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SLE and Rapidly Progressive Glomerulonephritis
KIDNEY BIOPSY TEACHING CASES
SLE and Rapidly Progressive Glomerulonephritis Naveed N. Masani, MD, Louis J. Imbriano, MD, Vivette D. D’Agati, MD, and Glen S. Markowitz, MD INDEX WORDS: Systemic lupus erythematosus (SLE); rapidly progressive glomerulonephritis (RPGN); antineutrophil cytoplasmic antibody (ANCA).
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YSTEMIC LUPUS erythematosus (SLE) is an autoimmune disease with dermatological, rheumatological, hematologic, and renal manifestations. Renal involvement occurs in the majority of patients with SLE and commonly manifests with hematuria, proteinuria, and/or renal insufficiency. Categorization of the multiple patterns of lupus nephritis (LN) recently was revisited in the 2003 International Society of Nephrology/Renal Pathology Society Classification of LN.1,2 Renal biopsy in patients with SLE is critical to define the pattern of renal disease, determine prognosis, and guide therapy.3 Rapidly progressive glomerulonephritis (RPGN) is a clinical term defined by the presence of accelerated renal failure caused by a proliferative inflammatory glomerular lesion. A common definition for RPGN is 50% loss of renal function over 3 months and clinical evidence of glomerular injury in the form of active urinary sediment including red blood cell (RBC) casts. In patients with RPGN, renal biopsy typically reveals crescentic glomerulonephritis (GN). Crescentic GN is a term that encompasses multiple patterns of glomerular disease that are best differentiated by immunofluorescence. Immunofluorescence typically shows 1 of 3 possible patterns: granular positivity consistent with immune complex–mediated GN, linear positivity From the Department of Medicine, Division of Nephrology and Hypertension, Winthrop University Hospital, Mineola, NY; and Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY. Received July 29, 2004; accepted in revised form August 11, 2004. Originally published online as doi:10.1053/j.ajkd.2004.08.047 on April 5, 2005. Address reprint requests to Glen S. Markowitz, MD, Department of Pathology, Columbia University, College of Physicians & Surgeons, 630 West 168th St, VC 14-224, New York, NY 10032. E-mail: [email protected] © 2005 by the National Kidney Foundation, Inc. 0272-6386/05/4505-0021$30.00/0 doi:10.1053/j.ajkd.2004.08.047 950
caused by anti–glomerular basement membrane disease, or no significant positivity, corresponding to “pauci-immune” crescentic GN. Most patients with pauci-immune crescentic GN have circulating antineutrophil cytoplasmic antibodies (ANCAs). A clinical presentation of RPGN may occur in patients with SLE, for which renal biopsy usually shows LN class IV with crescents. In rare cases, renal biopsy may show LN class III or even class V (eg, in association with renal vein thrombosis). We report the case of a patient with SLE and RPGN for whom renal biopsy findings and serological test results suggested an overlapping etiology of crescentic GN. CASE REPORT A 50-year-old white woman presented with acute renal failure. Her past medical history was significant for SLE for 2½ years, diabetes mellitus for 3 years (without retinopathy), hypertension, autoimmune hepatitis, primary biliary cirrhosis, pleurisy, pericarditis, and paroxysmal atrial fibrillation. There was no history of deep venous thrombosis, hemoptysis, or miscarriage. Her serum creatinine level 2 months before admission was 0.7 mg/dL (62 mol/L; normal, 0.6 to 1.5 mg/dL [53 to 133 mol/L]). At that time, urinalysis was significant for 1⫹ protein, 5 to 10 RBCs/highpower field, and the absence of RBC casts. Renal biopsy was postponed at the patient’s request. On admission, the patient’s serum creatinine level was 4.4 mg/dL (389 mol/L). She reported nausea, vomiting, decreased appetite, and generalized fatigue for 2 weeks before admission. Medications included aspirin, 325 mg once daily; digoxin, 0.125 mg once daily; sustained-release verapamil, 240 mg once daily; furosemide, 40 mg once daily; hydroxychloroquine, 200 mg once daily; omeprazole, 20 mg once daily; glyburide, 10 mg once daily; and prednisone, 10 mg once daily. She denied use of nonsteroidal anti-inflammatory medications or cyclooxygenase-2 inhibitors. Physical examination revealed an obese female with a blood pressure of 140/90 mm Hg, weight of 110 kg, malar rash, and 1⫹ bilateral pitting edema. Laboratory evaluation showed the following values: white blood cell count, 6.9 ⫻ 103/L (⫻ 109/L; normal, 3.9 to 11.0 ⫻ 103/L [⫻ 109/L]); hematocrit, 28% (normal, 36% to 48%); and platelet count, 175 ⫻ 103/L (⫻ 109/L; normal, 160 to 392 ⫻ 103/L [⫻ 109/L]). Chemistry panel showed the following values: serum sodium, 143 mEq/L (mmol/L; normal, 138 to 145 mEq/L [mmol/L]); potassium, 4.2 mEq/L (mmol/L; 3.7 to
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5.2 mEq/L [mmol/L]); chloride, 103 mEq/L (mmol/L; normal, 103 to 112 mEq/L [mmol/L]); carbon dioxide, 23 mEq/L (mmol/L; normal, 23 to 33 mEq/L [mmol/L]); blood urea nitrogen, 44 mg/dL (15.7 mmol/L; normal, 8 to 25 mg/dL [2.9 to 8.9 mmol/L]; creatinine, 4.4 mg/dL (389 mol/L); and albumin, 2.2 g/dL (22 g/L; normal, 3.5 to 5.5 g/dL [35 to 55 g/L]). Serological evaluation showed the following values: antinuclear antibody titer, 1:160; negative anti–double-stranded DNA; C3, 71 mg/dL (0.71 g/L; normal, 112 to 244 mg/dL [1.12 to 2.44 g/L]); C4, 10 mg/dL (0.1 g/L; normal, 14 to 40 mg/dL [0.14 to 0.4 g/L]); and erythrocyte sedimentation rate, 125 mm/h (normal, 1 to 35 mm/h). Cryoglobulins, hepatitis B surface antigen, and hepatitis C antibody test results were negative. Renal ultrasound showed a right kidney measuring 11.7 cm and a left kidney measuring 12.6 cm, without evidence of hydronephrosis. Urinalysis showed a specific gravity of 1.010, 2⫹ protein, 17 RBCs/high-power field, 7 white blood cells/high-power field, and the absence of cellular casts. A 24-hour urine collection contained 3.2 g of protein. Pulse methylprednisolone therapy was initiated, and a renal biopsy was performed.
