New Approaches to the Treatment of Dense Deposit Disease
Richard J.H. Smith*,
Jessy Alexander,
Paul N. Barlow,
Marina Botto,
Thomas L. Cassavant||,
H. Terence Cook,
Santiago Rodriguez de Córdoba¶,
Gregory S. Hageman**,
T. Sakari Jokiranta,
William J. Kimberling,
John D. Lambris,
Lynne D. Lanning||||,
Vicki Levidiotis¶¶,
Christoph Licht***,
Hans U. Lutz,
Seppo Meri,
Matthew C. Pickering,
Richard J. Quigg,
Angelique L. Rops,
David J. Salant,
Sanjeev Sethi||||||,
Joshua M. Thurman¶¶¶,
Hope F. Tully****,
Sean P. Tully****,
Johan van der Vlag,
Patrick D. Walker,
Reinhard Würzner,
Peter F. Zipfel Dense Deposit Disease Focus Group
Departments of * Internal Medicine and Otolaryngology and ** Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City, Iowa; Department of Internal Medicine, University of Chicago Hospitals, Chicago, Illinois; Institute of Structural and Molecular Biology, University of Edinburgh, King’s Buildings, Edinburgh, United Kingdom; Imperial College, Faculty of Medicine, London, England; || Center for Bioinformatics and Computational Biology, University of Iowa, Iowa City, Iowa; ¶ Centro de Investigaciones Biologicas, Madrid, Spain; Department of Bacteriology and Immunology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; Boys Town National Research Hospital, Omaha, Nebraska; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; |||| Kidneeds, Greater Cedar Rapids Community Foundation, Cedar Rapids, Iowa; ¶¶ Royal Prince Alfred Hospital, Sydney, Australia; *** Department of Pediatrics, Hospital for Sick Children, Toronto, Canada; Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland; Nephrology Research Laboratory, Division of Nephrology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; Department of Medicine, Boston University Medical Center, Boston, Massachusetts; |||||| Department of Pathology, The Mayo Clinic, Rochester, Minnesota; ¶¶¶ Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado; **** Milagros Research Fund, Chappaqua, New York, New York; Nephropathology Associates, Little Rock, Arkansas; Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria; and Leibniz Institute for Natural Product Research and Infection Biology and Friedrich Schiller University, Jena, Germany
Correspondence: Dr. Richard J.H. Smith, 200 Hawkins Drive, 21151 PFP, University of Iowa, Iowa City, IA 52242. Phone: 319-356-3612; Fax: 319-356-4108; E-mail: richard-smith{at}uiowa.edu
The development of clinical treatment protocols usually relieson evidence-based guidelines that focus on randomized, controlledtrials. For rare renal diseases, such stringent requirementscan represent a significant challenge. Dense deposit disease(DDD; also known as membranoproliferative glomerulonephritistype II) is a prototypical rare disease. It affects only twoto three people per million and leads to renal failure within10 yr in 50% of affected children. On the basis of pathophysiology,this article presents a diagnostic and treatment algorithm forpatients with DDD. Diagnostic tests should assess the alternativepathway of complement for abnormalities. Treatment options includeaggressive BP control and reduction of proteinuria, and on thebasis of pathophysiology, animal data, and human studies, plasmainfusion or exchange, rituximab, sulodexide, and eculizumabare additional options. Criteria for treatment success shouldbe prevention of progression as determined by maintenance orimprovement in renal function. A secondary criterion shouldbe normalization of activity levels of the alternative complementpathway as measured by C3/C3d ratios and C3NeF levels. Outcomesshould be reported to a central repository that is now accessibleto all clinicians. As the understanding of DDD increases, noveltherapies should be integrated into existing protocols for DDDand evaluated using an open-label Bayesian study design.
In the past two decades, the development of new clinical treatmentprotocols has revolved around evidence-based guidelines. Randomized,controlled trials have become the favored metric for assessingthe effectiveness of novel interventions, with anything fallingbelow this level of certainty running the danger of being discounted.1For rare diseases, this requirement represents a significantchallenge.
A rare disease makes the randomized, controlled study designimpractical for numerous reasons: Sample size is small and geographicallydispersed; the use of historical controls is often impossible;and randomization can be seen as unethical, especially in theface of significant disease morbidity.2 Because rarity, by definition,suggests an insubstantial public health care concern, one approachto this conundrum is to avoid rare diseases in favor of morecommon and substantial problems. However, this option is impracticalbecause rare diseases, in aggregate, still represent a substantialhealth care problem in the developed world.
