31. Mechanism of Tubulointerstitial Fibrosis

Mechanisms of Tubulointerstitial Fibrosis

Michael Zeisberg* and Eric G. Neilson

*Division of Matrix Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts;and Departments of Medicine and Cell and Developmental Biology, Vanderbilt University School of Medicine,Nashville, Tennessee

Progressive tubulointerstitial fibrosis isthe final common pathway for all kidneydiseases leading to chronic renal fail-ure.1,2 Its presence correlates with im-paired excretory function1 and the de-gree of fibrosis3,4 or fibroblast number5

are robust pathologic markers of pro-gression. The histopathology of tubulo-interstitial fibrosis features deposition ofinterstitial matrix in association with in-flammatory cells, tubular cell loss, fibro-blast accumulation, and rarefaction ofthe peritubularmicrovasculature.6 Thereis broad agreement that each of thesehallmarks contributes to relentless pro-gression, although priority and interrela-tionship among the various componentsincluding their genetic predispositionare still muddled by unresolved com-plexity (Figure 1).

Humans lose about 4500 nephronsper year per kidney,7 and although not allagree,8,9 classic inulin clearance studiessuggest GFR falls by 10 ml/min per de-cade of life.10 Therefore, the calculus forconceptualizing normal loss of renalfunction over time begins with a startingnumber of nephrons at birth integratedby the natural history of tissue involution

from a lifetime of subclinical vasculardisease, metabolic stress, cicatrization,andnephrosclerosis;1114 hence, the priv-ilege of aging provides a natural windowinto basic mechanisms of renal fibrogen-esis.

Aging disturbs normal structure-function relationships in the kidney formany reasons,15,16 including the influ-ence of telomere shortening on cell se-nescence,1618 aberrant DNA methyl-ation destabilizing the epigenome ofrenal cells,19 loss of self-renewing stemcells,2022 and diminished rates of tubu-lar cell proliferation.23 These nuclearconsequences of aging are compoundedby dysregulation of cellular energy sen-sors,24 oxidative stress,13,25,26 and mito-chondrial dysfunction26,27 fogging theintegrity of nephron structure with pro-gressive glomerular and tubulointersti-tial fibrosis.12,13,28 PPAR agonists atten-uate some of these aging effects byengaging klotho,29 stabilizing mitochon-drial deterioration,29 and reducing theactivity of TGF,29,30 all to suggest thataging renal tissues are susceptible to thelongitudinal affects of profibrotic sig-nals.19,23,28,31 Persistent subclinical injury

to the tubulointerstitium also affects thenormal physiology of many nephrons atonce, which explains the sensitivity ofGFR to the spread of pernicious fibro-genesis.1

Whereas the molecular mechanismsdriving fibrogenesis are better under-stood from 20 years ago1 and a variety ofpreclinical strategies to inhibit or evenreverse tubulointerstitial fibrosis are ef-fective in rodents, only a few antifibrotictherapies are in clinical use today.2,3234

Finding better targets for future therapyin humans will require deeper under-standing of the molecular signals modu-lating fibrogenic events.

THE EXTRACELLULAR MATRIX

Excessive deposition of extracellular ma-trix, particularly the presence of collage-nous fibers, is the most striking andname-lending feature of tubulointersti-tial fibrosis.6 By definition, fibrosis asso-ciates with both quantitative and qualita-

Published online ahead of print. Publication dateavailable at www.jasn.org.

Correspondence: Dr. Michael Zeisberg, HarvardMedical School, Division of Matrix Biology, BethIsrael Deaconess Medical Center, 330 BrooklineAvenue, RW 736C, Boston, MA 02215. Phone:617-667-3583; Fax: 617-667-0360; E-mail:[email protected]; or Dr. Eric G. Neil-son, Departments of Medicine and Cell and De-velopmental Biology, D-3100 MCN, VanderbiltUniversity School of Medicine, Nashville, TN37232-2358. Phone: 615-322-3146; Fax: 615-343-9391; E-mail: [email protected]

Copyright 2010 by the American Society ofNephrology

ABSTRACTThe pathologic paradigm for renal progression is advancing tubulointerstitialfibrosis. Whereas mechanisms underlying fibrogenesis have grown in scope andunderstanding in recent decades, effective human treatment to directly halt oreven reverse fibrosis remains elusive. Here, we examine key features mediating themolecular and cellular basis of tubulointerstitial fibrosis and highlight new insightsthat may lead to novel therapies. How to prevent chronic kidney disease fromprogressing to renal failure awaits even deeper biochemical understanding.

