1Department of Medicine, Division of Nephrology, University of Würzburg, 2Institute of Pathology, Schweinfurt, Germany and 3Immtech International, Inc., Vernon Hills, Illinois, USA
Correspondence and offprint requests to: Susanne B. Schwedler, MD, Department of Medicine, Division of Nephrology, University of Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany. Email: pelleas{at}t-online.de
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Abstract |
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Methods. Kidney biopsies from 20 diabetic patients, six with IgA nephropathy and six controls (absence of disease) were stained using a commercially available anti-CRP antibody (clone 8). We characterized this antibody by ELISA and found that it mainly recognized modified CRP (mCRP), the conformational isoform of CRP that occurs after dissociation of the pentameric isomer.
Results. A specific CRP signal was observed in the cytoplasma of tubules in 17 out of 20 kidney biopsies from diabetic patients, while all glomeruli, vessels and interstitium stained CRP-negative. This signal was absorbed against the mCRP protein suggesting that the detected tissue-based antigen is more closely related to the mCRP conformer than to the native CRP conformer. Almost all patients (eight out of nine) with severe chronic kidney disease [glomerular filtration rate (GFR) <30 ml/min/1.73 m2] strongly stained for the mCRP antigen, whereas only four out of 11 patients with mild and moderate chronic kidney disease (GFR 30 ml/min/1.73 m2) demonstrated a strong CRP signal. Normal renal tissue and most biopsies with IgA nephropathy were mCRP negative. Severity of histologic changes as assessed by histology score and mCRP staining correlated significantly, but no correlation was evident between tubular mCRP staining and serum levels of CRP or proteinuria.
Conclusions. The present group of diabetic patients showed progressive tubular mCRP staining with declining renal function and increasing severity of histological lesions. Further studies in less proteinuric patients should clarify whether tubular mCRP expression constitutes a progression factor. It also needs to be demonstrated whether mCRP accumulates in tubuli to further stimulate interstitial fibrosis or is mandatory for the resolution of the process. Since mCRP staining was independent of proteinuria we suggest that mCRP is locally produced.
Keywords: clone 8; C-reactive protein; diabetic nephropathy; immunohistochemistry inflammation; modified CRP
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Introduction |
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CRP occurs in at least two different conformationally distinct forms, native CRP (nCRP) and modified CRP (mCRP). nCRP is a cyclic disc composed of five identical, non-glycosylated subunits. It is a highly soluble serum protein that shows calcium-dependent affinity for phosphate monoesters, in particular PC. When nCRP is dissociated into subunits, a distinct conformational change occurs, hallmarked by a loss in aqueous solubility and the expression of neoantigenic epitopes [10]. Various monoclonal antibodies have been developed that specifically react with the nCRP and the mCRP proteins [11]. There is increasing evidence that each isomeric form of CRP induces different immunologic and inflammatory responses [12]. The classic acute phase reactant measured in serum is the cyclic pentameric nCRP. As with nCRP, mCRP is also a naturally occurring stable protein, found in the fibrous tissues of normal blood vessel intima [13]. The formation of mCRP from CRP is non-proteolytic and irreversible [14]. Whether mCRP is locally produced or represents degraded serum CRP needs to be clarified although reports have appeared describing extrahepatic synthesis of CRP expressing neoepitopes associated with the mCRP, rather than the nCRP molecules [15].
The aim of the present study was to assess immunohistochemical CRP localization in kidneys of diabetic patients. CRP staining was referred to renal function, severity of histological lesions and proteinuria. We used a commercially available, previously used antibody named clone 8, which is supposed to recognize both mCRP and nCRP [16]. We characterized this antibody again by performing ELISAs carefully constructed to differentiate nCRP antigen from mCRP antigen [10,17] and performed tissue blocking experiments to further elucidate which conformational form of CRP is found in tissues.
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Subjects and methods |
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The clinical charts of the remaining 24 patients were reviewed for the presence of diabetes mellitus (pathological oral glucose tolerance test, glucose values of 200 mg/dl or greater at any time or fasting glucose values 126 mg/dl, or the use of insulin or oral anti-diabetic drugs) as well as type and duration of diabetes mellitus.
Information about age, gender, body mass index, presence of hypertension, medical treatment, reason for hospitalization and kidney biopsy, creatinine, blood urea nitrogen (BUN), albumin, protein, serum CRP, HbA1c and 24 h urinary protein excretion were extracted. Since proteinuria was estimated to be an important parameter for later interpretation of the staining data four patients with missing proteinuria were excluded from the study. The remaining 20 patients were divided into two groups according to estimated GFR based on the MDRD study equation by Levey.
