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14. Colombo, G., et al. 1998. Appetite suppressionand weight loss after the cannabinoid antago-nist SR 141716.Life Sci.63:L113L117.

15. Di Marzo, V., et al. 2001. Leptin-regulated endo-cannabinoids are involved in maintaining foodintake.Nature.410:822825.

16. Shimada, M., Tritos, N.A., Lowell, B.B., Flier, J.S.,and Maratos-Flier, E. 1998. Mice lackingmelanin-concentrating hormone are hypophag-ic and lean.Nature.396:670674.

17.Qian, S., et al. 2002. Neither agouti-related pro-tein nor neuropeptide Y is critically required forthe regulation of energy homeostasis in mice.Mol. Cell. Biol.22:50275035.

18. Broberger, C., Landry, M., Wong, H., Walsh, J.N.,and Hokfelt, T. 1997. Subtypes Y1 and Y2 of theneuropeptide Y receptor are respectivelyexpressed in pro-opiomelanocortin- and neu-ropeptide-Y-containing neurons of the rat hypo-

thalamic arcuate nucleus. Neuroendocrinology.66:393408.

19. Broberger, C., Visser, T.J., Kuhar, M.J., and Hok-felt, T. 1999. Neuropeptide Y innervation andneuropeptide-Y-Y1-receptor-expressing neuronsin the paraventricular hypothalamic nucleus ofthe mouse.Neuroendocrinology.70:295305.

20. Jain, M.R., Horvath, T.L., Kalra, P.S., and Kalra,S.P. 2000. Evidence that NPY Y1 receptors areinvolved in stimulation of feeding by orexins

(hypocretins) in sated rats.Regul. Pept.87

:1924.21. Chaffer, C.L., and Morris, M.J. 2002. The feedingresponse to melanin-concentrating hormone isattenuated by antagonism of the NPY Y(1)-receptor in the rat.Endocrinology.143:191197.

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23. Kushi, A., et al. 1998. Obesity and mild hyperin-sulinemia found in neuropeptide Y-Y1 receptor-deficient mice. Proc. Natl. Acad. Sci. U. S. A.95:1565915664.

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Renal fibrosis: not just PAI-1 in the sky

Agnes B. Fogo

Department of Pathology, Vanderbilt University Medical Center, Nashville,Tennessee, USA

A delicate balance exists between ECM synthesis and degradation suchthat interruption of the corresponding pathways results in increasedplasminogen activator inhibitor-1 (PAI-1), pathological matrix accu-mulation, and glomerulosclerosis. A new study (see the related articlebeginning on page 379) demonstrates that therapy with a mutant PAI-1increases matrix turnover and reduces glomerulosclerosis by competingwith endogenous PAI-1, suggesting therapeutic utility in the treatmentof fibrotic renal disease.

J. Clin. Invest.112:326328 (2003). doi:10.1172/JCI200319375.

Progressive fibrosis leads to end-organ failure in multiple organs,including heart, lung, liver, and kid-ney. The process of substitution offunctional parenchymal cells byfibrotic tissue has been intensivelyinvestigated. Past progress over thelast few decades has resulted inremarkable gains in slowing the pro-gression of chronic kidney disease.The major emphasis has been on con-

trol of blood pressure, both systemi-

cally and within the glomerulus. Theefficacy of angiotensin Iconvertingenzyme inhibitors was linked to con-trol of glomerular pressure beyondsystemic pressure effects mediated bydilation of the efferent arterioles ofthe glomerulus (1). However, normal-ization of glomerular pressure didnot completely halt progression, norcould it regress existing fibrosis.Numerous additional factors thus

were implicated in progressive renalinjury, including modulation of ECMand parenchymal and infiltrating cellinteractions. In addition to angio-tensin, TGF- has been recognized asa key mediator of renal fibrogenesis.Interestingly, both angiotensin andTGF- induce plasminogen activatorinhibitor-1 (PAI-1) (2).

PAI-1 and fibrosis mechanismsHow might PAI-1 affect fibrosis?