J Am Soc Nephrol 21: 18191834, 2010. doi: 10.1681/ASN.2010080793

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J Am Soc Nephrol 21: 18191834, 2010 ISSN : 1046-6673/2111-1819 1819

tive changes in matrix.1 In fibrotickidneys the widened interstitial spacesfill with fibrillar material consisting ofpredominantly collagens type I and IIIand fibronectin.1,35 This fibrotic matrixalso contains residual fragments of colla-gen type IV, which are normally found inbasement membranes otherwise sup-porting intact endothelia or tubular epi-thelia36,37 as well as several fibronectinsplice variants that modulate fibrogenicpotential.38 The actual amount of colla-gen produced by renal fibrogenesisseems to attenuate fairly soon after itstarts, whereas the relative extent of fi-brosis seems to increase with time.39 Thisdynamic incongruity can be reconciledby recognizing that the volume of in-jured kidney also decreases as residual re-nal parenchyma collapses around inva-sive interstitial scars.40

Whereas fibrillar collagens spontane-ously assemble in vitro, this is not whathappens in vivo.41 During normal andabnormal tissue remodeling, interstitialcollagens have numerous binding part-ners and those critical for fibrillar assem-bly include fibronectin, collagen type V,fibronectin and collagen-binding inte-grins, and various fibrillins, including la-tent TGF-binding proteins (LTBP).42,43

Prototypical matrix deposition follows a

process where the initial appearance ofcollagen nucleators in interstitial fluidserve as scaffolding for fibrillar collagensproduced by local fibroblasts.43 Fi-bronectin, collagen type V, LTBP, andsecreted protein acidic and rich in cys-teine (SPARC) are interactive regulatorsof this matrix assembly:4244 fibronectinco-localizes with procollagen secretionto establish conformation-dependentrelationships with 51 integrins onfibroblasts43 and collagen type V oper-ates at the fibrillar core of collagen typeI assembly.45 LTBPs facilitate the secre-tion of pro-TGF and provide a mech-anism for its release and activation.42

SPARC is antiproliferative,46 stimu-lates the expression of matrix metallo-proteinases (MMPs)47 and plasmino-gen activator inhibitor-1 (PAI-1),48

and activates integrin-linked kinase(ILK),46 which engages epithelial-mes-enchymal (EMT) or endothelial-mes-enchymal transitions (EndMT) pro-ducing fibroblasts.49 51 All of theseprocesses shape the collagenous matrixfound in normal or disturbed tissues.

Generally speaking, fibrogenesis ini-tiates in small areas at random sites ofinflammation and then expands to be-come diffuse if profibrotic drivers per-sist.52 The accumulation of fibroblasts in

these damaged tissues is linked to the riskof fibrosis.5 Unfortunately, it is difficulttechnically to assign a definitive secre-tory role for collagen to any particularcell type found in these fibrogenic hotspots, and consequently, most in vivo ex-periments rely on surrogate markers ofcollagen secretion such as in situ hybrid-ization of mRNA encoding collagenchains,53 collagen promoter activity,54,55

or the presence of collagen-containingcells.56 Better still, the endoplasmic ex-pression of the collagen type I chaperone,HSP47, seems more related to secre-tion,5760 and cell-specific deletions thatassociate with decreasedmatrix accumu-lation strongly insinuate secretory func-tion.61

Fibroblasts use collagen fibrils as scaf-folding to crawl among damaged tissueplanes, and mathematical modeling sug-gests that fibroblast number and move-ment depends on their sensitivity to che-moattractant diffusion.62 These gradientsincrease collagen alignment in fibrogenictissue to facilitate the accrual of fibroblasts.The details of these events can be difficulttoassess in tissuesbiochemically, as impor-tant modulators of phenotypic change,particularly cytokines,mayexpress at levelsnot easily detectable, and in vitro activatorsor inhibitors may be artificially binary justby their presence in serum.63However, thepresence of PDGF and the absence ofTGF and FGF2/bFGF in fetal woundsthat do not scar compared with the pres-ence of all three cytokines in adult tissuethatdosuggestsdifferential expressionpat-terns of key growth factors may be neces-sary for enduring fibrogenesis.63 Prototyp-ical TGF signaling through Smadpathways64,65 under microRNA66 and epi-genetic control67,68 has one of the strongestprofibrotic effects on matrix produc-tion.69,70 The proximal action of angioten-sin II as a morphogenic cytokine stimulat-ing TGF71 and PAI-172 has also led to thewell-known renoprotective effects of re-nin-angiotensin inhibition.2,73 Whereascontainment of TGF signaling has beenthe centerpiece of various experimentalantifibrotic therapies,7480 knockdown ofTGF type II receptors along the collectingduct paradoxically accelerates renal fibro-sis associated with ureteral obstruction,81

Figure 1. Interactive relationships producing fibrosis. Renal fibrosis constitutively in-volves inflammation, fibroblast activation, injury to the tubular epithelium, and micro-vascular rarefaction. Our understanding of how inflammatory cells, fibroblasts, tubularepithelial cells (TECs), and endothelial cells actively contribute to fibrogenesis hasevolved considerably during the past 20 years and sustained growth factor pressurewithin the microenvironment and increased susceptibility to growth factormediatedstimulation emerge as the principal driving force of fibrosis. The molecular systems thatdetermine choice between pathologic fibrosis and physiologic repair however are stillevolving. Epidemiologic studies identify genetic polymorphisms, epigenetic modifica-tions, and aging as risk factors for chronic kidney disease. Linking these risk factorsmechanistically to fibrosis is a task for the future.


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all to suggest that too much or too little ofeven a single cytokine may be profibro-genic.82

TISSUE PROTEASES

Removal of extracellular matrix from theinterstitiumhingesonawell-wornbelief inthe role of various endogenous proteases.Most studies in this area focus on two fam-ilies of proteases: MMPs and members ofthe plasmin-dependent pathway. Bothclasses of proteases have the potential tofragment some extracellular matrix for re-moval, but also cleave nonmatrix sub-strates, releasing profibrotic growth factorsthat paradoxically trigger unwelcomeconsequences.

