Mechanisms of Disease Reversal in Focaland Segmental Glomerulosclerosis

Hai-Chun Yang and Agnes B. Fogo

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It iswell known that progression of chronic kidney disease can be ameliorated or stabilized by different interventions, butmorestudies indicate that it can even be reversed. Most data suggest that current therapy, especially renin-angiotensin systeminhibition alone, is not sufficient to initiate and maintain long-term regression of glomerular structural injury. In this article,we review the potential reversal of glomerulosclerosis and evidence from both human and animal studies. We discussmechanisms that involve matrix remodeling, capillary reorganization, and podocyte reconstitution. In the future, a multi-pronged strategy including novel anti-inflammatory and antifibrotic molecules should be considered to potentiate regressionof glomerulosclerosis.Q 2014 by the National Kidney Foundation, Inc. All rights reserved.Key words: Glomerulosclerosis, Regression, ECM, Capillary, Podocyte

IntroductionProgression of chronic kidney disease is a major healthproblem. Interventions have focused on control of bloodpressure and inhibition of the renin-angiotensin system(RAS) but have only resulted in slowing down progres-sion. Over the last years, multipronged intervention hasresulted in amelioration of progression and even stabili-zation of CKD. However, recent observations in humansand in experimental models point to the possibility ofregression of sclerosis, which led to a shift in paradigmsregarding progressive scarring from a view of sclerosisas a fixed, inevitable outcome in progressive kidney dis-eases to an understanding of sclerosis as a dynamic,ongoing process that may be modulated.1-5 Regressionof existing glomerulosclerosis requires degradationof extracellular matrix (ECM) accumulation andregeneration of parenchyma. The lesion of glomerularsclerosis is not only a phenomenon of primary focaland segmental glomerulosclerosis (FSGS) but is also aubiquitous secondary injury underlying the progressivedeterioration of many different types of kidneydiseases. We will review evidence and mechanisms ofregression of glomerulosclerosis, using the term“FSGS” to include this secondary progressive sclerosis.

The Potential Reversal of FSGSAlthough lower animals can regenerate nephrons afterinjury, inmammals, after term birth, no new nephron unitscan be generated.6 The tubular epithelium has ampleregenerative capacity. Thus, after acute kidney injury,restoration of parenchyma and function is possible. How-

m Department of Pathology, Microbiology and Immunology, Vanderbiltsity Medical Center, Nashville, TN.port: These studies were funded in part by the National Institutes ofDK 56942 and DK44757 (A.B.F).dress correspondence to Agnes B. Fogo, MD, Department of Pathology,iology and Immunology, 1161 21st Avenue South, Vanderbilt Universityl Center, MCN C3310, Nashville, TN 37232. E-mail: agnes.fogo@bilt.edu014 by the National Kidney Foundation, Inc. All rights reserved.8-5595/$36.00p://dx.doi.org/10.1053/j.ackd.2014.04.001

Advances in Chronic Kidney Disease, Vol 2

ever, even the tubule has limitations, and acute kidneyinjury has recently been recognized as a major risk factorfor CKD.7 This has been linked to loss of normal cell cycleprogression during repair, and loss of tubular epithelialcells, with fibrosis resulting rather than generation ofnew tubular epithelial cells. However, portions ofglomeruli can potentially be restored by capillary length-ening and/or branching. By using 3-dimensional recon-struction of individual glomerular capillary tufts,Remuzzi and colleagues8 found that after 10 weeks ofangiotensin-converting enzyme inhibition, at 60 weeks ofage, more than 20% of glomeruli were completely free ofsclerosis, whereas at 50 weeks of age practically allglomeruli had some degree of sclerosis. These data suggestthat space previously occupied by glomerulosclerosis wasnow occupied by new capillary tissue. Our mathematicalmodeling indicated that individual glomerular tufts withsclerosis occupying more than 50% of the capillaries aredoomed to progression. Conversely, glomeruli with lessthan 50% sclerosis of the tuft are capable of growing newcapillary loops.9 Not all glomerular cells have equal capac-ity for regeneration. Although endothelial cells and me-sangial cells readily proliferate after injury, the podocytehas limited, if any, regenerative capacity.