PAI-1 is the major inhibitor of tissue-type plasminogen activator (t-PA) and

urokinase-type plasminogen activator(u-PA), which activate plasminogen toyield plasmin, which in turn degradesfibrin. PAI-1 not only inhibits fibri-

nolysis but also has complex interac-tions with matrix, promoting net pro-teolysis (3). t-PA, u-PA, and plasmincan degrade a wide range of ECM pro-teins; they also activate latent MMPs,in particular MMP-1 and MMP-3,and indirectly activate MMP-2. Mod-els of glomerular sclerosis and/orinterstitial fibrosis have been devel-oped in both rats and mice to furtherexamine these mechanisms. Nobleand colleagues previously demon-

strated that direct manipulation ofthe plasmin/plasminogen activatorsystem with recombinant t-PA treat-ment decreased glomerular matrixaccumulation in the anti-Thy1 modelof glomerular matrix expansion (4).This model does not result in pro-gressive renal damage and shows noattendant interstitial fibrosis butallows in vivo determination of mech-anisms of glomerular matrix accu-mulation. Interstitial fibrosis can beinduced in vivo in either rats or mice

by unilateral ureteral obstruction(UUO). In contrast to the anti-Thy1model, UUO does not result in signif-icant glomerular lesions but demon-strates early macrophage infiltrationand robust interstitial fibrosis, alongwith tubular injury. The unobstruct-ed contralateral kidney serves as anideal control. The UUO model thusallows in-depth in vivo study of mech-anisms of interstitial fibrosis, andinteractions between epithelial and

infiltrating cells. The use of diversemodels of renal scarring thus allows

Address correspondence to:Agnes B. Fogo,MCN C3310, Department of Pathology,Vanderbilt University Medical Center,Nashville, Tennessee 37232, USA.Phone: (615) 322-3114; Fax: (615) 343-7023;E-mail: agnes.fogo@vanderbilt.edu.

Conflict of interest: The author has declaredthat no conflict of interest exists.

Nonstandard abbreviations used:plasminogen activator inhibitor-1 (PAI-1);tissue-type plasminogen activator (t-PA);urokinase-type plasminogen activator(u-PA); unilateral ureteral obstruction

(UUO); angiotensin type 1 (AT1);epithelial-mesenchymal transition (EMT).

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Figure 1PAI-1 modulates scarring in a complex manner, including effects on cell migration, matrixturnover, and macrophage infiltration. Whereas increased t-PA promotes dissolution ofmatrix and subsequently lessens glomerulosclerosis, this matrix breakdown facilitates cellmigration and epithelial-mesenchymal transition (EMT) and thus contributes to increasedinterstitial fibrosis. The effects of PAI-1 that facilitate enhanced macrophage infiltration invivo are complex and have not yet been established. Macrophages have beneficial effects

early in interstitial f ibrosis and promote fibrosis later. The increased number of glomeru-lar macrophages is linked to glomerulosclerosis. u-PA and its receptor and vitronectin alsointeract with PAI-1 and contribute to its final effects on fibrosis.

dissection of the divergent mecha-nisms that contribute to glomerularversus interstitial scarring (Figure 1).Thus, in contrast to the effects of t-PAon glomerular matrix accumulation,t-PA/ mice have reduced interstitialfibrosis after injury caused by UUO(see below) (5).

In normal, non-scarred kidneys,there is very little PAI-1 expression,but acute infusion of angiotensinmarkedly upregulates PAI-1 mRNAand protein via the angiotensin type 1(AT1) receptor by a nonpressure-dependent mechanism (6). Thisinduction has been shown in vitro tobe augmented by aldosterone via aputative glucocorticoid-response ele-ment in the PAI-1 promoter (7). Infibrotic renal diseases, PAI-1 is also

increased and localizes to areas ofglomerulosclerosis (8). Conversely,

inhibition of angiotensin or aldos-terone decreases PAI-1 and alsodecreases renal scarring (8, 9).Decreased glomerular PAI-1 may thuslessen severity of glomerulosclerosisby allowing increased proteolysis.PAI-1 also affects interstitial fibrosis.PAI-1/ mice had attenuated inter-

stitial fibrosis after either UUO orprotein overload compared withwild-type but were not completelyprotected from injury (10). In thisissue of theJCI, the novel and excitingstudies from Noble and colleaguesshow that inhibition of PAI-1 by amutant PAI-1, which binds matrix vit-ronectin but does not inhibit plas-minogen activators, results in signifi-cant, albeit incomplete, protectionagainst glomerular matrix accumula-

tion (11). Mechanisms demonstratedby the authors include increased

glomerular plasmin with direct degra-dation of accumulated excess matrix.