MMPs are a family of zinc-dependentendopeptidases that currently includes25 members with varying substrate spec-ificities controlled by tissue inhibitors ofmetalloproteinases 1-4.83 The overlap-ping activity and specificity of MMPsmake it difficult to dissect individual ac-tions in vivo. Most studies in the kidneyfocus on MMP-2 and MMP-9 (bothdegrade collagen type IV84 and perhapscollagen types I and III85), but fail todemonstrate a consistent antifibroticeffect in the interstitium.86,87 For exam-ple, combined pharmacologic inhibitionof MMP-2, MMP-3, and MMP-9 at ad-vanced stages of disease in Alport mice ac-celerates renal fibrogenesis, whereas com-bined MMP inhibition before onset oftubulointerstitial fibrosis is protective.88

More confusing, studies utilizing MMPnull mice show unaccelerated fibrogen-esis, and overexpression ofMMP-2 in tu-bular epithelial cells is sufficient to in-duce tubulointerstitial fibrosis.8789 Theprofibrotic effects of MMP-2 andMMP-9 on renal fibrosis, particularly theactivation of MMP-2 and MMP-14(MT1-MMP) by TGF associated withthe degradation of basement membranestimulating EMT,87 seem to outweightheir antifibrotic potential; in fact, micedeficient in TIMP-3 spontaneous de-velop interstitial fibrosis.90 MMP-14, amembrane-bound activator of secretedMMP-2 and MMP-9 in fibroblasts, iscritical for invasiveness into the intersti-

tial environment containing collagentypes I and III85 and its expression is un-der the control of Snail, which is a keyregulator of the EMTprogram.91,92 Thus,epithelial cells, engaged by TGF, ex-press essential MMPs for both basementmembrane degradation and interstitialinvasion that are ultimately required forsuccessful completion of EMTor detach-ment and loss into the tubular lumen(Figure 2).

The effects of the plasminogen-plas-min system on fibrosis are equally com-plex. Active plasmin is derived from pro-teolytic cleavage of plasminogen bytissue-type plasminogen activator (tPA)or urokinase-type plasminogen activator(uPA), which in turn are inhibited byPAI-1.93 Plasmin degrades some matrixconstituents, including laminin, entactin,perlecan, and fibronectin,93,94 whereasfibrillar collagen seems more resistant.95

Plasmin, perhaps more importantly, alsoaffects cell behavior and function in thefibrotic environment and promotes fi-

brogenesis by activating MMPs96 andstimulating EMT.97 tPA null mice withureteral obstruction have reduced localexpression of MMP-9 and preserved tu-bular basementmembrane with less EMT,and less interstitial fibrosis,98 whereasurokinase cellular receptor (uPAR) nullmice develop worse renal fibrosis withlower expression levels of the antifibroticcytokine, hepatocyte growth factor(HGF).99 Angiotensin II stimulates thePAI-1 promoter in tubular cells100 andtubulointerstitial fibrosis associates withTGF-stimulated PAI-1 expression,101

whereas the genetic deletion of PAI-1ameliorates renal fibrogenesis inmice.72,102,103 The small heterodimerpartner (SHP) represses the transcrip-tion of TGF/Smad3regulated PAI-1expression, and SHP null mice developmore renal fibrosis, whereas SHP over-expression inhibits its development;101

SHP may turn out to be an interestingtherapeutic candidate depending on itsmolecular specificity.

Figure 2. Tissue proteases and tubular decondensation. Whereas proteases are keymodifiers of interstitial matrix, they also are critical for the disruption of basementmembrane. Tubular epithelial cells receive signals from the microenvironment tochange phenotype. As they release from basement membrane, they can either roundup and fall into the tubular lumen or undergo epithelial-mesenchymal transition andinvade the interstitium through rents in damaged basement membrane. SPARCthrough ILK, angiotensin II, and TGF activate nuclear programs (SHP, Snail1, andSmads) that engage in EMT-forming fibroblasts. Part of this mechanism is to stimulatethe release of PAI-1, MMP-2, and MMP-9. MMP-2 and MMP-9 are activated bymembrane-bound MMP-14, which is also modulated by PAI-1, tPA, and plasmin effectson MMPs.

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The profibrotic action of these pro-teases is a paradigm shift in thinkingabout the dynamics of matrix depositionor its dissolution (Figure 2), illustratingthe complexity of balancing modifier ef-fects, timing and context, and weighingwhat seems to be true in vitrowithwhat isnot observed in vivo. Whereas currentevidence argues against MMPs or plas-min to degrade only complexmatrixes invivo, murine studies reporting regressionof tubulointerstitial fibrosis do demon-strate less matrix deposition.104107 Howthis matrix is removed biochemically, orfails to form, is still unclear. Involvementof other proteases, perhaps the lysosomalfamily of cysteine protease cathepsins,may be important.108112 The whole ap-proach to the regulation of collagen depo-sition in renal tissue is ripe for new insight.