Eleven patients with a GFR <90 and 30 ml/min/1.73 m2 constituted the group with mild and moderate chronic kidney disease (CKD) according to K/DOQI guidelines (high GFR group). The remaining nine patients with a GFR <30 ml/min/1.73 m2 had severe CKD or kidney failure (low GFR group). No patient had a GFR
90 ml/min/1.73 m2 (normal renal function).
Renal biopsies were carried out for the following reasons: in eight cases for high proteinuria despite relatively short duration of diabetes mellitus (<10 years) and/or because diabetic complications (especially retinopathy) were missing, in seven cases for rapid decrease of GFR and in three patients because of symptoms and/or laboratory parameters which were indicative of a vasculitis. In two patients, diabetes mellitus was unknown at the time of the biopsy and renal histology confirmed the diagnosis. Eleven patients were hospitalized for renal biopsy, five patients were hospitalized initially for acute renal failure and four patients were referred from other departments (after intravitreal haemorrhage, arterial bypass surgery, leucocytoclastic vasculitis, drug allergy) where a nephrotic syndrome was first detected.
Two patients had diabetes mellitus type 1 (duration: 3 and 11 years, both in the high GFR group), 12 patients had diabetes mellitus type 2 for which a duration was indicated in the clinical charts [mean duration in the high GFR group (n = 6) 106 ± 63 months, in the low GFR group (n = 6) 147 ± 93 months], in two patients diabetes mellitus type 2 was diagnosed at the time of kidney biopsy, in three patients diabetes mellitus was known but the duration could not be determined and one patient had a diabetes type MODY (duration: 5 years, low GFR group).
In addition, six kidney biopsies with IgA nephropathy were stained. Normal kidney tissue (four kidney biopsies and two tumor-free specimens from nephrectomized kidneys) served as controls.
Immunohistochemistry
Biopsies were stained for CRP using an avidinbiotinperoxidase system (Vectastain ABC Mouse IgG Kit, Vector laboratories, Burlingame, CA) and a monoclonal mouse anti-human CRP-antibody (clone 8, Sigma, Taufkirchen, Germany), which is supposed to recognize nCRP and mCRP according to the product description. Freshly cut paraffin-embedded 4-µm sections were mounted on silane-coated slides, deparaffinized and rehydrated through a graded ethanol series. Slides were incubated in 10% normal horse serum (30 min, room temperature) to block non-specific binding sites followed by an incubation with the clone 8 antibody (1:500 in PBS with 1% horse serum, 30 min, room temperature). Slides were then stepwise covered with biotin-conjugated anti-mouse antibody and avidinbiotinperoxidase reagent (each 30 min, room temperature). Between each incubation step slides were washed with PBS (5 min). Reaction products were visualized by immersion in diaminobenzidine tetrachloride (brown colour deposits). Finally, the slides were counterstained with haematoxylin and mounted. A scoring system (negative, 1+ to 3+) was used by two blinded investigators to quantify CRP expression in glomeruli, vessels, tubuli and interstitium. Each investigator scored the slides twice (with 1 week between first and second evaluation) leading to two means. The final scoring was then obtained by taking the mean of each investigators scoring.
In negative controls anti-CRP clone 8 was replaced by PBS. In addition, specific blocking experiments were performed by pre-incubating clone 8 (30 min, room temperature) with a 10-fold excess of mCRP antigen (43.5 µg mCRP/4.35 µg IgG) prior application to the slide. nCRP, mCRP and recombinant mCRP were prepared as described previously [12].
Characterization of the anti-CRP clone 8
For characterization of the anti-CRP clone 8, specific ELISAs were performed as described earlier [10]. For determination of nCRP reactivity, it is important to note that direct adsorption of nCRP onto plastic surfaces will result in the loss of nCRP antigenicity and the expression of mCRP antigenicity [10,17]. Therefore, to measure CRP antigenicity in solid-phase assays, nCRP must be captured on a specific ligand that is adsorbed onto the plastic surface. In these studies, nCRP was captured on PC-substituted KLH [17]. Briefly, to detect specificity to mCRP, 100 ng mCRP/well was directly adsorbed to the ELISA plate. After blocking and washing, wells were incubated with serial dilutions of anti-CRP clone 8 from 1:2500 to 1:160 000, diluted in a 1% albumin solution. Bound antibody was detected using F(ab'2) anti-mouse IgG-peroxidase reagent and ABTS substrate. Reaction was measured by absorbance at 405 nm. To detect specificity to nCRP, ELISA plates were first coated with 200 ng PC-KLH/well. After blocking and washing, 100 ng nCRP was bound in the presence of 2 mM CaCl2. Similar dilutions of the anti-CRP clone 8 were added as above. All washing and incubation buffers contained 2 mM CaCl2 to keep nCRP bound to PC-KLH. Bound antibody was detected with the same anti-mouse IgG-peroxidase conjugate and ABTS substrate as above.
