Deposition of the Membrane Attack Complex in Healthy and Diseased Human Kidneys

The membrane attack complex—also known as C5b-9—is the end-product of the classical, lectin, and alternative complement pathways. It is thought to play an important role in the pathogenesis of various kidney diseases by causing cellular injury and tissue inflammation, resulting in sclerosis and fibrosis. These deleterious effects are, consequently, targeted in the development of novel therapies that inhibit the formation of C5b-9, such as eculizumab. To clarify how C5b-9 contributes to kidney disease and to predict which patients benefit from such therapy, knowledge on deposition of C5b-9 in the kidney is essential. Because immunohistochemical staining of C5b-9 has not been routinely conducted and never been compared across studies, we provide a review of studies on deposition of C5b-9 in healthy and diseased human kidneys. We describe techniques to stain deposits and compare the occurrence of deposits in healthy kidneys and in a wide spectrum of kidney diseases, including hypertensive nephropathy, diabetic nephropathy, membranous nephropathy, IgA nephropathy, lupus nephritis, C3 glomerulopathy, and thrombotic microangiopathies such as the atypical hemolytic uremic syndrome, vasculitis, interstitial nephritis, acute tubular necrosis, kidney tumors, and rejection of kidney transplants. We summarize how these deposits are related with other histological lesions and clinical characteristics. We evaluate the prognostic relevance of these deposits in the light of possible treatment with complement inhibitors.


METHODS
We searched for relevant literature on Medline, PubMed, Web of Science, and Google Scholar, using the search terms "C5b-9", "C5b9", "C5b-C9", "membrane attack complex", or "terminal complement complex" in combination with "kidney" or "renal". We screened reference lists of included studies for additional relevant studies. We included 141 studies on deposition of C5b-9 in in vivo human kidney tissue. We excluded studies on in vitro kidney tissue, studies on animal kidney tissue, and studies that lacked an English abstract. The methodological characteristics of the included studies are summarized in Supplementary Table 1 below. The data reported by the included studies are listed in Supplementary Table 2 below.
We excluded small numbers of cases from our discussion that had unclear or unclassifiable kidney diseases without a definition, such as "angiitis" or "focal hyalinosis and sclerosis", which were predominantly found in older studies. Kidney diseases that were studied only incidentally are listed at the end of Supplementary Table 1 without a discussion in the text.
We followed the classifications as used in the original studies to regard kidney tissue as healthy and to group kidney diseases. We followed the terminologies as used in the original studies to describe the localization, pattern, and intensity of staining. Glomerular staining includes staining in the mesangium and/or along the glomerular capillary wall without further specification. We followed the classification of staining as absent or present as described in the original studies. We identified the antibodies used for staining according to their clone names given in the original studies and regarded them as unknown if a clone name was not given.
We cautiously interpreted studies that used a combination of antibodies against individual components of C5b-9, such as C6 and C9, instead of a selective antibody to stain deposits of C5b-9, because of the limitations discussed in the text.
Our findings derived from the included studies are summarized in Figure 1 and Table 1. For Figure 1, we calculated the proportions of studies that reported staining of deposits of C5b-9 to be either absent or present relative to the total number of these studies, we calculated the medians of the proportions of patients reported in the studies to exhibit staining, and we calculated the medians of the staining intensities reported in the studies, all separately for different localizations of the staining and for different kidney diseases. We omitted studies that did not report the respective data from each calculation. We omitted studies that included only one patient from the calculation of the medians of the proportions of patients reported to exhibit staining. We omitted patients exhibiting no staining from the calculation of the medians of the staining intensities. For comparability, we rescaled staining intensities that were reported on a scale from − to ++++ to the most commonly used scale from − to +++. In Table 1, we listed the histological lesions and clinical characteristics that were reported to correlate with staining of deposits of C5b-9 and are discussed in detail in the text, all separately for different localizations of the staining and for different kidney diseases.