INFLAMMATORY CELLS

Tubulointerstitial fibrosis is typically con-ditioned by the infiltration of inflamma-tory cells113 including dendritic cells,114

lymphocytes,1,115,116 macrophages,117 andmast cells.118 Whereas inflammation as awhole contributes significantly to the en-gagement of fibrogenesis, evidence in re-cent years also highlights the antifibroticeffects of various subsets of lymphocytesand macrophages, providing novel po-tential avenues for antifibrotic therapies.

Innate immunity through the activa-tion of toll-like receptors 4 (TLR4)119

andTLR9,120 unlike TLR2 receptors,121 isan important early mediator of renal fi-brogenesis. Lymphocytes precede the in-flux of macrophages.1 Rag-2 null mice,which lack bothmature B andT lympho-cytes, are protected from fibrosis, dem-onstrating that effector lymphocytes arean essential early component of fibro-genesis.116 Adoptive transfer of CD4

cells into Rag/ mice, but not CD8

cells so much, increases fibrogenesis.122

The contribution of CD3 T cells to re-nal fibrogenesis is well established, as ac-tivated cytotoxic T cells attack tubularepithelial cells.2 Administration of PGE1inhibits the effector arm of T lympho-cytes in interstitial nephritis that is over-come by competitive exposure to IL-1.123

Long-term renal fibrosis after ischemia-reperfusion injury also depends on per-sistent infiltration of effector-memory Tlymphocytes, which correlate with onsetof fibrosis in later stages.124 Transfer of Tlymphocytes from mice with prolongedischemia-reperfusion injury to nave re-cipients is sufficient to induce renal in-jury, further highlighting the relevance ofmemory lymphocytes in theprogressionoftubulointerstitial fibrosis.125 In contrast,FoxP3 regulatory T cells (Tregs) bluntkidney injury and are required for physi-ologic kidney regeneration.126 Whereasrecent studies suggest the presence ofCD20B cells are as common asCD3Tlymphocytes in established fibrosis, thefunctional contribution of B cells to renalfibrogenesis is uncertain.2,127,128

When monocytes are recruited todamage the interstitium, they transdif-ferentiate into macrophages.129 There isa strong correlation between macro-phage infiltration and the extent of fibro-sis.130 Activation of TRL9 on macro-phages by CpG-oligodeoxynucleotides isan accelerant for interstitial fibrogen-esis,120 suggesting the dispersion of nu-clear fragments after local cell death mayenhance inflammation. Depletion ofmacrophages also ameliorates fibrogen-esis in mice, highlighting the traditionalprofibrotic view of macrophages.131,132

Macrophages however can be catego-rized into classically activated M1 mac-rophages or alternatively activated M2macrophages. Unlike the M1 macro-phages, M2 macrophages are anti-in-flammatory and provide cues for tissuerepair.129 Infusion of cells enriched forM2 macrophages ameliorates renal fi-brosis in mice.133 Finally, in addition tomacrophages, dendritic cells are critical toantigen processing in tubulointerstitial in-jury;114 their proteasomal processing of al-bumin, for example, creates new antigenictargets.134 The balance or relative presenceof these subsets of cells in renal inflamma-tion over time needs more study.

Mast cells are well-established con-tributors to fibrosis in lung, liver, andpancreas where they release proinflam-matory cytokines, including chemokinesCCL2-5, TNF, and leukotrienes.118

Surprisingly, however, mast celldefi-

cient KitW-sh/KitW-sh mice display en-hanced fibrosis in the kidney.118,135

Such accelerated fibrosis associateswith increased levels of TGF, suggest-ing that mast cells in kidney play a rolein suppressing the fibrotic process.118

Several new biologic modifiers of theimmune system reduce interstitial fibro-sis. PPAR agonists reduce the numberof macrophages by attenuating TGFexpression,30 and three independentstudies report the administration of aCCR1 antagonist, anti-TNF blockingantibodies, or an IL-1 receptor antago-nist are sufficient to ameliorate renalfibrosis, even when treatment is initi-ated in advancing disease.136138 Thesetherapeutic studies corroborate therole of inflammation not only in theinitiation but also in perpetuation offibrogenesis.

FIBROBLASTS

Fibroblasts in the renal interstitium areconsidered the principal source of fibril-lar matrix (collagen types I and III), andtubulointerstitial fibrosis inevitably asso-ciates with a robust accumulation of fi-broblasts,5 displaying increased intrinsicproliferative activity139,140 or, in some fi-broblasts, what is called an activated phe-notype expressing -smooth muscle ac-tin (SMA).70,141,142 Tissue fibroblastsexhibit phenotypic heterogeneity,143145

more likely based on origin,54,146,147 butthis heterogeneity in tissue is also bimo-dal: those cells that are RhoA-dependentSMA148 and those that are not. Onegeneric dilemma with SMA is thatwhen used as a fibroblast marker in in-jury, it is not distinguishable fromSMA mural cellsvascular smoothmuscle cells or venular pericytes;149,150

nor does thismarker detect the larger popu-lation of SMA fibroblasts.58,146,147,151,152

The view of myofibroblasts as principal me-diators of renal fibrosis is also based on thesuggestion that fibrosis associates withde novo accumulation of SMA

cells.51,153,154 This notion, however, hasto be tempered by observations thatSMA fibroblasts do notmove,155 renalfibrogenesis persists despite decreasing



numbers of SMA myofibroblasts,121

SMA fibroblasts contain and likelyexpress interstitial collagens in vivo,61,152

and mice deficient of SMA developmore fibrosis and their fibroblasts pro-duce more collagen type I than SMA

fibroblasts.156 Unequivocal functionaldata to support the idea that nonmotileSMA myofibroblasts principally me-diate fibrogenesis is lacking.

