Phenotype/genotype correlations in the ultrastructure of monogenetic glomerular diseases

Electron microscopy defined classic patterns of hereditary glomerular disease long before genetics revealed an underlying specific mutation. Genetic analysis is now easier to perform in clinical practice but an earlier optimism that genetics would predict disease severity and phenotype is challenged. The classic paradigm is Alport nephritis in which only a subset of mutations may predict glomerular abnormalities and disease severity. Interpretation of ultrastructural pathology of monogenetic diseases like Alport nephritis is complicated when the proband is the first family member to be diagnosed or there is discrepancy between clinical presentation and ultrastructural changes. In this review the authors have selected a dozen cases representative of common monogenetic glomerular diseases as a platform to discuss the utility of diagnostic electron microscopy in the era of molecular genetics. The emphasis is on genotype/glomerular phenotype correlations.     

Lysis of the glomerular basement membrane in children with IgA nephropathy and Henoch-Shönlein nephritis

An electron-microscopic study of the glomerular basement membrane was made on 242 renal biopsies from 222 children with a variety of renal diseases. Lysis of the basement membrane was observed in 16 of the 25 children with Henoch-Sch?nlein nephritis, 36 of the 72 with IgA nephropathy and one with acute poststreptococcal glomerulonephritis. Lysis was frequently associated with subepithelial dense deposits and polymorphonuclear leukocytes, and these are thought to play a role in the lytic process. There was a significant correlation between the presence of lysis of the basement membrane, the degree of proteinuria and the severity of glomerular changes by light microscopy. These findings suggest that the degree of lysis is an indication of the severity of the disease and lysis is associated with progressive disease and a worse prognosis in Henoch-Sch?nlein nephritis and IgA nephropathy.     

Renal tubular basement membrane changes in tubulointerstitial damage in patients with glomerular diseases

Injury to renal tubules and interstitium occur in various glomerular diseases, leading to functional impairment. Tubular basement membrane (TBM) is an important component in maintaining tubular epithelial cell integrity. Because ultrastructural changes in these structures had not been studied in detail, the authors analyzed 30 patients with various types of glomerular diseases, including minimal change disease (MCD), focal segmental glomerular sclerosis, IgA nephropathy, diffuse proliferative glomerulonephritis, membranous nephropathy, and lupus nephritis, by light, electron, and immunofluorescence microscopy. Ultrastructural changes in the TBM were studied and morphometric measurements were performed. The tubular basement membranes showed membranous structures, lucent or lytic areas, and tubular epithelial detachment. There was significant linear correlation between these tubular basement membrane changes and terminal complement complex neoantigens. The interstitial widening was due to banded collagen fibers, with anchoring fibers in the TBM. The various glomerular diseases lead to tubulointerstitial damage via changes in the TBM, leading to renal dysfunction.     

Renal Tubular Basement Membrane Changes in Tubulointerstitial Damage in Patients with Glomerular Diseases

Injury to renal tubules and interstitium occur in various glomerular diseases, leading to functional impairment. Tubular basement membrane (TBM) is an important component in maintaining tubular epithelial cell integrity. Because ultrastructural changes in these structures had not been studied in detail, the authors analyzed 30 patients with various types of glomerular diseases, including minimal change disease (MCD), focal segmental glomerular sclerosis, IgA nephropathy, diffuse proliferative glomerulonephritis, membranous nephropathy, and lupus nephritis, by light, electron, and immunofluorescence microscopy. Ultrastructural changes in the TBM were studied and morphometric measurements were performed. The tubular basement membranes showed membranous structures, lucent or lytic areas, and tubular epithelial detachment. There was significant linear correlation between these tubular basement membrane changes and terminal complement complex neoantigens. The interstitial widening was due to banded collagen fibers, with anchoring fibers in the TBM. The various glomerular diseases lead to tubulointerstitial damage via changes in the TBM, leading to renal dysfunction.     

