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BASIC SCIENCE |
*Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Munich, Germany; Department of Immunology, Berlex Biosciences, Richmond, California; Department of Pathology, University of Washington, Seattle, Washington; and Department of Pathology, Hospital Ramon y Cajal, Universidad de Alcala, Madrid, Spain
Correspondence to Dr. H.-J. Anders, Medizinische Poliklinik der LMU, Pettenkoferstrasse 8a, 80336 Munich, Germany. Phone: ++49-89-5996846; Fax: ++49-89-5996860; E-mail: hjanders{at}med.uni-muenchen.de
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Abstract |
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. BX471 did not affect serum DNA autoantibodies, proteinuria, or markers of glomerular injury in MRLlpr/lpr mice. This is the first evidence that, in advanced chronic renal injury, blockade of CCR1 can halt disease progression and improve renal function by selective inhibition of interstitial leukocyte recruitment and fibrosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The leukocytic cell infiltrate is triggered by locally secreted chemokines (3). In vitro studies suggest a role for CCR1 in leukocyte adhesion and transendothelial migration (4), which may explain the beneficial effects of CCR1 antagonists in certain disease models, including pulmonary fibrosis (5) as well as in heart and renal transplant rejection (6,7). Using the model of unilateral ureteral obstruction in mice, we showed recently that blockade of CCR1 with the nonpeptide antagonist BX471 reduced leukocyte infiltration even when treatment was started at a time when renal fibrosis was already present (8). These data indicate that CCR1 blockade is a potential target for therapeutic intervention of progressive renal fibrosis. As chemokines are also involved in systemic immune responses (3), data from the unilateral ureteral obstruction model may not apply to renal manifestations of systemic autoimmunity, e.g., lupus nephritis. In fact, lack of CCR1 has been reported to modulate the course of nephrotoxic serum nephritis in mice in association with a Th1-like immune response (9). We therefore studied the effects of therapeutic CCR1 blockade in progressive renal injury of lupus-like nephritis in MRLlpr/lpr mice, an autoimmune disease that leads to progressive immune complex glomerulonephritis with tubulointerstitial disease resulting in end-stage renal failure that resembles human lupus nephritis. We recently characterized the expression of chemokines and chemokine receptors during the course of this model and found that, among other chemokine receptors, CCR1 and its chemokine ligand CCL3 are expressed in kidneys of MRLlpr/lpr mice (10). We hypothesized that the CCR1 antagonist BX471 might improve renal outcome during the progressive phase of disease by inhibiting renal leukocyte recruitment. This hypothesis proved to be correct for the interstitial compartment but not for the glomerular leukocyte recruitment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evaluation of Glomerulonephritis
Blood samples were collected from each animal at the end of the study by bleeding from the retro-orbital venous plexus under general anesthesia with inhaled ether. After centrifugation, all serum samples were stored at –80°C until analysis. Spot urine samples were collected from each animal at the end of the study for determination of proteinuria. The following parameters were determined using standard analytical protocols as described previously (10): Bradford assay for urine protein concentration, urease/glutamate dehydrogenase method for blood urea nitrogen (BUN) measurements (Merck Diagnostika), IgG ELISA for analysis of DNA autoantibodies using the following antibodies for detection: IgG1 (Pharmingen, Hamburg, Germany; 1:100) and IgG2a (Dianova, Hamburg, Germany; 1:100). The left kidney from each mouse was fixed in 4% buffered formalin, processed, and embedded in paraffin. Sections for Silver and hematoxylin-eosin stains were prepared as described (8,12). The severity of the renal lesions was graded using the indices for activity and chronicity as described for human lupus nephritis (13) and previously used for this murine model (10). All morphologic evaluations were performed by a renal pathologist who was unaware of the source of the tissue.
