C5b–9 and adhesion molecules in human idiopathic membranous nephropathy

Aikaterini A. Papagianni, Efstathios Alexopoulos, Maria Leontsini and Menelaos Papadimitriou

Department of Nephrology and Department of Pathology, Hippokration General Hospital, Thessaloniki, Greece



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Cellular immune responses and C5b–9 seem to play an important role in the pathogenesis and progression of idiopathic membranous nephropathy (IMN). The aim of the study was to investigate the role of C5b–9 and adhesion molecules in the pathogenesis of the disease.

Methods. The clinical and pathological data of 35 patients with biopsy-proven IMN were correlated with immunohistochemical findings using monoclonal antibodies against T lymphocytes, monocytes/macrophages (MM), HLA-DR antigens, C5b–9, and adhesion molecules such as {alpha}3ß1, LFA-1ß, and ICAM-1.

Results. In the glomeruli, C5b–9 deposits showed a significant correlation with the intensity of IgG and C3 deposition. The stage of the disease had a significant negative relationship with the glomerular {alpha}3ß1 expression. In the tubulointerstitium (TIN), the number of HLA-DR(+) cells was highly correlated with the numbers of total T lymphocytes, MM, and LFA-1ß(+) cells, as well as with the percentage of tubules with C5b–9 deposits. The extent of ICAM-1 expression in the TIN was significantly correlated with the numbers of interstitial MM, HLA-DR(+), and LFA-1ß(+) cells, as well as with the extent of tubular C5b–9 deposition. The severity of tubular atrophy and interstitial fibrosis had a relationship with the numbers of total T lymphocytes, MM, HLA-DR(+), and LFA-1ß(+) cells and with the extent of tubular C5b–9 deposition and ICAM-1 expression in the TIN. Serum creatinine (Scr) was highly correlated with the numbers of interstitial total T lymphocytes, MM, HLA-DR(+), and LFA-1ß(+) cells. Moreover, Scr had a significant relationship with the severity of tubular atrophy and interstitial fibrosis, as well as with the extent of tubular C5b–9 deposition and ICAM-1 expression in the TIN. Proteinuria was significantly correlated with the extent of tubular {alpha}3ß1 expression.

Conclusions. In IMN, C5b–9 formation may be secondary to IgG and C3 deposition. Proteinuria may contribute to the TIN damage by altering the expression of {alpha}3ß1 integrins in tubular cells. De novo ICAM-1 and C5b–9 expression within the TIN as well as the activated interstitial cells may be important factors leading to renal damage and renal function impairment.

Keywords: adhesion molecules; C5b–9; interstitial damage; membranous nephropathy



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Idiopathic membranous nephropathy (IMN) is the most common cause of nephrotic syndrome in adults. The disease has been considered to have a rather benign course with about 35% of the patients entering spontaneous remission, yet a significant proportion of cases progress gradually toward renal failure [13]. It is well documented that IMN is immune-mediated and is characterized by the presence of dense immune deposits containing immunoglobulin and usually complement in a subepithelial location adjacent to the glomerular basement membrane proper. Despite extensive investigation, the mechanisms of the initial glomerular injury and of the disease progression remain largely unknown.

During the last few years, several studies have demonstrated that the deposition of immune complexes in the renal tissue results in the activation of many mediator systems including leukocytes, complement, adhesion molecules, products of arachidonic acid metabolism, and a variety of cytokines and growth factors. Expression of C5b–9 and adhesion molecules has previously been reported in patients with IMN [49]. However, studies on the relationship between the expression of these molecules and the clinical or morphological activity of the disease are very limited. The aim of the present study was to investigate the mechanisms, which may be involved in the pathogenesis of the disease, with particular focus in the role of the terminal complement complex C5b–9 as well as in the role of adhesion molecules.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Thirty-five patients (23 male) with IMN were retrospectively studied. None of the patients had a history of disease or exposure to drugs that could induce membranous nephropathy. Serum tests for hepatitis B antigen and hepatitis C antibodies were negative. Serum cryoglobulins and complement components were normal. Immunological tests including detection of rheumatoid factor and anti-DNA antibodies, as well as anti-phospholipid, anti-thyroid, and anti-smooth muscle cell antibodies were negative. In addition, none of the patients had evidence of neoplasia or developed neoplasia during the follow-up. Clinical information was obtained by careful retrospective study of available hospital records.