There are 5000 to 6000 rare diseases, most of which are geneticin origin, and with the continued separation of broad diseasecategories into smaller, well-defined entities, approximately250 new rare diseases are described each year.3 For a diseaseto be considered rare in the United States, it must affect fewerthan 200,000 citizens, reflecting a prevalence of approximatelysix per 10,000, whereas in Europe, the definition is slightlystricter: Up to five per 10,000.4 Thus, an estimated 25 millionNorth Americans and 30 million Europeans are afflicted withrare diseases. How, then, are therapeutic advances to be developedfor these populations? This article focuses on dense depositdisease (DDD; also known as membranoproliferative glomerulonephritistype II), which is rare even among rare diseases, and uses DDDas a model for how new treatment guidelines can be proposedon the basis of evidence derived from animal studies and geneticand molecular data and how outcomes can be followed using Bayesiananalysis.
DDD affects an estimated two to three people per million. Itaccounts for <20% of all cases of membranoproliferative glomerulonephritisin children and only a fractional percentage of cases in adults.5,6The name itself is descriptive of the electron-dense transformationof the glomerular basement membranes (GBM) that occurs in asegmental, discontinuous, or diffuse pattern within the laminadensa (Figure 1). The precise composition of these altered areasremains unknown. The key complement protein, C3, is almost alwaysseen by immunofluorescence microscopy, usually in the absenceof Ig deposition. Its presence along the margins of the densedeposits produces a "railroad track" appearance, and where itoutlines the mesangium, rings are seen.7
Figure 1. Histopathology of dense deposit disease (DDD). (A) The classic light microscopic appearance showing a membranoproliferative pattern (seen in approximately 25% of patients; periodic acid-Schiff stain). (B) C3 in loops and mesangial areas. The prominent granular deposits in the mesangium result in rings of immunofluorescence that are characteristic of DDD (fluorescein-conjugated anti-C3 antibody stain). (C) Electron photomicrograph showing highly electron-dense transformation of the glomerular basement membranes diagnostic of DDD (unstained). Magnifications: x400 in A and B; x5000 in C.
The classic light microscopic appearance of a membranoproliferativeglomerulonephritis is seen in approximately 25% of patients.8,9Mild mesangial cell hypercellularity is the most common pattern(approximately 45%), but a crescentic pattern (approximately18%) or an acute proliferative and exudative pattern (12%) alsooccurs.9 In addition to glomerular dense deposits, patientsdevelop deposits along the choriocapillaris-Bruch’s membrane-retinalpigment epithelial interface, a region morphologically similarto the capillary tuft-GBM-podocyte interface (Figure 1).
As a histologically defined disease, DDD lacks unequivocal diagnosticserologic markers of disease activity, although most patientsare positive for C3 nephritic factor (C3NeF).6,10 This is anautoantibody that recognizes neoantigenic epitopes on C3bBb,the C3 convertase of the alternative pathway of complement.C3 convertases cleave C3 into C3b and C3a and thereby instigateand amplify the complement cascade. By stabilizing this normallylabile convertase, C3NeF impedes the physiologic regulationof C3bBb by the regulators of the complement activation familyand factor I. Nearly 80% of patients with DDD have evidenceof alternative pathway dysregulation as reflected by low C3levels and detectable C3 degradation products, such as C3d,in their serum.10
DDD affects female individuals slightly more frequently thanmale individuals. The DDD Database, a patient-parent–drivenepidemiologic study, reports a 3:2 female:male bias among the56 patients with DDD that it has accrued.11 This database alsoreports that progression to ESRD occurs in approximately halfof patients who have carried the diagnosis for at least 10 yr,in agreement with data reported by other investigators.12,13The North American Pediatric Renal Trials and CollaborativeStudies (NAPRTCS) database outcomes are similar. Of the 119registered children with DDD, 81 have progressed to ESRD (personalcommunication, William Harmon, MD, Children’s Hospital,Boston, MA; March 2, 2007). From the DDD Database, it seemsthat progression to ESRD develops rapidly, usually within 4yr of diagnosis, and is the more likely outcome in younger (10yr) than older patients (P = 0.006; Figure 2). Girls may havea more aggressive disease course than boys (P = 0.16).