Evidence of Disease Reversal in Human CKDThere are very few studies with repeated biopsies todirectly prove regression of sclerosis in humans, andnone specifically for primary idiopathic FSGS. However,some clinical observations support the possibility of re-modeling of sclerosis. Proof of principle of regression of ex-isting glomerular injury was shown in a small study ofdiabetic patients with moderately advanced diabetic ne-phropathy whose diabetes was cured by pancreas trans-plant.2 The severity of the diabetic nephropathy wasunchanged at 5 years; however, at 10 years, both glomer-ular lesions and tubulointerstitial lesions had regressed.10

The African American Study of Kidney Disease and Hy-pertension (AASK) showed that many CKD patientshave a nonlinear glomerular filtration rate (GFR) trajectoryor a prolonged period of nonprogression, which indicatesthat CKD need not be relentlessly progressive.11 Nondia-betic nephrotic patients who were treated with theangiotensin-converting enzyme inhibitor (ACEI) ramipril

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Mechanisms of Disease Reversal in FSGS 443

for 2 years as part of the REIN (Ramipril Efficacy in Ne-phropathy Core and Follow-up) study achieved stabiliza-tion of their rate of GFR decline, to a yearly loss similar tonormal aging. Interestingly, in a small subset of these pa-tients, GFR even improved, and thus, they have notreached ESRD.3 Full remission of proteinuria and stabi-lized kidney function in response to long-term ACEIwere also observed in a small number of patients inanother long-term follow-up study of diabetic patientswith nephropathy.12 Thesefindings support that remissionand even regression of the functional parameters of CKDcan occur in humans with diabetic or nondiabetic kidneydisease. However, whether these functional improve-ments were contributed to in part by any regression ofstructural injury remains unknown.

Mechanism of Disease Reversal in FSGS ModelsReversal of glomerulosclerosis may occur through varioussteps, including matrix remodeling, capillary reorganiza-tion, and podocyte reconstitution. We will review experi-mental evidence of mechanisms of such processes.

Effects on MatrixTo achieve regression of sclerosis, matrix degradation

CLINICAL SUMMARY

� Glomerulosclerosis at early, segmental stages can bereversed.

� Monotherapy with renin angiotensin blockade isinsufficient to effect regression of sclerosis.

� Mechanisms of regression of sclerosis involvescoordinated matrix remodeling, capillary reorganization/growth, and podocyte reconstitution.

must exceed matrix synthe-sis. A delicate balance be-tween ECM synthesis anddegradation affects progres-sion and potential regressionof glomerulosclerosis. Keyfactors that promote collagensynthesis include, but are notlimited to, blood pressure,angiotensin II, transforminggrowth factor-b (TGF-b),platelet-derived growthfactor-B, and numerousother growth factors. Angio-

tensin has been a central focus for mechanisms of progres-sion, linked not only to its effects on systemic andglomerular hypertension but effects on matrix synthesisand cell proliferation. ACEI has shown superior effectson kidney disease progression in various human diseasesand in animal models compared with other antihyperten-sive agents, even in conditions without systemic hyperten-sion. These findings suggest that angiotensin II may haveeffects beyond blood pressure in progressive kidney dis-ease, and conversely, the effects of ACEI or angiotensintype 1 receptor blocker (ARB) might extend beyond anti-hypertensive mechanisms.13 Aldosterone has bothgenomic and nongenomic actions to promote fibrosis, in-dependent of its actions to increase blood pressure bymediating salt retention.14 It enhances angiotensin induc-tion of plasminogen activator inhibitor-1 (PAI-1) and alsohas direct actions on fibrosis.15

Models of progressive glomerular disease in rodentshave shown the potential for regression. Indeed, weshowed that higher doses of ACEI than required tonormalize both systemic and glomerular hypertensionhad greater benefits on established glomerulosclerosis inthe remnant kidney, a secondary FSGS model, than usual

antihypertensive dose.4 Although there was no furtherimpact on systemic or glomerular pressures, as shownby micropuncture studies, two-thirds of these animalsachieved regression of glomerulosclerosis with high-doseangiotensin inhibition. Regression was evidenced by lessextensive and less severe sclerosis after 4 weeks of therapythan that seen at the time of biopsy, when interventionwasstarted, 8 weeks after injury was initiated. Animals withregression had better preserved podocytes, more capillarybranching, and less matrix accumulation. There was alsocorresponding less tubulointerstitial fibrosis.16 Aldoste-rone inhibition also resulted in regression of sclerosis inthis model.5

