1 Department of Pathology, King Abdulaziz University, Jeddah, Saudi Arabia and 2 Department of Pathology, University of Leicester, Leicester, UK
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Abstract |
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Methods. Frozen renal tissue from 18 cases of TBMD, 18 of mild IgAN and 18 cases with no detectable abnormality were studied: (i) by quantitative in situ zymography, to estimate the activity of glomerular collagenases; and (ii) by quantitative immunoelectron microscopy to estimate the amount of major basement membrane proteins per unit length and per unit area of glomerular basement membrane.
Results. Cases of IgAN showed considerably more glomerular collagenase activity than normal (P=0.001). Thin basement membrane disease showed no difference in collagenase activity. A count of LCA-positive cells in glomeruli confirmed that the IgAN cases did not show glomerular leukocyte infiltration. Conversely, cases of IgAN showed no difference in GBM composition from normal, nor was any difference in GBM thickness detected in this group. However, cases of TBMD showed considerably less laminin (P=0.0008), fibronectin (P=0.002) and type VI collagen (P=0.0005) per unit length of basement membrane. Collagen IV showed a smaller reduction per unit length (P=0.01), but unlike the other protein studies it appeared to be present in higher concentration per unit area (P=0.03), suggesting that it is more compact in TBMD disease.
Conclusions. Two distinct mechanisms of haematuria seem to be involved in these two conditions. In IgAN there is increased activity of enzymes that can degrade GBM, probably reflecting mesangial cell activation. In TBMD an abnormal composition of the thinned GBM is confirmed. When considered with published reports of genetic abnormalities in TBMD, these results raise the possibility of an abnormal interaction between collagen IV and laminin.
Keywords: IgA nephropathy; immunoelectron microscopy; thin basement membrane disease; type IV collagen; zymography
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Introduction |
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In forms of glomerulonephritis with leukocytes infiltrating the glomeruli, the presence of basement membrane defects can be explained readily, as the normal function of inflammatory cells requires them to penetrate vascular basement membranes to achieve exocytosis. They can produce inflammatory mediators, reactive oxygen products [1] and several types of proteinases, such as MMP1, MMP2, MMP3 and MMP9 [24]. They express membrane-type metalloproteinases (MT-MMPs) on their surface [5]. These substances have the ability to damage GBM matrix structure and may be expected to produce haematuria.
However, several forms of glomerular disease that are clinically characterized by haematuria lack significant leukocyte involvementsuch as mild cases of IgA nephropathy (IgAN) and thin basement membrane disease (TBMD), which are the commonest causes of isolated microscopic haematuria in young people [6]. The activity of leukocytes cannot readily explain glomerular injury and haematuria in these conditions. The presence of infrequent GBM defects has been confirmed in IgAN using scanning electron microscopy [7] and recently in TBMD using transmission electron microscopy [8]. However, the pathogenesis of these defects is not understood. In TBMD it is assumed that the molecular composition of the GBM is abnormal, leading to weakness. In some kindreds various abnormalities in the genes for collagen IV have been described [9,10], but it is clear that as with Alport syndrome [11], several different mutations in several different genes can cause the same phenotype. Indeed, there appears to be some genetic overlap between TBMD and Alport syndrome [10], though in TBMD the complete absence of the 35 chains of collagen IV cannot be demonstrated. The possibility that other basement membrane proteins could be involved in some kindreds has not been excluded.
In mild IgAN, without detectable glomerular infiltration by leukocytes, the explanation of haematuria is perhaps even more elusive. All the intrinsic glomerular cells have been shown to be capable of producing type IV collagenases in vitro, under the control of a variety of cytokines, including IL-1, TNF and TGF-ß1 [1215]. However, in histologically mild IgAN the morphological changes are confined to the mesangium and the peripheral glomerular capillary loops appear entirely normal on electron microscopy.
Our initial hypothesis was that, if defects occur in the GBM in the absence of infiltrating leukocytes, either there must be an abnormality of the composition of the GBM, making it abnormally weak, or there must be localized enzymatic dissolution of the GBM from a source other than inflammatory cells. In TBMD the assumption is that the composition is abnormal, but the nature of the abnormality is unknown. In IgAN the occasional observation of an abnormally thin GBM suggests that its composition may be abnormal, but this possibility has not been investigated.
