1 Departments of Experimental Surgery, and 2 Nephrology, University of Antwerp, Belgium
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Abstract |
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Methods. Uninephrectomized rats were submitted to 60 min renal ischaemia by clamping of renal vessels.
Results. Severe acute renal failure was observed, with maximum functional impairment on day 2. By 12 h after the ischaemic event, up to 80% of proximal tubular cells in the outer stripe of outer medulla (OSOM) were already severely damaged. Proliferation (proliferating cell nuclear antigen (PCNA) staining) started after 24 h, reaching maximum activity on day 3. Regeneration of tubular morphology started on the 3rd day, and after 10 days 50% of tubules had regenerated completely. Interstitial leukocytes (OX-1 immunohistochemical staining) were already prominent at day 1, thereafter gradually increasing with time. The so-called neutrophil-specific identification methods (myeloperoxidase (MPO), chloroacetate esterase, mAb HIS-48) proved to be non-specific, since they also stained for macrophages, as demonstrated by flow cytometry and the combination of these stainings with the macrophage-specific ED-1 staining. MPO activity was already significantly increased at 1 h post-I/R (439±34%, P<0.005), reaching its maximum activity after 12 h of I/R (1159±138%, P<0.0005), declining thereafter. On the other hand, neutrophil presence investigated by H&E staining revealed only a few neutrophils in glomeruli, medullary rays, and OSOM at 24 h after the ischaemic event (4.7±4.2 cells/mm2 vs controls=2.3±2.0 cells/mm2 (n.s.)), and remained unchanged over the next 10 days. In contrast, significant monocyte/macrophage adhesion/infiltration (ED-1 staining) occurred at the OSOM at 24 h post-ischaemia (at 24 h, 120±46 cells/mm2 vs sham=18±4 cells/mm2 (P<0.05)), became prominent at day 5 (1034±161 cells/mm2 vs sham=18±18 cells/mm2 (P<0.05)), and almost disappeared after 10 days. CD4+ cells (W3/25) gradually increased from day 5, reaching a maximum at day 10. A few CD8+ cells (OX-8) were apparent from days 3 until 10, but no B-cells (OX-33) were observed.
Conclusions. After severe warm I/R renal injury, a pronounced acute tubular necrosis occurs during the first 1224 h in the absence of a marked cellular infiltrate, but with an important renal MPO activity, reflecting the activation of the adhering inflammatory cells (polymorphonuclear cells (PMNs) and mainly monocytes/macrophages). Only later at the time and site (OSOM) of regeneration a sequential accumulation of monocytes/macrophages and T cells becomes prominent, in contrast with the low number of neutrophils found in the kidney during the 10-day post-ischaemic period. The non-specificity of the so-called neutrophil-specific identification methods (MPO activity, naphthol AS-D chloroacetate esterase, or mAb HIS-48 staining), cross-reacting with monocytes/macrophages, explains the controversy in literature concerning the number of PMNs in post-ischaemic injury.
Keywords: damage; kidney; macrophages; myeloperoxidase; neutrophils; rat; regeneration
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Introduction |
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Renal injury after ischaemia appears to be a consequence of tissue hypoxia from interrupted blood supply but also from the process of reperfusion, leading to an active inflammatory response [4]. Sublethal or even lethal injured proximal tubular and endothelial cells trigger this process through the release of cytokines and chemokines that will promote cellular infiltration. Leukocytes may play an important role in the mechanism of parenchymal injury after I/R as well as in the regeneration process, but their exact role is far from clear. Polymorphonuclear cells (PMNs) recruited during reperfusion have long been implicated as critical mediators of the early renal parenchymal injury in ischaemic ARF, as recently reviewed [5]. These assumptions were supported by morphological criteria (H&E stain [6]), enzymatic criteria (myeloperoxidase [79], chloroacetate esterase [1013], mAb HIS-48 [14]) and labelling techniques (e.g. 111Indium labelling [15]), in most cases suggesting robust PMN recruitment in the post-ischaemic kidney [1]. These PMNs may contribute by potentiating an inflammatory response that leads to the generation of vasoconstrictor agents, cytokines, and toxic mediators such as reactive oxygen species and proteases [16,17]. I/R in rat myocardium [18], liver [19], and brain [20] has been correlated with the number of granulocytes adherent at the capillary walls. Also in the kidney, PMNs have been put forward as inducing or amplifying additional damage in post-ischaemic renal injury [79,12,13,15,21]. Several investigators, however, found that infiltration of the renal parenchyma by PMNs was not a prominent feature of experimental or human post-ischaemic ARF when PMNs in renal sections were counted using routine histology (H&E) [1].