Immunohistology
Paraffin-embedded sections were prepared as described (12). As primary antibodies, a rat anti-ERHR-3 (1:50, monocytes/macrophages; DPC Biermann, Bad Nauheim, Germany), a rat anti-CD3 (1:100, T lymphocytes, clone CD3-12; Serotec, Raleigh, NC), a mouse anti-smooth muscle actin (SMA; 1:100, myofibroblasts, clone 1A4; Dako, Carpinteria, CA), an anti-collagen I (LF-67, 1:50; provided by Dr. L.W. Fischer, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD), an anti-CCL5 (1:50; Peprotech, Rocky Hill, NJ), a rabbit anti-CCL2 (1:20, rabbit antiserum, prepared as described (12)), anti-Ki-67 (1:25, cell proliferation; Dianova), anti-ssDNA (1:50, apoptotic cells; Chemicon, Hofheim, Germany) were applied. Staining for immunoglobulins was performed on acetone-fixed frozen section using anti-IgG1 (rabbit, 1:50; Dianova) and anti-IgG2a (rabbit, 1:100; Dianova) as detection antibodies. For quantitative analysis, glomerular cells were counted in 10 cortical glomeruli per section and interstitial cells in 10 high-power fields per section selected by uniform random sampling, from each animal. For the assessment of glomerular Ig and complement deposits, 15 cortical glomeruli were analyzed from each section. Glomerular signals were scored using a semiquantitative index as follows: 0 = no signal, 1 = low signal, 2 = moderate signal, and 3 = strong signal intensity. For the quantification of interstitial collagen I immunostaining, digital pictures of 10 random high-power fields were taken using a digital camera (DC 300F; Leica Microsystems, Cambridge, UK). The area of positive staining for collagen I was measured and expressed as percentage using image analysis software (Leica Imaging Solutions, Cambridge, UK).
In Situ Hybridization
In situ hybridization for murine TGF-1 was performed as described previously (14). The TGF-1 probe was a gift from H.L. Moses (Department of Cell Biology, Vanderbilt University, Nashville, TN). Negative controls included hybridization performed on replicate tissue sections using the sense riboprobe.
RNA Preparation and RNase Protection Assay
Renal tissue from each mouse was snap-frozen in liquid nitrogen and stored at –80°C. From each animal, total renal RNA was prepared as described (12). Multiprobe template sets (mouse CC chemokines; Pharmingen, San Diego, CA) and 20 µg of total kidney RNA were used to perform RNase protection assays as described (12). Efficacy of RNase digestion was ensured by a yeast t-RNA sample in each assay. Gels were dried and exposed on phosphor screens of a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Bands were quantified using the ImageQuant software (Molecular Dynamics).
Real-Time Quantitative (TaqMan) Reverse Transcription-PCR
Reverse transcription from total renal RNA was performed as described (12). Real time reverse transcription-PCR (RT-PCR) was performed on a TaqMan ABI 7700 Sequence Detection System (PE Biosystems, Weiterstadt, Germany) using a heat-activated TaqDNA polymerase (Amplitaq Gold; PE Biosystems) as described previously (11). Controls that were composed of ddH2O were negative for target and housekeeper genes. Primers and probes were from PE Biosystems. Oligonucleotide primer (300 nM) and probes (100 nM) were used as described: murine GAPDH (8); murine collagen I (8); murine CCR1: forward 5'-TTAGCTTCCATGCCTGCCTTATA-3', reverse 5'-TCCACTGCTTCAGGCTCTTGT-3'; internal fluorescence labeled probe (FAM): 5'-ACTCACCGTACCTGTA-GCCCTCAT-TTCCC-3'; murine CCR2: forward 5'-CCTTGG-GAATGAGTAACTGTGTGA-3', reverse 5'-ACAAAGGCATAAATGACAGGATTAATG-3'; FAM: 5'-TGACAAGCACTTAGAC-CAGGCCATGCA-3'; CCR5: forward 5'-CAAGACAATCCTGATCGTGCAA-3', reverse 5'-TCCTACTCCCAAGCTGCATAGAA-3'; FAM: 5'-TCTATACCCGATCCACAGGAG-AACATGAAGTTT-3'; murine TGF-1: forward 5'-CACAGT- ACAGCAAGGTCCTTGC-3', reverse 5'-AGTAGACGAT-GGGCA- GTGGCT-3'; fluorescence labeled probe (FAM): 5'-GCTTCGGCGTCACCGTGCT-3'; murine GAPDH: forward 5'-CATGGCCTTCCGT-GTTCCTA-3', reverse 5'-ATGCCTGCTTCACCACCTTCT-3'; internal fluorescence labeled probe (VIC): 5'-CCCAATGTGTCCGTCGT- GGATCTGA-3'.