Kidney Biopsy Findings Light microscopic examination disclosed 34 glomeruli, none of which were globally sclerotic. Glomeruli appeared normal in size and showed a mild to moderate global increase in mesangial cells (Fig 1A). In addition, 10 glomeruli showed mild segmental endocapillary proliferation with infiltrating monocytes (Fig 1B and C). Among the 10 glomeruli with segmental endocapillary proliferation, 3 glomeruli were compressed by overlying cellular crescents associated with fibrinoid necrosis and rupture of the glomerular basement membrane, and 4 additional glomeruli showed segmental fibrinoid necrosis (without crescents; Fig 1D). Proximal tubules displayed protein resorption droplets, prominent intratubular RBCs and RBC casts, and diffuse degenerative changes. There was diffuse interstitial edema and mild to moderate interstitial chronic inflammation composed of lymphocytes and less extensive monocytes. There was minimal tubular atrophy and interstitial fibrosis involving less than 10% of the cortex sampled. Vessels showed mild arteriosclerosis. No arteritis was identified. Immunofluorescence staining of 5 glomeruli showed granular global mesangial and segmental capillary wall positivity of 3⫹ intensity for immunoglobulin G (IgG), ⫹/⫺ for IgM, 1⫹ for IgA, 2⫹ for C3, 2⫹ for C1q, 3⫹ for light chain, and 3⫹ for light chain (scale of positivity: 0, ⫹/ ⫺, 1 to 3⫹; Fig 1E). Immunofluorescence positivity for IgG (2⫹), IgM (⫹/⫺), C3 (1⫹), (1⫹), and (1⫹) also was noted in blood vessel walls. Sampling for electron microscopy consisted of 4 glomeruli. Glomerular capillary lumina appeared patent. Abundant electron-dense deposits globally expanded the mesangial matrix, associated with increased mesangial cellularity (Fig 2A and B). Endothelial cells showed intact fenestrations and multiple tubuloreticular inclusions. Of note, only rare small subendothelial and subepithelial electron-dense deposits were identified. Visceral epithelial cells displayed lipid droplets and 10% foot-process effacement. No tubulointersti-
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tial electron-dense deposits were identified. Vessels were not sampled.
Pathological Diagnosis Light microscopy showed diffuse mesangial and segmental endocapillary proliferative GN, notable for the presence of prominent necrotizing features and multiple cellular crescents. Light microscopic findings were diagnostic of LN class III (LN “class III (A)” by the 2003 International Society of Nephrology/Renal Pathology Society classification of LN1,2). The diagnosis of LN was supported by the “full house” of staining for immunoglobulins and complement by immunofluorescence, the presence of vessel wall deposits, and the ultrastructural finding of multiple endothelial tubuloreticular inclusions. By the National Institutes of Health criteria, the findings corresponded to an activity index of 9 (scale, 0 to 24) and a chronicity index of 1 (scale, 0 to 12). Although biopsy findings were diagnostic of LN class III, some findings raised the possibility of an additional diagnosis. The degree of endocapillary proliferation was mild, and only rare subendothelial electron-dense deposits were noted on ultrastructural evaluation. Conversely, the majority of glomeruli with endocapillary hypercellularity also showed fibrinoid necrosis or cellular crescents. Overall, the degree of necrosis and crescent formation appeared out of proportion to the sparse peripheral capillary wall deposits and minimal endocapillary proliferation. This apparent discrepancy prompted testing for ANCAs.
Clinical Follow-Up Indirect immunofluorescence (IIF) showed perinuclear ANCA (p-ANCA) positivity. Further testing using enzymelinked immunosorbent assay (ELISA) confirmed the specificity of the p-ANCA for myeloperoxidase (MPO), with an index of 4.3 (normal, ⬍1.0). Given the findings of LN class III with an activity index of 9, the prominent necrotizing and crescentic features, the presentation with RPGN, and the MPO-ANCA seropositivity, the patient was treated with pulse methylprednisolone (500 mg/d intravenously for 3 days), followed by tapering oral prednisone doses and monthly intravenous cyclophosphamide (1 g/m2 body surface area) for 6 months, followed by quarterly doses. Losartan, 50 mg/d, was initiated for hypertension, and subcutaneous recombinant human erythropoietin (20,000 U/wk) was administered for anemia. After 1 month of therapy, serum creatinine level improved to 2.1 mg/dL (186 mol/L), 24-hour urine protein excretion decreased to 1 g/d, and serum complement levels normalized. After 5 months of therapy, she had a creatinine level of 1.5 mg/dL (133 mol/L), 24-hour urine protein level of 170 mg/d, and a negative MPO-ANCA. Fourteen months postbiopsy, she has a creatinine of 1.3 mg/dL (115 mol/L), 24-hour urine protein less than 200 mg/d, and negative MPO-ANCA. To maintain remission of extrarenal manifestations of SLE, she continues to require prednisone, 7 mg daily.
