 binds to proMMP9 to forms a specificcomplex [63], that may play a role in proMMP9 activa-tion. Also TIMP1 exhibit other cellular activities, suchas stimulation of cell proliferation of keratinocytes [64]and other cells [65]. However, the underlying mechan-ism by which TIMP1 involves in renal fibrosis remainsfurther investigation.Based on the evidence in literature, it is postulated thatCLU can restrain the transition of IRI to renal fibrosis inthe kidney by several mechanisms. First, complementactivation plays a role in the progression of fibrosis in thekidney, as previous studies have shown that inhibition ofcomplement activation reduces chronic inflammatoryinjury to renal tubular cells [66, 67] and endothelialcells [68], which is associated with reduced tissue fibro-sis [66, 68]. CLU is a major glycoprotein in all thephysiological fluids including the plasma [21], and thissecreted form of CLU inhibits lytic terminal comple-ment cytotoxicity in many early studies [29, 69, 70].Thus, CLU in the blood and/or locally secreted by renalepithelial cells in WT mice may inactivate the comple-ment during the transition of IRI to tissue fibrosis,which at least partially reduces leukocyte inflammation(both CD8 T cells and macrophagies) and tubularatrophy. Secondly, intracellular CLU is a cytoprotectiveprotein that inhibits cell apoptosis by interacting withBAX or GRP78 [71–73], or promotes cell survival byactivating Akt and NF-kB pathway [74, 75] and prosurvi-val autophagy [40, 76]. CLU expression has been con-firmed in cultured glomerular mesangial and glomerularepithelial cells, and tubular epithelial cells [26, 40, 77–79],and facilitates cell survival following the exposure to pro-inflammatory cytokines (IFN-γ and TNF-α) [26] and hyp-oxia [40]. Thus, any of these CLU-expressing kidney cellsin WT kidneys may have better survival than those inCLU KO counterparts under the chronic inflammatory at-tack and/or hypoxia, resulting in the prevention of neph-ron damage as seen in Fig. 6. Finally, the lack of CLUexpression in tubular epithelial cells causes cell cycle ar-rest at G2/M phase in vitro [33], and worsens fibrosis inthe kidneys after IRI in this study. These data may be con-sistent with a previous study, showing that c-jun NH2-ter-minal kinase (JNK) signaling mediates the cell cycle arrestat G2/M phase in proximal tubular cells, and treatmentwith a JNK inhibitor, or bypassing the G2/M arrest by ad-ministration of a p53 inhibitor rescues renal fibrosis afterIRI [58]. Therefore, the potential of the down-regulationof EGF, G2/M phase interruption and more cell deathprobably result in the poorer maintenance of epithelial in-tegrity of the nephron in the kidneys of CLU KO miceafter IRI, which may contribute to more function loss(Fig. 4) as well as more space for infiltrates and myofibro-blasts within the CLU null kidneys.Myofibroblasts are commonly considered as the pre-dominant effector cells in kidney fibrosis [80, 81], and arecent study using multiple genetically engineered micehas identified that a half of renal myofibroblasts arefrom local resident fibroblasts through proliferation, andthe other half are derive through differentiation frombone marrow (35 %), the endothelial-to-mesenchymaltransition (EndMT) (10 %) and EMT (5 %) [45], suggest-ing that renal fibrogenesis is involved in multiple celltypes and multiple biological processes. The presentstudy showed that more myofibroblasts were seen inCLU KO kidneys (Fig. 8), which is confirmed by PCR array,showing the higher transcript expression of Acta2 (α-SMA,1.3753 fold increase with P = 0.0848), collagen (Col3a1),and fibrotic MMP9 [62] along with up-regulation of CCL12(Table 3) and perhaps PDGF (a fibroblast growth factor).CCL12 stimulates the proliferation of infiltrating fibrocytes/bone marrow-derived myofibroblasts [82, 83], suggestingthat a high level of CCL12 benefits the expansion of thismyofibroblast population in CLU null kidneys. More in-terestingly, by immunohistochemical stain many tubules inthese CLU KO kidneys are stained positively with α-SMA(Fig. 8), suggesting an important role of EMT in renal fib-rosis in these kidneys. However, little is known about CLUexpression and its function in renal fibroblast proliferationGuo et al. BMC Nephrology  (2016) 17:133 Page 12 of 15and survival, and myofibroblast differentiation from all dif-ferent origins, which remain further investigation.ConclusionsThe transition