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Gordana Petrusevska

IMMUNOHISTOCHEMICAL STUDY OF THE INFLAMMATORY INFILTRATE AND THE PROLIFERATIVE INDEX IN THE TUBULOINTERSTITIUM IN HUMAN GLOMERULONEPHRITIDIES. Gordana Petrusevska Institute of pathology, Medical faculty, “University of Sv. Kiril and Metodij”, Skopje, R. Macedonia. INTRODUCTION.

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Gordana Petrusevska

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  1. IMMUNOHISTOCHEMICAL STUDY OF THE INFLAMMATORY INFILTRATE AND THE PROLIFERATIVE INDEX IN THE TUBULOINTERSTITIUM IN HUMAN GLOMERULONEPHRITIDIES Gordana Petrusevska Institute of pathology, Medical faculty, “University of Sv. Kiril and Metodij”, Skopje, R. Macedonia

  2. INTRODUCTION • Renal interstitium provides structural support to the functional renal units, mediates and regulates almost all processes in the renal parenchyma. • Changes in the tubulointerstitial compartment have been found in almost all types of primary glomerular diseases, except for minimal change nephropathy. They include interstitial inflammatory infiltrate, interstitial fibrosis and tubular atrophy. • The accumulation of the inflammatory infiltrate in the interstititum is stimulated by inflammatory cytokines from the damaged glomeruli, tubular epithelial cells or as a response to the deposition or local synthesis of immune complexes.

  3. INTRODUCTION • Lan et al. have shown that an early event after the glomerular damage is the perivascular inflammatory infiltration around the hilar arterioles. This is followed by a spread of the infiltrate through the periglomerular area to the whole cortical interstitium. The early inflammatory infiltrate is composed by T lymphocytes and macrophages. • All progressive renal dieases end in the process of destructive fibrosis in the tubulointerstitial compartment. The pathogenetic mechanisms of interstitial fibrosis in all glomerular diseases are similar and include changes in the tubular epithelium, peritubular capilaries, inflammatory cells, interstitial mesenchymal cells and various immunological and non-immunological diseases.

  4. INTRODUCTION • The process of interstitial fibrosis develops through three phases according to the localization and dominant type of affected cells: • Phase 1 – glomerular disease with active involvement of the resident and non-resident glomerular cells; • Phase 2 – tubular damage resulting in production of variety of cytokines, chemokines and growth factors that promote the renal damage progression; • Phase 3 – activation of the interstitial fibroblasts, resulting in interstitial fibrosis. • Inflammatory infiltrate modulates all three phases through production of various pro-inflammatory and pro-fibrogenic cytokines, growth factors and adhesive molecules.

  5. AIMS • The aims of this study were: • To determine the extent of interstitial fibrosis in renal tissues affected with glomerulonephritic lesions; • To define the composition of interstitial infiltration, and to analayse the intensity and distribution pattern of the inflammatory cells in renal tissues affected with glomerulonephritidies; • To determine a proliferative index with Ki67 in the tubulointerstitial compartment; • To corelate the results from the above mentioned parameters and with the serum creatinine level, as well as with the 24-hour total protein excretion.

  6. METHODS: study population • 50 archival renal biopsies from 35 male and 15 female patients with diagnosed primary glomerulopathy. • The mean age at the time of biopsy was 41,8 (range 15-80) years. • Urine and blood samples were analyzed for serum creatinine level in the serum and 24-hour total protein extraction. • The main demographic and clinical data are shown in Table 1. • Control group - 20 nephrectomised kidneys due to renal carcinoma, from 13 male and 7 female patients, at a mean age 55,2 (range 27-76), with regular renal histomorphology. Table 1.

  7. METHODS: Tissue preparation • Formalin fixed and paraffin embedded renal biopsies; • Tissue sections: H&E, PAS, trichrome Mason and PASM-Jones. • A portion of each tissue sample were fixed in 2.5% phosphate-buffered glutaraldehyde, postfixed in OsO4 and embedded in epoxy resin for semi-thin sections (1m); • Immunoperoxidase staining on paraffin sections for CD20cy CD43 , CD68 , HLA-DRa , LCA and Ki67 (Clone MIB1) was performed with the LSAB staining method (DAKO).