Renal biopsies
All renal samples had been taken by percutaneous biopsies using Tru-Cut needles (Travenol). Five normal biopsies obtained from kidney donor nephrectomies before transplantation were used as controls. The diagnosis of IMN was based on routine light (LM) and immunofluorescence (IF) microscopy examination. Glomerular stages were classified according to the criteria proposed by Ehrenreich and Churg [10]. Briefly, four evolutionary stages were defined: stage I, subepithelial deposits; stage II, spike formation; stage III, incorporation of spikes; and stage IV, disappearing deposit stage. The following alterations were evaluated semiquantitatively on a scale of 0 (absent),+ (mild), ++ (moderate), and +++ (severe): extent of tubular atrophy and interstitial fibrosis, intensity of IF findings and glomerular C5b–9 deposition, and extent of interstitial {alpha}3ß1 and ICAM-1 expression. The glomerular adhesion molecule {alpha}3ß1 and ICAM-1 expression was evaluated as normal, increased, or decreased in comparison with their expression in normal renal tissue.

Monoclonal antibodies
The monoclonal antibodies employed were specific for all T lymphocytes (M756, CD3) (DAKO), monocytes/macrophages (MM) (M718, CD68) (DAKO), HLA-DR-related antigen (M704, DK22) (DAKO), LFA-1ß, ß chain (M783, CD18) (DAKO), activated C9 (M777, C9 epitope) (DAKO), {alpha}3ß1, {alpha} chain (NCL-CDW49c, CD49) (NOVOCASTRA), and ICAM-1 (NCL-CD54, CD54) (NOVOCASTRA). An indirect immunoperoxidase method was used throughout the study as previously described [11]. Briefly, serial frozen sections (4 µm) were fixed in cold acetone at 4°C for 10 min. Sections were subsequently incubated for 10 min with 25% normal rabbit serum. This was followed by sequential 90 min incubation with the appropriate mouse anti-human monoclonal antibodies at the optimal dilutions. Finally, sections were revealed with diaminobenzidine tetrahydrochloride (0.5 mg/ml in phosphate-buffered saline (PBS)+0.01% H2O2) for 2 min. After extensive washing in PBS, sections were counterstained with Harris haematoxylin, dehydrated, and mounted in Entellan (Merck). An internal negative control was routinely processed.

Quantitative studies
Quantitative studies were performed in the absence of any clinical data. Positive cells were separately counted in the glomeruli and the interstitium.

Glomerular cells
For every section, both the number of intraglomerular positive cells and the number of glomeruli were counted. HLA-DR(+) cells were excluded from the enumeration of glomerular cells because epithelial and endothelial glomerular cells are normally stained by this antibody, from which infiltrating mononuclear cells could not readily be distinguished. Finally, the number of positive cells was expressed as the number of cells per glomerular cross-section.

Interstitial cells and deposits
Interstitial cells were counted using an eye-piece graticule to identify 10 microscopic fields, each 0.058 mm2. Hence, a total area of 0.58 mm2 was counted. The sections were counted in a sequence of adjacent fields and no adjustments were made except to avoid glomeruli or major vessels. Finally, the number of positive cells was expressed as cells per mm2. By using the same method the number of tubules with clearly identifiable C5b–9 deposits or ICAM-1 expression was counted. Finally, the intensity or positivity was expressed as percentage of tubules bearing C5b–9 deposits or expressing ICAM-1.

Clinicopathological correlations
Correlation coefficients were calculated between the numbers of individual types of intraglomerular or interstitial cells and the LM findings as well as the intensity of immune deposition on IF. In addition, the number of each type of cells as well as the extent of deposits were correlated with the degree of glomerular and tubulointerstitial lesions seen on LM and IF and with clinical data of the patients at the time of biopsy, including renal function parameters.

Statistical analysis
Values are expressed as means (±SD, ±SE, or with range). Student's t test for unpaired data, Pearson's, and Spearman's correlation coefficients were used for statistical analysis. Multiple linear regression analyses were also used to study interrelationships between the parameters. In view of the multiple batches of correlations undertaken a Bonferroni correction was formally applied and a resulted P-value <0.05 was accepted as significant. The calculations were performed using StatView v. 4.5 statistical software (Abacus Concepts Inc., Berkeley, CA, USA).



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The mean age of the patients was 50 years (range 23–71 years) and the mean serum creatinine (Scr) at the time of biopsy was 1.3±0.4 mg/dl. Nephrotic syndrome (24-h urine protein excretion of >3.5 g) was observed in 26 patients (74.3%) and microscopic haematuria in 12 patients (34.3%). Renal insufficiency at the time of biopsy (Scr >1.5 mg/dl) was observed in 11 patients (31.4%).

