Dense deposit disease (DDD) is a glomerular disease

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1 Original Articles Dense Deposit Disease: Clinicopathologic Study of 32 ediatric and Adult atients Samih H. Nasr,* Anthony M. Valeri, Gerald B. Appel, Julius Sherwinter, Michael B. Stokes,* Samar M. Said,* Glen S. Markowitz,* and Vivette D. D Agati* *Department of athology and Department of Medicine, Division of Nephrology, Columbia University, College of hysicians and Surgeons, New York, New York; and s Healthcare of Atlanta at Scottish Rite, Atlanta, Georgia Background and objectives: Dense deposit disease (DDD) is a rare disorder that most commonly affects children. This study reports the largest North American series addressing clinicopathologic and outcome differences in children and adults. Design, setting, participants, & measurements: Thirty-two patients with DDD were analyzed from the archives of Columbia University between 1977 and Characteristic intramembranous electron-dense deposits defined all diagnoses. Results: The cohort included 14 children (<16 yr) and 18 adults, with 39% of adults >60 yr. The female/male ratio was 1.9. At presentation, the mean 24-h urine protein was 4.6 g, nephrotic syndrome was present in 33%, renal insufficiency in 59%, and hematuria in 87% of patients. Compared with adults, children had lower incidence of renal insufficiency and were more likely to have reduced C3. Histologic pattern included membranoproliferative, mesangial, endocapillary, and crescentic glomerulonephritis. Treatment included immunosuppression (IS) alone in seven, renin angiotensin system (RAS) blockade alone in six, and combined IS/RAS blockade in 11. On follow-up (mean 63 mo) available in 27 patients, 26% had complete response, 48% had persistent renal dysfunction, and 26% had ESRD. Correlates of ESRD were older age and higher creatinine at biopsy, the absence of combined IS/RAS blockade therapy and the presence of subepithelial humps, but not histologic pattern. On multivariate analysis, age and creatinine emerged as the only independent predictors of ESRD. Conclusions: DDD is clinically and pathologically heterogeneous. have worse outcome than children, despite similar treatment. Combined IS/RAS blockade appears superior to either agent alone. Clin J Am Soc Nephrol 4: 22 32, doi: /CJN Dense deposit disease (DDD) is a glomerular disease defined at the electron microscopic level by a transformation of the lamina densa of the glomerular basement membrane by ribbon-like, highly electron-dense material, which by immunofluorescence stains predominantly for C3. The disease was first recognized in France in 1963 by Galle (1). For many years, DDD was also called membranoproliferative GN (MGN) type II. Because recent studies have indicated that the light microscopic pattern in most patients is not membranoproliferative, the modern trend is to consider DDD a distinct entity, rather than a variant of MGN (2,3). The rarity of DDD, which afflicts only two to three individuals per million population (4), has impeded studies into its clinical course and optimal treatment. The disease has a Caucasian racial predominance and is most commonly seen in children and adolescents (5,6). Its natural history is variable, but approximately 50% of patients progress to ESRD within 8 to 10 yr (6,7). Animal and human studies indicate that the pathophysiologic basis of DDD Received July 12, Accepted August 16, ublished online ahead of print. ublication date available at Correspondence: Dr. Samih H. Nasr, Department of athology, Columbia University, College of hysicians and Surgeons, 630 West 168th Street, VC14-224, New York, New York, hone: ; Fax: ; sn386@columbia.edu is an uncontrolled systemic activation of the alternative complement pathway because of the presence of an autoantibody to C3 convertase, mutations in the factor H gene, or the presence of an autoantibody to factor H (4). Why the glomerulus is preferentially affected is unknown, although the physical stresses of glomerular filtration are likely to play a role in local complement activation. Although several studies of DDD were performed before 1990 (5 14), there has been only a single study in recent years evaluating the clinical characteristics and course of the disease (15). articularly little is known about the course in adults, and only one study published in 1983 compared the features of DDD in children and adults (6). Herein, we report the largest North American series addressing the clinicopathologic