  8. METHODS: Morphometric analysis • Software color extraction of the interstitial fibrous tissue on tissue samples stained with Trichrome Mason was made with the Image Analyzing System Lucia M – Nikon. Images were sequentially taken on 10 consecutive high power fields (HPF), avoiding glomeruli and large vessels. The results were expressed as percentage of the total analyzed area.

  9. METHODS: Morphometric analysis • The morphometric method on sequentially taken images on sections stained with CD20, CD43, CD68 and LCA was also employed for counting the interstitial inflammatory cells, the results of which were expressed as number of cells per HPF, as well as for determination of the proliferative index in the tubulointerstitial compartment on the staining with Ki67, expresses as number of cells per 10 HPF.

  10. METHODS: Statistical analysis • The data were statistically analyzed with commercial statistical software (StatSoft, 2001). The results were expressed as mean values +/-SD. The Mann-Whitney test, Kruskal-Wallis test, chi square, Spearman`s coefficient of correlation (R) and modified Student`s t-test were used. Values of p equal or lower than 0,05 were considered to be statistically significant.

  11. RESULTS: Fibrosis • The extent of interstitial fibrosis was greater than 9% in all but one case from the analyzed group, with a mean value of 18,75%±5,04 (range 8,6%-32%). • In 27 of the analyzed biopsies the extent of the interstitial fibrosis was less than 20%; • In 22 cases it was more than 20%; • Only one case had interstitial fibrosis occupying more than 30% of the analyzed area.

  12. RESULTS: Interstitial inflammatory infiltrate • Inflammatory cells were present in the interstitium in all types of glomerulopathies , while the control group of biopsies contained only rare single interstitial inflammatory cells. • The number of interstitial inflammatory cells evaluated in 45 (90%) cases was significantly higher as compared to the control group (t-test: t=4,23; df=68; p<0,01). Figure 1: Total inflammatory cells in the interstitium in different types of glomerulopathies and in the control group of biopsies (cells/HPF)

  13. RESULTS: Interstitial inflammatory infiltrate • Focal distribution of the inflammatory substrate was present in 78% while it was diffusely dispersed throughout the interstitium in 22% of the cases. .

  14. RESULTS: Interstitial inflammatory infiltrate • Apart from being present in the interstitium, the inflammatory cells were also scattered among the TECs, between the TECs and the TBM as well as inside the delaminated TBM. The majority of these findings were allocated in cases with more intense inflammatory reaction.

  15. RESULTS: Interstitial inflammatory infiltrate • The statistical analyses revealed that T lymphocytes were predominant finding (58,26%)of the interstitial inflammatory infiltrate, followed by macrophages (22,92%) and B cells (18,62%). CD68 CD20 CD3

  16. RESULTS: Interstitial inflammatory infiltrate • High percentage (40,86±24,12) of the interstitial inflammatory cells in the analyzed group were positive for HLA-DRa, whereas the interstitial inflammatory infiltrate of the control group contained only 23,5% of cells positive for HLA-DRa. • We also noticed that TECs from atrophic tubules were positive for HLA-DRa more intensively than those from the control group and spared tubuli (p<0.01).

  17. RESULTS: Proliferative index • A mean number of 6.62±10.58 TECs/10HPF were positive for the proliferative marker in the biopsies of the analyzed group , while the control group of tissue samples had only 0,08±0,83 TECs/10HPF. • A weak proliferative activity was also noticed among the inflammatory cells in the interstitium.

