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Notes: Summary: Recent in vitro studies have shown the synthesis of interleukin-6 (IL-6) in glomerular mesangial and epithelial cells, and suggested the involvement of IL-6 in mesangial proliferative glomerulonephritis. However, the expression site of IL-6 mRNA in renal tissue of IgA nephropathy (IgAN), the most common chronic mesangial proliferative glomerulonephritis, remains obscure. to localize IL-6 mRNA in renal biopsy specimens of IgAN, we used nonradioactive in situ hybridization (ISH) developed in our laboratory, sensitive in detecting individual cells positive for a specific mRNA. In some sections, periodic acid-Schiff staining was performed after ISH in order to identify the topographical relation between IL-6 mRNA positive cells and glomerular basement membrane and mesangial area. In situ hybridization for IL-6 mRNA and immunohistochemistry for CD3 and CD68, markers for lymphocytes and monocytes, respectively, were also performed on serial sections to examine the contribution of infiltrated mononuclear cells to cells positive for IL-6 mRNA in glomeruli. Glomerular resident cells, including glomerular mesangial and epithelial cells and cells of Bowman's capsule, as well as tubular epithelial cells and infiltrated mononuclear cells expressed IL-6 mRNA. We also compared the localization of IL-6 mRNA and protein and showed different distribution between the gene product and protein. the expression of IL-6 mRNA correlated with the degree of mesangial cell proliferation and tubulointerstitial changes. Our results indicate that IL-6 is synthesized in renal tissues of IgAN and suggest that the increased IL-6 expression may be important in the pathogenesis of IgAN.

Notes: Summary: The relationship between renal expression of intercellular adhesion molecule-1 (ICAM-1), glomerular hypercellularity, renal function and renal tumour necrosis factor-α (TNF-α) expression was examined by immunohistochemistry staining in 64 cases of human glomerulonephritis. Glomerular anti-ICAM-1 antibody staining was increased in most cases of IgA nephropathy and lupus nephritis, but was unchanged compared to normal in membranous nephropathy and minimal change disease, and reduced in glomerular sclerosis. However, when taken together, patients with mild or no glomerular hypercellularity (group A) showed normal ICAM-1 expression, those with moderate to severe hypercellularity (group B) had increased glomerular ICAM-1 expression (P〈0.001), while those with glomerular sclerosis (group C) had reduced glomerular ICAM-1 expression. Patients in groups B and C also showed a significant increase in tubular ICAM-1 expression (P〈0.01) and interstitial infiltration of ICAM-1 + cells (P〈0.001). Indeed, tubular ICAM-1 expression correlated with decreased creatinine clearance (r= -0.352; P〈0.05). In situ hybridization demonstrated that increase in tubular ICAM-1 staining was due to de novo gene expression, rather than absorption of soluble ICAM-1 from the lumen. Focal expression of tumour necrosis factor-α was seen in areas of leucocyte infiltration and strong ICAM-1 expression. Indeed, TNF-α staining correlated with increased renal ICAM-1 expression in both glomerular and tubulointerstitial compartments (r=0.81; P〈0.01). to confirm that TNF-α can directly stimulate renal ICAM-1 expression, TNF-α was shown to transiently increase ICAM-1 mRNA synthesis for 4-8 h and cause a progressive increase in ICAM-1 protein on the surface of cultured human mesangial cells. In summary; (i) increased glomerular ICAM-1 expression was restricted to cases of moderate to severe hypercellularity; (ii) tubular ICAM-1 expression correlated with both creatinine clearance and interstitial infiltration of ICAM-1+ cells; and (iii) TNF-α expression was shown to correlate with the degree of renal ICAM-1 expression, suggesting that local TNF-α plays an important role in the up-regulation of ICAM-1 in human glomerulonephritis.

Notes: Summary: The traditional dogma that macrophages do not proliferate within inflammatory lesions has recently been challenged. We have addressed this issue in a study of experimental Goodpasture's syndrome (rat anti-glomerular basement membrane [GBM] disease). Monocyte and macrophage proliferation was assessed during the initation and evolution of this severe inflammatory disease by expression of the proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation using twocolour immunohistochemistry. This study found that: (i) the initial accumulation of ED1+ macrophages in the kiney and lung seen during the indcution of disease resulted from blood monocyte recruitment; however, large numbers of proliferating macrophages (up to 60% of total macrophages) were present in these tissues during subsequent disease evolution; (ii) macrophage proliferation was restricted to the kidney and lung as demonstrated by the complete lack of PCNA expression and BrdU uptake by circulating monocytes and unchanged levels of resident macrophage proliferation within the spleen and liver; (iii) local macrophage proliferation within inflamed tissues was confined to cells of an ED1+ED2−ED3− phenotype indicating that they were recently arrived monocytres and not resident tissue macrophages; and (iv) proliferating macrophages within inflamed tissues were localized in focal areas of severe tissue damage. In conclusion, this study has demonstrated that local proliferation of recuited monocytes makes a major contribution to macrophage accumulation within inflamed tissues during the evolution of rat anti-GBM disease. Furthermore, local macrophage proliferation may play an important role in the mediation of tissue injury in this disease model.