CCR1 Expression of Leukocytes and Intrinsic Renal Cells
For assessing CCR1 mRNA expression in macrophages, T cells or intrinsic renal cells were prepared from MRLlpr/lpr mice as follows: Macrophages and T cells were isolated from spleens by immunomagnetic selection as described (15). Tubular segments were microdissected from RNase inhibitor-treated tissue in ice-cold PBS, as described previously for human renal biopsies (16). For isolation of primary renal fibroblasts, small pieces of renal tissue were incubated in DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS (Invitrogen), penicillin, and streptomycin for 16 d. Adherent cells were obtained by treatment with 1.5 mM EDTA (Calbiochem-Novabiochem, San Diego, CA) and were depleted of leukocytes by immunomagnetic selection using FITC anti-mCD45 (Pharmingen) and anti-FITC MicroBeads as described (Miltenyi Biotec, Bergisch Gladbach, Germany). mRNA of isolated cells was prepared by standard methods (8). Baseline CCR1 mRNA expression was determined by real-time RT-PCR as above.
In Vivo Assay of Renal T Cell Infiltration
ERHR-3–positive macrophages and CD8 T cells were prepared from spleens of MRLlpr/lpr mice by a previously described isolation and labeling method (15). In brief, spleen cells were isolated by immunomagnetic selection using anti-CD8 (Ly-2) and anti-ERHR-3 MicroBeads (Miltenyi Biotec). Purity of isolated cells was verified by flow cytometry. Separated cells were labeled with PKH26 (Red Fluorescence Cell Linker Kit; Sigma-Aldrich Chemicals, Steinheim, Germany), and labeling efficacy was assessed by flow cytometry. Viability as assessed by trypan blue exclusion was >90%. Twenty-week-old MRLlpr/lpr mice received an injection of either 3.5 x 105 CD8-positive T cells or ERHR-3–positive macrophages in 200 µl of isotonic saline through tail vein. Two groups of mice received an injection of either labeled T cells or macrophages that were preincubated with 600 µM of the CCR1 antagonist BX471 for 30 min. Mice in these groups received a single subcutaneous injection of BX471 (50 mg/kg). Renal tissue was obtained after 3 h, snap-frozen, and prepared for microscopy.
Statistical Analyses
Data were expressed as mean ± SEM. Comparison of groups was performed using unpaired t test. P < 0.05 was considered to indicate statistical significance.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal Expression of CCR1 in MRLlpr/lpr Mice
As appropriate antibodies that allow detection of CCR1 protein by cell fluorescence or immunostaining in mice were not available, we used real-time RT-PCR to determine the expression of CCR1 mRNA in kidneys of MRLlpr/lpr mice. Kidneys of 24-wk-old MRLlpr/lpr mice showed a marked induction of CCR1 mRNA compared with MRL wild-type mice of the same age (Figure 2), a finding that is consistent with our previously reported analysis using RNase protection assays from kidneys of MRLlpr/lpr mice (10). For determining the source of renal CCR1 expression, tubular segments were microdissected manually from kidneys of the same MRLlpr/lpr mice and primary renal fibroblasts were isolated from kidneys of MRLlpr/lpr mice as described in Materials and Methods. Furthermore, we isolated macrophages and T cells from spleens of MRLlpr/lpr mice by magnetic bead isolation. Real-time RT-PCR for CCR1 was performed with RNA isolates from all types of cells prepared. Both macrophages and CD8 T cells expressed CCR1 mRNA, but CCR1 mRNA transcripts were not detected in tubular epithelial cells or renal fibroblasts (Figure 2B). These data indicate that renal CCR1 expression does not originate from renal tubular cells or interstitial fibroblasts; in contrast, CCR1 positive macrophages and T cells may contribute to renal CCR1 expression after infiltrating the kidney.
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CCR1 Blockade with BX471 Reduces Renal Damage in MRLlpr/lpr Mice
Renal Function.
Daily treatment with BX471 from 20 to 24 weeks of age significantly improved renal function as illustrated by a reduction of serum BUN levels compared to vehicle-treated MRLlpr/lpr mice (Table 1). In contrast, BX471-treatment did not affect proteinuria and body weight compared to vehicle-treated MRLlpr/lpr mice (Table 1).
