chronic kidney disease ( ckd ) is a major health problem that has reached epidemic proportions and may lead to end - stage renal disease or early cardiovascular death . moreover , the increasing incidence of diabetes , hypertension , and obesity will result in future increases in the number of patients with ckd . available therapy for ckd only delays , but does not prevent , disease progression . besides , there are still no valid biomarkers that more accurately reflect the severity of the underlying renal histopathological changes and predict ckd progression or death . among the potential biomarkers and therapeutic targets , the ccn family member 2 ( ccn2 ) has emerged as an interesting candidate . ccn2 was initially described as the major platelet derived growth factor - related mitogen secreted by human vascular endothelial cells and named connective tissue growth factor ( ctgf ) . this matricellular protein is a member of the ccn family of secreted , cysteine - rich regulatory proteins ; therefore , the term ccn2 is used , as a proposal for uniform nomenclature . ccn2 is a developmental gene , silenced in the adult kidney and reexpressed during kidney injury . ccn2 levels in plasma or urine have been proposed to behave as risk biomarkers for ckd [ 57 ] and for cardiac dysfunction in patients exhibiting myocardial fibrosis and chronic heart failure . initial studies showed that ccn2 contributed to fibrosis , and it was proposed as an antifibrotic target [ 10 , 11 ] . experimental studies have shown that inhibition of endogenous ccn2 by antisense oligonucleotides slows disease progression in experimental diabetic nephropathy , unilateral ureteral obstruction , and nephrectomized tgf-1 transgenic mice [ 3 , 1214 ] , suggesting that selective ccn2 blockade could be used to treat renal disease . ccn2 contains four functional modules than can be cleaved by proteases leading to several degradation products with biological activity . among these degradation fragments , the c - terminal cysteine knot module 4 of 11.2  kda , named here ccn2(iv ) ccn2 binds to the epidermal growth factor receptor ( egfr ) , through this c - terminal module . many studies have described that ccn2 and ccn2(iv ) share many biological responses , including regulation of fibrosis , activation of the egfr pathway , and downstream signalling , including mapks cascade . emerging experimental evidences support the novel concept of ccn2 as a proinflammatory cytokine [ 2 , 18 ] . ccn2 is a chemotactic factor for immune cells , promotes cell adhesion and migration , and upregulates proinflammatory factors , including cytokines , chemokines , and adhesion molecules in resident cells [ 2 , 1821 ] . we have previously demonstrated that systemic administration of recombinant ccn2(iv ) acutely induced th1/th2 cytokine production in the murine kidney after 24 hours , suggesting that ccn2 could induce inflammation in vivo . moreover , chronic ccn2(iv ) administration caused a sustained kidney proinflammatory response , mainly characterized by activation of the th17 immune response . ccn2 as a mediator or coactivator of tgf- mediated profibrotic responses [ 2 , 9 , 11 , 22 ] . ccn2 overproduction has been proposed to play a major role in pathways that lead to fibrosis [ 2 , 11 ] . indeed , the notion that ccn2 is a downstream profibrotic mediator of tgf- is the chief operating paradigm in the field , but there is no data on the effect of tgf- blockade in ccn2 actions in vivo . in this paper we have investigated the effect of tgf- blockade in experimental ccn2(iv)-induced renal damage , focusing on the regulation of inflammation and the modulation of th17/treg responses . studies were performed in adult male c57bl/6 mice ( 912 weeks old , 20  g ; obtained from harlan interfauna ibrica ) and maintained at the local animal facilities under special pathogen free conditions . all procedures on animals were performed according to the international and instituto de investigacin sanitaria fundacin jimnez daz animal research committee guidelines . mice received a single intraperitoneal injection ( i.p . ) of ccn2(iv ) at the dose of 