The most durable fibroblast markerin the kidney is fibroblast-specific pro-tein-1 (FSP1; S100A4).58,146,147,151 Theproclivity for FSP1 in fibroblasts, amem-ber of the S100 family of calcium-bind-ing proteins, was discovered in a differ-ential genetic screen between fibroblastsand epithelial cells,157 and renal fibrosisassociates with a robust accumulation ofFSP1 fibroblasts.5,61,157 A few studiessuggest FSP1 is expressed by macro-phages;55,158,159 however, this notion per-sists from studies using underdetectionmethods for FSP1/S100A4 that may en-hance nonspecific staining and/or fromimproper application of so-called anti-macrophage antibodies that share targetspecificities with fibroblasts and are notmacrophage-specific;158160 FSP1 pro-moter activity in fibroblasts driving aGFP reporter60,161 or Cre recombinase162

clearly discriminate tissue fibroblasts fromF4/80macrophages.FSP1, F4/80 fibro-blasts are also the only population in thekidney proven to contribute to fibrogen-esis, as their ablation in FSP1-tk trans-genic mice ameliorates fibrosis substan-tially.61 This kind of functional deletionexperiment is critical evidence not yet re-ported in FSP1 cells expressing SMA,particularly pericytes.54 For themoment,it is best to think of all tissue fibroblasts aspotentially fibrogenic.58,147,152

The true heterogeneity of fibroblastsin the kidney may be understood by ex-amining the different cellular origins ofthese cells.147 Pools of fibrogenic fibro-blasts comingle either by proliferation ofresident fibroblasts,163 some of whichmay be subject to local or time-depen-dent epigenetic effects;68,164 from localtubular epithelial cells51,59,157,165 or endo-thelial cells (simple squamous epithe-lia)51,166 after EMT or EndMTduring tis-sue injury; after recruitment of FSP1,

CD34 bone marrowderived fibro-cytes;59,146 and perhaps fromblood vesselshedding of SMA pericytes54,150 thatderive developmentally from mesothe-lium through EMT.167,168 It is not yetclear whether FSP1, SMA peri-cytes54,55 should be considered true fi-broblasts or a separate and unique phe-notype of collagen-producing cells.150 Asopposed to tissue-derived fibroblasts,CD34 bone marrowderived fibro-cytes59 may also have a unique lineagefromCD11b, CD115, Gr1 leukocyteprogenitors under the control of CD4Tcells;56 some studies suggest these circu-lating fibrocytes do not contribute to tis-sue fibrogenesis,53,55 whereas other ex-periments suggest they can to a limitedextent.50,56,59 For the moment, it isprobably best to view multiple lineagesas contributing to the final mix of fi-broblast populations in tissue. It is en-tirely unknown if these various lin-eages have priority among individualsor different diseases or if there is indi-vidual variation or preference as fibro-genesis progresses.

Depletion of interstitial fibroblasts byselectively corrupting their DNA replica-tion is one classic strategy for experimen-tal antifibrotic therapy.61 Conditionalexpression of an IB dominant-negativetransgene in fibroblasts also attenuatesfibrogenesis by inhibiting NF-B.60 Butbecause specific targeting of fibroblastshas not yet been achieved without ge-netic manipulation, current antifi-brotic therapies center on variousother approaches; these approaches in-clude blocking PDGF-C,169 PDGF-D,170 or TGF171,172 with neutralizingantibodies or using pirfenidone,173175

pyridoxamine,176 ALK5 receptor ki-nase inhibitors,177 paricalcitol,178 Rasinhibitors,68,179 or PPAR agonists.30

Blockade of AT1R by the small moleculeinhibitorCGP-48933 also attenuates inter-stitial fibrosis,180 as does inhibition of an-giotensin II73 or aldosterone action.32,33

TUBULAR EPITHELIUM

Tubular epithelial cells spark the pro-gression of fibrogenesis to their own

detriment. They do this by modulatingtheir proliferation,23,181 or by provid-ing proinflammatory chemokines andcytokines that induce mononuclear in-flammation and the formation of fi-broblasts (Figure 3).59,182184 These localcytokines provide critical signals mediat-ing primary interstitial injury.185 Sec-ondary interstitial damage after glomer-ulonephritis also depends on the tubulartoxicity of glomerular proteinuria,1,186

the filtration of protein-bound cytokinesfrom plasma to the tubular microenvi-ronment,184,187 or possible cytokine leak-age through bulk fluid flow from the af-ferent arteriole directly into the interstitialspaces associated with the juxtaglomerularapparatus.188 Hydrodynamic forces alongthe tubules themselves are also profi-brotic.189Tubular stretch fromobstructioninduces TGF and NF-B; increases insinglenephronGFRandcompensatory in-creases in fluid shear stress also decreasetPA and increase TGF.