DISCUSSION
ANCAs target cytoplasmic antigens in the granules of neutrophils and monocytes. ANCAs
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Fig 1. (A) A representative glomerulus with mild global mesangial proliferation. Glomerular capillary lumina appear patent, without evidence of endocapillary proliferation, fibrinoid necrosis, or crescent formation (hematoxylin and eosin). (B) In addition to mesangial proliferation, this glomerulus shows segmental endocapillary proliferation and fibrinoid necrosis (hematoxylin and eosin). (C) Jones methenamine silver stain demonstrates segmental necrosis with rupture of the glomerular basement membrane in the same glomerulus shown in (B). (D) A glomerulus with segmental endocapillary proliferation and an overlying segmental cellular crescent. Fibrinoid necrosis is also seen in the center of the field (periodic acid–Schiff). (E) Immunofluorescence staining for IgG shows global mesangial positivity. Only scant positivity is seen in peripheral capillary walls (original magnification ⴛ400).
are associated with multiple vasculitic conditions, including Wegener granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and idiopathic necrotizing GN. Recent experiments in mice suggested that ANCAs play a direct
pathogenic role in the mediation of the necrotizing GN and vasculitis.4 Serological testing for ANCA is performed by means of IIF and ELISA. IIF is used for initial screening and requires ethanol fixation of the
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Fig 2. (A) On ultrastructural evaluation, abundant mesangial deposits are seen. Only scant small peripheral capillary wall deposits are identified (original magnification ⴛ2,000). (B) Prominent mesangial and small subepithelial deposits are present in this field (original magnification ⴛ4,000).
substrate. With formalin fixation, both cytoplasmic ANCA (c-ANCA) and p-ANCA localize to the cytoplasm. However, when ethanol-fixed neutrophils are used as the substrate, p-ANCA artifactually redistributes to the perinuclear region. ELISA is used to determine the specificity of the ANCA. Typically, the target antigens of c-ANCA and p-ANCA are proteinase 3 (PR3) and MPO, respectively. Interpretation of ANCAs in the setting of SLE is considerably more complex. First, high-titer antinuclear antibodies may interfere with the interpretation of ANCAs by IIF, which has led to the suggestion that IIF may be an inappropriate screening test for ANCAs in patients with SLE.5 Second, multiple studies have documented the presence of a variety of p-ANCA target antigens other than MPO in patients with SLE, including lactoferrin (LF), cathepsin G, human leukocyte elastase (HLE), bacteriacidal/permeability-increasing protein (BPI), and lysozyme.4–10 Finally, a false-positive MPO-ANCA result may be found in patients with SLE because of the binding of cationic MPO substrate to DNA within the antigen-binding site of anti–double-stranded DNA antibodies.11 ANCA positivity by means of IIF is common in patients with SLE, with a reported incidence as high as 42%.6 The largest cohort studied consists of 566 patients with SLE drawn from 11 centers in Europe.7 The prevalence of ANCA positivity by means of IIF was 16.4%, of which 15.4% was perinuclear in distribution. ANCA target antigens, assessed by means of ELISA, included LF (14.3%), MPO (9.3%), lysozyme
(4.6%), and PR3 (1.7%). In this study, anti-LF ANCA was associated with serositis and livedo reticularis.7 Another study examined 55 patients in Bulgaria with SLE and found ANCA positivity by IIF in 16 patients (29.1%), 15 of whom had a perinuclear pattern of staining.8 By ELISA, ANCA target antigens included BPI (23.6%), LF (18.2%), PR3 (12.7%), MPO (10.9%), and HLE (1.8%). In 43.8% of patients with positive ANCA, no target antigen was identified by means of ELISA, emphasizing the difficulty in interpreting ANCA by means of IIF in the setting of SLE.8 Although BPI-, LF-, and PR3-ANCA levels correlated with disease activity, none of the antibodies tested correlated with the presence of renal disease. ANCA positivity by IIF did not correlate with any parameter of disease activity, leading the investigators to conclude that in the setting of SLE, ANCA testing should be performed strictly by means of ELISA.8 A study of patients with SLE in South Korea found ANCA positivity by IIF in 19 patients (37.3%), including c-ANCA in 3 patients and p-ANCA in 16 patients.9 ELISA showed anti-LF ANCA in 13 patients and anti-MPO in a single patient; ELISA testing for other target antigens was not performed. Importantly, p-ANCA positivity by IIF correlated with the presence of LN (particularly LN class IV) and with the presence of anti–double-stranded DNA antibodies. A study from China used ELISA to search for antibodies against ANCA target antigens in 95 patients with biopsy-proven LN.5 Anti-cathepsin G antibodies were present in 59 patients (62.1%), and their presence correlated with active renal lesions.