characteristics and outcome in patients with DDD. Thirty-two patients, including 14 children and 18 adults, were studied with particular emphasis on identifying clinical, pathologic, and outcome differences between these age groups. Materials and Methods Thirty-two patients with DDD were identified in a retrospective review of all native renal biopsies received at Columbia University Medical Center from 1977 to The diagnosis of DDD was based on the ultrastructural finding of a transformation of glomerular basement membranes by ribbon-like, highly electron-dense material and predominant immunofluorescence staining for C3. The Renal athology Labo- Copyright 2009 by the American Society of Nephrology ISSN: /

2 Clin J Am Soc Nephrol 4: 22 32, 2009 Dense Deposit Disease 23 ratory of Columbia University processed 15 of the 32 initial renal biopsies. The remaining seventeen were processed at local hospitals and sent to Columbia University for a second opinion at the request of the treating nephrologist. Renal biopsy samples were processed by standard techniques for light microscopy (LM), immunofluorescence (IF), and electron microscopy (EM). For each sample, multiple glass slides were stained with hematoxylin and eosin, periodic acid Schiff, Masson trichrome, and Jones methenamine silver. Ultrastructural evaluation was performed using a transmission electron microscope. IF was performed on 3- m cryostat sections using polyclonal FITC conjugated antibodies to IgG, IgM, IgA, C3, C1q, and kappa and lambda light chains. IF staining intensity was graded 0 to 3 on a semiquantitative scale. atients medical records were reviewed for demographic information, presenting clinical and laboratory findings, treatment, and outcome. For purposes of age stratification, patients 16 yr of age were considered as children and those 16 yr of age as adults. The following definitions were applied: nephrotic range proteinuria, 24 h urine protein 3 g/d or spot urine protein/creatinine ratio 3; hypoalbuminemia, serum albumin 3.5 g/dl in adults and 2.5 g/dl in children; renal insufficiency, serum creatinine 1.2 mg/dl in adults and more than the age-related normal values in children; and hematuria, 5 red blood cells per high power field on microscopic examination of the urinary sediment or positive blood by dipstick. Nephrotic syndrome was defined as nephrotic range proteinuria, hypoalbuminemia, and peripheral edema. Statistical analyses were performed using SSS for Windows, version 15.0 (SSS, Chicago, Illinois, USA) and StatXact for Windows, version 6.0 (Cytel Software Corporation, Cambridge, Massachusetts, USA). Continuous variables are reported as the mean SEM. Analyses were performed by nonparametric exact statistical methods using the Wilcoxon rank-sum test, the Kruskal Wallis test, and the Fisher exact test, as appropriate for variable type. Response was defined as follows: (1) complete response (CR), remission of proteinuria to 500 mg/d with normal renal function; (2) partial response (R), reduction in proteinuria by at least 50% and to 2 g/d with stable renal function (no more than a 20% increase in serum creatinine); and (3) no response (NR), failure to meet criteria for either CR or R, including patients with unremitting proteinuria, progressive chronic kidney disease, or progression to ESRD. Survival estimates (endpoint ESRD) were computed by the method of Kaplan and Meier. Multivariate survival analyses were performed using the Cox proportional hazards model (Cox regression). Statistical significance was assumed at The study was approved by the Institutional Review Board of Columbia University Medical Center. Results Clinical Features The cohort consisted of 14 children, all 5 yr of age, including nine (64.3%) who were 10 yr, and 18 adults including seven (39%) who were 60 yr of age (Table 1). The female/ male ratio was 1.9 and most patients were Caucasian (84.4%). There was no difference between children and adults with respect to gender or race/ethnicity. Only one child developed partial lipodystrophy and one child was known to have retinal involvement (drusen). Comorbid conditions were present in 11 adults: chronic hypertension in five, plasma cell dyscrasia in four (monoclonal gammopathy in three and multiple myeloma in one), ulcerative colitis in one, rheumatoid arthritis in one, and coronary artery disease in one. A single child had a history Table 1. Demographics (32 patients) Number of atients Female/male 21/11 (65.6/34.4) Age (yr; mean SD) (43.8) 16 to 30 4 (12.5) 31 