We have used quantitative immunoelectron microscopy to assess changes in the amount of the major structural GBM proteins in these two conditions. We used quantitative in situ zymography to estimate levels of activity of glomerular gelatinase activity, to test the alternative hypothesis that defects might be due to excess degradation. The absence of infiltrating leukocytes was confirmed by immunohistochemistry.
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Subjects and methods |
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Preliminary studies [17] indicated that the reproducibility of the measurements was relatively poor, for both immunoelectron microscopy and in situ zymography, but reproducibility was improved if samples for comparison were processed as a batch rather than sequentially. It was physically impossible to process all the biopsies in one batch, so cases were grouped into triplets. Each triplet contained one case of TBMD, one IgAN and one normal. The cases in each triplet group were closely matched for patient age, gender and the time the biopsies had spent in the liquid nitrogen store (although subsequent analysis failed to demonstrate any detectable effect from these factorsdata not included). Each triplet was handled as a single batch throughout the processing steps, thus, permitting statistical testing of the results from paired samples.
In situ zymography protocol
The principle of in situ zymography has been described elsewhere [18]. In brief, any gelatinase activity in frozen sections will be manifest as digestion of an overlay of autoradiographic emulsion; hence, areas with enzyme activity will be bright under transmission microscopy, against a background of black emulsion. The extent of this digestion over specific structures (such as glomeruli) can be estimated by measurement of the amount of light transmitted through the structures in question. The results are expressed on an arbitrary scale, but this suffices for comparison of enzyme activity between groups. Unlike gel zymography, tissue localization is achieved, but it is impossible to know which enzyme is responsible for the digestion.
Frozen tissue sections (3 µm) were cut on to uncoated clean glass slides. The sections were washed with PBS pH 7.3 for 5 min on a slow shaker, drained and wiped around the sections. Slides were dipped in autoradiographic emulsion (Type NTB-2, Kodak, catalogue no. 1654433), diluted 1:2 in filtered distilled water and melted in a water bath at 40°C. The slides were placed on a cold metal plate to gel for 2030 min. They were incubated in a humidified chamber for 24 h at room temperature to permit digestion of the gelatin. The slides were then developed in Kodak D19 at dilution of 1:1 for 4 min and fixed for 5 min, with washes with distilled water between steps.
Quantitation of in situ zymography results
Images were captured using a Ziess Axiophot light microscope with a Cohu integrating video camera connected to a Power Macintosh computer through a Scion LG-3 framegrabber. The software programme NIH Image (freeware, written by Wayne Rasband) was used for image analysis (Figure 1). A set of NIH Image macros was written to facilitate the analysis and is freely available on request to pnf1@le.ac.uk. All available glomeruli were examined in every case (up to six). After capture of the image of a glomerulus, its outline was delineated on screen by hand and the average intensity of pixels in the selected area was measured. The result was expressed on an arbitrary scale of 0 (black; no gelatinase activity) to 255 (white; maximal gelatinase activity).
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Immunoelectron microscopy
Tissue processing
The tissue remaining after in situ zymography was completed was fixed in freshly prepared 0.5% paraformaldehyde solution in 0.1 M PBS pH 7.3 for 2 h at 4°C and washed in several changes of PBS, then dehydrated in graded methanol (50%, 70% and 90%) at -25°C, for 45 min each step. The samples were infiltrated with LR gold monomer (Ted Pella Inc., no. 18183) at -25°C, following the manufacturer's instructions, and embedded in capped gelatin capsules filled with fresh 100% LR Gold monomer with added initiator. Polymerization was at -20°C under a projector lamp (12 V, 100 W) at a distance of 20 cm from the block for 24 h.
Selection of antibodies for immunoelectron microscopy
We initially studied a wide range of commercially available antibodies against basement membrane components and matrix metalloproteinases. Unfortunately, most proved to be unsuitable. Some did not work with the post-embedding immunoelectron microscopy method, but a common problem was that reproducibility studies [17] showed very variable results which, although morphologically relevant [18], were completely unsuited to quantitation. Pre-embedding methods were not used because of shortage of tissue and variability of results. After these preliminary studies, the only antibodies we considered suitable for quantitative work are listed in Table 1.