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Anti-LF and anti-BPI antibodies were shown in 8.4% and 1.1% of patients, respectively. ELISA failed to show MPO-, PR3-, or HLE-ANCA in any patient. Of note, by IIF only 22% of patients were ANCA positive. A report from Mexico of 50 children with SLE found ANCA positivity by IIF in 16%, all of whom had MPO-ANCA by ELISA.10 Not all studies have documented the significance of ANCA positivity in patients with SLE. One study examined 41 patients with SLE and compared serological test results with 2 control groups consisting of 12 individuals with systemic vasculitis and 15 healthy volunteers.12 By ELISA, ANCA prevalence and titers were similar between the SLE group and healthy controls and significantly less than in patients with vasculitis. The investigators concluded that in patients with SLE, ANCA testing does not provide important diagnostic or prognostic data.12 Sen and Isenberg6 reviewed the aforementioned and additional smaller studies on ANCA in patients with SLE and drew several important conclusions. Approximately 20% of patients with SLE are ANCA positive by IIF, overwhelmingly p-ANCA.6 The most common ANCA target antigens by ELISA appear to be LF, cathepsin G, and MPO. Targets of ANCA vary widely in different studies from varying geographic regions, suggesting a role for genetic factors. Approximately 8% of patients with SLE have MPO-ANCA by means of ELISA. Some studies suggest a correlation between ANCA positivity and SLE disease activity. MPO-ANCAs are particularly common in patients with drug-induced SLE. There is minimal information available on renal biopsy findings in patients with SLE and ANCA. Marshall et al13 described 2 cases of probable overlap between LN and ANCA-associated GN. Both patients had SLE and p-ANCA positivity by IIF; ELISA was available in 1 patient and showed MPO-ANCA. Renal biopsy showed membranous LN class V with mesangial and subepithelial electron-dense deposits and endothelial tubuloreticular inclusion. Despite the absence of endocapillary proliferation or subendothelial deposits, fibrinoid necrosis and cellular crescents were seen in 50% and 33% of glomeruli. These findings led the investigators to conclude that patients had an overlap of membranous LN and pauci-immune focal segmental ne-
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crotizing and crescentic GN in the setting of p-ANCA seropositivity.13 Another report described 2 patients with SLE and “pauci-immune necrotizing lupus nephritis,” although test results for ANCA were negative in both patients.14 ANCA positivity also may occur in the setting of other immune complex–mediated GNs, such as IgA nephropathy.15 Haas et al15 described 6 patients with severe crescentic GN, ANCA positivity, and mesangial deposits of IgA. ELISA showed PR3-ANCA in 4 patients, MPO-ANCA in 1 patient, and both PR3- and MPO-ANCA in 1 patient. Light microscopy showed relatively mild mesangial proliferation in the setting of extensive crescent formation involving 53% to 100% of glomeruli. The investigators concluded that the 6 cases resembled ANCA-associated GN more than IgA nephropathy with respect to histological findings, response to therapy, and clinical outcome.15 Similar to the case reported here, the immune complex load appeared insufficient to account for the extent of crescent formation, suggesting that ANCA seropositivity contributed to the necrotizing and crescentic phenotype. In conclusion, we report the case of a 50-yearold woman with SLE, RPGN, and LN class III. Renal biopsy showed only mild endocapillary proliferation and rare subendothelial deposits in the setting of prominent necrotizing and crescentic features. The disproportionate degree of necrosis and crescent formation led to the discovery of p-ANCA positivity by IIF and MPO-ANCA by ELISA and suggests that this GN represents an overlap of LN and “pauci-immune” focal segmental necrotizing and crescentic GN in the setting of p-ANCA seropositivity. The significance of ANCA positivity in patients with SLE, its role in modulating disease activity, and its implications for optimal treatment and prognosis remain to be fully defined. REFERENCES 1. Weening JJ, D’Agati VD, Schwartz MM, et al: The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 65:521-530, 2004 2. Weening JJ, D’Agati VD, Schwartz MM, et al: The classification of glomerulonephritis in systemic lupus erythematosus revisited. J Am Soc Nephrol 15:241-250, 2004
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3. Contreras G, Roth D, Pardo V, Striker LG, Schultz DR: Lupus nephritis: A clinical review for practicing nephrologists. Clin Nephrol 57:95-107, 2002 4. Xiao HP, Heeringa P, Hu P, et al: Evidence that ANCA-associated vasculitis is transferable. J Clin Invest 110:955-963, 2002 5. Zhao MH, Liu N, Zhang YK, Wang HY: Antineutrophil cytoplasmic autoantibodies (ANCA) and their target antigens in Chinese patients with lupus nephritis. Nephrol Dial Transplant 13:2821-2824, 1998 6. Sen D, Isenberg DA: Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus. Lupus 12:651-658, 2003 7. Galeazzi M, Morozzi G, Sebastiani GD, et al: Antineutrophil cytoplasmic antibodies in 566 European patients with systemic lupus erythematosus: Prevalence, clinical associations and correlation with other autoantibodies. Clin Exp Rheumatol 16:541-546, 1998 8. Manolova I, Dancheva M, Halacheva K: Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus: Prevalence, antigen specificity, and clinical associations. Rheumatol Int 20:197-204, 2001 9. Chin HJ, Ahn C, Lim CS, et al: Clinical implications of antineutrophil cytoplasmic antibody test in lupus nephritis. Am J Nephrol 20:57-63, 2000
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10. Faure-Fontenla MA, Rodriguez-Suarez RS, AriasVelasquez R, Garcia-Gonzalez JE: Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus in childhood. J Rheumatol 26:2480-2481, 1999 11. Jethwa HS, Nachman PH, Falk RJ, Jennette JC: False-positive myeloperoxidase binding activity due to DNA/ anti-DNA antibody complexes: A source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic antibodies. Clin Exp Immunol 121:544-550, 2000 12. Molnar K, Kovacs L, Kiss S, Husz S, Dobozy A, Pokorny G: Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus. Clin Exp Dermatol 27:59-61, 2002 13. Marshall S, Dressler R, D’Agati V: Membranous lupus nephritis with antineutrophil cytoplasmic antibodyassociated segmental necrotizing and crescenteric glomerulonephritis. Am J Kidney Dis 29:119-124, 1997 14. Aktar M, Al-Dalaan A, El-Ramahi KM: Pauciimmune necrotizing lupus nephritis: Report of two cases. Am J Kidney Dis 23:320-325, 1994 15. Haas M, Jafri J, Bartosh SM, Karp SL, Adler SG, Meehan SM: ANCA-associated crescentic glomerulonephritis with mesangial IgA deposits. Am J Kidney Dis 36:709718, 2000
SLE and Rapidly Progressive Glomerulonephritis Naveed N. Masani, MD, Louis J. Imbriano, MD, Vivette D. D’Agati, MD, and Glen S. Markowitz, MD INDEX WORDS: Systemic lupus erythematosus (SLE); rapidly progressive glomerulonephritis (RPGN); antineutrophil cytoplasmic antibody (ANCA).