to 45 5 (15.6) 46 to 60 2 (6.3) 60 7 (21.9) Race White 27 (84.4) Black 2 (6.3) Hispanic 2 (6.3) Asian 1 (3.1) of type 1 diabetes without evidence of diabetic nephropathy. The appearance of renal symptoms was preceded by an acute infection in eight children including pneumonia in four, upper respiratory tract infection in three, and bronchitis in one. Only one adult had a history of recent infection (urinary tract infection). Depressed C3 levels were universal in the pediatric group but detected significantly less in the adult group (100% versus 41.2%, 0.001). C4 was depressed in only a single patient (an adult). C3 nephritic factor was present in all six children and one of three adults tested ( 0.027). The single patient (an adult) in whom factor H gene screening was performed did not have disease-causing mutations but had tyrosine-402-histidine (Y402H) polymorphism, a preferentially DDD-associated allele variant (16). At presentation, all patients except two children had proteinuria (Table 2). The mean 24-h urine protein was 4.6 g. roteinuria was in the nephrotic range in 56.7% of patients, and 33.3% developed full nephrotic syndrome. Microhematuria was documented in all children and in 77.8% of adults, whereas gross hematuria was present in only 16.1% of patients. Erythrocyte casts on microscopic examination of urine sediment were uncommon but more frequent in children (46.2% versus 11.8%, 0.049). Just over half of the patients had elevated serum creatinine. The mean serum creatinine was 0.81 mg/dl and 3.2 mg/dl in children and adults, respectively. Adult patients had a higher incidence of renal insufficiency than children ( 0.029). The mean serum albumin was 2.8 g/dl, and peripheral edema was present in 37.5% of patients. athologic Features The most distinctive feature in LM was thickening of the glomerular basement membranes (GBM) by ribbon-like glassy intramembranous deposits. The deposits stained highly eosinophilic with hematoxylin and eosin, periodic acid Schiff-positive, trichrome-gray or -red, and nonargyrophilic (Figure 1A). Similar intramembranous ribbon-like deposits were also seen involving Bowman s capsule and tubular basement membranes in some patients. The most frequent histologic pattern of glomerular injury with LM was MGN, found in 43.8% of patients,

3 24 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 4: 22 32, 2009 Table 2. Clinical characteristics at presentation Characteristic n 32 n 14 n 18 eripheral edema 12 (37.5) 6 (42.8) 6 (33.3) NS a Mean 24-h urine protein 4.6 g 4.0 g 5.1 g roteinuria 1 g/24 h 5/30 (16.7) 2/13 (15.4) 3/17 (17.6) NS roteinuria 1 to 3 g/24 h 8/30 (26.7) 4/13 (30.8) 4/17 (23.5) NS roteinuria 3 g/24 h 17/30 (56.7) 7/13 (53.8) 10/17 (58.8) NS Full nephrotic syndrome 10/30 (33.3) 4/13 (30.7) 6/17 (35.3) NS Mean serum albumin 2.8 g/dl 2.7 g/dl 2.9 g/dl Hematuria (microscopic or macroscopic) 27/31 (87.1) 13/13 (100) 14 (77.8) NS Macroscopic hematuria 5/31 (16.1) 3/13 (23.1) 2 (11.1) NS Red blood cell casts 8/30 (26.7) 6/13 (46.2) 2/17 (11.8) Mean serum creatinine at biopsy 2.2 mg/dl 0.8 mg/dl 3.2 mg/dl Renal insufficiency at presentation 19 (59.4) 5 (35.7) 14 (77.8) Low C3 20/30 (66.7) 13/13 (100) 7/17 (41.2) Low C4 1/30 (3.3) 0/13 (0) 1/17 (5.9) NS ositive C3 nephritic factor 7/9 (77.8) 6/6 (100) 1/3 (33.3) receding infection 9 (28.1) 8 (57.1) b 1 (5.6) c a NS, not significant ( 0.05). b neumonia in four, upper respiratory tract infection in three, and bronchitis in one. c Urinary tract infection. Figure 1. Light microscopic patterns of dense deposit disease (DDD). (A) In this patient with membranoproliferative pattern, there are large nonargyrophilic deposits expanding the mesangium and thickening the glomerular capillary walls, with segmental duplications. (Jones methenamine silver, 400). (B) An example of mesangial pattern illustrates the mild increase in mesangial cell number and matrix, with preservation of luminal patency. (periodic acid Schiff, 600). (C) The endocapillary proliferative and exudative pattern features global occlusion of glomerular capillaries by endocapillary cells, including infiltrating neutrophils and monocytes, with lobular accentuation. Many of the glomerular basement membranes display ribbon-like thickening. (hematoxylin and eosin, 400). (D) An example of crescentic proliferation with membranoproliferative features in the underlying tuft. There is prominent ribbon-like thickening of the glomerular basement membranes and nodular mesangial expansion. (periodic acid Schiff, 400).