of AKI to CKD has major clinical signifi-cance in patients, but its molecular pathways are notfully understood. CLU is a chaperone-like protein andhas different activities in different cells or experimentalsystems [22]. Our previous studies have demonstratedthat CLU plays an important role in the maintenanceand restoration of renal tubular integrity during the earlyphase of IRI [26, 33]. The data from current study indi-cate that as compared to CLU-expressing controls CLUdeficiency has negative impacts on the preservation ofkidney function and its normal architecture during thetransition of IRI to renal fibrosis or CKD development.Also, our PCR array reveals that MMP9 but not TGF-βmay be a major fibrotic factor for CLU deficiency-inducedrenal fibrosis. However, further understanding of the mo-lecular mechanisms underlying the negative impacts ofCLU deficiency on the kidney is needed, which may leadto develop an effective strategy to reduce the incidence ofCKD after AKI.Additional filesAdditional file 1: Figure S1. Tubular dilatation and degeneration inouter area of renal cortex of some WT kidneys. The sections of WT kidneys(≥ 1.5 of fibrosis score) were stained with MT (top) or H&E (middle andbottom). Data were presented as a typical microscopic image of renalcortex in each type of stain (MT or H&E stained sections), showing thetubular dilatation and degeneration in the squared area of the renal cortex.(TIFF 9328 kb)Additional file 2: Table S1. Fibrosis-related gene expression in CLU KOkidneys compared to WT controls after 30 days of IRI. (DOCX 21 kb)AbbreviationsAKI: Acute kidney injury; ANOVA: Analysis of variance; BUN: Blood ureanitrogen; CCL12: Chemokine (C-C motif) ligand 12; CKD: Chronic kidneydisease; CLU: Clusterin; Col3a1: Collagen, Type III, alpha 1; CTGF: Connectivetissue growth factor; EDN: Endothelin; EGF: Epidermal growth factor;EMT: Epithelial-mesenchymal transition; ESRD: End-stage renal disease;FACS: Fluorescence-activated cell sorting; HE: Hematoxylin and eosin;IFN: Interferon; IL: Interleukin; IRI: Ischemia-reperfusion injury; JNK: c-jun NH2-terminal kinase; KO: Knockout; MMP: Matrix metallopeptidase; MT: Masson’strichrome; PAS: Periodic acid-Schiff; PBS: Phosphate buffered saline;PDGF: Platelet-derived growth factor; SD: Standard derivation; SMA: Smoothmuscle actin; TBS: Tris buffer saline; TGF: Transforming growth factor;TIMP1: Tissue inhibitor of metalloproteinase 1; TNF: Tumor necrosis factor;UUO: Unilateral ureteral obstruction; VEGF: Vascular endothelial growthfactor; WT: Wild typeAcknowledgementsThis study was supported by grants from the Kidney Foundation of Canada(MEG, CYCN and CD). JG and XL received funding support from Natural andScience Foundation of Hubei Province (No. 2012FFA096) and the FundamentalResearch Funds for the Central Universities (No. 2042014KF0115), and HW fromNational Natural Science Foundation of China (No. 81273257 and 81471584)and Tianjin Application Basis and Cutting-Edge Technology Research Grant(No. 14JCZDJC35700).Availability of data and materialsThe data were deposited in the website of figshare.com, and the materialsavailable upon request.Authors’ contributionsJG and QG performed the experiments and analyzed the data; XL, HW, MEG,CYCN and CD conceived and designed the experiments; MEG contributedreagents and mice; CD analyzed the data and JG, CYCN and CD contributedthe writing of the manuscript. All the authors read and approved the finalversion of the manuscript.Competing interestsThe authors declare that they have no competing interests.Ethics approval and consent to participateThe animal experiments in this study were performed in accordancewith the Canadian Council on Animal Care guidelines under the protocol(No: A11-0409) approved by the Animal Use Subcommittee at theUniversity of British Columbia (Vancouver, BC, Canada).Author details1Department of Urologic Sciences, University of British Columbia, Vancouver,BC, Canada. 2Department of Urology, Renmin Hospital of Wuhan University,Wuhan, Hubei, China. 3Department of General Surgery, Tianjin MedicalUniversity General Hospital, Tianjin, China. 4Vancouver Prostate Centre,Vancouver, BC, Canada. 5Department of Urologic Sciences, The University ofBritish Columbia, VGH-Jack Bell Research Centre, 2660 Oak St, Vancouver, BCV6H 3Z6, Canada.Received: 3 March 2016 Accepted: 12 September 2016References1. 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