S
YSTEMIC LUPUS erythematosus (SLE) is an autoimmune disease with dermatological, rheumatological, hematologic, and renal manifestations. Renal involvement occurs in the majority of patients with SLE and commonly manifests with hematuria, proteinuria, and/or renal insufficiency. Categorization of the multiple patterns of lupus nephritis (LN) recently was revisited in the 2003 International Society of Nephrology/Renal Pathology Society Classification of LN.1,2 Renal biopsy in patients with SLE is critical to define the pattern of renal disease, determine prognosis, and guide therapy.3 Rapidly progressive glomerulonephritis (RPGN) is a clinical term defined by the presence of accelerated renal failure caused by a proliferative inflammatory glomerular lesion. A common definition for RPGN is 50% loss of renal function over 3 months and clinical evidence of glomerular injury in the form of active urinary sediment including red blood cell (RBC) casts. In patients with RPGN, renal biopsy typically reveals crescentic glomerulonephritis (GN). Crescentic GN is a term that encompasses multiple patterns of glomerular disease that are best differentiated by immunofluorescence. Immunofluorescence typically shows 1 of 3 possible patterns: granular positivity consistent with immune complex–mediated GN, linear positivity From the Department of Medicine, Division of Nephrology and Hypertension, Winthrop University Hospital, Mineola, NY; and Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY. Received July 29, 2004; accepted in revised form August 11, 2004. Originally published online as doi:10.1053/j.ajkd.2004.08.047 on April 5, 2005. Address reprint requests to Glen S. Markowitz, MD, Department of Pathology, Columbia University, College of Physicians & Surgeons, 630 West 168th St, VC 14-224, New York, NY 10032. E-mail: [email protected] © 2005 by the National Kidney Foundation, Inc. 0272-6386/05/4505-0021$30.00/0 doi:10.1053/j.ajkd.2004.08.047 950
caused by anti–glomerular basement membrane disease, or no significant positivity, corresponding to “pauci-immune” crescentic GN. Most patients with pauci-immune crescentic GN have circulating antineutrophil cytoplasmic antibodies (ANCAs). A clinical presentation of RPGN may occur in patients with SLE, for which renal biopsy usually shows LN class IV with crescents. In rare cases, renal biopsy may show LN class III or even class V (eg, in association with renal vein thrombosis). We report the case of a patient with SLE and RPGN for whom renal biopsy findings and serological test results suggested an overlapping etiology of crescentic GN. CASE REPORT A 50-year-old white woman presented with acute renal failure. Her past medical history was significant for SLE for 2½ years, diabetes mellitus for 3 years (without retinopathy), hypertension, autoimmune hepatitis, primary biliary cirrhosis, pleurisy, pericarditis, and paroxysmal atrial fibrillation. There was no history of deep venous thrombosis, hemoptysis, or miscarriage. Her serum creatinine level 2 months before admission was 0.7 mg/dL (62 mol/L; normal, 0.6 to 1.5 mg/dL [53 to 133 mol/L]). At that time, urinalysis was significant for 1⫹ protein, 5 to 10 RBCs/highpower field, and the absence of RBC casts. Renal biopsy was postponed at the patient’s request. On admission, the patient’s serum creatinine level was 4.4 mg/dL (389 mol/L). She reported nausea, vomiting, decreased appetite, and generalized fatigue for 2 weeks before admission. Medications included aspirin, 325 mg once daily; digoxin, 0.125 mg once daily; sustained-release verapamil, 240 mg once daily; furosemide, 40 mg once daily; hydroxychloroquine, 200 mg once daily; omeprazole, 20 mg once daily; glyburide, 10 mg once daily; and prednisone, 10 mg once daily. She denied use of nonsteroidal anti-inflammatory medications or cyclooxygenase-2 inhibitors. Physical examination revealed an obese female with a blood pressure of 140/90 mm Hg, weight of 110 kg, malar rash, and 1⫹ bilateral pitting edema. Laboratory evaluation showed the following values: white blood cell count, 6.9 ⫻ 103/L (⫻ 109/L; normal, 3.9 to 11.0 ⫻ 103/L [⫻ 109/L]); hematocrit, 28% (normal, 36% to 48%); and platelet count, 175 ⫻ 103/L (⫻ 109/L; normal, 160 to 392 ⫻ 103/L [⫻ 109/L]). Chemistry panel showed the following values: serum sodium, 143 mEq/L (mmol/L; normal, 138 to 145 mEq/L [mmol/L]); potassium, 4.2 mEq/L (mmol/L; 3.7 to
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5.2 mEq/L [mmol/L]); chloride, 103 mEq/L (mmol/L; normal, 103 to 112 mEq/L [mmol/L]); carbon dioxide, 23 mEq/L (mmol/L; normal, 23 to 33 mEq/L [mmol/L]); blood urea nitrogen, 44 mg/dL (15.7 mmol/L; normal, 8 to 25 mg/dL [2.9 to 8.9 mmol/L]; creatinine, 4.4 mg/dL (389 mol/L); and albumin, 2.2 g/dL (22 g/L; normal, 3.5 to 5.5 g/dL [35 to 55 g/L]). Serological evaluation showed the following values: antinuclear antibody titer, 1:160; negative anti–double-stranded DNA; C3, 71 mg/dL (0.71 g/L; normal, 112 to 244 mg/dL [1.12 to 2.44 g/L]); C4, 10 mg/dL (0.1 g/L; normal, 14 to 40 mg/dL [0.14 to 0.4 g/L]); and erythrocyte sedimentation rate, 125 mm/h (normal, 1 to 35 mm/h). Cryoglobulins, hepatitis B surface antigen, and hepatitis C antibody test results were negative. Renal ultrasound showed a right kidney measuring 11.7 cm and a left kidney measuring 12.6 cm, without evidence of hydronephrosis. Urinalysis showed a specific gravity of 1.010, 2⫹ protein, 17 RBCs/high-power field, 7 white blood cells/high-power field, and the absence of cellular casts. A 24-hour urine collection contained 3.2 g of protein. Pulse methylprednisolone therapy was initiated, and a renal biopsy was performed.