Figure 2. Age at diagnosis versus outcome (stable or ESRD). Patients who are 10 yr of age are more likely to progress to ESRD than are older patients (P = 0.006). Progression to ESRD typically occurs within 4 yr of diagnosis.
There have been fewer than 200 transplants in patients withDDD.14 Five-year allograft survival approximates 50%, whichis significantly worse than the NAPRTCS database as a whole(P = 0.001). Living-related donor grafts fair better than deceased-donorgrafts (P < 0.005). Histologic evidence of recurrent DDDdevelops in nearly all grafts and is the predominant cause ofgraft failure in 15 to 50% of transplant recipients.6,15 Graftloss typically occurs within 2.5 years of transplantation. Thereare few data to suggest that any therapeutic interventions havean impact on reversing this course, although isolated reportshave described the use of plasmapheresis, which seems to beof equivocal benefit.16,17 The impact of genetics on graft survivalhas not yet been explored.
The first animal model in which DDD was described was the NorwegianYorkshire breed of piglets.18,19 Affected piglets seemed healthyat birth but after a few weeks failed to thrive as a resultof a rapidly progressive glomerulonephritis that inevitablyled to death (median 37 d; n = 25). Hegasy et al.20 showed thatthe molecular basis for kidney failure was a point mutationleading to an isoleucine-to-arginine change at amino acid position1166 (I1166R), which resulted in a nonfunctional factor H geneproduct. The factor H gene, CFH, encodes a soluble member ofthe regulators-of-complement-activation family that acts atthe level of the C3 and C5 convertases. The I1166R mutationeffectively impedes extracellular release of factor H, resultingin a decrease in serum factor H levels and unchecked and deregulatedactivation of the alternative pathway of complement.
Although the DDD Norwegian Yorkshire pigs are no longer available(sperm has been stored), a mouse with a targeted deletion ofFactor H (Cfh–/–) has been made.21 Deletion of factorH, like its intracellular retention, results in uncontrolledactivation of the alternative pathway of complement, evidencedin these mice by significantly reduced concentrations of C3and the presence of C3 breakdown products in the homozygousmutants. Cfh–/– mice also develop renal diseasecharacterized by the deposition of C3 on glomerular capillarywalls, mesangial hypercellularity with marked matrix expansion,peripheral capillary loop thickening with the deposition ofperiodic acid-Schiff–positive material, and double-contouringof the GBM, entirely consistent with the diagnosis of DDD andin concordance with the histology that develops in the porcinekidney. However, unlike the Norwegian Yorkshire pig, the factorH–deficient mouse has only 25% 8-mo mortality.
Mouse mutants that are null for both factors H and B (Cfh–/–.Cfb–/–)have a normal renal phenotype, as would be predicted from thealternative pathway complement cascade, because factor B isnecessary for the formation of C3bBb, the alternative pathwayconvertase (Figure 3). The absence of factor B in the Cfh–/–.Cfb–/–mutant precludes formation of this convertase, making the absenceof factor H inconsequential. This finding also suggests thatuncontrolled activation of C3 is an absolute requirement forthe development of DDD and is consistent with the observationthat C3 deposition in the GBM is evident before the appearanceof the dense deposits.21
Figure 3. The alternative pathway is constitutively active at low levels through the hydrolysis of the thioester in C3 to C3(H2O). Hydrolyzed C3 combines with factor B, and in the presence of factor D, C3(H2O)Bb is formed. This intermediate convertase leads to the production of C3a and C3b from C3, and C3b enters the C3bBb amplification loop. Amplification on soluble C3bBb occurs with low efficiency because free C3b is rapidly inactivated by factors H and I. However, if C3b binds covalently to surfaces or as a covalent dimer to fluid-phase IgG, then it is partially protected from inactivation. In its dimeric form (C3bC3bIgG), it is seven to 10 times more efficient in generating a C3 convertase than surface-bound monomeric C3b.71 The very same enzyme on surfaces or on IgG in the fluid phase becomes a C5 convertase by acquiring an additional C3b in its vicinity, which increases the affinity of the enzyme for C5. Here we show in red just one of the possible amplification routes, which seems to be the most relevant in DDD (see text). In the absence of factor H to control levels of C3b in the fluid phase, the Cfh–/– mouse mutant develops DDD. Because factor B is critical to the formation of C3bBb, its absence in the Cfh–/–.Cfb–/– mutant rescues the disease phenotype and DDD does not develop. In the Cfh–/–.C5–/– mutant and the Cfh–/– mutant treated with anti-C5 antibodies, the degree of kidney disease is decreased compared with the degree of kidney disease seen in the Cfh–/– mutant.