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Post-embedding immunogold labelling
Immunolabelling was performed on ultrathin sections using a post-embedding immunogold labelling technique. Different antibodies required some modifications of the protocol, as described in Table 2.
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Quantitation of immunoelectron microscopy
Image capture
The sections were examined using Philips CM100 transmission electron microscope. After screening the whole section to identify the glomeruli available for examination, areas of the peripheral capillary loops were selected at a low magnification of x2500, then photographed at x11 000 subject to the following criteria: (1) peripheral GBM was selected, not areas overlying mesangium; (2) the selected areas should contain GBM excluding any area with an obviously oblique cut; (3) the grid bars were avoided by at least the width of one field; (4) each area thus selected was photographed once without overlapping; and (5) at least 10 micrographs were taken from each sample, from as many glomeruli as were available in each sample (up to six).
The electron micrographs were then printed at the same final magnification. The contrast was also matched as closely as possible for each batch of pictures to facilitate the automated image analysis.
Image analysis
The micrographs were scanned into a Power Macintosh 8100/110 computer using the Apple Color OneScanner and the Ofoto software programme at a constant setting (greyscale scan, 150 dpi).
The images obtained were analysed using the public domain program NIH image for Apple Macintosh. For each image, the area occupied by the basement membrane was delineated manually on screen (Figure 2). A set of NIH-Image macros was written to automate the required image analysis functions; these are freely available on request to pnf1@le.ac.uk. The macros automatically count the number of gold particles (which appear black) in the defined area; they also measure the length and area of this feature. From these measurements the number of particles per unit area of basement membrane and per unit length of basement membrane were calculated, as was the average thickness of the section of basement membrane examined (not corrected for any oblique cutting). Calibration to give the measurements in µm was performed with the aid of a printed micron bar on the electron micrographs.
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Assessment of glomerular leukocytes
To confirm that the cases of IgAN did not contain a glomerular infiltrate of leukocytes, sections were cut from the corresponding formalin fixed, paraffin embedded tissue from each case. Immunolabelling for leukocyte common antigen (CD45, Dako) was performed using a routine indirect immunoperoxidase technique. Positive cells were counted manually in all glomeruli and the average number of positive cells per glomerular cross-section was calculated for each case.
Statistical analysis
Calculations were carried out using the Microsoft Excel analysis toolpack and the Minitab statistical package. The results for in situ zymography and immunoelectron microscopy were expressed as the mean value±95% confidence limit for each study group. Comparison between disease groups and normal was done using the paired t-test after appropriate checks confirmed an approximately normal distribution.
However, normal and TBMD are known to have a continuous range of thickness rather than representing two completely separate entities [19]. The measurements of GBM thickness made in this study were considerably more thorough than those on which the original diagnosis had been made. Repeating the photography and thickness measurement showed the result to be highly reproducible [17]. These measurements were not corrected for oblique sectioning and, therefore, cannot be taken as a true measurement of GBM thickness, but as all study groups had been treated in exactly the same way, the measurements could be used for comparisons between the groups. Consequently, for immunoelectron microscopy a second analysis was carried out, pooling the groups and using linear regression to compare measured thickness of GBM with immunolabelling particle density. In this analysis information was lost by abandoning the separation into triplets, but information was gained by using the actual measured GBM thickness of each case rather than accepting the prior diagnosis and ignoring the variation within each diagnostic group.
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Results |
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The intensity of gold particle labelling of the basement membrane was expressed in two different ways [20]. First, the total number of particles overlying a slice of basement membrane was determined. This was divided by the length of basement membrane examined to give the particles per unit length. When the results were averaged for all measurements on a single case, as overall glomerular size is not altered we take particles per unit length to be a representation of the total amount of the molecule in question in the glomerulus.
We also used the measured area of the basement membrane slices to calculate number of particles per unit area. We assume this to be proportional to the concentration of the protein in question in the basement membrane. We made no attempt to count particles at different levels of the basement membrane.