2.5  ng / g of body weight , dissolved in saline ( n = 10 mice ) , as previously described [ 17 , 18 ] and were studied 10 days later . the purity of ccn2(iv ) ( obtained from mbl / peprotech , bionova ) was confirmed by maldi - tof ( not shown ) . we have previously described that systemic ccn2(iv ) administration caused a sustained inflammatory response in the kidney that peaked at 10 days ; therefore , this time point was chosen for the experiments . for tgf- neutralization experiments , mice were injected with an anti - tgf- pan - specific neutralizing antibody ( 100  g / mouse ) or their corresponding igg control ( r&d , n = 10 mice per group ) , starting 24  h before ccn2(iv ) injection and every 72  h thereafter until sacrifice at 10 days , following a previously described neutralization protocol . one kidney from each mouse was fixed in buffered formalin , embedded in paraffin , and used for immunohistochemistry . the other kidney was snap - frozen in liquid nitrogen for gene and protein studies . immunostaining was carried out in 3  m thick tissue sections that were deparaffinized and antigen retrieved using the pt link system ( dako diagnsticos ) with sodium citrate buffer ( 10  mm ) adjusted to ph 6 or ph 9 depending on the immunohistochemical marker . the endogenous peroxidase was blocked and then sections were incubated for 30  min at room temperature with primary antibody : anti - cd3 and anti - cd4 ( dako ) or anti - f4/80 ( serotec ) . after washing , slides were treated with the envision duoflex doublestain system using 3,3-diaminobenzidine . for f4/80 staining , the total number of positive stained cells was quantitated in 5 randomly chosen fields ( 200x ) using image - pro plus software . negative controls were incubated with a nonspecific immunoglobulin of the same isotype as the primary antibody and without primary antibody . kidney extracts were lysed in lysis buffer [ 50  mm tris - hcl , ph 7.4 , 150  mm nacl , 2  mm edta , 2  mm egta , 0.2% triton x-100 , 0.3% np40 , 100  m phenylmethylsulfonyl fluoride , 1  mm dithiothreitol , 100  m na3vo4 , and 1  mm protease - inhibitor cocktail ( sigma ) ] . tissue protein extracts ( 30  g / lane ) were separated on 812% polyacrylamide - sds gels under reducing conditions . samples were then transferred onto pvdf membranes ( bio - rad ) , blocked in tbs with 0.05% tween-20 and 5% nonfat dry milk , and then incubated overnight at 4c with the primary antibodies and subsequently incubated with peroxidase - conjugated igg ( amersham ) and developed by ecl chemiluminescence ( ge healthcare ) . autoradiographs were scanned using the gs-800 calibrated densitometer ( quantity one , bio - rad ) . primary antibodies were affinity purified anti - mouse / human / rat foxp3 ( 1  :  1000 ) ( e - bioscience : 14 - 4774 ) , ngal ( 1  :  500 ) ( santa cruz , sc-18698 ) . the efficacy of protein loading and transfer to membranes was assessed by incubation with mouse anti - gapdh antibody ( 1  :  5000 ) ( chemicon : mab374 ) . cdna was synthesized using the high capacity cdna archive kit ( applied biosystems ) using 2  g of total rna primed with random hexamer primers , following the manufacturer 's instructions . multiplex rt - pcr was performed using fluorogenic ( fam ) taqman mgb probes and primers designed by assay - on - demand gene expression products ( applied biosystems ) : mcp-1 mm00441242_m1 and rantes mm01302428_m1 . the mrna copy numbers were calculated for each sample by the instrument software using ct value . results were expressed in copy numbers , calculated relative to control mice , after normalization against 18s ( 4210893e vic ) . in blood samples from different mice groups , circulating levels of t lymphocytes cell surface staining was performed using fitc - labeled anti - cd3 or anti - cd8 and pe - labeled anti - cd4 ( bd pharmingen ) . after cell surface staining , foxp3 was stained using foxp3 staining kit ( bd pharmingen ) according to manufacture instruction . flow cytometry analysis was conducted on a facscalibur ( bd biosciences ) with cell quest pro software . statistical analysis was done using the spss statistical