  18. RESULTS: Correlations • There was a strong positive correlation among the various inflammatory cell populations, with values of Spearman’s coefficient of correlation R above 0,85 at the level of significance of p<0,01. • We also confirmed a significant correlation between the extent of interstitial fibrosis at one, and the number of inflammatory cells of various population and total number of inflammatory cells on the other hand. Fig. 6: Correlation between the extent of interstitial fibrosis and the number of interstitial inflammatory cells. (Values of Spearman`s coefficient of correlation were: B cells: R=0,45, p<0,01; T cells: R=0,33, P<0,05; Makrophages: R=0,34, p<0,05; Total inflammatory infiltrate: R=0,35, p<0,05)

  19. RESULTS: Correlations • In addition, there was also significant correlation (p<0,01) between serum creatinine and the number of interstitial inflammatory cells, whereas proteinuria did not correlate with any of the inflammatory cells` population, or the total inflammatory cell count (p>0,05). Fig.7: Correlation between the creatinine level in patients` serum and the number of interstitial inflammatory cells. (Values of Spearman`s coefficient of correlation were: B cells: R=0,47; T cells: R=0,44; Makrophages: R=0,46; Total inflammatory infiltrate: R=0,465)

  20. RESULTS: Correlations • Serum creatinine positively correlated (p<0,01) also with the extent of interstitial fibrosis. Fig.8: Correlation between the concentration of the creatinine in patients` serum and the extent of interstitial fibrosis (Spearman’s coefficient of correlation R=0,54, p<0,01)

  21. RESULTS: Correlations • The proliferative index in the tubular compartment had strong positive correlation (p<0,05) with all subpopulations of inflammatory cells in the interstitium, as well as with the total inflammatory infiltrate, but not with the extent of fibrosis in the interstitium. Fig.9: Correlation between the proliferative index in the tubular compartment and the inflammatory infiltrate (Values for Spearman’s coefficient of correlation R were: B cells: 0,42; T cells: 0,48; Macrophages: 0,41; Total inflammatory infiltrate: 0,45)

  22. CONCLUSIONS • We have confirmed that in all types of primary glomerulopathies there is affection of the renal interstitium with some degree of interstitial fibrosis. • Our observations support the association of the interstitial inflammatory infiltrate with all processes that take part in the tubulointerstitial compartment during glomerular diseases. • Besides being present in the interstitium, the inflammatory cells, T cells, macrophages and B cells may be found among the tubular epithelial cells, between the tubular epithelial cells and the TBM, or within the TBM.

  23. CONCLUSIONS • Being the most prominent population, T lymphocytes modulate the activity of the tubular epithelial cells, the fibroblasts and the other types of inflammatory cells, thus promoting the development of interstitial fibrosis and from the other side may influence the process known as epithelo-mezenchymal transformation (EMT). • Negative corelation between the proliferation and interstitial fibrosis suggests that the process of EMT might preceed to the process of interstitial fibrosis and tubular atrophy CK/SMA CK/VIM

  24. CONCLUSIONS • Being the most prominent population, T lymphocytes modulate the activity of the tubular epithelial cells, the fibroblasts and the other types of inflammatory cells, thus promoting the development of interstitial fibrosis and from the other side may influence the process known as epithelo-mezenchymal transformation. • Positive corelation between the inflammatory cells and interstitial fibrosis as well as with serum creatinine level, imply its influences to the long-term prognosis of the disease. • The inflammatory cells may be used as a predictor of histological risk factors, which needs further investigations. • Also, the role of B cells remains to be completely elucidated in further prospective, long term studies.

  25. Matka lake Ohrid lake As.dr Slavica Kostadinova-Kunovska Prof.dr. Ladislava Grcevska As.dr. Rubens Jovanovic Prof.dr. Vesna Janevska Akad.prof. dr. Momir Polenakovic

  26. DISCUSSION • In addition, they produce various pro-inflammatory and pro-fibrogenic mediators (14, 15). By producing interferon g (IFN-g), they enhance the expression of MHC Class II molecules on the surface of the TECs and the expression of the intercellular adhesive molecule-1 (ICAM-1) (16). Although MHC Class II molecules are characteristic (specific?) for the B lymphocytes, dendritic cells and macrophages, as all being antigen presenting cells; the studies in this field have shown that the TECs also express them. The TECs acquire antigen presenting properties, thus contributing to further recruitment of inflammatory cells in the interstitium (6, 17, 18). In this respect, the results of our study have shown significant positive correlation between the number of T lymphocytes in the interstitial inflammatory infiltrate and the expression of HLA-DRa in the TECs.