Theepicenter forearlymolecularactionby tubular epithelial cells is through NF-B.190 Tubular cells toggle on NF-Bafter exposure to proteins from tubularfluid,134,191 activation of CD36 scavengerreceptors,25 or exposure to connective tis-sue growth factor,192 CD40 ligand,193 an-giotensin II,194,195 or aldosterone.32,33 Acti-vation ofNF-B is critical for initiating thedownstream release of PAI-1 or chemo-kines and growth factors,196 particularlyIL-1,137 IL-6,197MCP-1/CCL2,198,199RAN-TES/CCL5,113,200 or TNF.200 In opposi-tion, administration of Smad7,201,202 pari-calcitol,203 truncated IB,204 HGF,200,205

spironolactone,32,206 and decoyNF-Boli-godeoxynucleotides207 all attenuate tubu-lointerstitial injury by inhibiting the activ-ity of NF-B. Experimental inhibition ofproinflammatory chemokines such asTNF or CCR1 has also been successfulin preventing progressive interstitialinjury.113,138

With time, persistent tubulointersti-tial injury inevitably associates with mi-tochondrial dysfunction or loss,26,208 for-mation of aglomerular tubuli,209 tubularatrophy,27 and rampant interstitial fibro-sis,146 creating the classic pathologicpicture of renal progression.210 Majorepithelial mechanisms contributing to



chronic tubulointerstitial fibrosis areG2/M cell cycle arrest,181 cellular apo-ptosis,211 and EMT-forming fibro-blasts;147,151 all three processes aremodulated by the transcriptional re-pressor GLIS2.212 EMT is a fundamentalprocess to create cells that will move,155 amodification in lineage maturation thatdisturbs the stateofnucleardiapause in ep-ithelial andendothelial cells.59,213Althoughnot all agree,54,159,214,215 11 studies confirmthese transitions to fibroblasts in a varietyof lineage-tracing experiments fromdiffer-ent tissues, including kidney,50,59,60,165

intestine,216 liver,217 heart,166 lung,218220

and endothelium50,166,221 using 10 differ-ent cell fate reporter constructs.

The role of EMT in forming renal fi-broblasts has been recently reviewed49

and debated;160 several of the lineage-tracing studies unable to detect EMT orFSP1 fibroblasts are likely confoundedbyuse of nonpreferred anti-FSP1/S100A4 an-tibodies and/or inadequate antigen-re-trieval methods during tissue prepara-tion,54,159,215 by relying on a collagenpromoter driving green fluorescent pro-tein used to detect fibroblasts that per-haps may not express in all fibroblastsbecause of methylation or enhancer bi-as,160 and by overlooking lineage evi-dence that SMA mural cells/peri-

cytes may have emerged previouslyfrom EMT.167,168,222 It is increasinglyclear that fibroblasts derive from mul-tiple parental lineages, including epithe-lial cells, endothelial cells, and bonemar-row endosteal cells. The status ofpericytes in fibrogenesis remains an areaof great interest that will require morefunctional evidence of their role one wayor the other. Finally, although EMT isdifficult to assess in humans who are notbiopsied until there is clinical evidence ofdisease,5 early renal allograft biopsiessuggest changes in epithelial phenotypesupportive of transitions preceding fi-brosis,223225 although not all agree.226

Glis2 mutations producing autosomalrecessive NPHP7, a rare form ofnephronophthisis, may be the first sug-gestive evidence of spontaneous or dere-pressed EMT causing renal fibrosis inhumans.212,227

EMT induces a variety of intermedi-ate cell phenotypes, not all of which com-plete their transition to fibroblasts.147,225

This intermediacy and its reversibilitymay depend on the variable persiste-nce of endogenous perturbagens: hypo-xia,165,228230 TGF,231 EGF,231,232 PTH-RP,233 and plasmin97 all induce EMT, asdoes activation of ILK49 or RAGE.234 Vi-tamin D receptors downregulate the re-

nin-angiotensin system driving EMTand TGF and receptor absence acceler-ates fibrogenesis.235

After induction, a hierarchy of tran-scription factors program EMT, includ-ingGLIS2212 andCBF-A,236 amaster reg-ulator of Snail,237 Twist,238 HMGA2,LEF-1, and Ets-1.236 Overexpression ofSnail1237 induces EMT and fibrosis andrecessive mutations in Glis2 spontane-ously invoke EMT in tubular cells inmice with renal fibrosis;212 short inter-fering RNA knockdown of Snail1 alsoblocks EMT during vascular mural celldifferentiation.222 There appears to be agradualness to EMT events in the kid-ney,225 as indicated by the variably activesignaling of -catenin, Snail, or Twist inwatershed epithelial cells along chroni-cally injured tubules that is based on thetime-dependent permissiveness of localcytokine action. Whereas activation ofthe EMTprogram is ameans for parentalcells to resist proapoptotic stimuli,211,239

such tenuous protection from cell deathalso results in loss of parental cell pheno-type.59,240 The true frequency of tubularepithelial cells that complete EMT andbecome fibroblasts is unknown, butlikely depends on the persistence of in-flammation, epigenetic control of fi-brotic gene regulation,67 and ultimatelythe emergence of tubular atrophy withacellular scars that are the consequenceof profibrotic cytokine exhaustion andtubular cell disappearance.