Heterogeneity of the clinical syndrome in patients with systemic lupus erythematosus and genetic deficiency of the second complement component.

Two patients with systemic lupus erythematosus associated with homozygous deficiency of the second complement component (SLE-C2D) illustrate the different clinical disease patterns found in patients with this illness. Despite the differences in extent and severity of clinical manifestations and serological findings, the renal disease was similar and kidney function was well preserved in both patients. Renal microscopic changes were focal and segmental, deposits of immuno-globulins and complement components were present by immunofluorescent staining, and dense deposits were seen by electron microscopy. Tubulo-reticular inclusion bodies were found in glomerular endothelial cells and lymphocytes of both patients, but not in the lymphocytes of a clinically healthy C2D sibling. The findings in these two patients stress the importance of careful evaluation to determine the presence of systemic disease in patients with SLE-C2D and suggest that an intact classic complement pathway is important in the development of severe lupus, nephritis, but is not needed in the pathogenesis of lupus skin lesions.     

Lupus nephritis: When and how often to biopsy and what does it mean?

Renal disease is a frequent complication of SLE which can lead to significant illness and even death. Today, a baseline renal biopsy is highly recommended for all subjects with evidence of lupus nephritis. Biopsy allows the clinician to recognize and classify different forms of autoimmune lupus glomerulonephritis, and to detect other glomerular diseases with variable pathogenesis which are not directly related to autoimmune reactivity, such as lupus podocytopathy. Moreover, not only glomerular diseases, but other severe forms of renal involvement, such as tubulo-interstitial nephritis or thrombotic microangiopathy may be detected by biopsy in lupus patients. Thus, an accurate definition of the nature and severity of renal involvement is mandatory to assess the possible risk of progression and to establish an appropriate treatment.The indications to repeat biopsy are more controversial. Some physicians recommend protocol biopsies to recognize the possible transformation from one class to another one, or to identify silent progression of renal disease, others feel that good clinical monitoring is sufficient to assess prognosis and to make therapeutic decisions. At any rate, although any decision should always be taken by considering the clinical conditions of the patient, there are no doubts that repeat renal biopsy may represent a useful tool in difficult cases to evaluate the response to therapy, to modulate the intensity of treatment, and to predict the long-term renal outcome both in quiescent lupus and in flares of activity.     

Renal biopsy interpretation in Alport Syndrome

Alport Syndrome is a heritable progressive renal disease that, despite the large number of published studies, because of its genetic, clinical, immunohistochemical, and ultrastructural heterogeneity, still remains a diagnostic challenge. The focus of the discussion is on electron microscopy and immunohistochemistry Col (IV) chains. The differential diagnosis from thin glomerular basement membrane disease is discussed in depth, because both are familial, and can have similar clinical presentation and even ultrastructural pathology, but with a different outcome. The diagnostic role of molecular genetics, which identified the presence of collagen IV gene mutations and its relationship to the phenotypic expression of the renal damage, is also discussed.     

Hereditary nephritis mimicking immune complex-mediated glomerulonephritis

The defining ultrastructural features of hereditary nephritis are "basket weave" lamellation or thinning of glomerular basement membranes. Electron-dense deposits are not seen and immunofluorescence (IF) is generally negative. In this study, we report 5 cases of hereditary nephritis in which substantial amounts of glomerular electron-dense deposits were identified on electron microscopy, with corresponding positive IF staining in 4 cases, suggesting immune complex-mediated glomerulonephritis. However, no case had histological evidence of glomerular endocapillary or extracapillary proliferation or leukocyte infiltration typical of active glomerulonephritis. Four cases were diagnosed at outside institutions simply as forms of glomerulonephritis without considering the possibility of hereditary nephritis and were sent for consultation in contemplation of possible immunosuppressive therapy. All patients had negative serologies and no known underlying infectious or autoimmune disease; 4 patients had family history of hematuria or renal disease. The glomerular electron-dense deposits were predominantly mesangial (4 cases) and intramembranous (4 cases), as well as subepithelial (2 cases) or subendothelial (1 case). Corresponding IF positivity for immune reactants was identified in 4 cases, and IgG was the predominant immunoglobulin deposited. A characteristic feature was the tendency for deposits to form between the complex layers of glomerular basement membrane material, favoring a process of nonspecific entrapment of immune reactants within the thickened, lamellated basement membrane. In all cases, a diagnosis of hereditary nephritis was confirmed by demonstration of the characteristic loss of immunoreactivity for the alpha5 subunit of collagen IV (4 cases) or Goodpasture's antigen (1 case) in renal or epidermal basement membranes. These cases expand the spectrum of unusual pathological findings in hereditary nephritis and emphasize the potential for hereditary nephritis to mimic immune complex glomerulonephritis.     