Cleavage of C5 by C5 convertase is the last enzymatic step inthe complement cascade (Figure 3). Of the two forms of C5 convertase,one (the alternative pathway convertase) is formed by additionof C3b to the C3 convertase, C3bBb. This trimolecular C5 convertase(i.e., C3bC3bBb) converts C5 into C5a and C5b. C5b, in turn,complexes with C6 and C7, which recruit C8 and trigger bindingand polymerization of C9 to form C5b-9, the membrane attackcomplex (MAC). MAC creates pores in membranes that are not protectedby complement regulators and promote destruction of pathogenicorganisms or immune complex–coated cells.
Although serum convertases that are formed with monomeric C3bare inefficient in converting C5 into C5a and C5b, in DDD, thecontinued cleavage of C3 and the formation of C3b-C3b dimerson the GBM is a particularly effective mechanism for promotingthe formation of C5 convertase at this site.22 To determinethe effect of C5 and downstream proteins of the complement cascadein DDD, Pickering et al. studied the renal phenotype in Cfh–/–.C5–/–mice and in 12-mo-old animals observed less severe renal diseasewith reduced mortality and reduced glomerular cellularity ascompared with age-matched Cfh–/– mice. However,the proteinuria at 12 mo did not differ between the Cfh–/–and Cfh–/–.C5–/– mice, suggesting thatchronic deposition of C3 along the GBM alone is sufficient todisrupt the glomerular permeability barrier.23
Suspecting that renal inflammation during DDD flare-ups maycritically depend on C5 activation, they next explored the effectof C5 inhibition using a monoclonal anti-C5 antibody and foundthat it protected Cfh–/– mice that were exposedto a nephrotoxic insult triggered by nephrotoxic serum.23 Administrationof anti-C5 antibody completely prevented the development ofproteinuria and glomerular neutrophil influx. Similar experimentsperformed on Cfh–/–.C6–/– mice wereaccompanied by marked neutrophil infiltration and proteinurianot significantly different from that seen in Cfh–/–animals, indicating that it is cleavage of C5 to the anaphylatoxinC5a, as opposed to the generation of MAC, that accounts forthe glomerular neutrophil influx and albuminuria in Cfh–/–mice during heterologous nephritis.23
In aggregate, animal data firmly place fluid-phase dysregulationof the alternative pathway of complement as the triggering pathophysiologicevent in DDD. During disease progression, solid-phase activationof downstream complement proteins, in particular cleavage ofC5 to C5a and C5b, contributes to the injury.
DDD is a complex genetic disease. Only a few families in whichmore than one member has DDD have been identified, althoughthere are several families in which multiple members have avariety of other autoimmune diseases such as Celiac disease,thyroiditis, and type 1 diabetes.6 Included in the latter groupof families is one in which there are identical twins, one withDDD and the other with type 1 diabetes, suggesting that in thepresence of a permissive genotype, environmental factors maybe important determinants of disease phenotype.
Of the genes associated with DDD, the most robust data are availablefor factor H. Consistent with animal data implicating deletionof this gene in dysregulation of the alternative pathway andthe development of a DDD renal phenotype, one family of consanguineousparentage has been reported in which two siblings’ DDDwas diagnosed by renal biopsy.24 Both children were positivefor C3NeF and had low C3 and alternative pathway–mediatedhemolysis (APH) 50 levels with increased levels of the C3 degradationproduct C3d. (APH 50 measures total hemolytic activity of thealternative pathway.) Mutation screening of the factor H gene,CFH, showed that the affected children were homozygous for thedeletion of a lysine residue at position 224 (K224).