The evaluation of glomerular stages showed: stage I, two patients (5.7%); stage II, 14 patients (40%); stage III, 14 patients (40%); stage IV, five patients (14.3%). A variable degree of tubular atrophy and interstitial fibrosis was seen in 21 (60%) and 18 patients (51.4%) respectively. On IF, granular IgG deposits in the capillary wall were present in all patients and a weaker segmental IgM in 18 patients (51.4%). Granular C3 deposits were seen in 30 patients (85.7%). No immune deposits were found along the tubular basement membrane or within the interstitium. Control biopsies had no detectable immune deposits in the glomeruli or the interstitium.

Analysis of cells and deposits
Adequate frozen renal tissue for the evaluation of total T lymphocytes, MM, HLA-DR(+) cells, and C5b–9 deposits was available in all 35 cases and for the evaluation of LFA-1ß(+) cells in 27 cases. The mean number (±SD) of glomerular T lymphocytes and MM was 0.3±0.4 and 0.5±0.4 per glomerular cross-section, respectively. In addition, in the glomeruli a small number of cells (0.9±0.5 per glomerular cross-section) expressing the integrin LFA-1ß were found. The mean number (±SE) of interstitial T lymphocytes and MM was 147±11 and 192±13/mm2, respectively. In the interstitium a significant number of cells expressing HLA-DR antigens (240±15/mm2) were also observed. The number of interstitial cells expressing LFA-1, was 62±8/mm2 (Table 1 and Figure 1AGo).


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Table 1. Phenotypic analysis of cells infiltrating the glomeruli and TIN

 


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Fig. 1. (a) LFA-1ß expression by cells infiltrating the interstitium and the glomerulus in a patient with IMN (original magnification x100). (b) Prominent C5b–9 expression by a glomerulus in a patient with IMN. Note also the presence of C5b–9 positive tubules (original magnification x100). (c) {alpha}3ß1 integrin expression by the glomerulus and occasional tubules in a patient with IMN (original magnification x25). (d) ICAM-1 expression by glomeruli and occasional tubules in a patient with IMN (original magnification x100).

 
Glomerular C5b–9 deposition was noted in all cases. C5b–9 deposits were mainly distributed along the capillary wall in a similar pattern to the IgG and C3 deposition. Mesangial C5b–9 deposition was noted in eight cases (22.8%). In addition, tubular depositions of C5b–9 were seen mainly along the tubular basement membrane and usually had a granular or linear appearance (Figure 1BGo). Occasionally the tubular epithelial cells were also stained with the anti-C5b–9 antibody. The percentage of tubules with C5b–9 deposits varied from 10 to 88%. In 28 cases (80%), extraglomerular vascular C5b–9 deposition was also detected (Table 2Go). The IF study in control biopsies was negative in all specimens.


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Table 2. C5b–9 deposition in 35 patients with IMN

 

{alpha}3ß1 and ICAM-1 expression
Adequate frozen renal tissue for the evaluation of {alpha}3ß1 and ICAM-1 expression was available in 25 cases. Compared with the controls, the glomerular {alpha}3ß1 was decreased in 15 cases (60%). In addition, in six cases (24%) a segmental loss of the normal {alpha}3ß1 expression was found. Moreover, in 20 patients (80%) a variable degree of {alpha}3ß1 expression by the tubular cells and particularly the distal tubular cells was observed (Table 3 and Figure 1CGo). Control biopsies showed only occasional staining in distal tubular cells. In the normal kidney, ICAM-1 was expressed by the glomerular endothelial cells but not by the podocytes or the tubular cells. Decreased glomerular ICAM-1 expression was observed in 11 cases (44%) and no expression at all in seven cases (28%). Decreased glomerular ICAM-1 expression was noted in 42% of the patients with stage III disease and 50% of the patients with stage IV disease. Twenty patients (80%) showed de novo ICAM-1 expression by a small number of epithelial cells. This expression was restricted in few tubules and in only eight patients (32%) it involved >10% of tubules (Table 3Go and Figure 1DGo).


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Table 3. {alpha}3ß1 and ICAM-1 expression in 25 patients with IMN

 

Pathological correlations
In the glomeruli, C5b–9 deposits showed a significant correlation with the intensity of IgG and C3 deposition (P<0.001 and P<0.02, respectively). The stage of the disease had no relationship with the degree of glomerular infiltration by inflammatory cells and the extent of glomerular C5b–9 deposition but had a negative correlation with glomerular {alpha}3ß1 expression (P<0.001). No significant relationship was found between the extent of glomerular and tubular C5b–9 deposition.