Desmin-Related Myopathy: Ultrastructural Findings

While murine models of autoimmune (lupus-like) glomerulonephritis have been available for sometime, it is only recently that immune and inflammatory mechanisms and molecular genetics have been extensively investigated. Genes involved in murine and human lupus nephritis have been discovered and provide insight into this disease process and provide avenues for molecular-targeted therapy. Immune modulation of murine nephritis has provided insight into novel therapy that may attenuate this disease or halt disease progression. With the advances in understanding the pathogenesis of lupus nephritis using translational research modalities, including electron microscopy, and molecular genetics, many “designer” therapies have become available for clinical use and for clinical investigational trials. This paper reviews autoimmune (lupus-like) glomerulonephritis in murine models, candidate genes involved in lupus nephritis, adhesion molecules implicated in murine lupus-like nephritis, immune modulation of murine lupus-like nephritis, and novel and potential therapy for immune complex glomerulonephritis.     

Review of autoimmune (lupus-like) glomerulonephritis in murine models

While murine models of autoimmune (lupus-like) glomerulonephritis have been available for sometime, it is only recently that immune and inflammatory mechanisms and molecular genetics have been extensively investigated. Genes involved in murine and human lupus nephritis have been discovered and provide insight into this disease process and provide avenues for molecular-targeted therapy. Immune modulation of murine nephritis has provided insight into novel therapy that may attenuate this disease or halt disease progression. With the advances in understanding the pathogenesis of lupus nephritis using translational research modalities, including electron microscopy, and molecular genetics, many “designer” therapies have become available for clinical use and for clinical investigational trials. This paper reviews autoimmune (lupus-like) glomerulonephritis in murine models, candidate genes involved in lupus nephritis, adhesion molecules implicated in murine lupus-like nephritis, immune modulation of murine lupus-like nephritis, and novel and potential therapy for immune complex glomerulonephritis.     

Ultrastructural Study of Renal Involvement in Two Females with Anderson-Fabry Disease

Anderson-Fabry disease (AFD) is a rare X-linked lipid storage disorder due to a deficient lysosomal α-galactosidase A (α-Gal) activity. In males with the classic form of the disease the enzymatic defect leads to progressive accumulation of glycosphingolipids (GL) in different organs, mainly in the kidney, heart, and brain, causing severe multisystem failure. AFD is usually mild in heterozygous females, but severe cerebrovascular, renal, and cardiac manifestations have been rarely described. The aim of this study is to describe renal involvement of mild symptomatic female carriers by ultrastructural analysis focusing to microvascular lesions, considered to be one of the major causes of systemic disease in AFD. Resin-embedded renal biopsies from 2 sisters with isolated mild proteinuria and belonging to a family group with AFD were observed by light and electron microscopy. In spite of the mild clinical symptoms, diffuse GL storages were demonstrated in all types of glomerular cells and in interstitial endothelial cells. Moreover, platelets were frequently observed in glomerular vassels, a feature coherent with a possible role of prothrombotic state, and platelet activation, in early glomerular lesions.     