4 Clin J Am Soc Nephrol 4: 22 32, 2009 Dense Deposit Disease 25 Table 3. Histological pattern by light microscopy attern n 32 n 14 n 18 Mesangial proliferative GN 9 (28.1) 4 (28.6) 5 (27.8) NS a Endocapillary proliferative GN with or without 6 (18.8) 3 (21.4) 3 (16.7) NS exudative features Membranoproliferative GN 14 (43.8) 4 (28.6) 10 (55.6) NS Crescentic GN b 3 (9.4) 3 (21.4) 0 (0) NS a NS, not significant ( 0.05). b Defined by the presence of crescents affecting 50% of glomeruli. followed by mesangial proliferative GN (28.1%), endocapillary proliferative GN (18.8%) with (12.5%) or without (6.3%) exudative features (defined as neutrophil infiltration), and crescentic GN (defined by the presence of crescents in 50% of glomeruli) (9.4%) (Table 3) (Figures 1A-1D). Compared with other patterns, the MGN pattern was associated with more segmental sclerosis ( 0.003), interstitial inflammation ( 0.026), and foot process effacement ( 0.005). Crescentic GN was seen only in three children. Focal crescents, however, were common and present in over half of patients, whereas necrosis was rare (15.6%). Glomerular pattern of injury by LM did not correlate with age, gender, race/ethnicity, depression of C3, serum creatinine at biopsy, degree of proteinuria, serum albumin, the percentage of global or segmental glomerulosclerosis, or the degree of tubular atrophy/interstitial fibrosis, interstitial inflammation, or arteriosclerosis. Adult patients had a significantly higher percentage of global glomerulosclerosis ( 0.001) and a greater degree of tubular atrophy/interstitial fibrosis ( ) and arteriosclerosis ( ) than children. The proportion of glomeruli with segmental sclerosis, endocapillary hypercellularity, exudative features, crescents, and necrosis was not statistically different between children and adults (Table 4). IF was performed in 30 patients with available glomeruli Table 4. Light microscopic findings athologic Findings n 32 n 14 n 18 Mean number of glomeruli mean NS d ercent of globally sclerotic glomeruli ercent of segmentally sclerotic glomeruli NS ercent of cases with crescents a 19 (59.4) 11 (78.6) 8 (44.4) NS focal diffuse Number of cases with necrosis 5 (15.6) 1 (7.1) 4 (22.2) NS Number of cases with endocapillary 20 (62.5) 10 (71.4) 10 (55.6) NS hypercellularity a focal diffuse Number of cases with intracapillary neutrophil 14 (43.8) 8 (57.1) 6 (33.3) NS infiltration a focal diffuse Interstitial inflammation: none/focal/diffuse b 5/21/6 5/7/2 0/14/4 NS Tubular atrophy and interstitial fibrosis: none/ 8/13/7/4 8/5/1/0 0/8/6/ mild/moderate/severe c Arteriosclerosis and arteriolar hyalinosis: none/ mild/moderate/severe 15/7/8/2 13/1/0/0 2/6/8/ a Focal, 50% of glomeruli; diffuse, 50% of glomeruli. b Focal, 50% of cortical surface area; diffuse 50%. c Mild, (0 to 25% of cortical surface area); moderate (26 to 50%); severe ( 50%). d NS, not significant ( 0.05).

5 26 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 4: 22 32, 2009 Table 5. Immunofluorescence findings n 30 n 14 n 16 C3 30 (100) 14 (100) 16 (100) NS (2.5 ) (2.6 ) (2.3 ) C1q 3 (10) 3 (21.4) 0 (0) NS (1.8 ) (1.8 ) IgG 8 (26.7) 5 (35.7) 3 (18.8) NS (1.1 ) (1.3 ) (0.5 ) IgM 11 (36.7) 8 (57.1) 3 (18.8) (0.7 ) (0.9 ) (0.5 ) IgA 4 (13.3) 1 (7.1) 3 (18.8) NS (0.6 ) (1 ) (0.5 ) Data indicate the number (percentage) of positive patients and the mean intensity of staining when positive (scale, 0.5, 1 to 3 ). NS, not significant ( 0.05). (Table 5). Intense C3 staining was detected in all patients (mean intensity 2.5 ) in the mesangium as granular or ring-like deposits and along glomerular capillary walls (GCW) in a linear to semilinear pattern (Figures 2A, 2B). In some patients the linear staining appeared as narrow tramtracks outlining the inner and outer aspects of the thickened GCW (Figure 2A). Focal linear or semilinear C3 staining was also seen along tubular basement membranes in 60% of patients and along Bowman s capsule in 30% of patients. In 47% of patients, the glomerular deposits weakly stained for one or more additional immune reactants, including IgM (36.7% of patients), IgG (26.7%), IgA (13.3%), and C1q (10%) (Table 5). IgM positivity was more frequent in children than adults (57.1% versus 18.8%, 0.019). Ultrastructural evaluation, performed on all 32 patients, exhibited large highly electron-dense intramembranous deposits that irregularly thickened and transformed the lamina densa (Table 6) (Figure 3A). In most glomerular loops, the intramembranous deposits were interrupted, producing a sausage string appearance to the GBM. In some patients, they were particularly prominent involving the GBM reflection over the mesangium, with less involvement of the peripheral GCW. Similar intramembranous electron-dense deposits were identified focally in Bowman s capsule and tubular basement membranes in 44 and 50% of patients, respectively (Figure 3B). Segmental or global electron-dense deposits were also seen embedded in the expanded mesangium in all patients. In 56% of patients, the mesangial deposits formed large, rounded nodules ( ring forms ), whereas in the remaining 44% of patients they appeared small and granular (Figure 3C). Large humpshaped, subepithelial electron-dense deposits were identified in ten patients (31%), including eight adults and two children ( 0.068). The subepithelial humps were rare ( 2) in six patients and multiple in the remaining four patients. Half of the patients had few small subendothelial deposits, which in most instances appeared to represent extensions of intramembranous deposits into the subendothelial space. In a few patients, large, highly electron-dense deposits pooled in the subendothelial region with sparing of the lamina densa, a finding described by McEn- Figure 2. Immunofluorescence features of DDD. (A) Staining for C3 highlights the glomerular capillary walls globally in a semilinear pattern. In some capillaries, there appears to be a double layer of linear staining (arrows). A few ring forms are seen in the mesangium. Semilinear deposits are also present in Bowman s capsule and adjacent tubular basement membranes. ( 400). (B) In this example, the mesangium contains numerous characteristic ring forms staining for C3, with weaker linear staining of the glomerular capillary walls ( 400).