Kidney Biopsy Findings Light microscopic examination disclosed 34 glomeruli, none of which were globally sclerotic. Glomeruli appeared normal in size and showed a mild to moderate global increase in mesangial cells (Fig 1A). In addition, 10 glomeruli showed mild segmental endocapillary proliferation with infiltrating monocytes (Fig 1B and C). Among the 10 glomeruli with segmental endocapillary proliferation, 3 glomeruli were compressed by overlying cellular crescents associated with fibrinoid necrosis and rupture of the glomerular basement membrane, and 4 additional glomeruli showed segmental fibrinoid necrosis (without crescents; Fig 1D). Proximal tubules displayed protein resorption droplets, prominent intratubular RBCs and RBC casts, and diffuse degenerative changes. There was diffuse interstitial edema and mild to moderate interstitial chronic inflammation composed of lymphocytes and less extensive monocytes. There was minimal tubular atrophy and interstitial fibrosis involving less than 10% of the cortex sampled. Vessels showed mild arteriosclerosis. No arteritis was identified. Immunofluorescence staining of 5 glomeruli showed granular global mesangial and segmental capillary wall positivity of 3⫹ intensity for immunoglobulin G (IgG), ⫹/⫺ for IgM, 1⫹ for IgA, 2⫹ for C3, 2⫹ for C1q, 3⫹ for light chain, and 3⫹ for light chain (scale of positivity: 0, ⫹/ ⫺, 1 to 3⫹; Fig 1E). Immunofluorescence positivity for IgG (2⫹), IgM (⫹/⫺), C3 (1⫹), (1⫹), and (1⫹) also was noted in blood vessel walls. Sampling for electron microscopy consisted of 4 glomeruli. Glomerular capillary lumina appeared patent. Abundant electron-dense deposits globally expanded the mesangial matrix, associated with increased mesangial cellularity (Fig 2A and B). Endothelial cells showed intact fenestrations and multiple tubuloreticular inclusions. Of note, only rare small subendothelial and subepithelial electron-dense deposits were identified. Visceral epithelial cells displayed lipid droplets and 10% foot-process effacement. No tubulointersti-
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tial electron-dense deposits were identified. Vessels were not sampled.
Pathological Diagnosis Light microscopy showed diffuse mesangial and segmental endocapillary proliferative GN, notable for the presence of prominent necrotizing features and multiple cellular crescents. Light microscopic findings were diagnostic of LN class III (LN “class III (A)” by the 2003 International Society of Nephrology/Renal Pathology Society classification of LN1,2). The diagnosis of LN was supported by the “full house” of staining for immunoglobulins and complement by immunofluorescence, the presence of vessel wall deposits, and the ultrastructural finding of multiple endothelial tubuloreticular inclusions. By the National Institutes of Health criteria, the findings corresponded to an activity index of 9 (scale, 0 to 24) and a chronicity index of 1 (scale, 0 to 12). Although biopsy findings were diagnostic of LN class III, some findings raised the possibility of an additional diagnosis. The degree of endocapillary proliferation was mild, and only rare subendothelial electron-dense deposits were noted on ultrastructural evaluation. Conversely, the majority of glomeruli with endocapillary hypercellularity also showed fibrinoid necrosis or cellular crescents. Overall, the degree of necrosis and crescent formation appeared out of proportion to the sparse peripheral capillary wall deposits and minimal endocapillary proliferation. This apparent discrepancy prompted testing for ANCAs.
Clinical Follow-Up Indirect immunofluorescence (IIF) showed perinuclear ANCA (p-ANCA) positivity. Further testing using enzymelinked immunosorbent assay (ELISA) confirmed the specificity of the p-ANCA for myeloperoxidase (MPO), with an index of 4.3 (normal, ⬍1.0). Given the findings of LN class III with an activity index of 9, the prominent necrotizing and crescentic features, the presentation with RPGN, and the MPO-ANCA seropositivity, the patient was treated with pulse methylprednisolone (500 mg/d intravenously for 3 days), followed by tapering oral prednisone doses and monthly intravenous cyclophosphamide (1 g/m2 body surface area) for 6 months, followed by quarterly doses. Losartan, 50 mg/d, was initiated for hypertension, and subcutaneous recombinant human erythropoietin (20,000 U/wk) was administered for anemia. After 1 month of therapy, serum creatinine level improved to 2.1 mg/dL (186 mol/L), 24-hour urine protein excretion decreased to 1 g/d, and serum complement levels normalized. After 5 months of therapy, she had a creatinine level of 1.5 mg/dL (133 mol/L), 24-hour urine protein level of 170 mg/d, and a negative MPO-ANCA. Fourteen months postbiopsy, she has a creatinine of 1.3 mg/dL (115 mol/L), 24-hour urine protein less than 200 mg/d, and negative MPO-ANCA. To maintain remission of extrarenal manifestations of SLE, she continues to require prednisone, 7 mg daily.
