RENAL BIOPSIES in patients with the clinical

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1 RENAL BIOPSY TEACHING CASE Crescentic Glomerulonephritis With a Paucity of Glomerular Immunoglobulin Localization Alexis A. Harris, MD, Ronald J. Falk, MD, and J. Charles Jennette, MD RENAL BIOPSIES in patients with the clinical findings of rapidly progressive glomerulonephritis usually demonstrate crescentic glomerulonephritis. This light microscopic diagnosis alone is not adequate for accurate prognostication or for optimum design of treatment strategy. Light microscopy must be supplemented with immunohistologic and ultrastructural findings, and sometimes serologic data, to more precisely categorize the disease process. The major categories of crescentic glomerulonephritis are antiglomerular basement membrane (GBM) antibody crescentic glomerulonephritis, immune-complex crescentic glomerulonephritis, and pauci-immune crescentic glomerulonephritis. The latter category is often, but not always, associated with and possibly mediated by antineutrophil cytoplasmic autoantibodies (ANCAs). The following case report illustrates an approach to the pathologic evaluation of crescentic glomerulonephritis by integrating light, immunofluorescence, and electron microscopic findings and serologic data. The findings in this patient also demonstrate that we still have much to learn about the categorization of crescentic glomerulonephritis. CASE REPORT A 76-year-old white man developed flu-like symptoms, including a low-grade fever, chills, and diffuse myalgias. He visited a minor emergency clinic where laboratory tests demonstrated serum creatinine 2.0 mg/dl, blood urea nitrogen 35 mg/dl, mildly elevated liver enzymes, and an erythrocyte sedimentation rate of 115. Prednisone was administered to treat possible polymyalgia rheumatica. Over the next 3 days his symptoms did not improve and he developed nausea and problems with walking. Cephalexin was added to the treatment regimen for presumed urinary tract infection. The patient was referred to a regional medical center. Evaluation revealed no past history of renal disease in the patient or his family. There was no history of rash, abdominal pain, hemoptysis, bloody nasal discharge, or sinusitis. Physical examination demonstrated a temperature of 99.8 F, respiration 16 breaths/min, heart rate 80 beats/min, blood pressure 158/72 mm Hg, 1 pedal edema, and difficulty standing. Laboratory data included serum creatinine 3.3 mg/dl, blood urea nitrogen 64 mg/dl, 2 proteinuria, 4 hematuria, glucose 128 mg/dl, albumin 2.6 g/dl, cholesterol 127 mg/dl, total protein 6.2 g/dl, globulin 3.6 mg/dl, bilirubin 1.1 mg/dl, mildly elevated liver enzymes, hemoglobin 11.7 g/dl, normal platelet count, and normal coagulation test results. The patient was referred to a nephrologist. Additional laboratory data were ordered and the patient was scheduled for renal biopsy. By the time of biopsy, the following additional data were available: proteinuria 1.6 g/24 hr, creatinine clearance 23 ml/min, serum creatinine 3.6 mg/dl, and negative antinuclear antibody assay. The clinical diagnosis was rapidly progressive glomerulonephritis, possibly as a component of systemic small vessel vasculitis. A renal biopsy was performed. ANCA and anti-gbm test results were pending at the time of biopsy. RESULTS Renal Biopsy Findings Light microscopy revealed a necrotizing glomerulonephritis with crescents in over 50% of the up to 12 glomeruli per level of section (Fig 1). Foci of segmental fibrinoid necrosis contained scattered neutrophils, some undergoing leukocytoclasia (Fig 2). A few glomeruli had focal disruption of Bowman s capsule with continuity between glomerular and periglomerular inflammation (Fig 2). The nonnecrotic glomeruli had no hypercellularity and no thickening of capillary walls (Fig 3). There was mild to moderate interstitial fibrosis and tubular atrophy with an associated predominantly mononuclear leukocyte interstitial infiltrate. Except for rare contiguous extension of glomerular inflammation and necrosis into hilar arterioles, there were no necrotizing or inflammatory changes in arterioles or arteries (Fig 3). Immunofluorescence microscopy demonstrated only low-intensity (1 on a scale of 0 to 4 ) mesangial staining with antisera specific for im- From the Departments of Medicine and Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill. Received and accepted as submitted March 25, Address reprint requests to J. Charles Jennette, MD, CB# 7525, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC jcj@med.unc.edu 1998 by the National Kidney Foundation, Inc /98/ $3.00/0 American Journal of Kidney Diseases, Vol 32, No 1 (July), 1998: pp

2 180 HARRIS, FALK, AND JENNETTE Fig 1. Light microscopy showing a circumferential cellular crescent. There has been lytic necrosis of portions of the glomerular tuft, and there is scattered karyorrhectic debris. The periglomerular interstitium has slight edema and infiltration by mononuclear leukocytes. (Periodic acid-schiff stain; magnification 500.) munoglobulin G (IgG), IgA, and C3. There was no glomerular staining with antisera specific for IgM or C1q. There was no linear or granular staining of glomerular capillary walls with antisera specific for immunoglobulins or complement. There was focal staining of glomerular crescents and necrotic glomerular segments with an antiserum specific for fibrin. There was no staining of tubulointerstitial tissue or extraglomerular vessels. Electron microscopy demonstrated only a few scattered small mesangial dense deposits (Fig 4). There were no capillary wall dense deposits. No endothelial tubuloreticular inclusions were identified. Visceral epithelial cell foot processes had extensive effacement. There were focal gaps in Fig 2. Light microscopy showing a glomerulus with extensive segmental necrosis and disruption of the overlying portion of Bowman s capsule resulting in extension of the inflammation into the adjacent periglomerular interstitium. (Periodic acid- Schiff stain; magnification 500.)