K244 is located within the complement regulatory region in thefourth of the 20 short consensus repeats (SCR) of factor H.Functional studies of factor H K224 have shown that bindingto heparin, C3d, and human umbilical vein endothelial cellsis not altered, consistent with its intact C-terminal recognitionand cell-binding properties. However, binding to C3b is weak;consequently, both co-factor activity of factor H K224 in thepresence of factor I and decay-accelerating activity are markedlyreduced.24
Most patients with DDD do not have disease-causing mutationsin CFH; however, several alleles of both CFH and the complementfactor H–related 5 gene (CFHR5) are preferentially associatedwith DDD.25–27 Of these associations, one of the potentiallymost interesting is the tyrosine-402-histidine (Y402H) polymorphism.The frequency of the factor H H402 variant is increased in bothDDD and age-related macular degeneration, which may be germanebecause patients with DDD develop early-onset macular drusen.28–30
The Y402H polymorphism lies in SCR7. This SCR contributes toone of at least three glycosaminoglycan (GAG)-recognition sitesin factor H and participates in binding to C-reactive proteinand a number of pathogens that sequester factor H for protectionagainst complement. Structural studies have shown that the substitutionoccurs toward the center of SCR7, well away from boundarieswith SCR8 and 9, and that the three-dimensional structures ofboth allotypic variants are otherwise identical.31 Nevertheless,binding studies indicate that the Y402H change alters the specifictypes of GAG that are recognized by this particular site, whichis interesting in view of the fact that mutations disruptingSCR20 affect binding to C3d/C3b and are linked to another rarekidney disorder, atypical hemolytic uremic syndrome.32–34In vitro functional studies have shown that binding to bothhuman umbilical vein endothelial cells and C-reactive proteinis reduced for the H402 variant of factor H as compared withthe Y402 variant.33,34 Heparin-binding assays of the H402 andY402 variants produce equivocal results.
Significant associations with DDD have also been found withthe two common allotypes of C3, glycine 102 (G102) and arginine102 (R102), designated C3F (fast, G102) and C3S (slow, R102)on the basis of differences in electrophoretic motility.35,36C3F is the less common variant and is found in only 20% of white,5% of black, and 1% of Asian individuals.37–39 It is inlinkage disequilibrium with a second polymorphism of C3, leucine314proline(L314P): R102 preferentially segregates with P314 and G102 preferentiallysegregates with L314.40 An increased prevalence of C3F has beenlinked to a number of immune-mediated diseases, including IgAnephropathy,41 systemic vasculitis,42 and unspecified glomerulonephritis.43We have found that the uncommon C3 haplotype—C3 G102/P314—isassociated with DDD, consistent with other reports.39,40
To identify additional associations between DDD and other complement-relatedgenes, we completed a single-nucleotide polymorphism–basedfirst-pass analysis of approximately 80 genes in 20 patientswith DDD and more than 100 control subjects. For 17 genes, oneor more exonic and/or intronic SNP generated P < 0.05 with>10% association.
In aggregate, these data suggest that most patients with DDDsegregate particular variants of several complement or relatedgenes. The functional impact of these variants may be to alterthe kinetics of complement regulation or to expose novel aminoacid epitopes that facilitate formation of autoantibodies suchas C3NeF, with the common outcome being dysregulation of thealternative pathway of complement. The consequence is uncheckeddamage to unprotected extracellular matrices such as the GBMand Bruch’s membrane.
A renal biopsy is essential to diagnose DDD, with the pathognomonicfeature being electron-dense deposits along the GBM that areresolved by electron microscopy.6 Immunofluorescence stainingfor C3 is almost always positive in capillary loops and in mesangialareas; staining for Ig is usually negative.
Once a diagnosis of DDD is made, the status of the complementsystem should be documented by ordering CH50, APH 50, C3, C3d,C4, and FH; C3NeF should be measured; and CFH should be screenedfor mutations using bidirectional sequencing (Figure 4). Complementprotein measures in DDD are distinctive, with most patientshaving only low C3 levels, whereas properdin, C5, and otherterminal proteins are within the normal range. Factor H levelscan be low, as has been reported with missense mutations inthe coding sequence that block protein secretion from the endoplasmicreticulum.25 (For a list of laboratories providing these tests,please contact the correspondence author.)
