BRIEF REVIEW www.jasn.

org

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

ABSTRACT
The pathologic paradigm for renal progression is advancing tubulointerstitial to the tubulointerstitium also affects the
fibrosis. Whereas mechanisms underlying fibrogenesis have grown in scope and normal physiology of many nephrons at
understanding in recent decades, effective human treatment to directly halt or once, which explains the sensitivity of
even reverse fibrosis remains elusive. Here, we examine key features mediating the GFR to the spread of pernicious fibro-
molecular and cellular basis of tubulointerstitial fibrosis and highlight new insights genesis.1
that may lead to novel therapies. How to prevent chronic kidney disease from Whereas the molecular mechanisms
progressing to renal failure awaits even deeper biochemical understanding. driving fibrogenesis are better under-
stood from 20 years ago1 and a variety of
J Am Soc Nephrol 21: 1819 1834, 2010. doi: 10.1681/ASN.2010080793
preclinical strategies to inhibit or even
reverse tubulointerstitial fibrosis are ef-
fective in rodents, only a few antifibrotic
Progressive tubulointerstitial fibrosis is from a lifetime of subclinical vascular therapies are in clinical use today.2,3234
the final common pathway for all kidney disease, metabolic stress, cicatrization, Finding better targets for future therapy
diseases leading to chronic renal fail- and nephrosclerosis;1114 hence, the priv- in humans will require deeper under-
ure.1,2 Its presence correlates with im- ilege of aging provides a natural window standing of the molecular signals modu-
paired excretory function1 and the de- into basic mechanisms of renal fibrogen- lating fibrogenic events.
gree of fibrosis3,4 or fibroblast number5 esis.
are robust pathologic markers of pro- Aging disturbs normal structure-
gression. The histopathology of tubulo- function relationships in the kidney for THE EXTRACELLULAR MATRIX
interstitial fibrosis features deposition of many reasons,15,16 including the influ-
interstitial matrix in association with in- ence of telomere shortening on cell se- Excessive deposition of extracellular ma-
flammatory cells, tubular cell loss, fibro- nescence,16 18 aberrant DNA methyl- trix, particularly the presence of collage-
blast accumulation, and rarefaction of ation destabilizing the epigenome of nous fibers, is the most striking and
the peritubular microvasculature.6 There renal cells,19 loss of self-renewing stem name-lending feature of tubulointersti-
is broad agreement that each of these cells,20 22 and diminished rates of tubu- tial fibrosis.6 By definition, fibrosis asso-
hallmarks contributes to relentless pro- lar cell proliferation.23 These nuclear ciates with both quantitative and qualita-
gression, although priority and interrela- consequences of aging are compounded
tionship among the various components by dysregulation of cellular energy sen-
Published online ahead of print. Publication date
including their genetic predisposition sors,24 oxidative stress,13,25,26 and mito- available at www.jasn.org.
are still muddled by unresolved com- chondrial dysfunction26,27 fogging the
Correspondence: Dr. Michael Zeisberg, Harvard
plexity (Figure 1). integrity of nephron structure with pro- Medical School, Division of Matrix Biology, Beth
Humans lose about 4500 nephrons gressive glomerular and tubulointersti- Israel Deaconess Medical Center, 330 Brookline
per year per kidney,7 and although not all tial fibrosis.12,13,28 PPAR agonists atten- Avenue, RW 736C, Boston, MA 02215. Phone:
617-667-3583; Fax: 617-667-0360; E-mail:
agree,8,9 classic inulin clearance studies uate some of these aging effects by mzeisber@bidmc.harvard.edu; or Dr. Eric G. Neil-
suggest GFR falls by 10 ml/min per de- engaging klotho,29 stabilizing mitochon- son, Departments of Medicine and Cell and De-
cade of life.10 Therefore, the calculus for drial deterioration,29 and reducing the velopmental Biology, D-3100 MCN, Vanderbilt
University School of Medicine, Nashville, TN
conceptualizing normal loss of renal activity of TGF,29,30 all to suggest that 37232-2358. Phone: 615-322-3146; Fax: 615-343-
function over time begins with a starting aging renal tissues are susceptible to the 9391; E-mail: eric.neilson@vanderbilt.edu
number of nephrons at birth integrated longitudinal affects of profibrotic sig- Copyright 2010 by the American Society of
by the natural history of tissue involution nals.19,23,28,31 Persistent subclinical injury Nephrology

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

 BRIEF REVIEW www.jasn.org

these damaged tissues is linked to the risk

of fibrosis.5 Unfortunately, it is difficult
technically to assign a definitive secre-
tory role for collagen to any particular
cell type found in these fibrogenic hot
spots, and consequently, most in vivo ex-
periments rely on surrogate markers of
collagen secretion such as in situ hybrid-
ization of mRNA encoding collagen
chains,53 collagen promoter activity,54,55
or the presence of collagen-containing
Figure 1. Interactive relationships producing fibrosis. Renal fibrosis constitutively in- cells.56 Better still, the endoplasmic ex-
volves inflammation, fibroblast activation, injury to the tubular epithelium, and micro- pression of the collagen type I chaperone,
vascular rarefaction. Our understanding of how inflammatory cells, fibroblasts, tubular HSP47, seems more related to secre-
epithelial cells (TECs), and endothelial cells actively contribute to fibrogenesis has tion,57 60 and cell-specific deletions that
evolved considerably during the past 20 years and sustained growth factor pressure associate with decreased matrix accumu-
within the microenvironment and increased susceptibility to growth factormediated
lation strongly insinuate secretory func-
stimulation emerge as the principal driving force of fibrosis. The molecular systems that
determine choice between pathologic fibrosis and physiologic repair however are still
tion.61
evolving. Epidemiologic studies identify genetic polymorphisms, epigenetic modifica- Fibroblasts use collagen fibrils as scaf-
tions, and aging as risk factors for chronic kidney disease. Linking these risk factors folding to crawl among damaged tissue
mechanistically to fibrosis is a task for the future. planes, and mathematical modeling sug-
gests that fibroblast number and move-
ment depends on their sensitivity to che-
tive changes in matrix.1 In fibrotic process where the initial appearance of moattractant diffusion.62 These gradients
kidneys the widened interstitial spaces collagen nucleators in interstitial fluid increase collagen alignment in fibrogenic
fill with fibrillar material consisting of serve as scaffolding for fibrillar collagens tissue to facilitate the accrual of fibroblasts.