Histopathology of Lupus Nephritis

The spectrum of morphologic changes in lupus nephritis, either microscopic, ultrastructural, or immunohistological, closely reflects the great variety of immune complexes that are produced in the course of the disease. Every tissue component of the kidney can be affected, but glomeruli are the target structure in most patients. Several attempts have been made to correlate the clinical severity and the outcome of the nephritis with the pathologic features; the current classification and the six classes that resulted from an international study group are entirely based on glomerular changes. Major criteria of classification include the focal or diffuse involvement of the glomerulus, the site of hypercellularity, the site of immune complex deposition and the presence of active and/or sclerotic lesions. Even if less thoroughly investigated than the glomerulus, the interstitial compartment has revealed many interesting features as are vascular lesions, a common and often underestimated feature. Typing of subpopulation of lymphoid infiltrates supports the emerging evidence indicating that B cells are promoting autoimmunity in mechanisms other than autoAb secretion. Many aspects are still debated and/or poorly understood, such as the interpretation of the so-called “full house nephropathy” that closely mimic lupus nephritis in seronegative patients.     

Ultrastructural study of renal involvement in two females with Anderson-Fabry disease

Anderson-Fabry disease (AFD) is a rare X-linked lipid storage disorder due to a deficient lysosomal a-galactosidase A (a-Gal) activity. In males with the classic form of the disease the enzymatic defect leads to progressive accumulation of glycosphingolipids (GL) in different organs, mainly in the kidney, heart, and brain, causing severe multisystem failure. AFD is usually mild in heterozygous females, but severe cerebrovascular, renal, and cardiac manifestations have been rarely described. The aim of this study is to describe renal involvement of mild symptomatic female carriers by ultrastructural analysis focusing to microvascular lesions, considered to be one of the major causes of systemic disease in AFD. Resin-embedded renal biopsies from 2 sisters with isolated mild proteinuria and belonging to a family group with AFD were observed by light and electron microscopy. In spite of the mild clinical symptoms, diffuse GL storages were demonstrated in all types of glomerular cells and in interstitial endothelial cells. Moreover, platelets were frequently observed in glomerular vassels, a feature coherent with a possible role of prothrombotic state, and platelet activation, in early glomerular lesions.     

Anti-neutrophil cytoplasmic (ANCA) and anti-glomerular basement membrane (GBM) autoantibodies in necrotizing and crescentic glomerulonephritis

Anti-neutrophil cytoplasmic autoantibodies (ANCA) and anti-glomerular basement membrane (GBM) necrotizing and crescentic glomerulonephritis are aggressive and destructive glomerular diseases that are associated with and probably caused by circulating ANCA and anti-GBM antibodies. These necrotizing lesions are manifested by acute nephritis and deteriorating kidney function often accompanied by distinctive clinical features of systemic disease. Prompt diagnosis requires clinical acumen that allows for the prompt institution of therapy aimed at removing circulating autoantibodies and quelling the inflammatory process. Continuing exploration of the etiology and pathogenesis of these aggressive inflammatory diseases have gradually uncovered new paradigms for the cause of and more specific therapy for these particular glomerular disorders and for autoimmune glomerular diseases in general.     

Upregulated expression of cardiac ankyrin-repeated protein in renal podocytes is associated with proteinuria severity in lupus nephritis

Cardiac ankyrin-repeated protein (CARP) was originally identified as a protein specifically expressed in cardiomyocytes, but has recently been found to be upregulated in some muscle diseases including muscular dystrophy and myopathy, suggesting that CARP may be induced in some pathologic conditions. In this study, we immunohistochemically analyzed 69 renal biopsy samples from patients with glomerular diseases and 2 individuals with normal kidney. We found that CARP was expressed in renal podocytes at a high level in 10 of 13 cases of crescentic glomerulonephritis, 7 of 19 cases of diabetic nephropathy, and 12 of 20 cases of lupus nephritis, although it was not expressed in endocapillary glomerulonephritis, minimal change disease, thin basement membrane disease, membranous glomerulonephritis, and normal kidney. Interestingly, in lupus nephritis, CARP expression tended to be induced in cases exhibiting nephrotic syndrome, but less so in cases without nephrotic syndrome, suggesting that CARP expression is correlated with the severity of proteinuria. Furthermore, we found that CARP was not expressed in membranous glomerulonephritis but evidently expressed in most cases of membranous lupus nephritis. Although membranous glomerulonephritis and membranous lupus nephritis are sometimes morphologically indistinguishable, it is suggested that their pathologic mechanisms differ. Therefore, we propose that examination of CARP expression is useful for precise differential diagnosis of membranous glomerulonephritis and membranous lupus nephritis.     