software ( version 11.0 , chicago , il ) . after kolmogorov - smirnov test that determined the nonnormal sample distribution of the data , differences between groups were assessed by mann - whitney u test . to evaluate the effect of tgf- blockade on ccn2(iv)-induced renal damage , active tgf- was blocked using a pan - specific neutralizing anti - tgf- antibody or its corresponding isotype igg , and renal damage was evaluated by assessment of the biomarker of renal injury , neutrophil gelatinase - associated lipocalin ( ngal ) . in ccn2(iv)-injected mice , kidney ngal protein expression levels were elevated showing a significant upregulation in the ccn2(iv)-injected tgf--treated mice ( figure 1 ) . the main pathological feature of ccn2(iv ) administration to mice was kidney infiltration by leukocytes and upregulation of proinflammatory mediators , while renal fibrosis was not observed . treatment with a tgf- neutralizing antibody significantly increased the number of infiltrating inflammatory cells , mainly f4/80 monocytes - macrophages , in the kidney of ccn2(iv)-injected mice , compared to igg - treated ccn2(iv)-injected mice ( figures 2(a ) and 2(b ) ) . moreover , renal gene expression of several proinflammatory mediators was elevated in response to tgf- neutralization ( figure 3 ) . the recruitment of immune cells into damaged tissue is mainly regulated by chemokines . among them , mcp-1 is the main chemokine involved in monocytes - macrophages recruitment . in ccn2(iv)-injected mice , renal mcp-1 and rantes gene expression , mcp-1 protein levels were also upregulated in this group ( around 2-fold versus igg - treated ccn2(iv)-injected mice , data not shown ) . these data suggest an activation of the local inflammatory response when tgf- is blocked and confirm the role of tgf- as an anti - inflammatory cytokine . tgf- behaves as an anti - inflammatory factor in some conditions and it has been involved in the differentiation of treg cells , through activation of the transcription factor x - linked forkhead / winged helix ( foxp3 ) [ 26 , 27 ] . we have previously reported that th17 , but not treg , immune response participates in ccn2(iv)-mediated renal inflammation . our next aim was to evaluate whether the effect of tgf- blockade on the renal inflammatory response could be mediated by a th17/treg response imbalance . in ccn2(iv)-injected mice , tgf- blockade did not modify il17a ( figure 4(b ) ) or foxp3 ( figure 4(a ) ) renal levels . although circulating cd4/foxp3 treg cells were not modified in ccn2(iv)-injected mice , tgf- blockade dramatically decreased circulating cd4/foxp3 treg cells ( figure 5(a ) ) . in addition , tgf- blockade increased the number of t cytotoxic lymphocytes ( cd8 ) and decreased the cd4/cd8 ratio ( figures 5(b ) and 5(c ) ) . these data suggest that tgf- blockade could lead to a systemic inflammatory response that increases the kidney susceptibility to inflammation . in this report we described that tgf- blockade increased the kidney inflammatory response to ccn2(iv ) administration . this increased inflammatory response was mainly characterized by the local kidney upregulation of mcp-1 and proinflammatory chemokines and the infiltration of monocytes / macrophages as well as by the dysregulation of the systemic treg immune response . ccn2 regulates numerous cellular processes including cell differentiation , adhesion , proliferation , and , as we described here , inflammation . ccn2 is a multimodular protein of four functional modules : the n - terminal insulin - like growth factor - binding domain ( igfb ) , the cysteine - rich domain ( also called von willebrand type c domain ) , a thrombospondin type 1 repeat domain , and the c - terminal heparin - binding domain [ 2 , 28 ] . functional domains within the ccn2 modules interact with different growth factors , receptors , and matrix components and mediate specific ccn2 responses [ 2 , 28 ] . these modules may be cleaved by proteases to yield several biologically active degradation products . in chondrocytes , the n - terminal ccn2 module , but not the c - terminal module , had a direct interaction