Section 1 -

Pathology and Pathogenesis of Lupus Glomerulonephritis Terence Cook Hammersmith Hospital London, UK

Introduction Systemic lupus erythematosus (SLE) is an auto­immune disease of unknown cause that can occur at almost any age, although it affects mostly women in their 20s. SLE commonly involves the kidney. In unselected patients with lupus, 25-50% will have abnormalities of urine or renal function early in their disease course and 80% of children and 60% of adults will develop clinical renal problems at some stage [1]. Moreover, renal alterations are found in almost 90% of lupus patients at autopsy. The lowest 5-year survival has been reported for patients with central nervous system and renal involvement. Renal biopsy manifestations of lupus include glomerular, tubulointerstitial and vascular changes, but it is the glomerular changes that have been considered to have the most impact on outcome and that have been the basis of classification of biopsy appearances. Here I will focus on lupus glomerulonephritis and discuss firstly the pathogenesis followed by the classification of lupus nephritis with particular reference to the recent ISN/RPS revision of the WHO classification. Pathogenesis It is useful to think about this in several different stages. Firstly there is the generation of the autoimmune response, secondly the deposition of immune deposits in glomeruli and thirdly the inflammatory mediators that lead to the amplification of inflammation in the glomerulus and the consequent structural changes. Genetics Considering first the generation of the autoimmune response, there is clearly an important genetic predisposition [2]. Thus, estimates of concordance rates in monozygotic twins ranges from 25 to 69% while it is only 1-2% in dizygotic twins. 10-12% of patients with SLE have first or second degree relatives with the disease. Genetic studies in mice have identified a number of genetic loci that are implicated in murine models of SLE and suggest that these loci affect three distinct biological pathways controlling respectively: 1. A breach of tolerance to nuclear antigens; 2. Dysregulation of the immune system allowing amplification of the immune response; and 3. Mediators of end organ damage. Whether similar genes are involved in humans is the subject of active investigation. Generation of the autoimmune response SLE is characterised by the presence of a bewildering range of antibodies directed against self-antigens. These may be intracellular antigens including a variety of nucleoproteins, some containing DNA (nucleosomes) and some containing RNA (nuclear and cytoplasmic riboproteins); cell surface components including negatively charged phospholipids (phosphatidylserine); and a few plasma proteins such as C1q and 2-glycoprotein. Central to understanding the pathogenesis of SLE is to understand what is the source of these, particularly as many of them are intracellular antigens, and how B cells that react with these antigens are generated. A major insight into the first question came with the observation that a common link between many of the antigens in SLE is that they are translocated to the cell surface when cells undergo apoptosis [3]. This suggests that apoptotic cells are an important source of antigenic material in SLE. How then do these antigens drive the B cell response? Possible answers to this question have been provided by the study of mice lacking early components of the classical pathway of complement activation. The rationale for generating these mice was the very strong association in humans between genetic deficiencies of C1 and C4 and the presence of SLE (reviewed by Pickering and Walport [4]). In order to investigate this we generated mice with a targeted deletion of C1q [5]. In mouse strains with a predisposition to autoimmunity, lack of C1q markedly enhanced the production of autoantibodies and led to the development of proliferative immune complex glomerulonephritis. Strikingly there were large numbers of apoptotic bodies in the glomeruli of these mice, which was surprising given the speed with which apoptotic cells are usually removed from sites of inflammation. This suggested that the mice had a problem handling apoptotic debris and we hypothesized, therefore, that failure of efficient clearance of dying cells allowed the antigens expressed on their surfaces to be inappropriately accessible to cells of the immune system with the consequent generation of an autoimmune response. Following this, deficiencies of several other proteins that are involved in the clearance of apoptotic cells, or of waste products of cell destruction such as chromatin and DNA, have also been shown to lead to autoimmunity in mice. In keeping with this hypothesis, macrophages from patients with SLE also show impaired phagocytosis of apoptotic cells [6]. The challenge now is to define the mechanisms by which impaired clearance of cellular waste leads to autoantibody synthesis. Glomerulonephritis Glomerulonephritis in SLE is almost always associated with the deposition of immune complexes in glomeruli. The mechanisms by which these form in the glomerulus are still poorly understood although there is some evidence that binding to the glomerular basement membrane may be dependent on the binding of histones because of their charge followed by the binding of antihistone antibodies. The site where the immune complexes deposit is of primary concern in the pathogenesis of the disease because different glomerular localizations of complexes initiate different pathogenetic path­ways. The localization of the complexes is determined by the specificity, affinity, and avidity of the antibodies formed, their class and subclass, and the size and valence of the complexes. In general, when an immune reaction is characterized by the presence of relatively small amounts of stable, intermediate sized complexes formed with high affinity antibodies, it is likely to result in mesangial glomerulo­nephritis. With larger complexes formed by high-avidity antibodies, or with larger numbers of complexes, the capacity of the mesangium to handle macromolecules becomes overloaded, resulting in a subendothelial accumulation of these complexes as well. The subendothelial complexes are able to activate circulating inflammatory mediators, especially the complement system and the Fc receptors of leukocytes. Adhesion molecules are then upregulated and inflammatory cells arrested and activated. This leads to a more severe histopathologic lesion with cell proliferation and, possibly, necrosis. Alternatively, an immune response may lead to the occurrence of small, unstable immune complexes formed by low-avidity or low-affinity antibodies in the presence of antigen excess. These complexes may dissociate, followed by re-association subepithelially and the development of