3 CRESCENTIC GN LACKS GLOMERULAR IG LOCALIZATION 181 Fig 3. Light microscopy showing one of the few glomeruli in the specimen that did not have necrosis or crescent formation. The absence of hypercellularity or capillary wall thickening suggests, but does not prove, that the crescent formation is not caused by glomerular immune complex localization. Also note the absence of vasculitis in the arteriole and artery. (Periodic acid- Schiff stain; magnification 400.) GBMs. Foci of glomerular fibrinoid necrosis and crescents had accumulations of fibrin tactoids (Fig 4). Renal Biopsy Diagnosis The histologic diagnosis was necrotizing and crescentic glomerulonephritis. There was uncertainty whether to consider this to be pauciimmune crescentic glomerulonephritis or immune complex crescentic glomerulonephritis. Given the small amount (paucity) of immunoglobulin detected, a diagnosis of pauci-immune crescentic glomerulonephritis was favored. Clinical Course Serologic tests for anti-gbm, C-ANCA/PR3- ANCA, and P-ANCA/MPO-ANCA were all negative. The patient was treated with intravenous methylprednisolone and intravenous cyclophosphamide. The serum creatinine peaked at 4.8 mg/dl after a few days, then decreased to 2.6 mg/dl within 2 weeks. Fig 4. Electron micrograph showing one of the few small mesangial electron dense deposits that were identified (straight arrow) and an accumulation of fibrin tactoids within Bowman s space (curved arrow). (Magnification 5,000.)

4 182 HARRIS, FALK, AND JENNETTE DISCUSSION The patient under consideration had crescentic glomerulonephritis. This is not a specific disease, but rather is the most severe histologic grade of glomerular inflammation observed in patients with glomerulonephritis. Glomerular crescent formation results from a final common pathway of glomerular injury that can be initiated by different etiologies and different pathogenic mechanisms. Crescents are composed mostly of macrophages and epithelial cells that are layered onto Bowman s capsule. 1-3 Crescent formation is initiated by rupture of glomerular capillary walls, 4 which allows inflammatory mediators, such as coagulation factors, cytokines, and growth factors, to enter Bowman s space, where they recruit and activate macrophage and stimulate epithelial proliferation. The demonstration of fibrin in Bowman s space and in the interstices between the cells of crescents by immunofluorescence and electron microscopy (Fig 4) is indicative that plasma constituents, including coagulation proteins, have gained access to Bowman s space. Cellular crescents evolve into fibrocellular and fibrous crescents as chronicity increases and the cells are progressively replaced by collagen. This usually is accompanied by progressive scarring of the underlying injured glomerular tuft. The three major categories of crescentic glomerulonephritis are anti-gbm, immune complex, and pauci-immune (Fig 5). 5-7 The pauciimmune category is usually ANCA positive, 8,9 but also includes a small category of idiopathic pauci-immune crescentic glomerulonephritis in patients who do not have ANCAs. Idiopathic pauci-immune crescentic glomerulonephritis may be the most appropriate diagnosis for the patient described in the case report above. Light microscopic evaluation alone cannot accurately determine the category of crescentic glomerulonephritis; however, there are histologic hints. In general, immune complex crescentic glomerulonephritis has more endocapillary hypercellularity and thickening of capillary walls than anti-gbm or pauci-immune crescentic glomerulonephritis. This is because of the structural changes that are caused not only by the physical presence of immune complex accumulations in capillary walls and mesangium, but also by the Fig 5. Diagram depicting the immunopathologic categories of crescentic glomerulonephritis. Note that each of the three major categories has multiple, more specific subcategories. IF, immunofluorescence; CGN, crescentic glomerulonephritis; SLE, systemic lupus erythematosus; H-S, Henoch-Schönlein; GN, glomerulonephritis; MPGN, mesangioproliferative glomerulonephritis. (Modified and reprinted with permission. 6 ) proliferative changes in mesangial and endothelial cells, and the influx of leukocytes that are induced by the immune complexes. Compared with immune complex disease, anti-gbm and pauci-immune crescentic glomerulonephritis have more fibrinoid necrosis and more disruption of Bowman s capsule, which results in a greater degree of periglomerular inflammation. 