DISCUSSION
ANCAs target cytoplasmic antigens in the granules of neutrophils and monocytes. ANCAs
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Fig 1. (A) A representative glomerulus with mild global mesangial proliferation. Glomerular capillary lumina appear patent, without evidence of endocapillary proliferation, fibrinoid necrosis, or crescent formation (hematoxylin and eosin). (B) In addition to mesangial proliferation, this glomerulus shows segmental endocapillary proliferation and fibrinoid necrosis (hematoxylin and eosin). (C) Jones methenamine silver stain demonstrates segmental necrosis with rupture of the glomerular basement membrane in the same glomerulus shown in (B). (D) A glomerulus with segmental endocapillary proliferation and an overlying segmental cellular crescent. Fibrinoid necrosis is also seen in the center of the field (periodic acid–Schiff). (E) Immunofluorescence staining for IgG shows global mesangial positivity. Only scant positivity is seen in peripheral capillary walls (original magnification ⴛ400).
are associated with multiple vasculitic conditions, including Wegener granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and idiopathic necrotizing GN. Recent experiments in mice suggested that ANCAs play a direct
pathogenic role in the mediation of the necrotizing GN and vasculitis.4 Serological testing for ANCA is performed by means of IIF and ELISA. IIF is used for initial screening and requires ethanol fixation of the
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Fig 2. (A) On ultrastructural evaluation, abundant mesangial deposits are seen. Only scant small peripheral capillary wall deposits are identified (original magnification ⴛ2,000). (B) Prominent mesangial and small subepithelial deposits are present in this field (original magnification ⴛ4,000).
substrate. With formalin fixation, both cytoplasmic ANCA (c-ANCA) and p-ANCA localize to the cytoplasm. However, when ethanol-fixed neutrophils are used as the substrate, p-ANCA artifactually redistributes to the perinuclear region. ELISA is used to determine the specificity of the ANCA. Typically, the target antigens of c-ANCA and p-ANCA are proteinase 3 (PR3) and MPO, respectively. Interpretation of ANCAs in the setting of SLE is considerably more complex. First, high-titer antinuclear antibodies may interfere with the interpretation of ANCAs by IIF, which has led to the suggestion that IIF may be an inappropriate screening test for ANCAs in patients with SLE.5 Second, multiple studies have documented the presence of a variety of p-ANCA target antigens other than MPO in patients with SLE, including lactoferrin (LF), cathepsin G, human leukocyte elastase (HLE), bacteriacidal/permeability-increasing protein (BPI), and lysozyme.4–10 Finally, a false-positive MPO-ANCA result may be found in patients with SLE because of the binding of cationic MPO substrate to DNA within the antigen-binding site of anti–double-stranded DNA antibodies.11 ANCA positivity by means of IIF is common in patients with SLE, with a reported incidence as high as 42%.6 The largest cohort studied consists of 566 patients with SLE drawn from 11 centers in Europe.7 The prevalence of ANCA positivity by means of IIF was 16.4%, of which 15.4% was perinuclear in distribution. ANCA target antigens, assessed by means of ELISA, included LF (14.3%), MPO (9.3%), lysozyme
(4.6%), and PR3 (1.7%). In this study, anti-LF ANCA was associated with serositis and livedo reticularis.7 Another study examined 55 patients in Bulgaria with SLE and found ANCA positivity by IIF in 16 patients (29.1%), 15 of whom had a perinuclear pattern of staining.8 By ELISA, ANCA target antigens included BPI (23.6%), LF (18.2%), PR3 (12.7%), MPO (10.9%), and HLE (1.8%). In 43.8% of patients with positive ANCA, no target antigen was identified by means of ELISA, emphasizing the difficulty in interpreting ANCA by means of IIF in the setting of SLE.8 Although BPI-, LF-, and PR3-ANCA levels correlated with disease activity, none of the antibodies tested correlated with the presence of renal disease. ANCA positivity by IIF did not correlate with any parameter of disease activity, leading the investigators to conclude that in the setting of SLE, ANCA testing should be performed strictly by means of ELISA.8 A study of patients with SLE in South Korea found ANCA positivity by IIF in 19 patients (37.3%), including c-ANCA in 3 patients and p-ANCA in 16 patients.9 ELISA showed anti-LF ANCA in 13 patients and anti-MPO in a single patient; ELISA testing for other target antigens was not performed. Importantly, p-ANCA positivity by IIF correlated with the presence of LN (particularly LN class IV) and with the presence of anti–double-stranded DNA antibodies. A study from China used ELISA to search for antibodies against ANCA target antigens in 95 patients with biopsy-proven LN.5 Anti-cathepsin G antibodies were present in 59 patients (62.1%), and their presence correlated with active renal lesions.