Thus, if basement membranes are abnormally thin simply because of a decrease in the total amount of the molecule, particles per unit length would be reduced, but particles per unit area would be unchanged. If a molecule is reduced in concentration, particles per unit area would be decreased. If removal of one molecule led thinner membranes to have other molecules present at higher concentrations, one would expect particles per unit area to be increased.
The quantitative immunoelectron microscopy results are summarized in Table 3. There were no significant differences between IgAN and normal for any of the proteins studied.
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This implies that thicker GBMs contain slightly more total collagen, but it is present at lower concentration; in thinner membranes the collagen present appears to be more compact.
In contrast, the mean immunogold label for laminin per GBM area of TBMD cases was slightly lower than the normal group, though the difference did not reach statistical significance (P=0.1). The laminin immunolabelling density per GBM length in the TBMD group was considerably reduced, being approximately half the value recorded for the normal group (P=0.001) (Table 3). Correlation of laminin immunolabelling density with measured GBM thickness confirmed this result, with no significant association when immunolabelling was expressed per GBM area (P=0.9). Correlation of laminin immunolabelling per GBM length with the GBM thickness confirmed a very significant decrease (P=0.0008, r=0.56).
This implies a considerable decrease in the total laminin content of glomeruli in TBM disease, a decrease that is roughly proportionate to the degree of GBM thinning.
The results for fibronectin paralleled those for laminin, with less immunolabelling per GBM length in the TBMD group (P=0.03) and a positive correlation with the thickness of GBM (P=0.002).
The results for collagen VI immunolabelling also paralleled those for laminin. When analysed in the triplet groups a significant difference was not detected, but there was a strong correlation between immunolabelling per GBM length and measured GBM thickness (P=0.00005).
In situ zymography results
The intensity of gelatinolytic activity of glomeruli in the IgAN group was considerably higher than that of the normal group (P=0.001; Table 4) and the TBMD group (P=0.04). The technique of in situ zymography provides relatively limited spatial resolution, but most of the gelatinolytic activity appeared to be over the glomerular mesangium (Figure 1
). Capillary loops, Bowman's capsule and renal tubules showed much lower activity.
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Immunolabelling for leukocyte common antigen
Results of the LCA positive cell count in the normal and IgA nephropathy group are shown in Figure 3. There is no evidence of leukocyte infiltration in the glomeruli of the IgAN cases.
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Discussion |
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In IgAN, despite reports that the basement membranes may be abnormally thin, we found no evidence of any change in the amount or distribution of the major basement membrane components that we were able to study. In these mild cases there was also no evidence of basement membrane thinning. We did, however, find a very significant increase in the level of gelatolytic activity over the glomeruli.
This result produces several questions: which enzymes are causing it, which cells are producing it, and why?
Gelatinase activity is usually assumed to relate to the two enzymes that are mainly responsible for the degradation of type IV collagen, MMP2 and MMP9, although other metalloproteinases may have significant gelatolytic effects [21]. MMP2 and MMP9 are known to be present in normal human glomeruli [18]. Unfortunately, the method of in situ zymography does not permit identification of the enzyme responsible.