predominantly collagens type I and III produced by local fibroblasts.43 Fi- The details of these events can be difficult
and fibronectin.1,35 This fibrotic matrix bronectin, collagen type V, LTBP, and to assess in tissues biochemically, as impor-
also contains residual fragments of colla- secreted protein acidic and rich in cys- tant modulators of phenotypic change,
gen type IV, which are normally found in teine (SPARC) are interactive regulators particularly cytokines, may express at levels
basement membranes otherwise sup- of this matrix assembly:42 44 fibronectin not easily detectable, and in vitro activators
porting intact endothelia or tubular epi- co-localizes with procollagen secretion or inhibitors may be artificially binary just
thelia36,37 as well as several fibronectin to establish conformation-dependent by their presence in serum.63 However, the
splice variants that modulate fibrogenic relationships with 51 integrins on presence of PDGF and the absence of
potential.38 The actual amount of colla- fibroblasts43 and collagen type V oper- TGF and FGF2/bFGF in fetal wounds
gen produced by renal fibrogenesis ates at the fibrillar core of collagen type that do not scar compared with the pres-
seems to attenuate fairly soon after it I assembly.45 LTBPs facilitate the secre- ence of all three cytokines in adult tissue
starts, whereas the relative extent of fi- tion of pro-TGF and provide a mech- that do suggests differential expression pat-
brosis seems to increase with time.39 This anism for its release and activation.42 terns of key growth factors may be neces-
dynamic incongruity can be reconciled SPARC is antiproliferative, 46 stimu- sary for enduring fibrogenesis.63 Prototyp-
by recognizing that the volume of in- lates the expression of matrix metallo- ical TGF signaling through Smad
jured kidney also decreases as residual re- proteinases (MMPs)47 and plasmino- pathways64,65 under microRNA66 and epi-
nal parenchyma collapses around inva- gen activator inhibitor-1 (PAI-1), 48 genetic control67,68 has one of the strongest
sive interstitial scars.40 and activates integrin-linked kinase profibrotic effects on matrix produc-
Whereas fibrillar collagens spontane- (ILK),46 which engages epithelial-mes- tion.69,70 The proximal action of angioten-
ously assemble in vitro, this is not what enchymal (EMT) or endothelial-mes- sin II as a morphogenic cytokine stimulat-
happens in vivo.41 During normal and enchymal transitions (EndMT) pro- ing TGF71 and PAI-172 has also led to the
abnormal tissue remodeling, interstitial ducing fibroblasts. 49 51 All of these well-known renoprotective effects of re-
collagens have numerous binding part- processes shape the collagenous matrix nin-angiotensin inhibition.2,73 Whereas
ners and those critical for fibrillar assem- found in normal or disturbed tissues. containment of TGF signaling has been
bly include fibronectin, collagen type V, Generally speaking, fibrogenesis ini- the centerpiece of various experimental
fibronectin and collagen-binding inte- tiates in small areas at random sites of antifibrotic therapies,74 80 knockdown of
grins, and various fibrillins, including la- inflammation and then expands to be- TGF type II receptors along the collecting
tent TGF-binding proteins (LTBP).42,43 come diffuse if profibrotic drivers per- duct paradoxically accelerates renal fibro-
Prototypical matrix deposition follows a sist.52 The accumulation of fibroblasts in sis associated with ureteral obstruction,81

1820 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1819 1834, 2010

all to suggest that too much or too little of
even a single cytokine may be profibro-
genic.82
TISSUE PROTEASES
Removal of extracellular matrix from the
interstitium hinges on a well-worn belief in
the role of various endogenous proteases.