with the proteoglycan aggrecan and stimulated its production . in xenopus cells , the n - terminal module , through the cysteine - rich domain , can directly bind to tgf- in the extracellular space , potentiating tgf- receptor binding and smad signalling . in cultured mesangial cells , ccn2 , via its n - terminal module , antagonized tgf-1 binding to tgf- type iii receptor ( endoglin ) and inhibited smad pathway activation . the thrombospondin type 1 repeat domain can bind to extracellular matrix and vascular endothelial growth factor . some authors have hypothesized that ccn2 is a downstream mediator of tgf- profibrotic actions [ 10 , 11 ] . individual ccn2 domains interact with tgf- in a different manner : the c - terminal domain mediated tgf--induced fibroblast proliferation , whereas the n - terminal domain mediated myofibroblast differentiation and collagen synthesis . the c - terminal ccn2 module binds integrins and regulates signalling in fibrosis and inflammation [ 2 , 10 ] . we have recently demonstrated that the c - terminal module of ccn2 binding to egfr regulates renal and vascular inflammation [ 16 , 33 ] . in tubular epithelial cells , v3 integrin directly binds to the c - terminal ccn2 module but is not necessary for the binding of ccn2 to egfr and the subsequent complex formation . all these data show the complexity of ccn2 actions and the importance of the evaluation of ccn2 modules functional activities . although many in vitro studies have demonstrated that tgf-1 induces ccn2 synthesis [ 11 , 12 ] , there are scarce studies evaluating whether ccn2 could regulate tgf- expression or actions . in addition , tgf- synthesis is a complex process with multiple steps of regulation . our group has previously described that systemic angiotensin ii infusion upregulates tissue ccn2 as early as at 3 days , while elevated levels of active tgf-1 were observed later on , at 7 days . moreover , ccn2 induction was associated with inflammation , while tgf-1 overproduction correlated with fibrosis , as assessed by fibronectin and collagen deposition [ 3436 ] . accordingly , ccn2(iv ) administration in mice caused a sustained renal inflammation , but there was no increase in tgf- synthesis or matrix deposition . these data suggest that ccn2 in vivo could be involved in the induction of a proinflammatory or a profibrotic response depending on the presence / absence of tgf-1 . tgf- is a pleiotropic cytokine that has been involved in many human diseases , including proliferative disorders , fibrotic diseases , and immune - mediated pathologies [ 11 , 37 ] . tgf-1 is a key factor in fibrosis , including the kidney fibrosis [ 3843 ] . in cultured renal cells , tgf-1 stimulates extracellular matrix production , inhibits matrix degradation by the regulation of matrix metalloproteinases , and is a key factor in the induction of tubuloepithelial to mesenchymal transition [ 42 , 43 ] . increased levels of active circulating tgf-1 in mice caused renal fibrosis . tgf- blockade ameliorated experimental fibrosis in models of vascular restenosis , spontaneously hypertensive rats , and peritoneal damage . both in ckd patients and in experimental renal fibrosis , elevated renal levels of tgf-1 have been associated with fibrosis , characterized by excessive matrix accumulation in the glomerulus and in the interstitium [ 40 , 4450 ] . tgf-1 blockade by different approaches , including neutralizing antibodies , sirnas , or blockers such as decorin , inhibited fibrosis both in vitro and in experimental renal disease [ 40 , 5053 ] . interestingly , in some of these models , such as puromycin - induced nephrosis or diabetic nephropathy , tgf- blockade worsened both proteinuria and albuminuria [ 40 , 5053 ] . furthermore , in unilateral urethral obstruction , conditional deletion of tgf-1 ameliorated tubulointerstitial fibrosis but increased inflammation . in this paper , we have observed that tgf-1 neutralization increased experimental renal inflammation induced by ccn2(iv ) , and this was mainly characterized by a significant increase in local kidney proinflammatory chemokines and macrophage infiltration . beside its role as a profibrotic factor , tgf- has anti - inflammatory functions . indeed , mice deficient in tgf-1 develop a lethal multiorgan inflammatory disease and died at 3 - 4 weeks of age , and conditional deletion of tgf-1 or its type ii receptor in t cells induced autoimmune disease [ 56 , 57 ] . moreover , increased tgf- plasma levels as a consequence of either exogenous administration or overexpression protect from experimental inflammatory diseases , including arthritis , autoimmune encephalomyelitis , nonobese diabetic mice , and systemic lupus erythematous [ 5860 ] . moreover , mice overexpressing latent tgf- were protected against both renal inflammation and fibrosis in obstructive kidney disease models [ 41 , 61 ] . in experimental vascular damage , tgf- blockade caused local inflammation associated with no reduction in stent - induced neointima formation   or acceleration of atherosclerotic plaque formation [ 6365 ] . the anti - inflammatory actions of tgf-1 have been attributed to its role in the activation of treg response [ 66 , 67 ] . in experimental angiotensin ii infusion , intravenous administration of treg cells inhibited immune cell infiltration and decreased proinflammatory mediators in both renal and vascular tissues . treg cells injection inhibited experimental renal damage , including anti - gbm glomerulonephritis and adriamycin - induced nephropathy [ 70 , 71 ] . patients with lupus nephritis presented elevated th17 immune response and exhibited low systemic levels of tgf-1 and treg cells , compared to healthy subjects [ 72 , 73 ] . we have observed that neutralizing antibodies against tgf- downregulated circulating cd4/foxp3 treg cells in experimental ccn2(iv)-mediated renal damage , suggesting that tgf- blockade significantly impaired the protective effect of treg cells . we have previously described that systemic long - term ccn2(iv ) administration induced a local sustained th17 immune response , characterized by increased kidney il-6 production , rort levels , and stat3 activation , with no changes in renal levels of th1/th2 cytokines or treg - related factors ( tgf- and foxp3 ) , suggesting that th1/th2/treg responses were not modulated by ccn2(iv ) , at least in the murine kidney . now , we have observed that tgf- blockade did not modify th17 immune response , as shown by unaltered renal il-17a levels . moreover , our data indicates that tgf- blockade did not modify renal levels of th17/treg differentiation factors , suggesting that the anti - inflammatory effects could be mediated by the regulation of systemic treg levels . our experimental data support the idea that tgf- exerts anti - inflammatory actions in the kidney and suggest that tgf- blockade may not be an adequate therapeutic strategy for kidney disease .
the ccn family member 2 ( ccn2 , also known as 
 connective tissue growth factor ) may behave as a risk 
 biomarker and a potential therapeutic target for renal 
 disease . 
 ccn2 participates in the regulation of 
 inflammation and fibrosis . 
 tgf- is considered 
 the main fibrogenic cytokine ; however , in some 
 pathological settings tgf- also has 
 anti - inflammatory properties . 
 ccn2 has been proposed 
 as a downstream profibrotic mediator of tgf- , 
 but data on tgf- role in ccn2 actions are 
 scarce . 
 our aim was to evaluate the effect of 
 tgf- blockade in ccn2-mediated experimental 
 renal damage . 
 systemic administration of the 
 c - terminal module of ccn2 to mice caused sustained 
 renal inflammation . in these mice , tgf- 
 blockade , using an anti - tgf- neutralizing 
 antibody , significantly increased renal expression of 
 the ngal ( a kidney injury biomarker ) , kidney 
 infiltration by monocytes / macrophages , and 
 upregulation of mcp-1 expression . 
 the 
 anti - inflammatory effect of tgf- seems to be 
 mediated by a dysregulation of the systemic treg 
 immune response , shown by decreased levels of 
 circulating cd4+/foxp3+treg 
 cells . 
 our experimental data support the idea that 
 tgf- exerts anti - inflammatory actions in the 
 kidney and suggest that it is not an optimal 
 therapeutic target .