Glomerular Injury.
Daily treatment with BX471 did not significantly affect the extent of mesangioproliferative glomerulonephritis, the number of ERHR-3–positive glomerular macrophages, and the number of Ki-67–positive proliferating glomerular cells compared with vehicle-treated controls (Table 1). Apoptotic cells were rarely detected in glomeruli of both groups by immunostaining with an anti-ssDNA antibody (not shown). No significant difference in the activity index was found between the two groups. This index includes mostly histopathologic abnormalities of the glomerular compartment in lupus nephritis, such as proliferation of the mesangium, glomerular leukocyte infiltration, mesangial matrix, focal glomerular necrosis, and cellular crescents (13).
Interstitial Injury.
In contrast to the lack of effect on glomerular inflammation and proteinuria and consistent with improved serum BUN levels, BX471 markedly reduced the extent of tubulointerstitial disease compared with vehicle-treated control mice. In BX471-treated mice, no tubular atrophy or confluent areas of interstitial fibrosis were observed. Furthermore, there was a marked reduction in periglomerular and interstitial accumulation of ERHR-3–positive macrophages and CD3 lymphocytes compared with vehicle-treated control mice (Figure 1). Immunostaining for KI-67–positive proliferating cells and ssDNA-positive apoptotic cells was performed as markers of cell turnover in the renal tubulointerstitium. BX471-treated mice showed a marked reduction of KI-67– and ssDNA-positive tubular cells as well as interstitial cells compared with vehicle-treated mice (Table 1). Immunostaining for SMA-positive myofibroblasts and interstitial collagen I deposits was performed as additional markers of interstitial fibrosis. BX471-treated mice showed a marked reduction of SMA-positive cells and collagen I deposits in the interstitium compared with those in vehicle-treated mice (Figure 3A). The latter finding was confirmed by real-time RT-PCR for renal collagen I mRNA expression, which showed a 10-fold reduction by treatment with BX471 (Figure 3B). Quantitative analysis of interstitial immunostaining for collagen I by automated digital evaluation of the collagen I-positive area per high-power field demonstrated a 2.5-fold reduction in the BX471-treated group compared with vehicle-treated MRLlpr/lpr mice (Figure 3C). These findings are also illustrated by a significant reduction of the chronicity index between the two groups (Table 1). This index evaluates histopathologic abnormalities of the interstitial compartment in lupus nephritis, such as the extent of tubular atrophy and interstitial fibrosis (13).
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1 mRNA Expression. We therefore determined the expression of TGF- mRNA in total renal RNA by real-time RT-PCR. Kidneys from BX471-treated mice had an 85% reduction of TGF- mRNA expression compared with vehicle-treated MRLlpr/lpr mice (Figure 7A). These data show that at a late stage of nephritis in MRLlpr/lpr mice, BX471 reduces renal leukocyte infiltration and TGF- mRNA expression, a cytokine that has been implicated in renal fibrosis. To determine the source of renal TGF-, we performed in situ hybridization for TGF-. In sense control kidneys of vehicle-treated MRLlpr/lpr mice, the in situ hybridization yielded only a weak diffuse deposition of silver grains (not shown). The strongest signal for TGF- mRNA was found in areas of tubulointerstitial infiltrates (Figure 7B). The resolution of the in situ hybridization was not sufficient to assign the signal in the infiltrate to specific cells. In areas without prominent cell infiltration, only background signal was present, similar to incubation with sense controls. No clear tubular expression of TGF- mRNA was apparent. These results suggest that the source of TGF- is in the interstitial infiltrate and not the tubular cells. Furthermore, the reduction of interstitial leukocyte infiltration observed in BX471-treated MRLlpr/lpr mice is associated with a decrease in levels of TGF- mRNA and protein, a cytokine that can stimulate epithelial-mesenchymal transformation, apoptosis, and collagen secretion by renal fibroblasts.