These inhibitors of the renin-angiotensin aldosterone sys-tem (RAAS) also decreased expression of PAI-1. PAI-1, amember of the superfamily of serine protease inhibitors, in-hibits tissue-type and urokinase-type plasminogen activa-tors and, thus, prevents activation of plasminogen toplasmin. Plasmin not only lyses fibrin but also can degradeECM. PAI-1 is produced from multiple sources, includingendothelium, vascular smooth muscle cells, liver, platelets,and tubular epithelial cells.17 Angiotensin induces PAI-1,via the angiotensin type 1 (AT1) receptor, independentlyof blood pressure effects. High levels of PAI-1 have been

linked to excess fibrosis inboth humans and in experi-mental models. We foundelevated PAI-1 expressionat sites of sclerosis andabsence of PAI-1 whenregression was achieved. Incontrast, TGF-b messengerRNA was not decreasedwhen regression wasachieved, and its localexpression level was notlinked to sclerosis. Howev-er, these findings do not

rule out a role for TGF-b signaling in progression, throughfor instance, connective tissue growth factor or phosphory-lation of Smad2, or a role for its inhibition in regression ofsclerosis. Interestingly, 2 key matrix metalloproteases,MMP-2 and MMP-9, were not linked to regression. Incontrast, the high level of PAI-1 in animalswithprogressiveglomerulosclerosis was associated with low levels ofplasmin.HighdosesofACEIorARB that resulted in regres-sion restored plasmin levels toward normal. These datasupport that RAAS inhibitors may in part modulate glo-merulosclerosis by effects to degrade ECM.Regression by high-dose RAAS inhibition was not only

limited to this remnant kidney model but also wasobserved in the primary podocyte injury–induced FSGSmodel of puromycin aminonucleoside nephropathy andin age-related glomerular and vascular sclerosis. In thechronic puromycin aminonucleoside model, regressionof sclerosis with intervention with either ACEI or lowprotein diet was inferred by comparisons of differentgroups of rats at various time points.18 We also showedregression of early biopsy-proven glomerulosclerosis le-sions in this model, with less sclerosis observed in kidneysat sacrifice in the same rats after treatment with high-dose

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ACEI.19 The sclerosis occurring in aged rats could beremodeled by inhibiting angiotensin II with high-dose ARB for 6 months, starting at a time point ofmoderate injury at 18 months of age.20

More recently, elegant studies by Adamczak and col-leagues21 and Remuzzi and others22 have confirmedregression in the remnant kidney model and in thespontaneous nephropathy occurring in the Munich Wis-tar Fromter model, with high-dose ACEI and combina-tion of ACEI and ARB, respectively. The data byAdamczak and others revealed that remodeling ofvascular sclerosis, tubulointerstitial fibrosis, and exist-ing glomerulosclerosis is feasible. Regression has alsobeen observed in experimental hypertensive nephropa-thy by high-dose ARB.23

To maximize the potential for regression, multiprongedapproaches will probably be necessary. The efficacy ofcombining ARB with ACEI has been explored in severalstudies as discussed earlier. Remuzzi and others showedthat ACEI and ARB, with or without added statins, led tofurther regression than monotherapy in an experimentalmodel.24 However, no advantage of combination of ARBand ACEI has been demonstrated in previous animalstudies, beyond that achieved with greater blood pres-sure control.25,26 Several clinical trials using dual RAASblockade failed to show cardiovascular or kidneyprotection, but showed more adverse events,suggesting that dual RAAS blockade for the treatmentof CKD cannot currently be recommended.27-29 Theangiotensin type 2 (AT2) receptor counteracts theclassic AT1 receptor actions, especially by inducingvasodilation instead of vasoconstriction and also withcell-specific effects and, thus, might play a beneficialrole in remodeling of sclerosis. There is now unequivocalevidence that the AT2 receptor mediates microvascularvasodilation by nitric oxide (NO) generation in abradykinin-dependent or independent manner.30,31