As shown in Figure 1 the anti-CRP clone 8 was highly specific for mCRP. No signal was detected for nCRP even if >10-fold more nCRP (i.e. 1 µg/well) was captured onto PC-KLH. The binding of nCRP to PC-KLH in this experiment was verified using the specific anti-nCRP monoclonal antibodies 1D6 and 8D8 [11] (data not shown).
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Results |
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Laboratory values of diabetic patients, of patients with IgA nephropathy and four controls are shown in Table 1. Clinical data of two patients who were nephrectomized were not available. Compared with patients in the low GFR group [median GFR 12 (423) ml/min/1.73 m2], patients in the high GFR group [median GFR 45 (3279) ml/min/1.73 m2] had a significantly lower CRP score (mean 1.2 ± 1.0 vs 2.3 ± 1.0, P < 0.05) and lower creatinine [median 1.4 (0.91.8) vs 4.9 (2.610.3) mg/dl, P < 0.001] and BUN levels [median 30 (1536) vs 68 (41127) mg/dl, P < 0.001]. Serum CRP levels were significantly elevated in the low GFR group [median 2.5 (1.08.5) vs 0.6 (0.16.7) mg/dl, P < 0.05]. They showed a variation up to 8-fold in the two groups. Gender distribution and mean values of age, BMI, albumin, total protein, proteinuria and HbA1c did not differ among the two groups. In both groups were two severely nephrotic patients (proteinuria of 25 g/day in the high GFR and 20 g/day in the low GFR group). Interestingly, the CRP scores of these patients were 0.5 and 3, respectively. Albuminuria and alpha 1-microglobulin excretion per day as tubular marker were equal in the high and low GFR groups.
In patients with IgA nephropathy, mCRP antigen was either absent or very weak (mean CRP score 0.3 ± 0.4). The median GFR was 68 (12102) ml/min/1.73 m2. One patient had a normal renal function, three patients a GFR between 30 and 90 ml/min/1.73 m2 while two patients had a GFR <30 ml/min/1.73 m2. While one patient had no proteinuria, the other five patients had a proteinuria >2 g/day.
Patients with normal biopsies had normal creatinine and BUN values and were younger than the other patients [median 20 (1230) years]. No proteinuria was found and they had normal serum CRP levels [median 0.3 (0.10.4) mg/dl].
In diabetic patients significant correlations were found between age and histology score (R = 0.50, P < 0.05), CRP and histology scores (R = 0.62, P < 0.01) as well as serum CRP levels and histology score (R = 0.52, P<0.05). An inverse relation was present between albumin levels and proteinuria (R = 0.57, P = 0.01) as well as GFR and histology (R = 0.71, P < 0.001). There was no correlation between proteinuria and CRP score (Figure 4), or between serum CRP levels and CRP score. Albuminuria and alpha1-microglobulin excretion per day did not correlate with the CRP score either.
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Discussion |
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Tubular staining was specific for mCRP as the reaction of anti-CRP clone 8 could be completely eliminated by pre-incubating the antibody with mCRP antigen. Since we did not see any staining in glomeruli, vessels or interstitium, our findings are in contrast to a recent report by Nakahara et al. [16] where CRP deposition was found along the capillary walls of glomeruli, in peritubular capillaries and small vessels of the interstitium in juvenile kidneys with various proliferative and non-proliferative diseases. Furthermore, these authors found no difference in histological severity between cases with and without CRP immunoreactivity. Of note, both this study and that reported herein by us used the same commercially available antibody (clone 8). The presence of paraffin may mask the expression of the mCRP antigen (personal observation). However, in the Nakahara report tissues were equally processed, namely fixed in 10% buffered formaldehyde and embedded in paraffin. One explanation for the difference in results may be that even though our study population exhibited a broad spectrum of diabetic kidney lesions and clear differences in renal function most of the patients had overt nephropathy with a proteinuria >0.5g/day. Therefore, our conclusions are restricted to patients with advanced diabetic nephropathy. Since all patients entering the study were thought to warrant renal biopsy they represent an atypical selection of diabetic patients.