6 Clin J Am Soc Nephrol 4: 22 32, 2009 Dense Deposit Disease 27 Table 6. Electron microscopic findings Location of Highly Electron-Dense Deposits n 32 n 14 n 18 Lamina densa of GBM a 32 (100) 14 (100) 18 (100) NS c segmental b global b Mesangial 32 (100) 14 (100) 18 (100) NS segmental global Subepithelial hump-shaped deposits 10 (31.3) 2 (14.3) 8 (44.4) NS Subendothelial 16 (50) 5 (35.7) 11 (61.1) NS Bowman s capsule 14 (43.8) 6 (42.9) 8 (44.4) NS Tubular basement membranes 16 (50) 6 (42.9) 10 (55.6) NS a GBM, glomerular basement membranes. b Segmental, involving 50% of the total glomerular loops; global, involving 50% of the total glomerular loops. c NS, not significant ( 0.05). ery and McAdams as the dropping off phenomenon (17) (Figure 3D). In two patients (6%), a few electron-dense deposits were identified in arteriolar walls. Repeat biopsies were performed in five children and one adult (three repeat biopsies in three patients and two in three patients) at 34 mo to 22 yr after the initial biopsy. six patients undergoing repeat biopsies were treated with steroids [in addition to renin angiotensin system (RAS) blockade in three, cyclosporine in one, and tacrolimus in one]. six patients were NR, including five with persistent renal dysfunction and one who progressed to ESRD. Repeat biopsies showed similar degrees of mesangial sclerosis but greater global glomerulosclerosis (five patients), tubular atrophy, and interstitial fibrosis (four patients), consistent with disease progression. Endocapillary hypercellularity including intracapillary neutrophils, present in three of these six patients on the initial biopsy, resolved in two patients and persisted in one patient on subsequent biopsies. The histologic pattern with LM changed in four patients (mesangial proliferative GN to MGN in three and crescentic GN to mesangial proliferative GN in one). The IF and EM findings on repeat biopsies were not significantly different from those seen on the initial biopsy. Outcome Clinical follow-up was available in 27 patients (84.4%) including 13 children and 14 adults (Table 7). The mean duration of follow-up for the entire cohort was 63.4 mo (range 2 mo to 24 yr). Treatment included immunosuppression (IS) alone in seven patients, RAS blockade (angiotensin-converting enzyme inhibitor and/or angiotensin II receptor blocker) alone in six patients, and combined IS/RAS blockade in eleven patients. IS consisted of steroids in all 18 patients: five received a second agent including mycophenolate mofetil in two, cyclosporine in two, and tacrolimus in one. Two patients were not treated with RAS blockade or IS. No information about treatment was available in the remaining patient. atients who received IS therapy (with or without RAS blockade) had a significantly lower serum albumin ( 0.015), a higher percent of crescents ( 0.008), and less arteriosclerosis ( 0.037) than the patients who did not receive IS. IS-treated and untreated patients were not different with respect to 24-h urine protein, serum creatinine at biopsy, percent global or segmental sclerosis, tubular atrophy/interstitial fibrosis, or interstitial inflammation. On follow-up, 25.9% of patients (7.1% of adults and 46.1% of children, 0.033) had a CR and the remaining 74.1% had NR including 48.1% who had persistent renal dysfunction and 25.9% (7.7% of children and 42.9% of adults, 0.033) who progressed to ESRD. No patient met criteria for R. Two patients underwent renal transplantation, one of whom developed recurrent DDD on a background of chronic rejection diagnosed in two successive allograft biopsies performed for graft dysfunction at 4 and 8 yr posttransplant. The recurrent DDD exhibited mild mesangial proliferative GN by LM (different from the MGN pattern seen in the native kidney), but characteristic features by IF and EM. The mild severity of the recurrence suggested that it was not contributing significantly to the allograft dysfunction. Three adults who were on permanent hemodialysis died, one because of congestive heart failure, one because of myocardial infarction, and one of undetermined cause. On univariate analysis, the correlates of reaching ESRD were: older age ( 0.005), higher creatinine at biopsy ( 0.003), the degree of arteriosclerosis on biopsy ( 0.041), and the presence of subepithelial humps by EM ( 0.023). IS (with or without RAS blockade therapy) was protective but did not quite reach statistical significance ( 0.051). Combined therapy (IS with RAS blockade) was more efficacious than IS or RAS blockade therapy alone ( 0.031). Gender, race/ethnicity, depression of C3, the degree of proteinuria, serum albumin, the glomerular pattern by LM, the percentage of global or segmental sclerosis, the presence of crescents, and the degree of tubular atrophy/interstitial fibrosis and interstitial inflammation did not correlate significantly with ESRD outcome.