Vazquez and others32 found a time-dependent increasein AT2 receptor expression at 7, 15, and 30 days after 5/6 nephrectomy. Animals pretreated with ARB showed afurther increase in AT2 receptor expression. WhetherAT2 contributes to ARB-induced regression of glomeru-losclerosis is of interest. We directly explored this hy-pothesis by adding blockade of the AT2 receptor toAT1 blocker in the remnant kidney model.33 The addedinhibition of AT2 blockade completely prevented thebeneficial effects of the ARB. This progressive sclerosisin rats treated with AT2 receptor antagonist was associ-ated with increased PAI-1 in contrast to decrease withARB. AT1 and AT2 blockers also affected podocytesand collagen synthesis. Cultured wild-type podocytes,but not PAI-1 knockout cells, responded to angiotensinII with increased collagen, an effect that was completelyblocked by ARB with lesser effect of the AT2 receptorantagonist. We conclude that the beneficial effects onglomerular injury achieved with ARB are contributednot only by blockade of the AT1 receptor but also byincreasing angiotensin effects transduced through theAT2 receptor and that these effects include effects bothon matrix and podocytes.

Effects on CapillariesAlthough regression of chronic kidney diseases cannot beachieved by growing new glomeruli, we postulate, basedon elegant studies by Nyengaard and others, that seg-ments of glomeruli can regenerate by capillary length-ening and/or branching, whereas the sclerosed segmentmay be largely reabsorbed.34

In the remnant kidney model, regression by high-doseACEI was manifest by decreased sclerosis volume andincreasedvolumeof intact capillaries byup to 40%.8Mesan-gial and endothelial cell proliferation was reversed, but thenumber of podocytes per glomerulus was not changedby ACEI treatment.35 In our studies by confocal 3-dimensional imaging and graph theory analysis, we alsofound that glomeruli in rats with progressive sclerosiswere enlarged but had reduced number of capillarysegments and capillary branch points and decreasedcomplexity of the glomerular network compared withnormal glomeruli. In contrast, in ratswith regression of scle-rosis induced by high-dose ARB, glomerular enlargementwas not because of increased matrix but because of theincreased number of glomerular capillary segments andcapillarybranchpoints and restored complexityof the capil-lary network.36

Therapeutic efforts beyond RAAS inhibition mustinclude mechanisms to protect and restore the podocyte.The podocyte is a key for glomerular endothelial cell sur-vival, by secreting key angiogenic factors such as vascularendothelial-derived growth factor and angiopoietin-1.Elegant experiments have shown that podocyte vascularendothelial-derived growth factor expression is necessaryto maintain the endothelium of the glomerulus. In vitro,we showed that injured podocytes treated with ARB hadrestoration of their angiogenic capability.37 Thus, endothe-lial cell health and angiogenesis are crucially inter-relatedwith podocyte health.

Glomerular Epithelial CellsPodocytes have limited, if any, ability to proliferatebecause of high expression of a cyclin-dependentkinase inhibitor, p27kip1.38 Podocytes are key to thestructural integrity of the glomerulus and to its permselec-tivity functions and to maintain the glomerular endothe-lium (as mentioned previously). Podocyte loss is closelycorrelated with progressive glomerulosclerosis. Directpodocyte injury in genetically engineered rodents demon-strates that injury initially limited to only this cell canresult in fully developed sclerosis. In dosing experimentsfrom the group of Wiggins, a threshold for about 20%loss was shown to determine whether repair could occuror if progressive sclerosis ensued.39 Interestingly, evenwhen only some podocytes are initially injured, injurycan spread to other podocytes, as demonstrated usingtransgenic chimericmicewhere a toxin receptor for humanCD25 was expressed in only a subset of podocytes. Injec-tion of toxin that binds to hCD25 resulted in injury evento wild-type podocytes and also increasingly severe scle-rosis as injury spread.40

Podocytes can be regenerated from local progenitorcells. Parietal epithelial cells (PECs) are postulated to serve

Mechanisms of Disease Reversal in FSGS 445

as niche stem cells for podocytes, based onmarker studiesin humans and lineage tracing cells in animal models.41