Most studies in this area focus on two fam-
ilies of proteases: MMPs and members of
the plasmin-dependent pathway. Both
classes of proteases have the potential to
fragment some extracellular matrix for re-
moval, but also cleave nonmatrix sub-
strates, releasing profibrotic growth factors
that paradoxically trigger unwelcome
consequences.
MMPs are a family of zinc-dependent
endopeptidases that currently includes
25 members with varying substrate spec-
ificities controlled by tissue inhibitors of
metalloproteinases 1-4.83 The overlap-
ping activity and specificity of MMPs
make it difficult to dissect individual ac-
tions in vivo. Most studies in the kidney
focus on MMP-2 and MMP-9 (both
degrade collagen type IV84 and perhaps
collagen types I and III85), but fail to
demonstrate a consistent antifibrotic
effect in the interstitium.86,87 For exam-
ple, combined pharmacologic inhibition
of 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 of
tubulointerstitial fibrosis is protective.88
More confusing, studies utilizing MMP
null mice show unaccelerated fibrogen-
esis, and overexpression of MMP-2 in tu-
bular epithelial cells is sufficient to in-
duce tubulointerstitial fibrosis.87 89 The
profibrotic effects of MMP-2 and
MMP-9 on renal fibrosis, particularly the
activation of MMP-2 and MMP-14
(MT1-MMP) by TGF associated with
the degradation of basement membrane
stimulating EMT,87 seem to outweigh
their antifibrotic potential; in fact, mice
deficient in TIMP-3 spontaneous de-
velop interstitial fibrosis.90 MMP-14, a
membrane-bound activator of secreted
MMP-2 and MMP-9 in fibroblasts, is
critical for invasiveness into the intersti-
J Am Soc Nephrol 21: 1819 1834, 2010
www.jasn.org BRIEF REVIEW
tial environment containing collagen brogenesis by activating MMPs96 and
types I and III85 and its expression is un- stimulating EMT.97 tPA null mice with
der the control of Snail, which is a key ureteral obstruction have reduced local
regulator of the EMT program.91,92 Thus, expression of MMP-9 and preserved tu-
epithelial cells, engaged by TGF, ex- bular basement membrane with less EMT,
press essential MMPs for both basement and less interstitial fibrosis,98 whereas
membrane degradation and interstitial urokinase cellular receptor (uPAR) null
invasion that are ultimately required for mice develop worse renal fibrosis with
successful completion of EMT or detach- lower expression levels of the antifibrotic
ment and loss into the tubular lumen cytokine, hepatocyte growth factor
(Figure 2). (HGF).99 Angiotensin II stimulates the
The effects of the plasminogen-plas- PAI-1 promoter in tubular cells100 and
min system on fibrosis are equally com- tubulointerstitial fibrosis associates with
plex. Active plasmin is derived from pro- TGF-stimulated PAI-1 expression,101
teolytic cleavage of plasminogen by whereas the genetic deletion of PAI-1
tissue-type plasminogen activator (tPA) ameliorates renal fibrogenesis in
or urokinase-type plasminogen activator mice.72,102,103 The small heterodimer
(uPA), which in turn are inhibited by partner (SHP) represses the transcrip-
PAI-1.93 Plasmin degrades some matrix tion of TGF/Smad3regulated PAI-1
constituents, including laminin, entactin, expression, and SHP null mice develop
perlecan, and fibronectin,93,94 whereas more renal fibrosis, whereas SHP over-
fibrillar collagen seems more resistant.95 expression inhibits its development;101
Plasmin, perhaps more importantly, also SHP may turn out to be an interesting
affects cell behavior and function in the therapeutic candidate depending on its
fibrotic environment and promotes fi- molecular specificity.
Figure 2. Tissue proteases and tubular decondensation. Whereas proteases are key
modifiers of interstitial matrix, they also are critical for the disruption of basement
membrane. Tubular epithelial cells receive signals from the microenvironment to
change phenotype. As they release from basement membrane, they can either round
up and fall into the tubular lumen or undergo epithelial-mesenchymal transition and
invade the interstitium through rents in damaged basement membrane. SPARC
through ILK, angiotensin II, and TGF activate nuclear programs (SHP, Snail1, and
Smads) that engage in EMT-forming fibroblasts. Part of this mechanism is to stimulate
the release of PAI-1, MMP-2, and MMP-9. MMP-2 and MMP-9 are activated by
membrane-bound MMP-14, which is also modulated by PAI-1, tPA, and plasmin effects
on MMPs.