7 28 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 4: 22 32, 2009 Figure 3. Electron microscopic features of DDD. (A) Highly electron-dense intramembranous deposits thicken the glomerular basement membranes. In areas, the deposits are interrupted, producing sausage shapes. There is circumferential mesangial interposition ( 4000). (B) Intramembranous interrupted electron-dense deposits are present within a tubular basement membrane ( 3000). (C) The ring forms seen by immunofluorescence correspond to rounded electron densities within the mesangium, shown here. A few small intramembranous deposits are also present in the glomerular basement membranes ( 2000). (D) Some of the glomerular capillary wall deposits drop off the lamina densa, forming inframembranous densities ( 4000).

8 Clin J Am Soc Nephrol 4: 22 32, 2009 Dense Deposit Disease 29 Table 7. Clinical follow-up n 27 n 13 n 14 Duration of follow-up: mean (range) in months 63.4 (2 to 288) 79.4 (2 to 288) 48.5 (4 to 156) NS Treatment None 2 (7.4) 0 (0) 2 (14.3) NS RAS a blockade alone 6 (22.2) 2 (15.4) 4 (28.6) NS IS a alone 7 (25.9) 3 (23.1) 4 (28.6) NS Combined IS/RAS blockade 11 (40.7) 8 (61.5) 3 (21.4) NS Not available 1 (3.7) 0 (0) 1 (7.1) NS Outcome b complete response 7 (25.9) 6 (46.1) 1 (7.1) persistent renal dysfunction 13 (48.1) 6 (46.1) 7 (50) NS ESRD 7 (25.9) 1 (7.7) 6 (42.9) death 3 (11.1) 0 (0) 3 (21.4) NS a RAS, renin angiotensin system; IS, immunosuppressive agents (steroids with without a second agent). b Overall comparison, complete response/persistent renal dysfunction/esrd in children versus adults, By Kaplan Meier survival estimates (endpoint progression to ESRD), predictors of ESRD were adult age (using age 16 yr as the cutoff) at biopsy, absence of combined IS/RAS blockade, the presence of subepithelial humps, and the presence of arteriosclerosis (any degree) (Table 8) (Figure 4). Gender, race/ ethnicity, nephrotic range proteinuria, and the degree of tubular atrophy/interstitial fibrosis or interstitial inflammation did not predict renal survival. Using the Cox proportional hazards model, the only independent predictors of progression to ESRD on multivariate analyses were older age [hazard ratio (HR) 1.081, 95% confidence interval (CI) to 1.176), 0.07] and serum creatinine at biopsy (HR 2.885, 95% CI to 6.924, 0.018). When serum creatinine at biopsy was not included in the model, the only independent predictor of progression to ESRD was age (HR 1.052, 95% CI to 1.091, 0.008). Discussion This study reports our experience with a series of 32 patients with DDD. To our knowledge, this is the largest North American series dealing with the clinicopathologic characteristics and outcome of DDD. We particularly focused on the clinical and outcome differences between children and adults and sought to identify the features associated with poor renal outcome, aspects of the disease that are not adequately addressed in the literature. The percentage of adult patients older than 60 yr in our study was 39%, compared with 0% in the 1975 French study by Habib et al., the 1975 French-Belgian study by Galle and Mahieu, the 1983 U.K. study by Cameron et al., and several other smaller studies (5,6,8,10,13,14). The reason for the larger representation of elderly patients in our study is unclear. Because all but one of the patients 60 yr of age in our cohort were diagnosed within the current decade, contributing factors may be our center s increasingly large referral base for adult nephrology, the aging population trends in the United States, and the aggressive modern use of renal biopsy in the elderly. In our patient population there was a female predominance (1.9:1), similar to the reports by Cameron et al., Bennett et al., and Little et al. (6,13,15). In other studies, the female/male ratio ap- Table 8. redictors of ESRD by Kaplan and Meier survival estimates Factor Mean Time from Biopsy to ESRD in Months SEM Age ediatric Adult IS therapy (with or without RAS blockade) Yes No Any therapy RAS blockade IS Combined No endpoints Subepithelial humps Yes No resence of arteriosclerosis (any degree) Yes No

9 30 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 4: 22 32, 2009 Figure 4. Kaplan Meier survival analysis in childhood and adulthood DDD. proached unity (2,8). The gender and racial composition were not significantly different between children and adults. By contrast, in the cohort reported by Cameron et al. (6), children were more likely to be girls, and adults were more likely to be men. In 57% of affected children in our study, the clinical onset of disease was preceded by an upper or lower respiratory tract infection, mimicking acute postinfectious GN, whereas only 6% of adults (1 patient) had preceding urinary tract infection and none had a respiratory tract infection ( 0.004). Importantly, all children in our cohort had a depressed serum complement C3 compared with only 42% of adults ( 0.001). Furthermore, C3 