In addition, we drew figures to illustrate how deposition of C5b-9 was related with histological lesions or clinical characteristics in various kidney diseases. We reproduced figures from their original publications or newly drew figures based on individual data reported in the original publications, as indicated in the captions of the figures. For this, we selected all available published figures and data that revealed a possible correlation between staining of C5b-9 and histological lesions or clinical characteristics.
In the text, we describe relevant correlation coefficients and other statistical measures as reported in the original studies. As two exceptions in the section on lupus nephritis, we present an odds ratio of 0.60 that we calculated with logistic regression from individual data reported in the original study and an odds ratio of 0.22 that we inverted for clarity from the ratio reported in the original study. Throughout, we indicate Pearson's correlation coefficient as r and Spearman's correlation coefficient as ρ. We considered p < 0.05 as statistically significant, but took into account that p values might be overestimated in studies that included only small numbers of patients. None a Ages are given as ranges in years. b The method of staining of C5b-9 is specified according to the antibody and immunohistochemical technique. The antibodies are indicated according to their clone names as reported in the original studies or indicated as unknown if a clone name was not given. The antibodies' names correspond with those specified in Table 2. The immunohistochemical techniques are described as direct (dir.) or indirect (indir.), as fixed (fix.), frozen (fr.), or both, and as immunofluorescence (fluor.) or immunoperoxidase (perox.) and indicate the additional use of immunoelectron microscopy (imm.electr.).
(1) SUPPLEMENTARY FIGURE 1 | Deposits of C5b-9 in healthy human kidneys as detected with different antibodies. Pie charts show the proportion of studies that reported staining of C5b-9 as absent (light) or present (dark). Bar charts show the medians of the proportions of patients reported to exhibit staining. Scatter charts show the median staining intensities in these patients. All charts show data separately for staining in the glomerulus as a whole (glom.), in the mesangium (mes.), along the glomerular capillary wall (cap.), along the tubular basement membrane (tub.), or in the extraglomerular vascular wall (vas.). Error bars show the lowest and highest reported values. Numbers of studies are indicated between brackets. Some studies reported only part of the data shown, explaining differences in the numbers of studies between pie, bar, and scatter charts. Nothing is indicated if the data were never reported. Detailed data per study are listed in Supplementary Table 3. Data on antibodies used in only one original study are only listed in Supplementary Table 2. Antibodies' names correspond with those specified in Table 2. Glom. Mes. (1) Cap. Tub. Vas. (1) Glom. (2) Mes. (1) Cap. Tub. (1) Vas. (2) Glom. (2) Mes. (1) Cap. (1) Tub. (1) Vas. Glom. ( Mes. Cap. Tub. Vas. ( 2) Glom.
(1) SUPPLEMENTARY FIGURE 2 | Deposits of C5b-9 in diseased human kidneys as detected with differrent antibodies. Pie charts show the proportion of studies that reported staining of C5b-9 as absent (light) or present (dark). Bar charts show the medians of the proportions of patients reported to exhibit staining. Scatter charts show the median staining intensities in these patients. All charts show data separately for staining in the glomerulus as a whole (glom.), in the mesangium (mes.), along the glomerular capillary wall (cap.), along the tubular basement membrane (tub.), or in the extraglomerular vascular wall (vas.). Error bars show the lowest and highest reported values. Numbers of studies are indicated between brackets. Some studies reported only part of the data shown, explaining differences in the numbers of studies between pie, bar, and scatter charts. Nothing is indicated if the data were never reported. Detailed data per study are listed in Supplementary Table 3. Data on antibodies used in only one original study are only listed in Supplementary Table 2. Antibodies' names correspond with those specified in Table 2. Note that the data reflect various kidney diseases. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub. Vas. Glom. Mes. (2) Cap. (2) Tub. (2) Vas. (1) Glom. (1) Mes. (2) Cap.