a membranous type of glomerulopathy. Subepithelial complexes can activate complement, but chemotaxis is frustrated by the inability of inflammatory cells to pass the glomerular basement mem­brane. Thus a prolonged, chronic inflammation occurs, even­tually leading to abnormal basement membrane production, increased basement membrane permeability, proteinuria, and nephrotic syndrome. Classification of lupus glomerulonephritis Classification of the renal pathology of lupus patients is based on light microscopic, and immunohistochemical changes with ultrastructural observations providing important supporting data. As discussed above the site of immune complex deposition influences the glomerular response. Because of their clinical consequences, the classifi­cation of lupus nephritis is based on these distinctive patho­genetic pathways with their accompanying morph­ological patterns. Classification systems for lupus glomerulonephritis have evolved considerably over the years; the original 1974 World Health Organisation (WHO) classification [7] was modified in 1982 [8] and again in 1995 [9]. In 2004, a working party set up by the International Society of Nephrology and the Renal Pathology Society (ISN/RPS) proposed a revised classification which aimed to clarify the definitions of glomerular lesions and to standardise the reporting of renal biopsies in lupus nephritis thus providing a basis for further clinicopathological studies [10] (Tables I,II). An adequate tissue specimen and good histological technique are essential for reliable histological classification. The tissue should be processed by a skilled technician, cut at 3 microns and examined at multiple levels. In order to reasonably exclude a focal lesion the biopsy should contain a minimum of 10 glomeruli for light microscopic analysis. The ISN/RPS Consensus Conference recommended that, in addition to specifying the class, the biopsy report should also include a summary of the percentage of glomeruli with severe active lesions (fibrinoid necrosis, crescents) and of glomeruli with other active and chronic lesions. The extent, severity and type of tubulointerstitial (tubular atrophy, interstitial inflammation and fibrosis) and vascular disease should also be documented and graded. It has been generally accepted that use of the lupus nephritis classification facilitates the ease and reliability with which nephrologists and nephropatholo­gists communicate information, and that it has greatly improved standardization and reproducibility of biopsy interpretation. In contrast, the prognostic value of the so-called activity and chronicity indices used by some in lupus nephritis is subject to discussion and the utility of these indices is limited by concerns about irreproducibility. Nevertheless, distinguishing 'active' and 'sclerosing' lesions may help determine prognosis and sensitivity to treatment in both lupus and other glomerulonephritides. In general lesions that are potentially sensitive to treatment and reversible show "activity", characterized by hypercellularity, leukocyte exudation, necrosis/karyorrhexis, cellular crescents, hyalin deposits, and interstitial inflam­matory infiltrate. More permanent lesions less sensitive to treatment are glomerulosclerosis, fibrous crescents, tubular atrophy, and interstitial fibrosis. At the ultrastructural level the presence of subendothelial deposits is generally regarded as unfavorable. The persistence of subendothelial deposits has been associated with the progression of lupus nephritis and the decrease in the amount of subendothelial and mesangial deposits with a lower risk for renal impairment in SLE. Table I. 2003 ISN/RPS Classification of Lupus Glomerulonephritis (J Am Soc Nephrol 2004;15:241-250) Class I Minimal mesangial lupus nephritis (LGN) Normal glomeruli by LM, but mesangial immune deposits by IF Class II Mesangial proliferative lupus nephritis Purely mesangial hypercellularity of any degree or mesangial matrix expansion by LM with mesangial immune deposits May be a few isolated subepithelial or subendothelial deposits visible by IF or EM but not by LM Class III Focal lupus nephritis (involving less than 50% of the total number of glomeruli) Active or inactive focal, segmental or global endo- or extracapillary GN, typically with focal, subendothelial immune deposits, with or without focal or diffuse mesangial alterations - III (A) Purely active lesions: focal proliferative lupus nephritis - III (A/C) Active and chronic lesions: focal proliferative and sclerosing lupus nephritis - III (C) Chronic inactive with glomerular scars: focal sclerosing lupus nephritis Indicate the proportion of glomeruli with active and with sclerotic lesions Indicate the proportion of glomeruli with fibrinoid necrosis and/or cellular crescents Class IV Diffuse lupus nephritis (involving 50% or more of the total number of glomeruli either segmentally or globally) Active or inactive diffuse, segmental or global endo- or extracapillary GN, typically with diffuse subendothelial immune deposits, with or without mesangial alterations . This class is divided into diffuse segmental (IV-S) when >50% of the involved glomeruli have segmental lesions, and diffuse global (IV-G) when >50% of the involved glomeruli have global lesions. Segmental is defined as a lesion that involves less than half of the glomerular tuft - IV (A) Active lesions: diffuse segmental or global proliferative lupus nephritis - IV (A/C) Active and chronic lesions: diffuse segmental or global proliferative and sclerosing lupus nephritis - IV (C) Inactive with glomerular scars: diffuse segmental or global sclerosing lupus nephritis Indicate the proportion of glomeruli with active and with sclerotic lesions Indicate the proportion of glomeruli with fibrinoid necrosis and/or cellular crescents Class V Membranous lupus nephritis Global or segmental subepithelial immune deposits or their morphologic sequelae by LM and by IF or EM with or without mesangial alterations May occur in combination with III or IV in which case both will be diagnosed Class VI Advanced sclerosing lupus nephritis >90% of glomeruli globally sclerosed without residual activity Table II Abbreviated International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification of lupus nephritis Class I Minimal mesangial lupus nephritis Class II Mesangial proliferative lupus nephritis Class III Focal lupus nephritis a Class IV Diffuse segmental (IV-S) or global (IV-G) lupus nephritis a Class V Membranous lupus nephritis b Class VI Advanced sclerosing lupus nephritis Indicate and grade (mild, moderate, severe) tubular atrophy, interstitial inflammation and fibrosis, severity of arteriosclerosis or other vascular lesions. a Indicate whether lesions are active (A), chronic (C) or both (A/C). 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