Mechanisms of Tubulointerstitial Fibrosis 1821
 BRIEF REVIEW www.jasn.org
The profibrotic action of these pro-
teases is a paradigm shift in thinking
about the dynamics of matrix deposition
or its dissolution (Figure 2), illustrating
the complexity of balancing modifier ef-
fects, timing and context, and weighing
what seems to be true in vitro with what is
not observed in vivo. Whereas current
evidence argues against MMPs or plas-
min to degrade only complex matrixes in
vivo, murine studies reporting regression
of tubulointerstitial fibrosis do demon-
strate less matrix deposition.104 107 How
this matrix is removed biochemically, or
fails to form, is still unclear. Involvement
of other proteases, perhaps the lysosomal
family of cysteine protease cathepsins,
may be important.108 112 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 and
mast cells.118 Whereas inflammation as a
whole contributes significantly to the en-
gagement of fibrogenesis, evidence in re-
cent years also highlights the antifibrotic
effects of various subsets of lymphocytes
and macrophages, providing novel po-
tential avenues for antifibrotic therapies.
Innate immunity through the activa-
tion of toll-like receptors 4 (TLR4)119
and TLR9,120 unlike TLR2 receptors,121 is
an important early mediator of renal fi-
brogenesis. Lymphocytes precede the in-
flux of macrophages.1 Rag-2 null mice,
which lack both mature B and T lympho-
cytes, are protected from fibrosis, dem-
onstrating that effector lymphocytes are
an 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 tubular
epithelial cells.2 Administration of PGE1
inhibits the effector arm of T lympho-
cytes in interstitial nephritis that is over-
come by competitive exposure to IL-1.123
1822 Journal of the American Society of Nephrology
Long-term renal fibrosis after ischemia-
reperfusion injury also depends on per-
sistent infiltration of effector-memory T
lymphocytes, which correlate with onset
of fibrosis in later stages.124 Transfer of T
lymphocytes from mice with prolonged
ischemia-reperfusion injury to nave re-
cipients is sufficient to induce renal in-
jury, further highlighting the relevance of
memory lymphocytes in the progression of
tubulointerstitial fibrosis.125 In contrast,
FoxP3 regulatory T cells (Tregs) blunt
kidney injury and are required for physi-
ologic kidney regeneration.126 Whereas
recent studies suggest the presence of
CD20 B cells are as common as CD3 T
lymphocytes in established fibrosis, the
functional contribution of B cells to renal
fibrogenesis is uncertain.2,127,128
When monocytes are recruited to
damage the interstitium, they transdif-
ferentiate into macrophages.129 There is
a strong correlation between macro-
phage infiltration and the extent of fibro-
sis.130 Activation of TRL9 on macro-
phages by CpG-oligodeoxynucleotides is
an accelerant for interstitial fibrogen-
esis,120 suggesting the dispersion of nu-
clear fragments after local cell death may
enhance inflammation. Depletion of
macrophages also ameliorates fibrogen-
esis in mice, highlighting the traditional
profibrotic view of macrophages.131,132
Macrophages however can be catego-
rized into classically activated M1 mac-
rophages or alternatively activated M2
macrophages. Unlike the M1 macro-
phages, M2 macrophages are anti-in-
flammatory and provide cues for tissue
repair.129 Infusion of cells enriched for
M2 macrophages ameliorates renal fi-
brosis in mice.133 Finally, in addition to
macrophages, dendritic cells are critical to
antigen processing in tubulointerstitial in-
jury;114 their proteasomal processing of al-
bumin, for example, creates new antigenic
targets.134 The balance or relative presence
of 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, and
pancreas where they release proinflam-
matory cytokines, including chemokines
CCL2-5, TNF, and leukotrienes.118
Surprisingly, however, mast cell defi-
cient KitW-sh/KitW-sh mice display en-
hanced fibrosis in the kidney. 118,135
Such accelerated fibrosis associates
with increased levels of TGF, suggest-
ing that mast cells in kidney play a role
in suppressing the fibrotic process.118
Several new biologic modifiers of the
immune system reduce interstitial fibro-
sis. PPAR agonists reduce the number
of macrophages by attenuating TGF
expression,30 and three independent
studies report the administration of a
CCR1 antagonist, anti-TNF blocking
antibodies, or an IL-1 receptor antago-
nist are sufficient to ameliorate renal
fibrosis, even when treatment is initi-
ated in advancing disease.136 138 These
therapeutic studies corroborate the
role of inflammation not only in the
initiation but also in perpetuation of
fibrogenesis.
FIBROBLASTS
Fibroblasts in the renal interstitium are
considered the principal source of fibril-
lar matrix (collagen types I and III), and
tubulointerstitial fibrosis inevitably asso-
ciates with a robust accumulation of fi-
broblasts,5 displaying increased intrinsic
proliferative activity139,140 or, in some fi-
broblasts, what is called an activated phe-
notype expressing -smooth muscle ac-
tin (SMA).70,141,142 Tissue fibroblasts
exhibit phenotypic heterogeneity,143145
more likely based on origin,54,146,147 but
this heterogeneity in tissue is also bimo-
dal: those cells that are RhoA-dependent
SMA148 and those that are not. One
generic dilemma with SMA is that
when used as a fibroblast marker in in-
jury, it is not distinguishable from
SMA mural cellsvascular smooth
muscle cells or venular pericytes;149,150
nor does this marker 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 the
suggestion that fibrosis associates with
de novo accumulation of SMA
cells.51,153,154 This notion, however, has
to be tempered by observations that
SMA fibroblasts do not move,155 renal
fibrogenesis persists despite decreasing
J Am Soc Nephrol 21: 1819 1834, 2010

numbers of SMA myofibroblasts,121
SMA fibroblasts contain and likely
express interstitial collagens in vivo,61,152
and mice deficient of SMA develop
more fibrosis and their fibroblasts pro-
duce more collagen type I than SMA
fibroblasts.156 Unequivocal functional
data to support the idea that nonmotile
SMA myofibroblasts principally me-
diate fibrogenesis is lacking.