These enzymes can be produced by circulating leukocytes and (in vitro) by all the three main intrinsic glomerular cell types. We have demonstrated that glomerular leukocytes were not increased in number over controls in the samples we studied, which suggests that leukocytes are not responsible. The few leukocytes present could be more active. It has been reported that monocytes from the peripheral blood of IgAN patients have higher levels of MMP9 mRNA compared with similar cells from normal individuals [22] and in vitro monocytes can be activated by IgA molecules via cell surface receptors for IgA [23]. It might be possible to investigate this further using more specific markers of leukocyte activation, but we suggest that the absence of any recruitment of leukocytes to the glomeruli makes this mechanism unlikely. The most plausible explanation is that the extracellular deposits of IgA in the mesangium are having an effect on the adjacent mesangial cells. Mesangial cells in vitro bind IgA complexes, either through Fc receptors [24,25] or by other mechanisms [26]. Activated mesangial cells, as seen in experimental IgAN, increase their production of MMPs [2729]. Moreover, the active form of MMP2 has been reported to influence the activity of mesangial cells [30]; specific inhibition of MMP2 activity resulted in a decrease of mesangial cell activation in vitro [31]. It remains a little difficult to explain how cells in the mesangium can digest peripheral glomerular capillary basement membrane. The in situ zymography did suggest maximal enzyme activity to be over the mesangium (Figure 1
), but it does not have the resolution to determine the location of enzyme activity within the glomerulus with complete confidence, so other possibilities should be considered. Endothelial cells abut the mesangium and can produce some MMPs [13]. In some cases of IgAN a few subepithelial deposits are seen, and these could perhaps influence the activity of podocytes [12], but subepithelial deposits are very rarely seen in the milder cases of the disease such as those we studied, and indeed we saw none during the systematic immunoelectron microscopic examination. It might be possible to investigate this further using immunohistochemistry or in situ hybridization, but such methods take no account of the extent of activation of the proenzyme, nor are they subject to reliable quantification. On the evidence available, we suggest that mesangial cell activation is the most likely explanation.
Thin basement membrane disease in contrast showed no change in gelatolytic activity, but changes in basement membrane composition were detected. These implied that the type IV collagen in the thin basement membrane was slightly more compact, but all the other components that we were able to study were present in considerably reduced amounts, at least proportionate to the extent of GMB thinning. The impression from these results is of a basement membrane with a near-normal scaffold of type IV collagen, but a scaffold that has partly collapsed because many of the intervening non-collagenous molecules are missing.
The genetic cause of TBMD is poorly understood, but it is clearly a heterogeneous condition. In some kindreds it is associated with alterations in the gene for the 4 gene of type IV collagen [9]. Other mutations in this gene are known to be responsible for some cases of autosomal recessive Alport syndrome [11]. Thinning of the GBM is an early feature of Alport syndrome, which has led to suggestions that TBMD represents the carrier state or forme fruste of autosomal recessive Alport syndrome. However, TBMD, like Alport syndrome, appears to be genetically heterogeneous. Haematuria in TBMD was found to segregate with known loci for Alport syndrome (collagen IV
3 or
4) in 55% of kindreds in a recent Australian study [32], but no link was found in the other 45%. In some cases of TBMD mutations of
3 and
4 have been effectively excluded [33,34] and the condition is not sex-linked, which effectively excludes the
5 gene. So what is causing the abnormality?
If we follow the example of Alport syndrome, we may suggest that in TBMD a variety of genetic changes may produce a common phenotype by interfering with the molecular auto-assembly of the basement membrane. Since abnormal 3 can produce TBMD in some cases, we should ask what molecules interact with this molecule during assembly? Our results for collagen IV imply that total collagen IV is slightly reduced in TBMD, but that which is present is in a more compact arrangement. This could be explained by a reduction in other basement membrane components with which the collagen IV is normally connectedsuch as laminin. Our finding of particularly low levels of laminin in TBMD hints at the possibility that this interaction between collagen and laminin is being disrupted, perhaps by an abnormality in either molecule. However, in none of our cases was laminin completely absent from the GBM, and other molecules (notably nidogen) are known to be involved in the self-assembly process. It is of course unfortunate that because of inconsistent immunostaining we were unable to employ a wider battery of antibodies against basement membrane components, especially against nidogen, laminin subtypes and the various
chains of type IV collagen.
Our results do not provide an explanation of the mechanism of haematuria in TBMD, beyond the original assumption that the thinner basement membranes are abnormally weak. However, they do suggest thatas in Alport syndromethe underlying genetic abnormality causes a defect in assembly which influences incorporation of several molecules in addition to the one that harbours the genetic defect.
In summary, we have demonstrated increased glomerular gelatolytic activity, probably arising from activated mesangial cells, in mild IgAN but not in TBMD. In TBMD we found type IV collagen in the GBM to be more compact, while the other basement membrane components examined (especially laminin) were reduced, but not absent. Our results indicate different mechanisms of microscopic haematuria in these two conditions and provide suggestions for the direction of future study.
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Acknowledgments |
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Notes |
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References |
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