Thus, the presence of CD241/CD133 1 cells liningBowman capsule supports a stem cell function.42 Both po-docytes and PECs aremobile cells, and their cytoskeletonscan respond rapidly to stimuli and allow migration. Ininjury models, migration of PECs on to the tuft, eitheralong the vascular pole or by extension along synechiae,has been associated with local increase of matrix and,thus, postulated to rather be an injurious profibroticresponse. Aberrant Notch-1 expression may contributeto such adverse PEC migration.43 However, it is pos-sible that migration of such cells could give rise topodocyte-like cells, and perhaps even restore podocytesafter injury, depending on the local matrix context. Thus,tissue regeneration may, as in other wound healing set-tings, depend on the local balance of ECM accumulationand the degradation and resolution of a provisional ma-trix. Interestingly, the proliferating visceral epithelial cellswere observed in collapsing glomerulopathy stain withCD44, a marker of activated PECs. In the early phase ofrecurrence of FSGS in the transplant, when standard mi-croscopy and electron microscopy document only footprocess effacement, but no sclerosis, such activatedCD441 cells were increased in the glomerular tuft andalong Bowman capsule. In contrast, native kidney mini-mal change disease showed significantly less such acti-vated PECs.44 Insufficient PEC migration has also beenpostulated to contribute to age-related sclerosis.45 How-ever, by using lineage-tracing models, PEC migrationonto the vascular tuft and differentiation into podocytesonly was detected in juvenile mice but not in aging miceand adult mice with injury induced by subtotal nephrec-tomy.46 The biology and pathophysiologic functions ofPECs are still controversial. PECs might be reparative orinjurious, depending on the type of injury and age ofthe mouse or patient.47 In addition to PECs, cells ofrenin lineage may also enhance glomerular regenerationby serving as progenitors for glomerular epithelial cellsand podocytes.48

Podocyte injury can be protected, and its regenerationalso can be induced by some interventions. High-doseACEI could reverse the spontaneously occurring FSGS le-sions in aged Munich Wistar Fromter rats, linked to po-docyte proliferation. These “podocytes” are postulatedto result from regeneration from the parietal epithe-lium.49 ARB increased glomerular transitional cells inseveral models of glomerulosclerosis and proteinuria,with cells double staining for podocyte and PECmarkers, with resulting increased number of podocytes.50

Podocytes may also be directly protected by glucocorti-coids, and this treatment also increased progenitorcells in experimental FSGS induced by anti-podocyteantibody injection. Glucocortocoid also protected podo-cytes from apoptosis and, thus, promoted less injuryand increased repair, with ultimately less sclerosis intreated mice.51 Maintenance of microRNA-30, whichdecreased Notch-1 activation and inhibited the cell cyclemolecule p53, may contribute to these reparative effectsof glucocorticoids.52

Novel Strategies to Reverse FSGSAdditional preliminary studies have shown that combina-tion of peroxisome proliferator–activated receptor gammaagonists and ARB induced more regression of glomerulo-sclerosis in the 5/6 nephrectomymodel than monotherapywith either agent. These additional effects were related toless inflammation and more podocyte protection.53 Com-bination therapy with an ACEI, ARB, and statin alsoachieved even better results on sclerosis than monother-apy with any one of these classes of drug.24 Impressively,combination of ARBwith the diuretic hydrochlorothiazideafforded additional protection of function compared withARB alone when treatment was started at advanced stagesof injury in the 5/6 nephrectomy model. Other RAAS tar-gets also provide additional avenues for tissue protectionand potential regeneration. Renin inhibition reversed thebalance of profibrotic vs antifibrotic mediators in renintransgenic mice, with restoration of tubular epithelial celland podocyte function and structure.54 Novel targetsinclude periostin, also linked to disease progression.55

Although sclerosis was not regressed, progression wascompletely halted, indicating a potential for modulationeven of advanced injury.

SummaryReversal of glomerulosclerosis can be achieved. High-dose ACEI or ARB can slow the progression of chronickidney diseases or even partially reverse glomeruloscle-rosis. However, it appears unlikely that RAS inhibitionalone will be sufficient to initiate and maintain long-term regression of glomerular structural injury. Futureapproaches include consideration and targeting addi-tional mechanisms to optimize matrix remodeling,capillary reorganization, and podocyte reconstitution.Thus, a multipronged strategy including novel anti-inflammatory and antifibrotic molecules, in addition tostandard RAS inhibition, should be considered to poten-tiate regression of glomerulosclerosis.

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