The most durable fibroblast marker
in the kidney is fibroblast-specific pro-
tein-1 (FSP1; S100A4).58,146,147,151 The
proclivity for FSP1 in fibroblasts, a mem-
ber of the S100 family of calcium-bind-
ing proteins, was discovered in a differ-
ential genetic screen between fibroblasts
and epithelial cells,157 and renal fibrosis
associates with a robust accumulation of
FSP1 fibroblasts.5,61,157 A few studies
suggest FSP1 is expressed by macro-
phages;55,158,159 however, this notion per-
sists from studies using underdetection
methods for FSP1/S100A4 that may en-
hance nonspecific staining and/or from
improper application of so-called anti-
macrophage antibodies that share target
specificities with fibroblasts and are not
macrophage-specific;158 160 FSP1 pro-
moter activity in fibroblasts driving a
GFP reporter60,161 or Cre recombinase162
clearly discriminate tissue fibroblasts from
F4/80 macrophages. FSP1, F4/80 fibro-
blasts are also the only population in the
kidney proven to contribute to fibrogen-
esis, as their ablation in FSP1-tk trans-
genic mice ameliorates fibrosis substan-
tially.61 This kind of functional deletion
experiment is critical evidence not yet re-
ported in FSP1 cells expressing SMA,
particularly pericytes.54 For the moment,
it is best to think of all tissue fibroblasts as
potentially fibrogenic.58,147,152
The true heterogeneity of fibroblasts
in the kidney may be understood by ex-
amining the different cellular origins of
these cells.147 Pools of fibrogenic fibro-
blasts comingle either by proliferation of
resident fibroblasts,163 some of which
may be subject to local or time-depen-
dent epigenetic effects;68,164 from local
tubular epithelial cells51,59,157,165 or endo-
thelial cells (simple squamous epithe-
lia)51,166 after EMT or EndMT during tis-
sue injury; after recruitment of FSP1,
J Am Soc Nephrol 21: 1819 1834, 2010
CD34 bone marrow derived fibro-
cytes;59,146 and perhaps from blood vessel
shedding of SMA pericytes54,150 that
derive developmentally from mesothe-
lium through EMT.167,168 It is not yet
clear whether FSP1, SMA peri-
cytes54,55 should be considered true fi-
broblasts or a separate and unique phe-
notype of collagen-producing cells.150 As
opposed to tissue-derived fibroblasts,
CD34 bone marrow derived fibro-
cytes59 may also have a unique lineage
from CD11b, CD115, Gr1 leukocyte
progenitors under the control of CD4 T
cells;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 limited
extent.50,56,59 For the moment, it is
probably best to view multiple lineages
as contributing to the final mix of fi-
broblast populations in tissue. It is en-
tirely unknown if these various lin-
eages have priority among individuals
or different diseases or if there is indi-
vidual variation or preference as fibro-
genesis progresses.
Depletion of interstitial fibroblasts by
selectively corrupting their DNA replica-
tion is one classic strategy for experimen-
tal antifibrotic therapy.61 Conditional
expression of an IB dominant-negative
transgene in fibroblasts also attenuates
fibrogenesis by inhibiting NF-B.60 But
because specific targeting of fibroblasts
has not yet been achieved without ge-
netic manipulation, current antifi-
brotic therapies center on various
other approaches; these approaches in-
clude blocking PDGF-C, 169 PDGF-
D,170 or TGF171,172 with neutralizing
antibodies or using pirfenidone,173175
pyridoxamine, 176 ALK5 receptor ki-
nase inhibitors,177 paricalcitol,178 Ras
inhibitors,68,179 or PPAR agonists.30
Blockade of AT1R by the small molecule
inhibitor CGP-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
www.jasn.org BRIEF REVIEW
detriment. They do this by modulating
their proliferation,23,181 or by provid-
ing proinflammatory chemokines and
cytokines that induce mononuclear in-
flammation and the formation of fi-
broblasts (Figure 3).59,182184 These local
cytokines provide critical signals mediat-
ing primary interstitial injury.185 Sec-
ondary interstitial damage after glomer-
ulonephritis also depends on the tubular
toxicity of glomerular proteinuria,1,186
the filtration of protein-bound cytokines
from 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 interstitial
spaces associated with the juxtaglomerular
apparatus.188 Hydrodynamic forces along
the tubules themselves are also profi-
brotic.189 Tubular stretch from obstruction
induces TGF and NF-B; increases in
single nephron GFR and compensatory in-
creases in fluid shear stress also decrease
tPA and increase TGF.