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Discussion |
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BX471 Reduces Renal Damage in MRLlpr/lpr Mice by Blocking CCR1-Mediated Leukocyte Recruitment to the Renal Interstitium
Leukocyte migration to sites of tissue injury involves concerted interaction of adhesion molecules and chemokines and their receptors. Our data demonstrate that CCR1 is involved in interstitial macrophage and T cell infiltration in MRLlpr/lpr mice. The evidence comes from the results of the histologic evaluation and from the transfer experiments using a technique of injecting labeled macrophages and T cells in vivo. These studies confirm that the reduction of renal leukocytes and chemokine receptor expression in BX471-treated MRLlpr/lpr mice relates to impaired leukocyte recruitment into the kidney. As we have recently shown that CCR1 antagonism reduces the amount of interstitial leukocytes in the mouse kidney after unilateral ureteral obstruction (8), these data indicate that interstitial leukocyte infiltration may be CCR1 dependent in other mouse models of renal disease as well. CCR1 blockade has also been shown to have beneficial effects on the outcome of other disease models and in other species. For example, BX471 improved survival and renal function after kidney transplantation in rabbits (7), delayed heart transplant rejection in rats (6), and improved functional performance of rats with experimental encephalomyelitis (18). Our data show that renal CCR1 is expressed only on the infiltrating leukocytes, and in vitro studies confirmed the role of CCR1 for leukocyte adhesion and transmigration through activated endothelium (4). Furthermore, these data suggest that proteinuria-induced activation of proximal tubular cells may not be sufficient to maintain progression of tubulointerstitial injury in the absence of interstitial inflammatory cell infiltrates (19). Apparently, additional signals from the infiltrating leukocytes such as proinflammatory and profibrotic cytokines are required for progression of interstitial injury. This hypothesis is supported by numerous experimental and human biopsy studies indicating roles for T cells and macrophages in local cytokine and chemokine expression and in progressive tubulointerstitial injury (20–22). Our study shows that even in chronic renal injury, preventing tubulointerstitial leukocyte infiltrate improves the disease despite unaltered glomerular damage and proteinuria. Although of interest, from this study it remains unclear whether CCR1 blockade halts or just slows disease progression in MRLlpr/lpr mice. However, these data indicate that CCR1-mediated leukocyte recruitment is important for interstitial inflammation in the kidney as well as in other model systems and that therapeutic blockade of CCR1 with a small molecule antagonist late in the course of immune complex glomerulonephritis can have beneficial effects on disease progression.
BX471 Reduces Renal Fibrosis in MRLlpr/lpr Mice
How could the reduction in mononuclear leukocyte infiltration in BX471-treated MRLlpr/lpr mice relate to the concomitant reduction in interstitial fibroblasts and fibrosis? The infiltrating leukocytes, via secretion of cytokines such as TGF-, EGF, PDGF, or fibroblast growth factor, could contribute to epithelial-mesenchymal transformation, fibroblast proliferation, and collagen production (2). In fact, BX471-treated MRLlpr/lpr mice showed a marked reduction of renal TGF- mRNA expression, a key cytokine for the induction of fibroblast proliferation and the development of renal fibrosis (23). To localize the site of TGF- production, we performed in situ hybridization for TGF- and localized the TGF- production to the interstitial cell infiltrate. Similar to the real-time RT-PCR data, in situ hybridization signals for TGF- were reduced in kidneys of BX471-treated MRLlpr/lpr mice. The resolution of the latter method did not allow assignment of the signal to specific cells in the infiltrate, but the reduction of renal TGF- mRNA expression with BX471 treatment corresponds to the reduction of interstitial macrophages. It therefore seems reasonable to assign the TGF- signals to inflammatory cells, i.e., the infiltrating mononuclear leukocytes, although low-level TGF- mRNA expression may be beyond the sensitivity of the in situ hybridization method as the reported data about tubular TGF- expression in mice are conflicting (24,25). TGF- can also mediate a suppressive effect on systemic immune responses (26). In our study, reduced renal TGF- mRNA levels were not associated with a change of systemic autoimmunity in MRLlpr/lpr mice. It therefore seems that the reduction in TGF- mRNA in BX471-treated MRLlpr/lpr mice relates directly to the reduced renal leukocyte recruitment observed under these conditions.