nephritic factor, tested in nine patients, was positive in six of six children but in only one of three adults ( 0.027). These findings suggest that the pathophysiology of DDD in adults may not involve the same levels of sustained activation of the alternative complement pathway that have been recognized in children, possibly reflecting different pathogenetic mechanisms. Unfortunately, the more recently developed diagnostic tests for DDD, such as factor H serum level and screening for mutations in the factor H gene, were performed in only one adult, who was found to have Y402H polymorphism in the factor H gene, an allele variant associated with DDD (16). Four adults (22.2%), aged 42, 72, 80, and 80, had a history of dysproteinemia. Their renal biopsies showed typical ultrastructural features of DDD (which may be difficult to distinguish from monoclonal Ig deposition disease in some patients) and sole or dominant staining for C3 without light chain restriction. On follow-up, available in three patients, two progressed to ESRD and one had persistent renal dysfunction. Recently Sukov et al. identified nine patients 55 yr of age with DDD, six of which had a history of monoclonal gammopathy of undetermined significance (MGUS) (18). The outcome of these six patients was poor, with four patients progressing to ESRD and two to chronic kidney disease. In these six patients, the glomerular deposits stained by IF for C3, not Ig light chains. Furthermore, proteomic analysis of the glomerular deposits by gas chromatography mass spectroscopy showed mainly complement components, without significant Ig light chains. The above data suggest that DDD occurring in patients with MGUS is associated with a poor prognosis (which would have been predicted by our study on the basis of age alone) and does not represent a form of renal involvement by dysproteinemia. Taking into account the high prevalence of MGUS in the elderly (3% in persons 50 of age and 5% in persons 70 yr of age) (19), the concurrence of DDD and MGUS may be coincidental. On the other hand, it is likely that the detection of M-spike provided a compelling indication for workup of the underlying renal disease, which proved to be DDD. Further investigation is needed to determine whether the monoclonal protein could promote complement activation or block complement inhibitors, such as factor H or I (20, 21). Recently, Walker et al. reviewed the renal biopsy findings of 69 patients of DDD from around the world (2). They recognized five histologic patterns of glomerular injury by LM: mesangial proliferative GN (seen in 45% of patients), MGN (25%), crescentic GN (18%), acute proliferative and exudative GN (12%), and unclassified (3%) (2). In our experience, MGN was the most common pattern by LM, although it was present in only 44% of patients. Kashtan et al. found that the finding of MGN pattern by LM is associated with chronic renal insufficiency and ESRD (14). In contrast, the pattern of glomerular injury by LM in our series did not correlate with outcome or age. We identified several features that correlated with poor outcome, including older age, higher creatinine at biopsy, and the presence of subepithelial humps. Only 7.1% of our adult patients had CR compared with 46.1% of children ( 0.033). Conversely, 42.9% of adults progressed to ESRD compared with 7.7% of children ( 0.033). In contrast to previous studies (6,13,14), which included mostly children, the presence of nephrotic syndrome, gross hematuria, persistent hypocomplementemia, and crescents or glomerulosclerosis on initial biopsy did not portend a poor prognosis in our cohort. The lack of correlation between ESRD and the percentage of glomeruli with crescents in our study is likely because patients who had a higher percentage of glomeruli with crescents were more likely to have received immunosuppressive therapy ( 0.008). Bennett et al. found that crescents affecting 50% of glomeruli on initial biopsy correlated with progressive renal disease (13). Crescents were detected in over half of the patients in our cohort, but were focal in the majority. Of note, among the three children with crescents involving 50% of glomeruli, two achieved CR and one had worsening renal function. The optimal treatment of DDD remains undefined because of the disease s rarity and the lack of large, prospective, randomized clinical trials. Several therapeutic regimens have been used, including RAS blockade, anticoagulants, steroids and other immunosuppressive agents, and plasmapheresis/plasma exchange, singly or in combination (22). roposed new treatments include ectulizumub (an anti-c5 antibody) and sulodexide (a mixture of low-molecular weight heparin and dermatan sulfate that inhibits heparanase) (4). The effectiveness of steroids is controversial. A randomized placebo-controlled study by the International Study of Kidney Disease in of 80 children with MGN (including 14 with DDD) demonstrated that a regimen of long-term alternate-day prednisone improved