Figure 4. Flow diagram illustrating the diagnostic evaluation and treatment of a patient with DDD. The diagnosis is made by renal biopsy. Serologic tests of complement should be obtained, C3NeF should be assayed, and CFH should be screened for mutations. In the presence of C3NeF, removal or dilution of the autoantibody should be considered via plasma exchange or infusion, and anti–B cell agents such as rituximab might be valuable. In the presence of pathologic mutations in CFH that lead to absent or dysfunctional factor H protein, plasma infusion should be considered (with the use of recombinant factor H in the future). In addition, nonspecific treatment should be aimed at controlling BP and proteinuria. Other treatments that should be considered include eculizumab (an anti-C5 antibody [see Figure 3]) and sulodexide (a heparanase inhibitor [see Figure 5]). The criterion for treatment success should be prevention of disease progression as determined by maintenance of or prevention of decrease in renal function. The secondary criterion should be normalization of activity levels of the alternative complement pathway as measured by C3/C3d ratios and C3NeF levels. After having reached a clinical steady state, reasonable follow-up steps could be monthly for the first 3 to 6 mo, every 2 mo for the rest of the first year, and subsequently every 6 mo, adjusting clinical monitoring if a flare in disease activity occurs.
Most treatment guidelines for DDD are primarily based on caseseries before 1995.44–48 Recent animal and genetic data,however, suggest that novel interventions should be coupledwith nonspecific treatments to retard progression of glomerulardisease. Treatment options should reflect and be driven by diagnostictest results.
Nonspecific Treatments
Nonspecific measures that are effective in slowing progressionof numerous chronic glomerular diseases include aggressive BPcontrol and reduction of proteinuria.6 Angiotensin-convertingenzyme (ACE) inhibitors and angiotensin II type 1 receptor blockersare first-line agents to decrease proteinuria, improve renalhemodynamics, and possibly limit leukocyte infiltration in thekidney.49,50 Although not widely used in children, in the presenceof hyperlipidemia, lipid-lowering agents such as hydroxymethylglutarylCoA reductase inhibitors may also delay progression of renaldisease, correct endothelial cell dysfunction, and alter long-termatherosclerotic risks.51,52
The use of steroid therapy is probably not effective in DDD,6although it is extremely effective in a form of glomerulonephritiscalled juvenile acute nonproliferative glomerulonephritis, whichcan be confused with DDD.53 The two diseases can be distinguishedclinically, because DDD is typically associated with C3NeF-inducedhypocomplementemia, often with nephrotic syndrome and hypertension,whereas in juvenile acute nonproliferative glomerulonephritis,C3 levels remain at the lower limit of normal.
Strategies to reduce C3NeF in DDD using mycophenolate mofetilto inhibit differentiation, maturation, and allostimulatoryfunction of B and T lymphocytes or rituximab, a chimeric IgG1mAb that specifically targets the CD20 surface antigen expressedon B lymphocytes, have not been studied.6 The use of rituximabmay be justified in patients who are positive for C3NeF, donot have a mutation in CFH, and show evidence of C3 consumption(Figure 4). Standard rituximab protocols for the treatment ofrenal disease should be used, following C3NeF levels and complementassays to document any response.54
Disease-Specific Treatments
In patients with defined pathologic mutations of CFH (and perhapsthose carrying CFH risk alleles), specific treatment guidelinesshould include infusion of fresh frozen plasma or plasmapheresisand exchange with plasma, rather than albumin, to provide functionallyintact factor H (recombinant factor H is not currently available).The siblings reported by Licht et al.24 were treated with infusionsof 10 to 15 ml of fresh frozen plasma per kg body weight at14-d intervals, a dosing schedule based on the measured half-lifeof factor H of 6 d.55 Except for one episode of mild hypotensionand a few episodes of nonspecific abdominal pain that was responsiveto antihistamines, the treatment has been well tolerated andkidney function has been shown to be preserved. Of historicalnote, transfusion of normal porcine plasma to affected NorwegianYorkshire piglets also inhibited complement activation and increasedsurvival.56
An additional DDD-specific treatment that is supported by animaldata is the use of an anti-C5 antibody such as eculizumab (Soliris;Alexion Pharmaceuticals, Cheshire, CT) to decrease C5a-mediatedglomerular damage. Its development was based on a murine prototype(N19-8), which almost completely inhibits terminal complementcomplex formation and C5a release in vitro.57 Safety and efficacyof eculizumab have been tested by Hillmen et al.58 in patientswith paroxysmal nocturnal hemoglobinuria (PNH). In a double-blind,randomized, placebo-controlled, multicenter phase III trialinvolving 87 patients, these investigators observed stabilizationof hemoglobin levels in nearly 50% of patients who were on eculizumab(21 of 43) versus none in the placebo group (0 of 44). Patientsin the treatment group received infusions of 600 mg of eculizumabevery week for 4 wk, followed thereafter by a maintenance doseof 900 mg of eculizumab every 2 wk for the duration of the study.Serious adverse events were reported in four patients in theeculizumab group and nine patients in the placebo group butwere not considered to be treatment related. The most commonadverse events reported in the eculizumab group were headache,nasopharyngitis, back pain, and nausea, with headache and backpain occurring more frequently in the eculizumab group thanin the placebo group (Figure 5). (Note: Eculizumab has now beenapproved by the Food and Drug Administration for PNH.)