The relevance of EMT for progressionof renal fibrosis is highlighted by studies inwhich administration of bonemorphogenicprotein-7 (BMP7),80 HGF,241,242 the small-molecule inhibitor ILK, QLT-0267,243 orY-27632,aninhibitorofRho/ROCK244all in-hibit tubular EMT and ameliorate fibrogen-esis. Trps1 operates downstream ofBMP7 in the kidney,245 and the availabilityof Smad7controlling thephosphorylationofSmad3 is normally assured by Trps1 inhibi-tion of ubiquintination through Arkadia;246

Trps1 haploinsufficiency raises the levels ofArkadia,decreasingtheavailabilityofSmad7,and thus facilitating fibrosis through TGF/Smad3mediated EMT. Induction of heatshock protein-72 (HSP72) also protects tu-bular epithelia from apoptosis, EMT, and fi-brosis,239 and overexpression of klotho247 or

Figure 3. The cytokine milieu in fibrogenesis. NF-B activators and inhibitors competefor the engagement of mammalian target of rapamycin and release of chemokines andproinflammatory cytokines that draw macrophages, dendritic cells, and T lymphocytesto the tubulointerstitium. These mononuclear cells injure the tubular epithelia and activatefibroblasts. The transcriptional activation of EMT and EndMT is engaged by hypoxia andvarious cytokines, particularly TGF, EGF, and ILK. EMT inhibitors block the developmentof fibroblasts that deposit extracellular matrix into the fibrogenic interstitium.



inhibition of themammalian target of rapa-mycin248 attenuates fibrogenesis in a varietyof experimentalmodels.

Apivotal determinant in the failure to re-coverfrominterstitial injuryistherelativeab-sence of nephron regeneration in kidneysthatbecomefibrotic.181Onereasonfor this isthat glomerulotubular disconnections limitrepair of nephrons.107,209 The glomerulotu-bularneck at the antivascular pole of the glo-merular tuft is an at-risk break point for theformation of aglomerular tubuli249 and sur-prisingly angiotensin inhibition protectsthese unwanted separations.250 Another rea-son is that cell cycle arrest of tubular epithe-lium during acute kidney injurymay stimu-lateprofibrotic signaling.181Whereas tubularcells along atrophic tubules variably increasetheir proliferative activity in advanced tubu-lointerstitialdisease,210 this restorativepoten-tialmaydeclinewithagingthroughthemod-ulation of the adipokine, zinc-2-glycoprotein, knownasZag.23

The regenerative capacity of injured tu-bules absent persistent inflammation is likelythe result of self-renewing tubular cells orperhaps progenitor cells within other nichesalong the nephron.31 A CD24, CD133

progenitor cell niche has been identified inthe parietal epitheliumof Bowmans capsulenear the vascular pole,21,22,251 and the adop-tive transfer of these cells at the timeof tubu-lar injury obviates the appearance of intersti-tial fibrosis.252 The glomerulotubular neckregionisalsowhereexvivodifferentiatedem-bryonic stemcells traffic after adoptive trans-fer during development253 andmay be a tu-bular niche not unlike the vascular pole.21

Expansion of renal progenitor cells is underthe control of histone deacetylases and theirenzymatic inhibition may offer restorativepotential to the kidney.254

THE MICROVASCULATURE

Rarefaction of the peritubular vascula-ture with ensuing chronic hypoxia arehallmarks of tubulointerstitial fibro-sis.107 In early stages of injury the peritu-bular vasculature is damaged by proapop-totic stimuli, particularly transientischemia.255,256Progressive fibrosis thenremodels the peritubular endothelialnetwork governed by imbalance among

pro- and antiangiogenic stimuli257,258 orloss of peritubular endothelial cells byEndMT-forming fibroblasts.50,259

The fibrotic tubulointerstitium is a hy-poxic environment.260,261 Hypoxia resultsfrommicrovascular rarefaction made worsebymatrix expansion throughout the intersti-tium,258 requiring theoxygengradient todif-fuse greater distances from vasoconstrictedor attenuated microvessels to adjacent isch-emic tubules.230,260 Hypoxia also contributesdirectly to the progression of tubulointersti-tial fibrosis by simulating EMTandpromot-ing matrix accumulation from new fibro-blasts.165

Different strategies to improve thischronic hypoxia during renal progres-sion show some promise in experimentalmodels of renal fibrosis. Angiotensin IIinhibition increases capillary blood flowand cortical oxygen levels in normal re-nal tissues,262 and long-acting calciumchannel blockers attenuates the hypoxiaof angiotensin IIinduced tubulointer-stitial injury.263 Inhibition of the vaso-constrictor, endothelin, is also renopro-tective,264 particularly in combinationwith ACE inhibition.265 Activation of thehypoxia-inducible factor (HIF) pathwayor stabilization of HIF proteins attenu-ates renal fibrosis aswell.230,261,266 The ul-timate goal of reversing microvascularrarefaction, however, has been difficultto achieve. Whereas the HIF pathway isan important inducer of angiogenic vas-cular endothelial growth factor (VEGF)proteins,230 systemic administration ofVEGF121 does not improve renal micro-vascular disease.267 Whereas administra-tion of the antifibrotic molecule, BMP7,blocks EMT in the kidney,80 its affect onEndMT in the kidney is unknown; how-ever, BMP7 does inhibit EndMT, endo-thelial loss, and fibrogenesis in experi-mental cardiac fibrosis.166