The mitochondrial and kidney disease phenotypes of kd/kd mice under germfree conditions

Interstitial nephritis occurs spontaneously in kd/kd mice, but the mechanisms leading to this disease have not been fully elucidated. The earliest manifestation of a phenotype is the appearance of ultrastructural defects in the mitochondria of mice as young as 42 days of age. To examine the influence of the environment on the phenotype, homozygous B6.kd/kd mice were transferred from specific pathogen-free (SPF) conditions to a germfree (GF) environment, and the development of the disease was observed. The GF state resulted in a highly significant reduction in the frequency of tubulointerstitial nephritis. In addition, GF conditions markedly reduced the appearance of the mitochondrial phenotype, with no sign of mitochondrial abnormalities in GF mice of up to 155 days of age. These results suggest that environmental factors are involved in the progression of all known manifestations of this disease phenotype.     

The pathology of mesangial IgA nephritis with clinical correlation

Of 710 patients in whom renal biopsies with immunofluorescence, light and electron microscopic and clinical data were available, 239 had idiopathic mesangial IgA nephritis. In these 239 cases IgA was found alone in 45.7%, accompanied by IgG in 50.1%, IgM in 21.4%, C3 in 82.4% and fibrin in 37.2%. Serum immunoglobulin levels including IgA were not significantly raised, and complement C3 activation was via the alternative pathway. There was a wide range of glomerular lesions with minor change, minor change with focal and segmental lesions including sclerosis and mesangial cell hypercellularity, diffuse mesangial cell proliferation and, infrequently, diffuse sclerosing glomerulonephritis. The glomerular lesions were related to the stage, duration and severity of the disease. There was also a wide variability of clinical presentations, with asymptomatic and symptomatic microscopic haematuria-proteinuria, macroscopic haematuria, recurrent proteinuria, nephrotic syndrome, acute nephritis, hypertension, and uncommonly as acute renal failure and potassium losing nephritis. No aetiological agent was found, and both the streptococcus and HBsAg could not be identified. The disease(s) was the commonest type of primary glomerulonephritis (33.7%) in Singapore, another geographic area in addition to Japan and France, where this lesion has an apparent high incidence.     

Hepatitis B virus-related nephropathy and lupus nephritis: morphologic similarities of two clinical entities.

We compared the clinicopathologic features of 22 patients with hepatitis B virus-related membranous nephropathy, all with detectable glomerular hepatitis B e antigen, and of 26 patients with lupus nephritis class V. Both groups of patients similarly presented with heavy proteinuria or nephrotic syndrome; however, the patients with hepatitis B virus-related membranous nephropathy, who were predominantly male, did not have the extrarenal manifestations and autoantibodies seen in systemic lupus erythematosus. The glomerular lesions in both clinical entities were similar and at times indistinguishable, demonstrating polyclonal immunoglobulins and polytypic complements in similar subepithelial ultrastructural distribution. No morphologic feature, single or combined, carrying a high positive predictive value for the diagnosis of either nephritis was identified. Lesions such as hematoxyphil bodies and fingerprint dense deposits, distinctive of systemic lupus erythematosus, were rarely found. At the time of biopsy, when systemic lupus erythematosus is not clinically suspected, the diagnosis between hepatitis B virus-related membranous nephropathy and lupus nephritis may be difficult or impossible to differentiate, especially in geographic areas where both lupus nephritis and hepatitis B surface antigen carriers are common. This study focused on the use of specific monoclonal antisera to detect glomerular hepatitis B virus antigens, which contribute to the diagnosis of hepatitis B virus-related nephritis.     