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Anti-LF and anti-BPI antibodies were shown in 8.4% and 1.1% of patients, respectively. ELISA failed to show MPO-, PR3-, or HLE-ANCA in any patient. Of note, by IIF only 22% of patients were ANCA positive. A report from Mexico of 50 children with SLE found ANCA positivity by IIF in 16%, all of whom had MPO-ANCA by ELISA.10 Not all studies have documented the significance of ANCA positivity in patients with SLE. One study examined 41 patients with SLE and compared serological test results with 2 control groups consisting of 12 individuals with systemic vasculitis and 15 healthy volunteers.12 By ELISA, ANCA prevalence and titers were similar between the SLE group and healthy controls and significantly less than in patients with vasculitis. The investigators concluded that in patients with SLE, ANCA testing does not provide important diagnostic or prognostic data.12 Sen and Isenberg6 reviewed the aforementioned and additional smaller studies on ANCA in patients with SLE and drew several important conclusions. Approximately 20% of patients with SLE are ANCA positive by IIF, overwhelmingly p-ANCA.6 The most common ANCA target antigens by ELISA appear to be LF, cathepsin G, and MPO. Targets of ANCA vary widely in different studies from varying geographic regions, suggesting a role for genetic factors. Approximately 8% of patients with SLE have MPO-ANCA by means of ELISA. Some studies suggest a correlation between ANCA positivity and SLE disease activity. MPO-ANCAs are particularly common in patients with drug-induced SLE. There is minimal information available on renal biopsy findings in patients with SLE and ANCA. Marshall et al13 described 2 cases of probable overlap between LN and ANCA-associated GN. Both patients had SLE and p-ANCA positivity by IIF; ELISA was available in 1 patient and showed MPO-ANCA. Renal biopsy showed membranous LN class V with mesangial and subepithelial electron-dense deposits and endothelial tubuloreticular inclusion. Despite the absence of endocapillary proliferation or subendothelial deposits, fibrinoid necrosis and cellular crescents were seen in 50% and 33% of glomeruli. These findings led the investigators to conclude that patients had an overlap of membranous LN and pauci-immune focal segmental ne-
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crotizing and crescentic GN in the setting of p-ANCA seropositivity.13 Another report described 2 patients with SLE and “pauci-immune necrotizing lupus nephritis,” although test results for ANCA were negative in both patients.14 ANCA positivity also may occur in the setting of other immune complex–mediated GNs, such as IgA nephropathy.15 Haas et al15 described 6 patients with severe crescentic GN, ANCA positivity, and mesangial deposits of IgA. ELISA showed PR3-ANCA in 4 patients, MPO-ANCA in 1 patient, and both PR3- and MPO-ANCA in 1 patient. Light microscopy showed relatively mild mesangial proliferation in the setting of extensive crescent formation involving 53% to 100% of glomeruli. The investigators concluded that the 6 cases resembled ANCA-associated GN more than IgA nephropathy with respect to histological findings, response to therapy, and clinical outcome.15 Similar to the case reported here, the immune complex load appeared insufficient to account for the extent of crescent formation, suggesting that ANCA seropositivity contributed to the necrotizing and crescentic phenotype. In conclusion, we report the case of a 50-yearold woman with SLE, RPGN, and LN class III. Renal biopsy showed only mild endocapillary proliferation and rare subendothelial deposits in the setting of prominent necrotizing and crescentic features. The disproportionate degree of necrosis and crescent formation led to the discovery of p-ANCA positivity by IIF and MPO-ANCA by ELISA and suggests that this GN represents an overlap of LN and “pauci-immune” focal segmental necrotizing and crescentic GN in the setting of p-ANCA seropositivity. The significance of ANCA positivity in patients with SLE, its role in modulating disease activity, and its implications for optimal treatment and prognosis remain to be fully defined. REFERENCES 1. Weening JJ, D’Agati VD, Schwartz MM, et al: The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 65:521-530, 2004 2. Weening JJ, D’Agati VD, Schwartz MM, et al: The classification of glomerulonephritis in systemic lupus erythematosus revisited. J Am Soc Nephrol 15:241-250, 2004
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3. Contreras G, Roth D, Pardo V, Striker LG, Schultz DR: Lupus nephritis: A clinical review for practicing nephrologists. Clin Nephrol 57:95-107, 2002 4. Xiao HP, Heeringa P, Hu P, et al: Evidence that ANCA-associated vasculitis is transferable. J Clin Invest 110:955-963, 2002 5. Zhao MH, Liu N, Zhang YK, Wang HY: Antineutrophil cytoplasmic autoantibodies (ANCA) and their target antigens in Chinese patients with lupus nephritis. Nephrol Dial Transplant 13:2821-2824, 1998 6. Sen D, Isenberg DA: Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus. Lupus 12:651-658, 2003 7. Galeazzi M, Morozzi G, Sebastiani GD, et al: Antineutrophil cytoplasmic antibodies in 566 European patients with systemic lupus erythematosus: Prevalence, clinical associations and correlation with other autoantibodies. Clin Exp Rheumatol 16:541-546, 1998 8. Manolova I, Dancheva M, Halacheva K: Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus: Prevalence, antigen specificity, and clinical associations. Rheumatol Int 20:197-204, 2001 9. Chin HJ, Ahn C, Lim CS, et al: Clinical implications of antineutrophil cytoplasmic antibody test in lupus nephritis. Am J Nephrol 20:57-63, 2000
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10. Faure-Fontenla MA, Rodriguez-Suarez RS, AriasVelasquez R, Garcia-Gonzalez JE: Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus in childhood. J Rheumatol 26:2480-2481, 1999 11. Jethwa HS, Nachman PH, Falk RJ, Jennette JC: False-positive myeloperoxidase binding activity due to DNA/ anti-DNA antibody complexes: A source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic antibodies. Clin Exp Immunol 121:544-550, 2000 12. Molnar K, Kovacs L, Kiss S, Husz S, Dobozy A, Pokorny G: Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus. Clin Exp Dermatol 27:59-61, 2002 13. Marshall S, Dressler R, D’Agati V: Membranous lupus nephritis with antineutrophil cytoplasmic antibodyassociated segmental necrotizing and crescenteric glomerulonephritis. Am J Kidney Dis 29:119-124, 1997 14. Aktar M, Al-Dalaan A, El-Ramahi KM: Pauciimmune necrotizing lupus nephritis: Report of two cases. Am J Kidney Dis 23:320-325, 1994 15. Haas M, Jafri J, Bartosh SM, Karp SL, Adler SG, Meehan SM: ANCA-associated crescentic glomerulonephritis with mesangial IgA deposits. Am J Kidney Dis 36:709718, 2000