The epicenter for early molecular action
by tubular epithelial cells is through NF-
B.190 Tubular cells toggle on NF-B
after exposure to proteins from tubular
fluid,134,191 activation of CD36 scavenger
receptors,25 or exposure to connective tis-
sue growth factor,192 CD40 ligand,193 an-
giotensin II,194,195 or aldosterone.32,33 Acti-
vation of NF-B is critical for initiating the
downstream release of PAI-1 or chemo-
kines and growth factors,196 particularly
IL-1,137 IL-6,197 MCP-1/CCL2,198,199 RAN-
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 decoy NF-B oli-
godeoxynucleotides207 all attenuate tubu-
lointerstitial injury by inhibiting the activ-
ity of NF-B. Experimental inhibition of
proinflammatory chemokines such as
TNF or CCR1 has also been successful
in preventing progressive interstitial
injury.113,138
With time, persistent tubulointersti-
tial injury inevitably associates with mi-
tochondrial dysfunction or loss,26,208 for-
mation of aglomerular tubuli,209 tubular
atrophy,27 and rampant interstitial fibro-
sis,146 creating the classic pathologic
picture of renal progression.210 Major
epithelial mechanisms contributing to
Mechanisms of Tubulointerstitial Fibrosis 1823
 BRIEF REVIEW www.jasn.org

nin-angiotensin system driving EMT

and TGF and receptor absence acceler-
ates fibrogenesis.235
After induction, a hierarchy of tran-
scription factors program EMT, includ-
ing GLIS2212 and CBF-A,236 a master reg-
ulator of Snail,237 Twist,238 HMGA2,
LEF-1, and Ets-1.236 Overexpression of
Snail1237 induces EMT and fibrosis and
recessive mutations in Glis2 spontane-
ously invoke EMT in tubular cells in
mice with renal fibrosis;212 short inter-
fering RNA knockdown of Snail1 also
blocks EMT during vascular mural cell
differentiation.222 There appears to be a
gradualness to EMT events in the kid-
Figure 3. The cytokine milieu in fibrogenesis. NF-B activators and inhibitors compete
for the engagement of mammalian target of rapamycin and release of chemokines and ney,225 as indicated by the variably active
proinflammatory cytokines that draw macrophages, dendritic cells, and T lymphocytes signaling of -catenin, Snail, or Twist in
to the tubulointerstitium. These mononuclear cells injure the tubular epithelia and activate watershed epithelial cells along chroni-
fibroblasts. The transcriptional activation of EMT and EndMT is engaged by hypoxia and cally injured tubules that is based on the
various cytokines, particularly TGF, EGF, and ILK. EMT inhibitors block the development time-dependent permissiveness of local
of fibroblasts that deposit extracellular matrix into the fibrogenic interstitium. cytokine action. Whereas activation of
the EMT program is a means for parental
chronic tubulointerstitial fibrosis are cytes may have emerged previously cells to resist proapoptotic stimuli,211,239
G2/M cell cycle arrest,181 cellular apo- from EMT.167,168,222 It is increasingly such tenuous protection from cell death
ptosis, 211 and EMT-forming fibro- clear that fibroblasts derive from mul- also results in loss of parental cell pheno-
blasts; 147,151 all three processes are tiple parental lineages, including epithe- type.59,240 The true frequency of tubular
modulated by the transcriptional re- lial cells, endothelial cells, and bone mar- epithelial cells that complete EMT and
pressor GLIS2.212 EMT is a fundamental row endosteal cells. The status of become fibroblasts is unknown, but
process to create cells that will move,155 a pericytes in fibrogenesis remains an area likely depends on the persistence of in-
modification in lineage maturation that of great interest that will require more flammation, epigenetic control of fi-
disturbs the state of nuclear diapause in ep- functional evidence of their role one way brotic gene regulation,67 and ultimately
ithelial and endothelial cells.59,213 Although or the other. Finally, although EMT is the emergence of tubular atrophy with
not all agree,54,159,214,215 11 studies confirm difficult to assess in humans who are not acellular scars that are the consequence
these transitions to fibroblasts in a variety biopsied until there is clinical evidence of of profibrotic cytokine exhaustion and
of lineage-tracing experiments from differ- disease,5 early renal allograft biopsies tubular cell disappearance.