SUPPLEMENTARY FIGURE 3 | Deposits of C5b-9 in healthy human kidneys as detected with different staining techniques. Pie charts show the proportion of studies that reported staining of C5b-9 as absent (light) or present (dark). Bar charts show the medians of the proportions of patients reported to exhibit staining. Scatter charts show the median staining intensities in these patients. All charts show data separately for staining in the glomerulus as a whole (glom.), in the mesangium (mes.), along the glomerular capillary wall (cap.), along the tubular basement membrane (tub.), or in the extraglomerular vascular wall (vas.). Error bars show the lowest and highest reported values. Numbers of studies are indicated between brackets. Some studies reported only part of the data shown, explaining differences in the numbers of studies between pie, bar, and scatter charts. Nothing is indicated if the data were never reported. Detailed data per study are listed in Supplementary Table 2. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub.
SUPPLEMENTARY FIGURE 4 | Deposits of C5b-9 in diseased human kidneys as detected with differrent staining techniques. Pie charts show the proportion of studies that reported staining of C5b-9 as absent (light) or present (dark). Bar charts show the medians of the proportions of patients reported to exhibit staining. Scatter charts show the median staining intensities in these patients. All charts show data separately for staining in the glomerulus as a whole (glom.), in the mesangium (mes.), along the glomerular capillary wall (cap.), along the tubular basement membrane (tub.), or in the extraglomerular vascular wall (vas.). Error bars show the lowest and highest reported values. Numbers of studies are indicated between brackets. Some studies reported only part of the data shown, explaining differences in the numbers of studies between pie, bar, and scatter charts. Nothing is indicated if the data were never reported. Detailed data per study are listed in Supplementary Table 2. Note that the data reflect various kidney diseases. Vas. Glom. (1) Mes. (1) Cap. Tub. Vas. Glom. Mes. Cap. Tub. (1) Vas. (1) Vas. Glom. Mes. ( Cap. Tub. (2) Vas. Glom. Mes. Cap. Tub. Vas. Vas.
Tub. Proportion of studies reporting deposits of C5b-9 as absent ( ) vs present ( ) Proportion of patients with deposits of C5b-9 Glom.
(3) SUPPLEMENTARY FIGURE 5 | Deposits of C5b-9 in healthy human kidneys as detected in different tissue sources. Pie charts show the proportion of studies that reported staining of C5b-9 as absent (light) or present (dark). Bar charts show the medians of the proportions of patients reported to exhibit staining. Scatter charts show the median staining intensities in these patients. All charts show data separately for staining in the glomerulus as a whole (glom.), in the mesangium (mes.), along the glomerular capillary wall (cap.), along the tubular basement membrane (tub.), or in the extraglomerular vascular wall (vas.). Error bars show the lowest and highest reported values. Numbers of studies are indicated between brackets. Some studies reported only part of the data shown, explaining differences in the numbers of studies between pie, bar, and scatter charts. Nothing is indicated if the data were never reported. Detailed data per study are listed in Supplementary Tables 2 and 3. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub. Vas. Glom. Mes. Cap. Tub. Vas. Vas.
(2) SUPPLEMENTARY FIGURE 6 | Deposits of C5b-9 in diseased human kidneys as detected in different tissue sources. Pie charts show the proportion of studies that reported staining of C5b-9 as absent (light) or present (dark). Bar charts show the medians of the proportions of patients reported to exhibit stai-ning. Scatter charts show the median staining intensities in these patients. All charts show data separately for staining in the glomerulus as a whole (glom.), in the mesangium (mes.), along the glomerular capillary wall (cap.), along the tubular basement membrane (tub.), or in the extraglomerular vascular wall (vas.). Error bars show the lowest and highest reported values. Numbers of studies are indicated between brackets. Some studies reported only part of the data shown, explaining differences in the numbers of studies between pie, bar, and scatter charts. Nothing is indicated if the data were never reported. Detailed data per study are listed in Supplementary Tables 2 and 3. Note that the data reflect various kidney diseases.