It is possible that the mCRP antigen is found in kidney tubules by a reabsorption process of CRP removed from circulating blood. Such a possibility may reflect on an interaction between phospholipids and CRP from serum, or from CRP produced by infiltrating cells. Alternatively, CRP could be directly produced by tubular cells. The lack of correlation between intensity of tubular staining and proteinuria suggests that tubular CRP staining is independent of glomerular protein filtration. Since there was also no correlation between serum CRP levels and tubular expression of CRP (i.e. the CRP score), our results suggest that local processes in diabetic kidneys may be independent of the clinically measurable systemic CRP response that is a hallmark of the acute phase, inflammatory response. This conclusion may partly be noticed with caution since one single CRP measurement as done in this study may not reflect the cumulative tubular load of CRP in patients with long standing chronic inflammatory disease like diabetes.
The tissue-associated, non-soluble characteristic of the detected CRP antigen is consistent with our conclusion that it is mCRP rather than nCRP that is expressed. While one report documented CRP could be found in urine (together with alpha2-macroglobulin), these results were observed in patients with acute interstitial rejection of the renal allograft [18]. In normal courses, urinary CRP was not increased. The same group found recently that human renal tubular cells expressed CRP mRNA and protein after stimulation with conditioned medium or IL-6 [19]. Our results would support this finding and add that the CRP locally produced is expressed in its modified form. While nCRP found in the serum is produced predominantly by hepatocytes, extrahepatic CRP production has been reported in lymphocytes [20,21] and macrophages [15], islet cells of the pancreas [22], and epithelial cells of the respiratory tract [23]. Most of the described CRP proteins synthesized in these cells express poor solubility (i.e. the protein is not secreted, is expressed intracellularly, or is bound to a membrane), and express the neo-CRP antigens that are expessed on the mCRP protein. Since granulocytes and macrophages are reported to express CRP receptors [24,25], it is possible that the CRP antigen expressed in tubular cells may be involved in the development of interstitial fibrosis by promoting pro-inflammatory activities, possibly involving the complement system. Such a possibility would be consistent with the study reporting that binding of CRP to cellular membranes enhanced opsonization and phagocytosis of apoptotic cells by macrophages. CRP and the classical complement components acted in concert to promote non-inflammatory clearance of the apoptotic cells [26]. In our study, however, no CRP was found deposited on infiltrating cells. In addition, in the groups with mild diabetic nephropathy mCRP signal was localized at the apical membrane rather than the basal membrane indicating its probable origin from the tubular cell. Whether mCRP staining in the tubules is an indicator of the perpetuation of the disease or rather represents a part of the resolution process cannot be clarified at this point.
Our results show clearly that mCRP and not nCRP accounted for the tubular staining. The distinction between these isoforms of CRP is important in light of the opposite biological effects (pro-inflammatory vs anti-inflammatory) recently delineated for the mCRP and nCRP proteins, respectively [12]. The loss of the pentameric symmetry in CRP was associated with the appearance of novel pro-inflammatory activities in human neutrophils and endothelial cells. mCRP activated neutrophil Erk via the Ras/Raf-1/MEK cascade, leading to up-regulation of CD11b/CD18 expression, promoting neutrophil adhesion to human coronary artery endothelial cells via a CD18-dependent mechanism. It was suggested that endothelial injury might result in exposure of mCRP with activation, attachment and emigration of neutrophils in injured tissues. Alternatively, mCRP may also be formed at sites of injury or infection as part of the acute activation and then resolution of the inflammatory process.
The epitope recognized by anti-CRP clone 8 is of particular relevance to this, and all other studies using this reagent. The supplier described its reactivity against native and denatured-reduced CRP as assessed using ELISA, dot-blot and immunoblotting techniques. Our results show essentially all of the specificity of this reagent is for the mCRP antigen. Equally important in this context of working with CRP is the observation that prolonged storage of purified CRP in the absence of calcium, or in the presence of chelating agents, will cause a spontaneous conversion of nCRP to mCRP [12]. As expression of mCRP from the nCRP does not require proteolysis, SDSPAGE an-alysis would not be sufficient to show the presence of the mCRP antigen in a particular working lot of nCRP.
In summary, tubular mCRP staining increased with declining renal function and increasing severity of histological lesions in patients with advanced diabetic nephropathy. Whether it may serve as a marker of progression deserves further immunohistochemical analysis in patients with early nephropathy. Since mCRP staining was independent of proteinuria and was tightly associated within tubular cytoplasma, we postulate this CRP antigen is locally produced. Further studies are needed to elucidate the origin and function of this isoform of CRP and its relevance to the processes of diabetic kidney lesions.
Conflict of interest statement. L. A. Potempa holds stocks in NextEra Therapeutics, Inc., a developmental stage company that holds patents on recombinant mCRP. The remaining authors declared no conflicts of interest.
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References |
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