ent tissues, including kidney,50,59,60,165 suggest changes in epithelial phenotype The relevance of EMT for progression
intestine,216 liver,217 heart,166 lung,218 220 supportive of transitions preceding fi- of renal fibrosis is highlighted by studies in
and endothelium50,166,221 using 10 differ- brosis,223225 although not all agree.226 which administration of bone morphogenic
ent cell fate reporter constructs. Glis2 mutations producing autosomal protein-7 (BMP7),80 HGF,241,242 the small-
The role of EMT in forming renal fi- recessive NPHP7, a rare form of molecule inhibitor ILK, QLT-0267,243 or
broblasts has been recently reviewed49 nephronophthisis, may be the first sug- Y-27632,aninhibitorofRho/ROCK244 allin-
and debated;160 several of the lineage- gestive evidence of spontaneous or dere- hibit tubular EMT and ameliorate fibrogen-
tracing studies unable to detect EMT or pressed EMT causing renal fibrosis in esis. Trps1 operates downstream of
FSP1 fibroblasts are likely confounded by humans.212,227 BMP7 in the kidney,245 and the availability
use of nonpreferred anti-FSP1/S100A4 an- EMT induces a variety of intermedi- of Smad7 controlling the phosphorylation of
tibodies and/or inadequate antigen-re- ate cell phenotypes, not all of which com- Smad3 is normally assured by Trps1 inhibi-
trieval methods during tissue prepara- plete their transition to fibroblasts.147,225 tion of ubiquintination through Arkadia;246
tion,54,159,215 by relying on a collagen This intermediacy and its reversibility Trps1 haploinsufficiency raises the levels of
promoter driving green fluorescent pro- may depend on the variable persiste- Arkadia,decreasingtheavailabilityofSmad7,
tein used to detect fibroblasts that per- nce of endogenous perturbagens: hypo- and thus facilitating fibrosis through TGF/
haps may not express in all fibroblasts xia,165,228 230 TGF,231 EGF,231,232 PTH- Smad3mediated EMT. Induction of heat
because of methylation or enhancer bi- RP,233 and plasmin97 all induce EMT, as shock protein-72 (HSP72) also protects tu-
as,160 and by overlooking lineage evi- does activation of ILK49 or RAGE.234 Vi- bular epithelia from apoptosis, EMT, and fi-
dence that SMA mural cells/peri- tamin D receptors downregulate the re- brosis,239 and overexpression of klotho247 or

1824 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1819 1834, 2010

inhibition of the mammalian target of rapa-
mycin248 attenuates fibrogenesis in a variety
of experimental models.
A pivotal determinant in the failure to re-
coverfrominterstitialinjuryistherelativeab-
sence of nephron regeneration in kidneys
that become fibrotic.181 One reason for this is
that glomerulotubular disconnections limit
repair of nephrons.107,209 The glomerulotu-
bular neck at the antivascular pole of the glo-
merular tuft is an at-risk break point for the
formation of aglomerular tubuli249 and sur-
prisingly angiotensin inhibition protects
these unwanted separations.250 Another rea-
son is that cell cycle arrest of tubular epithe-
lium during acute kidney injury may stimu-
late profibrotic signaling.181 Whereas tubular
cells along atrophic tubules variably increase
their proliferative activity in advanced tubu-
lointerstitial disease,210 this restorative poten-
tial may decline with aging through the mod-
ulation of the adipokine, zinc-2-
glycoprotein, known as Zag.23
The regenerative capacity of injured tu-
bules absent persistent inflammation is likely
the result of self-renewing tubular cells or
perhaps progenitor cells within other niches
along the nephron.31 A CD24, CD133
progenitor cell niche has been identified in
the parietal epithelium of Bowmans capsule
near the vascular pole,21,22,251 and the adop-
tive transfer of these cells at the time of tubu-
lar injury obviates the appearance of intersti-
tial fibrosis.252 The glomerulotubular neck
region is also where ex vivo differentiated em-
bryonic stem cells traffic after adoptive trans-
fer during development253 and may be a tu-
bular niche not unlike the vascular pole.21
Expansion of renal progenitor cells is under
the control of histone deacetylases and their
enzymatic inhibition may offer restorative
potential to the kidney.254
THE MICROVASCULATURE
Rarefaction of the peritubular vascula-
ture with ensuing chronic hypoxia are
hallmarks of tubulointerstitial fibro-
sis.107 In early stages of injury the peritu-
bular vasculature is damaged by proapop-
totic stimuli, particularly transient
ischemia.255,256Progressive fibrosis then
remodels the peritubular endothelial
network governed by imbalance among
J Am Soc Nephrol 21: 1819 1834, 2010
pro- and antiangiogenic stimuli257,258 or
loss of peritubular endothelial cells by
EndMT-forming fibroblasts.50,259
The fibrotic tubulointerstitium is a hy-
poxic environment.260,261 Hypoxia results
from microvascular rarefaction made worse
by matrix expansion throughout the intersti-
tium,258 requiring the oxygen gradient to dif-
fuse greater distances from vasoconstricted
or attenuated microvessels to adjacent isch-
emic tubules.230,260 Hypoxia also contributes
directly to the progression of tubulointersti-
tial fibrosis by simulating EMT and promot-
ing matrix accumulation from new fibro-
blasts.165
Different strategies to improve this
chronic hypoxia during renal progres-
sion show some promise in experimental
models of renal fibrosis. Angiotensin II
inhibition increases capillary blood flow
and cortical oxygen levels in normal re-