In the tubulointerstitium (TIN), the number of HLA-DR(+) cells was significantly correlated with the number of total T lymphocytes (r=0.69, P<0.001), MM (r=0.76, P<0.001), and LFA-1ß(+) cells (r=0.57, P<0.02) (Figure 2Go).



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Fig. 2. Correlation between the numbers of interstitial HLA-DR(+) and LFA-1ß(+) cells in 27 patients with IMN.

 
The severity of tubular atrophy and interstitial fibrosis showed a positive association with the number of interstitial total T lymphocytes (P<0.001 and P<0.001, respectively), MM (P<0.002 and P<0.002, respectively), HLA-DR(+) (P<0.001 and P<0.01, respectively), and LFA-1ß(+) cells (P<0.01 and P<0.01, respectively).

There was a significant association between the percentage of tubules with C5b–9 deposits and the numbers of interstitial total T lymphocytes (r=0.58, P<0.002), MM (r=0.63, P<0.001), HLA-DR(+) (r=0.58, P<0.002), and LFA-1ß(+) cells (r=0.67, P<0.001) (Figure 3Go). The severity of tubular atrophy and interstitial fibrosis also showed a correlation with the intensity of tubular C5b–9 deposition (P<0.002 and P<0.01, respectively).



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Fig. 3. Correlation between the numbers of interstitial LFA-1ß(+) cells and the extent of C5b–9 deposition in the tubules in 27 patients with IMN.

 
Tubular {alpha}3ß1 expression was not associated with the degree of interstitial infiltration by inflammatory cells or the extent of tubulointerstitial damage.

The extent of de novo ICAM-1 expression in the TIN had a highly significant correlation with the number of interstitial MM, HLA-DR(+), and LFA-1ß(+) cells (P<0.004, P<0.003, and P<0.004, respectively), as well as with the extent of tubular C5b–9 deposition (P<0.002).

The severity of tubular atrophy and interstitial fibrosis showed a positive correlation with the extent of ICAM-1 expression in the TIN (P<0.003 and P<0.001, respectively).

In a multiple linear regression analysis using the number of interstitial LFA-1ß(+) cells as the dependent variable, the correlations with ICAM-1 expression in the TIN and tubular C5b–9 deposition were not significant. In the same analysis where the percentage of tubules with C5b–9 deposits was the dependent variable only the correlations with the number of interstitial T lymphocytes and ICAM-1 expression were significant (P=0.01 and P=0.001, respectively).

Clinical correlations
The severity of proteinuria and the presence or not of nephrotic syndrome was not related with any of the glomerular deposits or the intensity of the expression of different adhesion molecules. However, proteinuria was significantly related with the extent of tubular {alpha}3ß1 expression (P<0.02). Scr at the time of biopsy was significantly correlated with the numbers of interstitial T lymphocytes (r=0.59, P<0.002), MM (r=0.69, P<0.001), HLA-DR(+) (r=0.77, P<0.001), and LFA-1ß(+) cells (r=0.71, P<0.001) (Figure 4Go). Scr also had a positive relationship with the severity of tubular atrophy and interstitial fibrosis (P<0.001 and P<0.001, respectively) as well as with the extent of tubular C5b–9 deposition (r=0.73, P<0.001) (Figure 5Go) and ICAM-1 expression in the TIN (P<0.001). In addition, mean Scr in the eight patients showing ICAM-1 expression in >10% of the tubules was significantly greater compared with the mean Scr of all other patients (1.8±0.4 vs 1.0±0.2 mg/dl) (t=6.33, P<0.0001).



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Fig. 4. Correlation between the numbers of interstitial LFA-1ß(+) cells and Scr at the time of biopsy in 27 patients with IMN.

 


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Fig. 5. Correlation between Scr at the time of biopsy and the extent of tubular C5b–9 deposition in 27 patients with IMN.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Despite extensive research the pathogenesis of IMN is still poorly understood. This study showed that glomerular C5b–9 formation in IMN may be secondary to immune complex deposition but is not related with the severity of proteinuria or the presence of nephrotic syndrome. In addition, the weaker glomerular {alpha}3ß1 and ICAM-1 expression seen in the more advanced stages of the disease are probably related to the progressive damage of normal glomerular elements.