10,11 This suggests that the pathogenic mechanisms that are initiated by anti-gbm and ANCAs induce more lytic injury than the mechanism initiated by immune complexes. Because of their histologic similarities, it is not possible to distinguish anti- GBM crescentic glomerulonephritis from pauciimmune crescentic glomerulonephritis by light microscopy, unless there is an accompanying vasculitis in the biopsy specimen. Small vessel vasculitis is much more frequent with pauciimmune crescentic glomerulonephritis, especially when the patient has ANCAs. Therefore, necrotizing vasculitis affecting the cortical arteries or arterioles, or the medullary vase recta (medullary angiitis), in a renal biopsy specimen increases the likelihood of ANCA-positive pauciimmune crescentic glomerulonephritis. Immunofluorescence and electron microscopy

5 CRESCENTIC GN LACKS GLOMERULAR IG LOCALIZATION 183 Table 1. Frequency of the Three Immunopathologic Categories of Crescentic Glomerulonephritis in Patients of Different Ages (n 20) Age Group, yr (%) (n 42) (n 61) 65 (n 65) Anti-GBM CGN Immune complex CGN Pauci-immune CGN NOTE. All patients had crescents in 50% of glomeruli. Abbreviation: CGN, crescentic glomerulonephritis. Reprinted with permission. 7 are required for optimum categorization of crescentic glomerulonephritis into anti-gbm, immune complex, or pauci-immune disease. Table 1 gives the relative frequency of immunopathologic categories of crescentic glomerulonephritis (defined as 50% of glomeruli with crescents) with respect to age in patients whose renal biopsy specimens were evaluated in the University of North Carolina Nephropathology Laboratory. 7 Note the very high frequency of pauci-immune crescentic glomerulonephritis in the elderly. The comparatively high frequency of immune complex crescentic glomerulonephritis in younger patients is not surprising given the fact that most forms of immune complex proliferative glomerulonephritis, from which crescentic immune complex glomerulonephritis arises, are more frequent in children and young adults, such as IgA nephropathy, lupus nephritis, membranoproliferative glomerulonephritis, and postinfectious glomerulonephritis. Most patients with pauci-immune crescentic glomerulonephritis have evidence of systemic small vessel vasculitis, such as microscopic polyangiitis or Wegener s granulomatosis. 9,12 The patient described in the case report above had arthralgias, myalgias, hepatic dysfunction, and possible neuropathy, which suggest a systemic vasculitis. If the patient has systemic small vessel vasculitis affecting vessels in the muscle, liver, and nerves, as well as glomeruli, the most appropriate diagnosis is microscopic polyangiitis, because there is no evidence of respiratory tract granulomatous disease or of eosinophilia and asthma (Fig 5). 12 Although ANCAs are most frequent in patients with pauci-immune crescentic glomerulonephritis, one quarter to one third of patients with anti-gbm crescentic glomerulonephritis have ANCAs A less well-recognized observation is that patients with immune complex crescentic glomerulonephritis have a higher frequency of ANCAs than healthy controls, and that the likelihood of ANCA positivity is inversely proportional to the intensity of glomerular staining for immunoglobulins. 16,17 Figure 6 demonstrates that although the frequency of ANCAs is greatest in patients with pauci-immune glomerulonephritis, the proportion of patients who are positive or negative for ANCAs depends on how pauci-immune the glomerular disease is, that is, how little staining for immunoglobulins is observed by immunofluorescence microscopy. A biopsy specimen from a patient with crescentic glomerulonephritis that has no staining for immunoglobulin indicates an approximately 80% to 90% likelihood of the patient being ANCApositive, whereas 1 staining, as was the case for the patient described earlier in this article, indicates that the patient has an approximately 70% to 80% chance of being positive and thus a 20% to 30% chance of being negative, as our patient was. Patients with crescentic glomerulonephritis who have clear-cut evidence of immune complex localization in glomeruli have a higher likelihood of having ANCAs than patients with immune complex disease who have no glomerular crescents. This suggests that in some patients, ANCAs can be synergistic with immune complexes in causing severe glomerular injury. Fig 6. Graph showing the frequency of ANCA positivity as a function of the intensity of glomerular staining for immunoglobulin. The data were obtained form 213 patients with glomerular crescent formation. Anti- GBM and lupus glomerulonephritis patients were excluded.