However, the results of Noble andcolleagues (11) suggest that PAI-1also has more far-reaching effects,including indirect effects on matrixsynthesis. Thus, mRNA expression

for matrix molecules was alsodecreased, perhaps because of thereduced macrophage infiltrationobserved with PAI-1 inhibition. Themechanisms for decreased macro-phage infiltration with PAI-1 inhibi-tion remain to be determined (Figure1). The macrophage has a particular-ly critical role after injury. Macro-phages produce numerous profibrot-ic factors, which increase matrixsynthesis by resident parenchymalcells. However, in the absence of

TGF- signaling, macrophages maybe ineffectual in promoting fibrosis.This has been illustrated experimen-tally in 6 integrin knockout mice.The heterodimeric integrin v6,expressed in epithelia in the skin,lung, and kidney, is one key local acti-vator of TGF-. TGF- circulates inan inactive form, linked to latency-associated peptide (LAP). 6 integrinbinds to this TGF-LAP inactivecomplex, effecting local TGF- acti-

vation. 6/

mice were resistant tolung fibrosis induced by bleomycin,despite robust numbers of infiltrat-ing macrophages (12). We haveshown similar protection in 6/

mice in the renal fibrosis model ofUUO (13). Thus, macrophages maynot be profibrotic in the absence oflocal TGF- activation.

Macrophages and fibrosisOf note, glomerular and interstitialmacrophage infiltration may not acti-

vate identical injury responses. Thispossibility is supported by our obser-vation of differing phenotype ofglomerular versus interstitial macro-phages in human diabetic nephropa-thy; only the latter expressed PPAR-(14). It is also possible that macro-phage infiltration, especially intersti-tial, has an early, beneficial role afterinjury, whereas persistent macro-phage infiltration, whether glomeru-lar or interstitial, is profibrotic.

Recently, we have further demon-strated that infiltrating macrophages

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even play a beneficial role in earlyinjury after UUO, and this salutaryeffect is dependent on intact AT1receptor (15). Wild-type mice wereradiated and underwent bone mar-row reconstitution withAT1a/ mar-row. Surprisingly, these mice had

more severe fibrosis after UUO thanidentically treated mice reconstitutedwith wild-type marrow. These benefi-cial effects of macrophages are similarto the well-established role of macro-phages in early wound healing, inwhich phagocytosis is a key elementin resolution of injury. In Noble andcolleagues study (11), macrophageinfiltrate was decreased, which couldbe causal in decreasing glomerularinjury. Whether PAI-1 antibody alsodecreases interstitial macrophages

and thereby contributes to decreasedinterstitial fibrosis remains to bedetermined. Even those remainingglomerular macrophages could beinhibited in transducing fibroticeffects, because TGF- content ofglomeruli was also reduced by themutant PAI-1, thus reducing down-stream fibrosis-effector mechanisms.This decrease in TGF- possiblycould result from increased clearanceof TGF- by PAI-1 mutant protein

binding to vitronectin.

PAI-1 and cell migrationPAI-1 also importantly modulatescellular adhesion and migration andthus has impact on inflammation,wound healing, angiogenesis and cellmigration, and tumor cell metastasis(3). PAI-1 competes with the u-PAreceptor (u-PAR) for the NH2 termi-nusbinding domain on vitronectin.When PAI-1 binds to vitronectin,u-PAR interaction with the vit-

ronectin and surrounding ECM isprevented, thus inhibiting vitro-nectin-dependent cellular adhesionand migration. This effect is demon-strated in PAI-1/ mice, which showenhanced smooth muscle cell migra-tion, while PAI-1 overexpression invitro inhibits this process (16, 17).However, in vivo PAI-1 enhances cellmigration in some settings. IncreasedPAI-1 is linked to macrophage infil-tration, and more aggressive tumor

cell metastasis, pointing to complex

interactions with vitronectin andmatrix in vivo, and possible directchemotactic actions of PAI-1 (Figure1) (3). The mutant PAI-1 used byNoble and colleagues (11) maintainsinteractions with vitronectin andthus can have cell migration

inhibitory effects independent of itsproteolytic activity. This might notonly decrease macrophage infiltra-tion but also affect epithelial-mes-enchymal transition (EMT). EMT ischaracterized by migration of epithe-lial cells into the interstitium, wherethey become mesenchymal, myofi-broblast-type cells. Neilson and col-leagues have recently elegantlydemonstrated that this process con-tributes to renal fibrosis in vivo afterUUO (18). The importance of EMT

in interstitial fibrosis was also sup-ported by a decrease in interstitialcollagen after UUO in t-PA/ mice.This protection was linked todecreased MMP-9 induction and pre-served tubular basement membraneintegrity, postulated to prevent EMT.These findings contrast with theeffects of increased t-PA to protectagainst glomerular matrix accumula-tion, discussed above. The mutantPAI-1 antibody used by Noble and

colleagues could possibly inhibit thismigration by its persistent interac-tion with vitronectin. However,effects on interstitial fibrosis cannotbe extrapolated from their studies inthe anti-Thy1 model, which has onlymesangial proliferation and matrixexpansion and no fibrosis.