BX471 Does Not Affect DNA Autoantibodies, Glomerular Macrophage Recruitment, and Proteinuria in MRLlpr/lpr Mice
The unchanged serum anti-DNA IgG isotype titers or glomerular IgG isotype deposits in BX471-treated mice compared with vehicle-treated controls indicate that BX471 does not induce a major shift in the Th1/Th2 balance of the systemic immune response in our lupus model. Such a shift toward the Th1 response was observed by Topham et al. (9) when nephrotoxic serum glomerulonephritis was induced in CCR1-deficient mice. This discrepancy may relate to different pathomechanisms of the nephrotoxic serum nephritis versus the MRLlpr/lpr lupus model. In nephrotoxic serum nephritis, a specific immune response against the planted glomerular antigen is required, whereas the MRLlpr/lpr lupus mouse model shows a broad polyclonal unregulated antibody production. Given this difference in pathoimmunology of the two models, the discrepancy seems less surprising, especially as in our model, the CCR1 blockade occurs after the establishment of the immune complex disease rather then during the generation of the immune response. It is interesting that we found that BX471 does not affect glomerular macrophage or T cell recruitment compared with vehicle-treated control mice, despite reduced interstitial macrophage counts with BX471. Surprising is that after injection, labeled and vehicle-treated macrophages did not localize to glomeruli, although there are clearly macrophages in glomeruli of lupus mice. Whether this observation relates to a different temporal interaction of circulating macrophages with glomerular endothelium remains unclear. Nevertheless, these data indicate that CCR1 is involved in interstitial but not in glomerular leukocyte recruitment. These data add to the growing body of evidence indicating marked differences in the mechanisms of leukocyte recruitment to the glomerular and tubulointerstitial compartment. For example, Tesch et al. (27) reported that CCL2/MCP-1–deficient mice were protected from tubulointerstitial but not from glomerular injury after induction of nephrotoxic serum nephritis. Furthermore, we recently showed in a model of Apoferritin-induced immune complex glomerulonephritis in mice that Met-RANTES, a presumed CCR5 antagonist, reduced glomerular macrophage infiltration by 
50% (28). Conversely, Met-RANTES or CCR5-deficient mice showed no impairment of interstitial macrophage recruitment after unilateral ureteral ligation in mice (11). Together, these data support different roles for CCR1 and CCR5 in renal leukocyte recruitment with CCR5 involved in the glomerulus, while CCR1 is involved in the interstitial compartment. This phenomenon may relate to different adhesion molecules present on these different vascular beds, which modulate chemokine-mediated leukocyte adhesion and transmigration differentially (29,30). Further evidence for this hypothesis comes from the observation that CD3–positive lymphocytes are rarely found in the glomerulus in murine or human glomerulopathies, suggesting that glomerular endothelia may not support lymphocyte recruitment in this compartment, which again is demonstrated by the lack of recruitment of labeled CD8 T cells after intravenous injection in the present study. Together, CCR1 blockade with BX471 did not affect serum anti-DNA IgG isotype titers, glomerular immune complex deposition, glomerular macrophage recruitment, and proteinuria in MRLlpr/lpr mice. These data indicate that CCR1 blockade does not interfere with the humoral immune response in systemic autoimmunity of MRLlpr/lpr mice and that the recruitment and activation state of glomerular macrophages is not mediated by CCR1.
In summary, CCR1 mediates interstitial but not glomerular recruitment of mononuclear cells in the mouse kidney. When given late in the course of progressive renal injury in MRLlpr/lpr mice, BX471 improved renal function and diminished interstitial injury but did not affect glomerular damage, proteinuria, and systemic autoimmunity. These data signify the importance of interstitial injury for progressive renal dysfunction and provide the first evidence that blockade of CCR1—late in the course of chronic renal failure—can halt disease progression and improve renal function by selective inhibition of interstitial macrophage and T cell recruitment. Therefore, we propose that CCR1 blockade, currently evaluated for the treatment of multiple sclerosis in Phase II trials (31), may offer a new therapeutic strategy for lupus nephritis and perhaps for other chronic nephropathies that lead to end-stage renal failure.17
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Acknowledgments |
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We thank P.J. Nelson for assistance with BX471.
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1-deficiency in mice is dependent on MHC class II antigen expression. Nature 359: 693–699, 1992[CrossRef][Medline]
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) and BX471-treated (