6 184 HARRIS, FALK, AND JENNETTE The patient described in this case report does not have evidence of anti-gbm antibodies or ANCAs. There is evidence of only a very minor degree of immune complex accumulation, which would not be expected to cause the severe glomerular inflammation and necrosis that was observed. This raises the specter of additional idiopathic pathogenic factors which have yet to be identified that can cause crescentic glomerulonephritis. Based on the data presented in Fig 6, this category of idiopathic crescentic glomerulonephritis that is not anti-gbm disease, not immune complex disease, and not ANCA disease may account for approximately 10% to 20% of patients with rapidly progressive glomerulonephritis. REFERENCES 1. Sarno EN, Alvarenga FB, Ruzany F, Gattass CR: Distribution of mononuclear phagocytes in glomerulonephritis with crescents. Nephron 32:265, 1982 (letter) 2. Magil AB: Histogenesis of glomerular crescents. Immunohistochemical demonstration of cytokeratin in crescent cells. Am J Pathol 120: , Jennette JC, Hipp CG: The epithelial antigen phenotype of glomerular crescent cells. Am J Clin Pathol 86: , Bonsib SM: Glomerular basement membrane necrosis and crescent organization. Kidney Int 33: , Couser WG: Rapidly progressive glomerulonephritis: Classification, pathogenetic mechanisms, and therapy. Am J Kidney Dis 11: , Jennette JC, Falk RJ: Diagnosis and management of glomerular diseases. Med Clin North Am 81: , Jennette JC, Falk RJ: Crescentic glomerulonephritis, in Massry SG, Glassock RJ (eds): Textbook of Nephrology (ed 3). Baltimore, MD, Williams & Wilkins, 1995, pp Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 318: , Jennette JC: Anti-neutrophil cytoplasmic autoantibodyassociated disease: A pathologist s perspective. Am J Kidney Dis 18: , Bhathena DB, Migdal SD, Julian BA, McMorrow RG, Baehler R, Baehler RW: Morphologic and immunohistochemical observations in granulomatous glomerulonephritis. Am J Pathol 126: , Ferrario F, Tadros MT, Napodano P, Sinico RA, Fellin G, D Amico G: Critical reevaluation of 41 cases of idiopathic crescentic glomerulonephritis. Clin Nephrol 41:1-9, Jennette JC, Falk RJ: Small vessel vasculitis. N Engl J Med 337: , Jayne DR, Marshall PD, Jones SJ, Lockwood CM: Autoantibodies to GBM and neutrophil cytoplasm in rapidly progressive glomerulonephritis. Kidney Int 37: , Bosch X, Mirapeix E, Font J, Borrellas X, Rodriguez RX, Lopez-Soto A, Ingelmo M, Revert L: Prognostic implication of anti-neutrophil cytoplasmic autoantibodies with myeloperoxidase specificity in anti-glomerular basement membrane disease. Clin Nephrol 36: , Short AK, Esnault VL, Lockwood CM: Anti-neutrophil cytoplasm antibodies and anti-glomerular basement membrane antibodies: Two coexisting distinct autoreactivities detectable in patients with rapidly progressive glomerulonephritis. Am J Kidney Dis 26: , Jennette JC, Wilkman AS, Tuttle RH, Falk RJ: How pauci-immune is ANCA-associated crescentic glomerulonephritis (CGN)? J Am Soc Nephrol 7:1735, 1996 (abstr) 17. Falk RJ, Jennette JC: ANCA small-vessel vasculitis. J Am Soc Nephrol 8: , 1997