GENETIC PREDISPOSITION TORENAL PROGRESSION

Whereas revelations regarding new cellularmechanismsofrenalfibrogenesiscontinuetoflourish, the identification of key risk factorsunderlying the true biologic fate of renal tis-sue is far from clear. Risk factors for chronic

kidneydiseaseare rapidly emerging fromep-idemiologic studies, including age,268,269

nephronmass,7,270 gender,268,271 levels of cal-cidiol,272 sympathetic activity,273 obesity anddiabetes,274 cardiovascular disease,275 geneticloci276 and polymorphisms,273,277279 andepigenetic modifications.19 But how theserisk factors modulate fibrogenesis is still un-certain.

Genetic polymorphisms in cytokine riskprofiles in some instances can be explainedusingexistingknowledgeof fibrotic signalingpathways.278 In other cases, it is unclearwhy genetic polymorphisms in uro-modulin280,281 or methenyltetrahydro-folate synthetase282 increase suscepti-bility to fibrogenesis. Insights into therole of epigenetics in renal progressionand fibrogenesis are also just starting toemerge. For example, aberrant hyper-methylation of selected genes includingRASAL1, a suppressor of Ras signaling,contributes to perpetual fibroblast acti-vation and fibrogenesis.68 Furthermore,there are large families of small, naturallyoccurring, noncoding RNAs that inter-fere post-transcriptionally with the stabil-ity or translation of coding mRNA;283 threeprototypical families of interfering RNAsinclude short interfering RNAs (siRNA),microRNAs (miRNAs), and piwi-interact-ing RNAs (piRNA).284 miRNA-192, forexample, promotes the activation ofTGF/Smad3-mediated fibrosis in ure-teral obstruction and 5/6 nephrec-tomy,285 and in diabetic glomeruli,TGF stimulates miRNA-192 to syner-gize with deltaEF1 short hairpin RNAsthat increase Col1a2 E-box activity inmesangial cells.286 Oddly enough, lowlevels of miRNA-192 in human kidneybiopsies of diabetic nephropathy cor-relate with late presentation and less fi-brosis.164 Thus, the interfering RNAstory becomes complicated quickly,largely because the network effect ofmultiple families is not addressed bystudying the action of a single familymember.

CONCLUSIONS

Major progress has been made duringthe last 20 years in characterizing the cel-



lular and molecular mechanisms of in-terstitial injury. The molecular eventsdetermining choice between self-limitedrepair of the tubulointerstitium or pro-gressive fibrogenesis suggest that persis-tent cytokine pressure and the accumu-lation of unwanted inflammatory cellsare the principal effectors of chronicity.There remains a gap in understandingthe genetic drivers of this cytokine pres-sure or sensitivity, and our knowledge ofhow genetic polymorphisms and epige-netic modifications facilitate fibrogen-esis are in their infancy.

Modulating the pivotal role of tubularepithelial cells in their provision of pro-fibrotic stimuli, and the difficulty in pre-venting microvascular rarefaction, re-main new challenges for the future. Therole of determining cells, particularlymyofibroblasts or pericytes, in renal fi-brogenesis needs more clarification incomparison to the known functionalityof FSP1 fibroblasts. A deeper under-standing of the molecular basis for fibro-blast expansion and collagen accumula-tion also requires more work. Newtherapies to abrogate EMT are on the ho-rizon but the chromatin biology modu-lating these phenotypic changes are un-clear; determining the point of no returnfor intermediate states of transition isstill an open question.

Today, the distinction between profi-brotic and antifibrotic stimuli also seemsincreasingly muddledsome macro-phage, T cell subpopulations, and mastcells, many of which were once viewed asunequivocally pernicious, now seemrenoprotective. Tissue proteases, alsoonce considered renoprotective, inmanyrespects now look as if they encouragefibrogenesis. The role of progenitor cellsin regeneration and the depletion of stemcell niches along the adult nephron isalso an underdeveloped area of investi-gation.

Most importantly, although the pres-ence of tubulointerstitial fibrosis has al-ways implied an unalterable fate, thisperception is changing in new preclinicalstudies using unique biologic modifiers.We have renewed optimism that in thenot too distant future some of thesenovel insights will become clinically rel-

evant for patients with chronic kidneydisease.

ACKNOWLEDGMENTS

Some of the cited work came from NIH

grants DK-46282 (E.G.N.) and DK-81576,

DK-81687, and DK-74558 (M.Z.). We thank

Youhua Liu, Raymond Harris, Robert

Colvin, and Yash Kanwar for helpful com-

ments regarding earlier drafts of this manu-

script, and we apologize for the lack of space

to cite many other interesting findings.

DISCLOSURESNone.

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31. Mechanism of Tubulointerstitial Fibrosis