In conclusion, the studies of Nobleand her colleagues (11) show us thatmodulating fibrosis is a goal withinreach, and inhibition of these mech-anisms could confer further benefits

beyond existing therapies in treat-ment of chronic kidney diseases. It isimportant to note that disease wasnot completely prevented, indicat-ing that additional targets might yetremain for optimal intervention infibrosis. Further understanding ofthe complex interactions of paren-chymal cells, infiltrating cells, andmatrix will allow optimal targetingand development of novel antifi-brotic therapies. Synergistic thera-

pies could halt progression of

lesions and even potentially regressexisting scarring (19).

1. Brenner, B.M. 2002. Remission of renal disease:recounting the challenge, acquiring the goal.J. Clin. Invest. 110:17531758. doi:10.1172/JCI200217531.

2. Ma, L.-J., and Fogo, A.B. 2001. Angiotensin asinducer of plasminogen activator inhibitor-1and fibrosis. Contrib. Nephrol.135:161170.

3. Eddy, A.A. 2002. Plasminogen activatorinhibitor-1 and the kidney.Am. J. Physiol. RenalPhysiol.283:F209F220.

4. Haraguchi, M., Border, W.A., Huang, Y., andNoble, N.A. 2001. t-PA promotes glomerularplasmin generation and matrix degradation inexperimental glomerulonephritis. Kidney Int.59:21462155.

5. Yang, J., et al. 2002. Disruption of tissue-typeplasminogen activator gene in mice reducesrenal interstitial fibrosis in obstructivenephropathy. J. Clin. Invest. 110:15251538.doi:10.1172/JCI200216219.

6. Nakamura, S., Nakamura, I., Ma, L., Vaughan,D.E., and Fogo, A.B. 2000. Plasminogen activa-tor inhibitor-1 expression is regulated by theangiotensin type 1 receptor in vivo. Kidney Int.

58:251259.7.Brown, N.J., et al. 2000. Aldosterone modulatesplasminogen activator inhibitor-1 and glomeru-losclerosis in vivo.Kidney Int.58:12191227.

8. Oikawa, T., Freeman, M., Lo, W., Vaughan, D.E.,and Fogo, A. 1997. Modulation of plasminogenactivator inhibitor-1 in vivo: a new mechanismfor the anti-fibrotic effect of renin-angiotensininhibition.Kidney Int.51:164172.

9. Brown, N.J., et al. 2000. Synergistic effect ofadrenal steroids and angiotensin II on plas-minogen activator inhibitor-1 production.J. Clin. Endocrinol. Metab.85:336344.

10. Oda, T., et al. 2001. PAI-1 deficiency attenuatesthe fibrogenic response to ureteral obstruction.Kidney Int.60:587596.

11. Huang, Y., et al. 2003. A mutant, noninhibitoryplasminogen activator inhibitor type 1 decreas-es matrix accumulation in experimentalglomerulonephritis.J. Clin. Invest.112:379388.doi:10.1172/JCI200318038.

12. Munger, J.S., et al. 1999. The integrin alpha vbeta 6 binds and activates latent TGF beta 1: amechanism for regulating pulmonary inflam-mation and fibrosis. Cell.96:319328.

13. Ma, L.-J., et al. 2003. Transforming growth fac-tor dependent and independent pathways ofinduction of tubulointerstitial fibrosis in 6/

mice.Am. J. Pathol. In press.14. Paueksakon, P., Revelo, M.P., Ma, L.J., Marcan-

toni, C., and Fogo, A.B. 2002. Microangiopath-ic injury and augmented PAI-1 in human dia-betic nephropathy. Kidney Int. 61:21422148.

15. Nishida, M., et al. 2002. Absence of angiotensinII type 1 receptor in bone marrowderived cells

is detrimental in the evolution of renal fibrosis.J. Clin. Invest. 110:18591868. doi:10.1172/JCI200215045.

16. Redmond, E.M., et al. 2001. Endothelial cellsinhibit flow-induced smooth muscle cell migra-tion: role of plasminogen activator inhibitor-1.Circulation.103:597603.

17.Proia, R.R., et al. 2002. The effect of endothelialcell overexpression of plasminogen activatorinhibitor-1 on smooth muscle cell migration.J. Vasc. Surg.36:164171.

18. Iwano, M., et al. 2002. Evidence that fibroblastsderive from epithelium during tissue fibrosis.J. Clin. Invest. 110:341350. doi:10.1172/JCI200215518.

19. Fogo, A. 2001. Progression and potential regres-sion of glomerulosclerosis. Nephrology Forum.

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