Figure 5. In DDD, glomerular basement membrane staining of heparan sulfate is decreased and heparanase expression is enhanced. Staining for the agrin core protein remains unchanged. Tubular expression of heparanase is high in both DDD and controls.
The use of sulodexide is another treatment that may slow diseaseprogression in DDD. Sulodexide is a combination of two GAGs—anelectrophoretically fast-moving low molecular weight heparin(80% by weight) and dermatan sulfate (20%)—and can begiven orally, subcutaneously, or by intravenous injection. Ithas profibrinolytic and antithrombotic properties and is aneffective inhibitor of heparanase, a -D-endoglycosidase.59 Glomerularheparanase expression is increased in DDD and contributes todisease pathogenesis by selectively degrading the negativelycharged GAG side chains of heparan sulfate proteoglycans withinthe GBM or at the surface of podocytes and the glomerular endothelium(Figure 5). This leads to altered permselective properties,loss of glomerular epithelial and endothelial cell anchor points,or liberation of heparan sulfate–bound factors, such asgrowth factors, chemokines, and cytokines.60,61 Desulfationof critical GAG also weakens interactions with factor H, whichmay prove pathogenic in individuals with factor H mutationsthat attenuate GAG binding.
Upregulation of glomerular heparanase expression has been observedin several other experimental and human glomerular diseases,62–64and its inhibition seems to be beneficial at least in animalmodels.60,61 Glomerular heparanase expression is augmented byreactive oxygen species, angiotensin II, and proinflammatorycytokines.65 In in vitro models of activated glomerular endothelialcells, heparanase expression not only is increased but alsois associated with structural changes to cell surface heparansulfates.66 Heparan sulfates on glomerular endothelium alsoplay a prominent role during inflammation and in local complementactivation and regulation.66,67
Sulodexide may therefore have multiple effects that could makeit effective in DDD, including inhibition of glomerular heparanaseactivity and interference with binding of leukocytes and/oractivated complement components to glomerular endothelium. Itis approved in Europe to treat vascular thrombotic conditions,and there are recent data to support its use in diabetes. Ithas been used in several small phase II studies to treat earlydiabetic nephropathy and can induce an additional 40 to 70%reduction in albuminuria in individuals with tight glycemicand BP control. There are two ongoing clinical trials to evaluateits effect in diabetes (Phase III: http://www.clinicaltrials.gov/ct/gui/show/NCT00130208;Phase IV: http://www.clinicaltrials.gov/ct/show/NCT00130312).At dosages of 200 mg/d, sulodexide has no anticoagulant propertiesand has an excellent safety profile.68 An international studyto test its efficacy in DDD is planned, and as newer structurallywell-defined GAG-based therapeutics are made available, it maybe possible to opt for agents with specific anti-heparanaseor GAG-replacing functions. Participation, although open toall, will require patient consent and institutional review boardapproval.
Treatment with rituximab, plasma exchange or infusion, eculizumab,or sulodexide should be initiated in the presence of end organdamage (proteinuria/hematuria) and be continuous for 6 to 12wk. The primary criterion for success at the end of this periodshould be prevention of disease progression (either maintenanceof or prevention of decrease in renal function) as measuredby the degree of proteinuria/hematuria. Secondary criteria forsuccess should be normalization of activity levels of the alternativecomplement pathway and reduction in C3NeF levels (Figure 4).If these outcomes are achieved, then treatment should be continuedwith adjustment to clinical monitoring in response to flaresin disease activity. Assessing efficacy, however, will be difficult,if not impossible, if a prospective, double-blinded study designis used; there are simply too few patients. Other investigatorshave considered this problem and concluded that a Bayesian approachis a reasonable alternative to evaluate treatment outcomes forrare and orphan diseases.2
There are two essential differences between the Bayesian andthe double-blinded approach. The first difference is that aBayesian study design allows the investigator to have some opinionabout the probable outcome of the trial. This preconceptionis expressed in a terms of a prior probability of a successfuloutcome (something greater than 0.5). The traditional clinicaltrial, in contrast, assumes the likelihood of a successful oran unsuccessful outcome to be equal (i.e., 50:50).