  27. DISCUSSION • T cells also produce one of the most important fibrosis promoting cytokines, the transforming growth factor-b (TGF-b) which is a potent stimulator of the production of collagen, fibronectin and proteoglycan by the fibroblasts (15, 19, 20). Its role is realized through the activation of the Smad signaling pathway in the cells, which gives an opportunity for a new research with regard to the therapeutic purposes (7, 21, 22). At the same time, it is strong inhibitor of the matrix degrading enzymes. Apart from that, the T cells also produce interleukin 4 (IL-4) which directly stimulates the activation of fibroblasts. • In our study, the presence of T lymphocytes in the interstitial inflammatory infiltrate correlated positively with the extent of interstitial fibrosis (R=0,33; p<0,05), confirming the previous reports for the role of T cells in the process of interstitial fibrosis. • By synthesising IFN-g and interleukin-2 (IL-2), T lymphocytes stimulate the accumulation of macrophages in the interstitium. Their presence, on the other hand, results with production of various cytokines, such as platelet derived growth factor - PDGF, tumor necrotizing factor alpha - TNF-a, interleukin-1 - IL-1, fibroblast growth factor - FGF as well as the above mentioned TGF-b (23). The target of these mediators are the fibroblasts, which proliferate and produce larger amount of fibronectin, collagen I and III, as well as the TECs (24) which enter the cell cycle and either proliferate or undergo other changes.

  28. DISCUSSION • We noticed enhanced proliferation rate of TECs stimulated by various cytokines and growth factors, mainly produced by T cells and macrophages. Interestingly, the proliferative index in the tubular compartment correlated positively with all inflammatory cell types. • The importance of the presence of B cells in the interstitium in glomerulonephritides has emerged lately. Namely, the attention for this lymphocytic subpopulation has been increased after the meaningful improvement in patients with renal manifestations of autoimmune diseases, one of them being the membranous glomerulonephritis, after the treatment with monoclonal antibodies against CD20 (Rituximab) (25). • In our study, we noticed higher percentage of B cells than described by other authors (4, 10, 14, 15) who analyzed the composition of the interstitial inflammatory infiltrate. Results similar to ours were presented in recent studies (11, 26, 27), some of which indicated probable early accumulation of the B cells in the course of the disease.

  29. DISCUSSION • Little is known about the role of B cells in the process of renal interstitial fibrosis. • The B cells act as antigen presenting cells of tubular proteins, augment the T-cell responses, co-stimulate the macrophages and take part in the neolymphangiogenesis (28, Martin, 2006; 29Kerjaschki, 2004). In this regard are the data from our study showing strong positive correlations between the B cells and other populations of inflammatory cells. • The correlation between the inflammatory infiltrate and the degree of renal damage at the time of the biopsy has been well established and documented (10, 14, 17). Our findings of positive correlation between each of the analyzed subpopulation of inflammatory cells at one, and the concentration of creatinine in the serum of the patients on the other hand support these statements and confirm the importance of the interstitial inflammatory infiltrate in the course of the renal disease, which is a target for therapeutic interventions in order to slow the progression of the disease.

  30. DISCUSSION • Destructive fibrosis in the tubulointerstitial compartment is considered to be the common final consequence of all progressive renal diseases (6, 7) which initiates vicious circle and worsens the renal function. 98% of our analyzed cases had interstitial fibrosis with extent of more than 9%, which corresponds to other studies (2, 3, 4). Besides being correlated to the inflammatory infiltrate, the interstitial fibrosis is strongly correlated to the renal function, which is also supported by the results from our study. Indeed, a strong correlation was found between the extent of interstitial fibrosis and the concentration of the serum creatinine. In contrast, there was no significant correlation with the proteinuria.

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