Section 2 -

Electron Microscopy in the Morphologic Classification of Lupus Nephritis Karlene Hewan-Lowe Brody School of Medicine at East Carolina University Greenville, North Carolina

Introduction: Systemic lupus erythematosus (SLE) is a severe, multisystem autoimmune disease. Loss of self tolerance, which is due to inadequate control or elimination of autoreactive lymphocyte clones, results in multiple autoantibodies with differing specificities [1]. Dysregulated apoptosis and ineffective removal of nuclear debris are additional events that contribute to the development of autoimmunity [1, 2]. Genetic predisposition, hormonal factors, altered immune regulatory factors and environmental stimuli can trigger the development of SLE in susceptible individuals. Within the first few years of the disease onset, about 50% of patients, who have SLE, develop clinical features of lupus nephritis [1]. Lupus nephritis affects all compartments of the kidney. Renal deposition of immune complexes, complement activation and release of chemokines are the basis for the heterogenous morphologic expression of lupus nephritis. The intrinsic renal targets of the immune complexes are the extracellular matrix and cell surface glycoproteins. Anti-DNA antibodies have a broad reactivity range that include the constituents of the glomerulus. The primary glomerular patterns of injury in lupus nephritis are mesangial, endothelial and epithelial. Tubulointerstitial and vascular lesions are also seen in lupus nephritis. Ethnicity, age and gender as well as the severity of the renal disease influence the prognosis of lupus nephritis. Clinical indicators of renal involvement, such as proteinuria, hypocomplimentemia and an active urinary sediment, are insensitive indicators of class and activity of lupus nephritis. Since renal biopsies are common and they are safe procedures in the hands of an experienced nephrologist, the optimal criterion for the diagnosis of lupus nephritis is renal histopathology [3, 4]. The renal biopsy is the most accurate means of predicting prognosis and guiding therapy in patients with lupus nephritis. An experienced nephropathologist requires integration of the renal biopsy findings on light, immunofluorescence and electron microscopy to render an accurate classification of lupus nephritis and quantify the degree of activity as well as chronicity. Electron microscopy has a critical role in the morphologic classification of lupus nephritis because it detects mild forms of glomerular involvement that are not seen on light microscopy, it allows the precise classification of the heterogenous lesions in lupus nephritis and it can suggest the transformation from a mild to a more aggressive class of lupus nephritis [5, 6]. Electron microscopy can detect submicroscopic features that suggest lupus nephritis. Information that is useful in the prognosis and therapy of lupus nephritis can also be obtained through ultrastructural examination of the renal biopsy. Classification of Glomerular Lesions The current ISN/RPS classification of the glomerulonephritis in lupus nephritis provides a clear description of the heterogenous, glomerular lesions in lupus nephritis (Table1). Class I, minimal mesangial lupus nephritis is defined as a normal renal biopsy in a patient who fulfills the American Rheumatism Association (ARA) criteria for the diagnosis of SLE. The glomeruli are normal and small; mesangial deposits are detected on immunofluorescence microscopy. Ultrastructural examination of this biopsy demonstrates small, scattered electron dense deposits within the mesangium. Class II, mesangial proliferative lupus nephritis is defined as pure mesangial proliferation that may range from mild to moderate. Loop closure is absent. Granular, mesangial immune complexes are present on immunofluorescence microscopy. Electron microscopy demonstrates a variable amount of electron dense, immune complex type deposits within the mesangium. Focal segmental or global glomerular hypercellularity is the light microscopic hallmark of Class III, focal proliferative lupus nephritis. Hypercellularity is seen in less than 50% of the glomeruli. Involvement of 50% or more glomeruli by diffuse segmental or global, glomerular hypercellularity is the diagnostic light microscopic feature of diffuse lupus nephritis, Class IV. Immunofluorescence shows focal and segmental subendothelial (peripheral capillary wall) deposits in Class III lupus nephritis. In contrast, the subendothelial deposits are more global and diffuse in Class IV lupus nephritis. Mesangial