If one assumes, for example, that eculizumab has a measurablebeneficial effect on patients with PNH 75% of the time, thenone might expect the effect of eculizumab in DDD to be similar,making the prior probability of success 0.75. This assumptionmoves the expected distribution of outcomes to the positiveside and allows the investigator to make decisions with fewerobservations when comparing eculizumab-treated with non–eculizumab-treatedpatients with DDD. Thus, the investigator, being limited inavailable patients, takes advantage of the fact that treatmentsare offered with a reasonable expectation of a positive outcome.
Because the estimation of prior probability is subjective andcan be affected by animal data and results of trials for relateddisorders, different investigators will assume different priorprobabilities. Some investigators may assume that the effectof eculizumab on DDD is 0.80, whereas others might assume itto be 0.50 (no effect). One approach to addressing variabilityin prior probability is to take the average suggested from agroup of investigators who are familiar with DDD or in dealingwith the drug being tested.
The second difference between the Bayesian and traditional double-blindedclinical trial is that the Bayesian trial is open-ended. Decisionsabout continuance and efficacy are made as every data pointis collected. This concept is a natural outgrowth of Wald’smethod of sequential analysis, which minimizes the sample sizerequired for decision making.69,70 The Bayesian approach codifiesthe intuitive decision making of an investigator when treatinga rare disease. If, for example, the first three treatmentsare a success, then one would be inclined to continue; if theyfail, then one would be inclined to stop treatment.
Because DDD is very rare, treatments and outcomes should bereported on all patients on the DDD Outcome Database (http://genome.uiowa.edu/ddd).This resource, which is available to all health care personnel,is intended to provide an up-to-date assessment of outcomesas related to treatment protocols. Ultimately, by registeringpatients on the DDD Outcome Database, physicians will be ableto offer care on the basis of a collective experience with alarge number of cases.
DDD is the prototypical rare disease, affecting only two tothree people per million. It causes significant morbidity, leadingto ESRD within 10 yr in 50% of people who are younger than 10yr at diagnosis. Renal transplantation is not a reliable treatmentoption, because up to 50% of recipients eventually lose theirgraft as a result of disease recurrence.
All patients who receive a diagnosis of DDD should undergo astandard battery of tests, including review of renal biopsies,serum markers of complement activity, screening for C3NeF, andgenetic testing of CFH. These tests focus on assessing the alternativepathway of complement for abnormalities. Treatments and outcomesshould be followed by monitoring indices of renal function andserum levels of complement activity.
Data should be reported to a central repository that is accessiblein real time to all clinicians. This reporting system will allow"best available" therapies to be used in patient treatment.As our understanding of DDD increases and novel treatments develop(e.g., recombinant factor H), the use of these treatments shouldbe integrated into DDD protocols that are continually analyzedand evaluated in an open-label Bayesian study design.
This Hinxton Retreat of the Dense Deposit Disease Focus Groupwas organized by KIDNEEDS and supported by grants from the WellcomeTrust, National Institutes of Health (NIDDK R13 DK077416-01),and the Milagros Research Fund. The views expressed in thisreport are the sole responsibility of the authors and do notnecessarily reflect the views of the funders.
This article is dedicated to patients with DDD with the hopeand expectation that the ideas contained herein will lead tothe eventual development of therapies to treat this disease.We hope that this model for studying DDD can be applied to otherrare diseases as well.
We are grateful to Dr. N. van de Kar for providing the biopsymaterial used in Figure 5.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
10 yr) than older patients (P = 0.006; Figure 2). Girls may have a more aggressive disease course than boys (P = 0.16). 
K224). 
-D-endoglycosidase.59 Glomerular heparanase expression is increased in DDD and contributes to disease pathogenesis by selectively degrading the negatively charged GAG side chains of heparan sulfate proteoglycans within the GBM or at the surface of podocytes and the glomerular endothelium (Figure 5). This leads to altered permselective properties, loss of glomerular epithelial and endothelial cell anchor points, or liberation of heparan sulfate–bound factors, such as growth factors, chemokines, and cytokines.60,61 Desulfation of critical GAG also weakens interactions with factor H, which may prove pathogenic in individuals with factor H mutations that attenuate GAG binding. 