Notes: Summary: Epidermal growth factor (EGF) is a potent mesangial cell and tubular epithelial cell mitogen. Based upon the novel finding that rat mesangial cells express EGF mRNA and protein in vitro, we investigated whether renal EGF production was involved in mesangial proliferation and concomitant tubular epithelial proliferation in rat anti-Thy-1 mesangial proliferative nephritis. During the period of mesangial proliferation in anti-Thy-1 nephritis (days 4–14) no EGF immunoreactive material was detected within the glomerulus. Epidermal growth factor-receptor (EGF-R) expression, which is strong on podocytes in normal glomeruli, was notably absent from focal areas of proliferating mesangial cells, suggesting that EGF available from the circulation was not involved in mesangial cell proliferation. Concomitant with the transient decline in creatinine clearance on day 8 of disease, there was mild tubular injury and a significant increase in cortical tubular proliferation as assessed by expression of the proliferating cell nuclear antigen (PCNA). Double immunohistochemistry staining found that the increased cortical tubular proliferation on day 8 occurred in EGF− tubules, but not EGF+ tubules. In contrast, there was an increase in proliferation of EGF+ tubules, but not EGF− tubules, on day 28. Renal EGF mRNA and protein expression was down-regulated over days 1-14, with a rebound in expression on day 28 which correlated with proliferation of EGF+ tubules. Tubular EGF-R expression, which is most clearly seen on EGF+ tubules in normal rat kidney, was unchanged over the disease course. the potential role of EGF in tubular proliferation in normal and diseased states is discussed. In summary, this study finds no evidence to implicate EGF in mesangial cell proliferation in rat anti-Thy-1 nephritis, even though mesangial cells can express EGF in vitro, and suggests that EGF may regulate proliferation of tubular epithelial cells in different stages of disease.

Notes: SUMMARY: To evaluate the effect of several cytokines on the total production of insoluble ECM, we performed immunoperoxidase staining directly on rat mesangial cells cultured on flat-bottomed 96-well plates. the peroxidase activity was demonstrated by orthophenilenediamine (OPD) and measured directly as optic density at 490 nm (OD490) by a microplate reader. After this procedure, cell number in each well was determined by crystal violet staining of which intensity was measured at 540 nm (OD540). the amount of ECM measured as OD490 was corrected by OD540 (OD490/OD540). OD540 was linearly correlated with actual cell number in the well and OD490 for each ECM was firmly correlated with cell number in the well. By this method, dose dependent decrease of fibronectin (FN) and laminin (LM) was observed in the presence of rat recombinant interferon gamma (IFNγ). Human recombinant platelet derived growth factor (PDGF) increased the total production of LM and type IV collagen (Col IV) as well as cell number. This method would also be useful in evaluation for other proteins of insoluble form as well as ECM produced by attached form of cultured cells.

Notes: Summary: Understanding the complex mechanisms involved in the induction and progression of glomerulonephritis requires a detailed analysis of the disease at the molecular level. This has become possible with the advent of antibody-based, and in particular, DNA-based techologies. This paper provides an overview of the methods of molecular analysis currently in use in the study of human glomerulonephritis. Although largely restricted to the research laboratories at present, these techniques of molecular analysis are now entering the mainstream of clinical diagnostic testing.

Notes: Summary: It is now widely accepted that tubulointerstitial lesions correlate with renal function in glomerulonephritis and that the severity of such lesions predicts disease progression. Interstitial leucocytic infiltration is a prominent feature of tubulointerstitial lesions which also correlates with renal function and outcome in human glomerulonephritis, while intervention studies in animal disease models have demonstrated a causal role of these cells in progressive tubulointerstitial injury. This paper focuses on two aspects of immune-mediated tubulointerstitial injury. First, the development of interstitial leucocytic infiltration and the relationship between periglomerular leucocytes and Bownman's capsule integrity, and second, the concept that tubular cells are not only passive targets of injury in glomerulonephritis, but that they actively participate in the immune/inflammatory process.

Notes: Summary: A number of studies have demonstrated an important role for macrophages (Mo) in lipid induced glomerular injury; however, little is known of the mechanisms which facilitate Mo infiltration in this disease. the present study examined the expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) during the development of glomerular Mo infiltration in ExHC rats; a strain which is susceptible to lipid induced glomerular injury. Twenty-five male 6 week old ExHC rats were placed on a normal diet supplemented with 3% cholesterol, 0.6% sodium cholate and 15% olive oil (high-cholesterol diet, HCD). Groups of five rats were killed prior to the beginning of the HCD or after 3 days, 1, 2 and 6 weeks on a HCD. A group of five matched ExHC rats on a normal diet served as a control. ExHC rats fed a HCD showed marked hypercholesterolaemia in the absence of any increase in plasma triglyceride levels from day 3 (190 ± 14 vs 42 ± 2 mg/dL in control; mean ± s.e.m., P〈0.01), and developed mild proteinuria (21.9 ± 2.7 vs 5.2 ± 0.5 mg/24 h in control; P〈0.01) and segmental glomerular lesions at week 6. Immunoperoxidase staining identified a significant increase in glomerular ED1+Mo at week 1 (2.0 ± 0.2 vs 1.0 ± 0.1 ED1+Mo/glomerular cross-section in control, P〈0.01) which was further increased at week 6 (6.9 ± 0.4 ED1+Mo/gcs). There was also a significant increase in glomerular cells expressing the adhesion molecule ligands lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4). Coincident with Mo infiltration, there was an increase in the intensity of glomerular ICAM-1 protein expression as shown by antibody staining. In addition, northern blot analysis of cortical RNA and in situ hybridization demonstrated an increase in glomerular ICAM-1 and VCAM-1 mRNA expression from day 3 onwards. In conclusion, these results suggest that both ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions play an important role in Mo recruitment and accumulation during the development of lipid induced glomerular injury.