deposits are present in Class III and IV lupus nephritis. With electron microscopy, subendothelial and mesangial deposits are seen in both Class III and Class IV lupus nephritis. In Class III lupus nephritis, the subendothelial deposits are focal and segmental. Subendothelial deposits in Class IV lupus nephritis are diffuse and global. The number and size of the subendothelial deposits correlate with the activity of the lupus nephritis. The ultrastructural finding in Class V, membranous lupus nephritis is the presence of subepithelial deposits in at least 50% of glomeruli and that the subepithelial deposits are present on at least 50% of the surface of the glomerular capillary. Thus a few scattered small deposits in one or two glomeruli are insufficient for a diagnosis of membranous lupus nephritis. The subepithelial deposits, which are of varying sizes, may be associated with no basement membrane alteration (Stage I), associated with lateral spikes (Stage II) or advanced thickening of the basement membrane with intramembranous deposits of varying electron density (Stage III and IV). Patients with mild membranous lupus nephritis (Stage I and II) have a better prognosis than patients with advanced (Stage III or IV) changes [7]. Membranous lupus nephropathy differs from idiopathic membranous glomerulopathy in that there is a background of mesangial, electron dense, immune complex deposits. Sparse, small subendothelial deposits may also be seen in membranous lupus nephropathy. The light microscopic correlate is the diffuse thickening of the glomerular basement membranes and a background of mesangial hypercellularity. Silver positive spikes or a chain-like thickening of the glomerular basement membrane are seen with the Jone's methenamine silver stain. Class VI, advanced sclerosing lupus nephritis shows significant, glomerulosclerosis that involves more than 90% of the glomeruli. However, even in the sclerosed glomeruli small, mesangial and intramembranous, electron dense deposits can be detected in the sclerosed tuft, the blood vessel wall, interstitium and tubular basement membranes. Tubulointerstitial Lesions A spectrum of vascular and tubulointerstitial lesions are also a component of lupus nephritis (Table 2). Although interstitial inflammation and tubulointerstitial immune complex deposits may accompany any lupus nephritis, Class II through V, the tubulointerstitial changes are more severe and prevalent in Class IV lupus nephritis. Deposits are present in the interstitium and along the tubular basement membranes as well as the capillary basement membranes. Tubulointerstitial deposits do not play a major role in the pathogenesis of the tubulointerstitial lesions and they do not correlate with the severity of the interstitial inflammation [8, 9, 10]. Uncomplicated, non-occlusive, vascular immune complex deposits, which are a specific feature of lupus nephritis, are most common in Class III and Class IV lupus nephritis. Vascular immune complex deposits have also been seen in Class II and Class V lupus nephritis. Massive vascular, immune complex deposits, detectable on light microscopy, has been reported in association with ischemic type collapse of the glomeruli, severe renal dysfunction and death [11]. This type of renal involvement was unresponsive to steroid therapy. Patterned Deposits Glomerular and extraglomerular deposits in lupus nephritis are usually electron dense. Substructural organization of immune complex deposits consists of parallel or semicircular arrays of alternating light and dark lines [5, 6, 12]. These organized, electron dense, "fingerprint" or patterned deposits, which represent crystalized DNA, have been reported in 6 to 10% of cases of lupus nephritis [6]. Similar fingerprint type deposits are seen in patients with cryoglobulinemia. The relationship between the "fingerprint" deposits in lupus nephritis and cryoglobulinemia is controversial [6, 13]. The observation of fingerprint deposits in conjunction with immunotactoid, microtubular deposits has raised the possibility of a relationship between the syndrome of immunotactoid glomerulopathy and lupus nephritis [14]. "Fingerprint" deposits are most commonly present as subendothelial deposits and are typically associated with diffuse proliferative, lupus nephritis. Other Ultrastructural Features Frequent tubuloreticular structures (TRSs) are seen in the glomerular and peritubular capillary endothelial cells. Cylindric confronting cisternae (CCC) are predominantly present in interstitial cells. TRSs and CCCs are associated with