10 Clin J Am Soc Nephrol 4: 22 32, 2009 Dense Deposit Disease 31 outcome in a heterogeneous group of patients with MGN I, II, and III, collectively, but not in those with DDD specifically (23). Conversely, McEnery and McAdams reported improvement in glomerular morphology and good clinical outcomes in six patients who were treated with long-term alternate-day prednisone (17). No studies have compared the efficacy of steroids alone versus steroids/ras blockade in patients with DDD. Most patients in our cohort were treated (by nephrologists from 11 different states representing a broad geographic region) with RAS blockade, IS (steroids alone or with a second agent), or both, for variable periods of time. In this large retrospective study we found that combined IS/RAS blockade was superior to either agent alone. A prospective, controlled study is needed to confirm these findings. None of our patients received disease specific treatments, except for one child who was on RAS blockade and was just started on sulodexide at the time of last follow-up. In summary, our study indicates that DDD is clinically and pathologically heterogeneous. Contrary to its stereotypic characterization as a childhood disease, DDD can present over a broad age range. Because adults are less likely than children to exhibit the usual heralds of a preceding infection and low serum complement, the renal pathologist must be particularly careful not to overlook the possibility of DDD in the older patient with atypical presentation. redictors of poor prognosis in DDD are older age and higher serum creatinine at biopsy. Combined IS and RAS blockade appears superior to either agent alone, but requires validation in prospective controlled studies. In the future, routine testing for etiologic factors, such as the presence of a disease-causing autoantibody versus genetic deficiency, should allow the design of more rational, individualized therapies. Disclosures None. References 1. Galle : A description of a unique lesion of the glomerular basement membranes seen as a hyaline substance with the electron microscope [in French] le sion singulie re des membranes basales du rein et de la substance hyaline [Thesis]. aris, Medical School, University of aris, Walker D, Ferrario F, Joh K, Bonsib SM: Dense deposit disease is not a membranoproliferative glomerulonephritis. Mod athol 20: , Walker D: Dense deposit disease: New insights. Curr Opin Nephrol Hypertens 16: , Smith RJ, Alexander J, Barlow N, Botto M, Cassavant TL, Cook HT, de Córdoba SR, Hageman GS, Jokiranta TS, Kimberling WJ, Lambris JD, Lanning LD, Levidiotis V, Licht C, Lutz HU, Meri S, ickering MC, Quigg RJ, Rops AL, Salant DJ, Sethi S, Thurman JM, Tully HF, Tully S, van der Vlag J, Walker D, Würzner R, Zipfel F, Dense Deposit Disease Focus Group: New approaches to the treatment of dense deposit disease. J Am Soc Nephrol 18: , Galle, Mahieu : Electron dense alteration of kidney basement membranes. A renal lesion specific of a systemic disease. Am J Med 58: , Cameron JS, Turner DR, Heaton J, Williams DG, Ogg CS, Chantler C, Haycock GB, Hicks J: Idiopathic mesangiocapillary glomerulonephritis. Comparison of types I and II in children and adults and long-term prognosis. Am J Med 74: , Swainson C, Robson JS, Thomson D, MacDonald MK: Mesangiocapillary glomerulonephritis: A long-term study of 40 cases. 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Am J Kidney Dis 12: , Sukov WR, Gamez J, Lager DJ, Dogan A, Miller DV: Clinicopathologic and proteomic assessment of dense deposit disease associated with monoclonal gammopathy of undetermined significance [Abstract]. Modern athology 21: 295A, Kyle RA, Rajkumar SV: Monoclonal gammopathy of undetermined significance and smouldering multiple myeloma: Emphasis on risk factors for progression. Br J Haematol 139: , Jokiranta TS, Solomon A, angburn MK, Zipfel F, Meri S: Nephritogenic lambda light chain dimer: a unique human miniautoantibody against complement factor H. J Immunol 63: , 1999

11 32 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 4: 22 32, Meri S, Koistinen V, Miettinen A, Törnroth T, Seppälä IJ: Activation of the alternative pathway of complement by monoclonal lambda light chains in membranoproliferative glomerulonephritis. J Exp Med 175: , Appel GB, Cook HT, Hageman G, Jennette JC, Kashgarian M, Kirschfink M, Lambris JD, Lanning L, Lutz HU, Meri S, Rose NR, Salant DJ, Sethi S, Smith RJ, Smoyer W, Tully HF, Tully S, Walker, Welsh M, Würzner R, Zipfel F: Membranoproliferative glomerulonephritis type II (dense deposit disease): An update. J Am Soc Nephrol 16: , Tarshish, Bernstein J, Tobin JN, Edelmann CM Jr: Treatment of mesangiocapillary glomerulonephritis with alternate-day prednisone: A report of the International Study of Kidney Disease in. ediatr Nephrol 6: , 1992 Access to UpToDate on-line is available for additional clinical information at