nal tissues,262 and long-acting calcium
channel blockers attenuates the hypoxia
of angiotensin IIinduced tubulointer-
stitial injury.263 Inhibition of the vaso-
constrictor, endothelin, is also renopro-
tective,264 particularly in combination
with ACE inhibition.265 Activation of the
hypoxia-inducible factor (HIF) pathway
or stabilization of HIF proteins attenu-
ates renal fibrosis as well.230,261,266 The ul-
timate goal of reversing microvascular
rarefaction, however, has been difficult
to achieve. Whereas the HIF pathway is
an important inducer of angiogenic vas-
cular endothelial growth factor (VEGF)
proteins,230 systemic administration of
VEGF121 does not improve renal micro-
vascular disease.267 Whereas administra-
tion of the antifibrotic molecule, BMP7,
blocks EMT in the kidney,80 its affect on
EndMT in the kidney is unknown; how-
ever, BMP7 does inhibit EndMT, endo-
thelial loss, and fibrogenesis in experi-
mental cardiac fibrosis.166
GENETIC PREDISPOSITION TO
RENAL PROGRESSION
Whereas revelations regarding new cellular
mechanismsofrenalfibrogenesiscontinueto
flourish, the identification of key risk factors
underlying the true biologic fate of renal tis-
sue is far from clear. Risk factors for chronic
Mechanisms of Tubulointerstitial Fibrosis
www.jasn.org BRIEF REVIEW
kidney disease are rapidly emerging from ep-
idemiologic studies, including age,268,269
nephron mass,7,270 gender,268,271 levels of cal-
cidiol,272 sympathetic activity,273 obesity and
diabetes,274 cardiovascular disease,275 genetic
loci276 and polymorphisms,273,277279 and
epigenetic modifications.19 But how these
risk factors modulate fibrogenesis is still un-
certain.
Genetic polymorphisms in cytokine risk
profiles in some instances can be explained
using existing knowledge of fibrotic signaling
pathways.278 In other cases, it is unclear
why genetic polymorphisms in uro-
modulin280,281 or methenyltetrahydro-
folate synthetase282 increase suscepti-
bility to fibrogenesis. Insights into the
role of epigenetics in renal progression
and fibrogenesis are also just starting to
emerge. For example, aberrant hyper-
methylation of selected genes including
RASAL1, a suppressor of Ras signaling,
contributes to perpetual fibroblast acti-
vation and fibrogenesis.68 Furthermore,
there are large families of small, naturally
occurring, noncoding RNAs that inter-
fere post-transcriptionally with the stabil-
ity or translation of coding mRNA;283 three
prototypical families of interfering RNAs
include short interfering RNAs (siRNA),
microRNAs (miRNAs), and piwi-interact-
ing RNAs (piRNA).284 miRNA-192, for
example, promotes the activation of
TGF/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 RNAs
that increase Col1a2 E-box activity in
mesangial cells.286 Oddly enough, low
levels of miRNA-192 in human kidney
biopsies of diabetic nephropathy cor-
relate with late presentation and less fi-
brosis. 164 Thus, the interfering RNA
story becomes complicated quickly,
largely because the network effect of
multiple families is not addressed by
studying the action of a single family
member.
CONCLUSIONS
Major progress has been made during
the last 20 years in characterizing the cel-
1825
 BRIEF REVIEW www.jasn.org
lular and molecular mechanisms of in-
terstitial injury. The molecular events
determining choice between self-limited
repair of the tubulointerstitium or pro-
gressive fibrogenesis suggest that persis-
tent cytokine pressure and the accumu-
lation of unwanted inflammatory cells
are the principal effectors of chronicity.
There remains a gap in understanding
the genetic drivers of this cytokine pres-
sure or sensitivity, and our knowledge of
how genetic polymorphisms and epige-
netic modifications facilitate fibrogen-
esis are in their infancy.
Modulating the pivotal role of tubular
epithelial cells in their provision of pro-
fibrotic stimuli, and the difficulty in pre-
venting microvascular rarefaction, re-
main new challenges for the future. The
role of determining cells, particularly
myofibroblasts or pericytes, in renal fi-
brogenesis needs more clarification in
comparison to the known functionality
of FSP1 fibroblasts. A deeper under-
standing of the molecular basis for fibro-
blast expansion and collagen accumula-
tion also requires more work. New
therapies 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 return
for intermediate states of transition is
still an open question.
Today, the distinction between profi-
brotic and antifibrotic stimuli also seems
increasingly muddledsome macro-
phage, T cell subpopulations, and mast
cells, many of which were once viewed as
unequivocally pernicious, now seem
renoprotective. Tissue proteases, also
once considered renoprotective, in many
respects now look as if they encourage
fibrogenesis. The role of progenitor cells
in regeneration and the depletion of stem
cell niches along the adult nephron is
also an underdeveloped area of investi-
gation.
Most importantly, although the pres-
ence of tubulointerstitial fibrosis has al-
ways implied an unalterable fate, this
perception is changing in new preclinical
studies using unique biologic modifiers.
We have renewed optimism that in the
not too distant future some of these
novel insights will become clinically rel-
1826 Journal of the American Society of Nephrology
evant for patients with chronic kidney
disease.
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.
DISCLOSURES
None.
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