clinically active, minimally treated lupus nephritis. Diffuse, active glomerular disease and acute interstitial lupus nephritis are often present. However, TRSs and CCCs, interferon foot prints, are not specific for lupus nephritis. Hematoxyphil bodies are rare and are seen predominantly in necrotizing, proliferative glomerular lesions of lupus nephritis. The ultrastructural feature of the hematoxyphil body consists of a mixture of clumps of chromatin and cytoplasmic material [6]. Foot process effacement is the only ultrastructural finding in a subset of patients with lupus nephritis. These patients develop nephrotic syndrome at the onset of SLE. Initially, this finding was felt to be minimal change glomerulopathy occurring in lupus nephritis. However, the observation that the nephrotic syndrome frequently correlates with the onset of SLE favors the diagnosis of lupus related podocytopathy [15]. Discordant Light and Ultrastructural Findings In some cases of lupus nephritis, the light microscopy features are discordant with the ultrastructural findings. Examples of these non conforming cases are presented below. Rare, small subendothelial or subepithelial deposits may be seen in Class II lupus nephritis. The presence of small subendothelial deposits is worrisome for the possibility of an unsampled focal lupus nephritis (Class III lupus nephritis) or transformation from Class II to Class III or IV lupus nephritis. Large or numerous subendothelial deposits warrant a designation of Class III or Class IV lupus nephritis. Subendothelial deposits may be sparse or absent in Class III or Class IV lupus nephritis. These rare cases represent examples of pauci-immune lupus nephritis [1]. Sparse small subendothelial deposits are seen in Class V, membranous, lupus nephritis. These subendothelial deposits are not accompanied by the glomerular hypercellularity that is characteristic of Class III or Class IV lupus nephritis. Ultrastructural Findings that are Predictive for Lupus Nephritis There are several ultrastructural features, which in the appropriate clinical context suggest a diagnosis of lupus nephritis [6]. Fingerprint deposits, are highly suggestive of lupus nephritis. Tubuloreticular inclusions, immune complexes along tubular basement membranes and interstitial immune complex deposits are also suggestive for lupus nephritis. Conclusion Electron microscopy has a significant role in the classification of lupus nephritis. When EM is integrated with the light microscopic and immunofluorescence features of the renal biopsy, it optimizes the classification of lupus nephritis and provides useful prognostic and therapeutic information. Table 1: Notes 1. In Class III lupus nephritis, indicate the proportion of glomeruli with active and with sclerotic lesions. 2. In Class IV lupus nephritis, indicate the proportion of glomeruli with fibrinoid necrosis and/or cellular crescents. 3. Class V lupus nephritis may occur in combination with Class III or Class IV. In this case, each class of lupus nephritis is reported. Source for Tables 1 and 2: Weening JJ, D'Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, Balow JE, Bruijn JA, Cook T, Ferrario F, Fogo AB, Ginzler EM, Hebert L, Hill G, Hill P, Jennette JC, Kong NC, Lesavre P, Lockshin M, Looi LM, Makino H, Moura LA, Nagata M; International Society of Nephrology Working Group on the Classification of Lupus Nephritis; Renal Pathology Society Working Group on the Classification of Lupus Nephritis. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int. 2004; 65: 521-30. * Modified from 1. Pirani CL, Olesnicky L. Role of electron microscopy in the classification of lupus nephritis. Am J Kidney Dis. 1982 Jul;2(1 Suppl 1):150-63 2. D'Agati V. Systemic Lupus Erythematosus. In: Silva FG, D'Agati VD, Nadasdy T. eds. Renal Biopsy Interpretation. New York; Churchill Livingstone; 1996: 181-220. Table Legend: Electron Microscopy and Immunofluorescence: 0 = absent, + = small or scattered, ++ = large or numerous, +/- = variable, * = intramembranous Light Microscopy: 0 = normal, + = mild proliferation, ++ = moderate or marked proliferation, +/- = variable * Electron dense deposits in Class VI, sclerosing lupus nephritis consist of scattered, small, intramembranous deposits. References: D'Agati V, Jenette JC, Silva FG. Systemic Lupus Erythematosus and other Rheumatologic Diseases. In: D'Agati V, Jenette JC, Silva FG. Atlas of Non Tumor Pathology. Non-neoplastic Kidney Diseases. Washington DC: American Registry of Pathology; 2004: 323-83. 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