Again this study confirmed the pathogenetic importance of the events occurring within the TIN. The infiltration of TIN by activated mononuclear cells suggests that cell-mediated immune mechanisms may actively participate in the pathogenesis of tubulointerstitial lesions in IMN [11]. This is further supported by the highly significant correlation between the severity of tubular atrophy and interstitial fibrosis with the numbers of all types of interstitial infiltrating cells.

Our results also provide insights into the role of C5b–9 in the pathogenesis of TIN lesions in IMN. Previous studies have shown tubular deposits of C5b–9 in the majority of patients with IMN [4,5]. However, the exact mechanisms of tubular C5b–9 deposition are not known. An antibody-dependent complement deposition should be excluded because in our own study, as in other studies, no tubular immune deposits were found [11,12]. Filtration of plasma C5b–9 is also unlikely as its molecular weight is very large and is not filtered by the glomerulus, even in the presence of nephrotic syndrome [13]. This study has shown that the number of interstitial T lymphocytes, MM, HLA-DR(+), and LFA-1ß(+) cells was highly correlated with the extent of tubular C5b–9 deposition. The importance of the interaction between cells and tubules is further supported by the strong relationship of the de novo ICAM-1 expression by the tubules and the severity of interstitial infiltration as well as with the severity of TIN damage. The above findings suggest that activated interstitial cells may play a decisive part in upregulating local complement synthesis and support previous reports in other forms of glomerular diseases [14,15]. However, these results do not establish a causal relationship. More work is needed to confirm these findings and to extend these studies to a broader range of physiological and pathological functions.

An interesting finding of our study was the association between the ICAM-1 expression in the TIN and the tubular C5b–9 deposition. This relationship has never been previously investigated in any type of glomerular injury and the mechanism linking these two phenomena is not completely understood. It is possible that cytokines, secreted by activated interstitial mononuclear cells, induce both tubular injury with consecutive complement activation and de novo ICAM-1 expression in resident and infiltrating cells [1618]. In addition, it has been shown that ICAM-1 expression enhances the cytotoxicity of immune reactive cells and it is likely to promote tubular injury and consequently local complement activation and C5b–9 formation [19]. Another interesting finding was the relationship of tubular {alpha}3ß1 expression with the degree of proteinuria. Interestingly, a recent study demonstrated that albumin increases {alpha}vß5 integrin synthesis and expression by tubular epithelial cells in culture [20]. Upregulation of the ß5 chain in tubular cells was confirmed in biopsies from proteinuric glomerulonephritis including membranous nephropathy but not in non-proteinuric kidneys [20]. Whether the upregulation of tubular ß1-integrin expression contributes to the tubulointerstitial damage needs further clarification.

The putative link between tubular C5b–9 and chronic TIN damage is an interesting one. Previous reports suggested that complement activation on the renal tubule might contribute to tubular damage [21]. However, the results obtained by the multiple regression analysis showed no correlation between tubular C5b–9 deposition and the degree of TIN damage. This could be partially explained by the fact that in biopsies taken at an early stage of IMN, significant TIN lesions are not a prominent feature. In addition, recent reports have emphasized that in protracted glomerular diseases persistent overproduction of complement by the tubules seems to have a more important influence on the evolution of chronic tubule injury than on the initial stage of the disease [22]. The functional consequences of the above findings seem to be important. Scr was highly correlated with the number of interstitial inflammatory cells as well as with the degree of tubular atrophy and interstitial fibrosis and this is in accordance with previous reports in patients with various forms of glomerulonephritis [11,23,24]. Scr was also found to be related with the extent of tubular C5b–9 and ICAM-1 expression but not with any of the deposits or other parameters studied in the glomeruli.

In conclusion, the role of immune deposits seems to be important in the glomerular formation of C5b–9 in IMN. Within the TIN, the interaction between activated mononuclear cells and tubular cells may play a significant role in the pathogenesis of TIN lesions and renal function impairment at the time of biopsy. Proteinuria may amplify this process by altering integrin expression and cellular behaviour of tubular cells. Further elucidation of the mechanisms by which the activation of those mediator systems perpetuate and amplify renal injury may establish new therapeutic strategies aiming to inhibit disease progression [25].



   Notes
 
Correspondence and offprint requests to: Aikaterini A. Papagianni, MD, Department of Nephrology, Hippokration General Hospital, 50 Papanastasiou Str, 54642 Thessaloniki, Greece. Email: aikpapag{at}otenet.gr Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 26. 4.01
Revision received 4. 8.01.