Pathology of glomerular deposition diseases

In routine diagnosis on renal biopsy, one of the confusing fields for pathological diagnoses is the glomerulopathies with fibrillary structure. The primary glomerulopathies with a deposit of ultrastructural fibrillary structure, which are negative for Congo-red stain but positive for immunoglobulins, include fibrillary glomerulonephritis and immunotactoid glomerulopathy. Several paraproteinemias including cryoglobulinemia, monoclonal gammopathy, and light chain deposition disease as well as hematopoietic disorders including plasmacytoma, plasma cell dyscrasia, and B cell lymphoproliferative disorders involve glomerulopathy with an ultrastructural fibrillary structure. A rare glomerulopathy with fibrillary structure that stains negative for Congo-red as well as for immunoglobulins has been also reported. The pathological diagnoses of these glomerulopathies can include either glomerular diseases, or paraproteinemic diseases, or hematopoietic diseases. The terminology is still confusing when glomerular diseases can be combined with paraproteinemic diseases and/or hematopoietic diseases. Therefore, the generic term, 'glomerular deposition disease' (GDD), has been proposed by pathologists with a requirement for clinicians to detect autoantibodies, paraproteins as well as to carry out a bone marrow check. An attempt has been made to rearrange the diseases with related disorders of fibrillary deposits, based on detailed clinical and pathological finding and to elucidate the correlation between GDD, paraproteinemia, and hematopoietic disorder.     

Markers of complement-dependent and complement-independent glomerular visceral epithelial cell injury in vivo. Expression of antiadhesive proteins and cytoskeletal changes.

BACKGROUND: Visceral glomerular epithelial cells (GEC) are an important component of the glomerular filtration barrier to proteins. While ultrastructural GEC changes have frequently been observed in proteinuric states, no suitable light microscopic markers of GEC injury have yet been identified. EXPERIMENTAL DESIGN: We have analyzed in vivo the GEC expression of proteins known to be involved in cell shape changes. SPARC (osteonectin, BM-40) and tenascin (cytotactin, J1, hexabrachion) belong to a group of anti-adhesive glycoproteins, that modulate cell-matrix interactions. We also studied cytoskeletal intermediate filament proteins, including desmin and vimentin. The GEC expression of SPARC, tenascin, desmin, and vimentin was analyzed in various types of GEC injury in the rat, including complement-mediated injury (passive Heymann nephritis, autologous immune complex nephritis, conA anti-conA nephritis), complement-independent injury (nephrotoxic nephritis), toxic injury (aminonucleoside nephrosis) and hypertensive injury (5/6 nephrectomy, angiotensin-II infusion). A complement-mediated model of mesangial cell injury (anti-Thy 1.1 mesangial proliferative nephritis) served as a control. RESULTS: SPARC mRNA and protein were constitutively expressed in normal rat glomeruli. Immunostaining and immunoelectron microscopy primarily localized SPARC to the cytoplasm of GEC. Markedly increased glomerular SPARC synthesis and GEC immunostaining was observed in all instances of complement-mediated GEC injury but in none of the other conditions. In contrast, glomerular immunostaining for tenascin, that also stained in a GEC pattern, either remained unchanged or increased to a minor degree (complement-mediated models). GEC immunostaining for desmin in normal rats was low and variable, and increased significantly in any form of GEC injury but not in anti-Thy 1.1 nephritis. No concomitant increase of GEC immunostaining for vimentin was detectable, which could have been due to the constitutively high expression of vimentin in GEC. CONCLUSIONS: SPARC and desmin, but not tenascin or vimentin, are suitable light microscopic markers of GEC injury. The combined staining for these proteins may be useful in differentiating the mechanisms of GEC injury.