The first step in unravelling this controversy consists in the careful analysis of the identification of these adhering/infiltrating cells and their kinetics and distribution, related to the course of tubular injury and repair after severe warm I/R.
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Subjects and methods |
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Post-operative animals were allowed to recover, each in a separate cage, at constant temperature (18°C) and humidity (45%) on a 12-h light/dark cycle. They received free tap water ad libitum, and standard laboratory rat pellets by the paired-feeding method. In this way non-ischaemic animals received the same protein and salt intake as post-ischaemic animals, which show greater post-operative anorexia. Animals were weighed and inspected daily. Sacrifice of 4 animals per experimental time point was done at hours 1, 2, 6 and 12 and on days 1, 2, 3, 5, 7, and 10. Blood samples were taken by heart puncture. All procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals No. 8523 (1985), and with approval of the Ethical Committee of the University of Antwerp.
Biochemical determinations:
Blood samples were allowed to clot and were centrifuged at high speed for 15 min. Serum was obtained and stored at -20°C until use. Serum creatinine values were determined in duplicate using a colorimetric method as modified by Jaffé (Creatinine Merckotest, Diagnostica Merck, Germany).
Tissue collection and fixation:
Immediately after sacrifice, tissue for analysis was collected from the left kidney. After dissection of the capsular fat, the kidney was weighed. Five sagittal tissue sections (1 mm thick) were made and fixed in formalin calcium, methacarn and Dubosq Brazil fixative. Two sections were stored in liquid nitrogen.
Morphological analysis of tubular injury, regeneration and cell proliferation:
The degree of injury in different tubular compartments was established on periodic acid-Schiff reagent (PAS)-haematoxylin-PCNA stained sections of methacarn-fixed and paraffin-embedded renal tissue. Proliferation was determined by immunohistochemical staining for the PCNA using the PC10 monoclonal antibody (Dako, Denmark). Sections were counterstained with PAS. Nuclei were stained with methyl green. Histological damage of the kidney was scored semi-quantitatively: 50 tubules in the outer stripe of the outer medulla (most sensitive zone for ischaemic injury) were assigned using a score-system ranging from 0 to 6 (score 0, normal tubule; score 1, (limited to) loss of brush border; score 2, <50% tubular damage, meaning less than 50% of naked basal membrane; score 3, >50% tubular damage; score 4, total destruction of all epithelial cells, naked basement membrane; score 5, small rim of cytoplasm with large dark nuclei (PCNA+); score 6, increase in cytoplasm volume; evolution to score 1, uncompleted brush border and score 0, complete regenerated brush border). The proliferation was measured by counting the number of PCNA-immunoreactive nuclei in 25 circular-shaped proximal tubules.
Renal adhesion/infiltrationleukocyte cell markers:
Sections (4 µm) were mounted on poly-L-lysine-coated microscope slides and treated for 5 min with 0.003% trypsin III (Sigma Chemical Co., St Louis, USA) in 10 mmol/l TrisHCl buffer pH7.3. After washing in Tris saline buffer (TSB, 0.01 mol/l TrisHCl pH 7.6 in 0.9% NaCl) and treatment with normal horse serum (1/5), the sections were incubated overnight with the primary antibody OX-1 (1/6000). This OX-1 mAb recognizes the rat leukocyte common antigen CD45, which is present on all marrow-derived leukocytes [24]. Positive cells were counted in 20 fields of 0.16 mm2 and data were expressed as leukocytes/mm2. Immunohistochemical detection of monocytes/macrophages was performed on methacarn-fixed, paraffin-embedded renal-tissue sections using the ED1 monoclonal antibody (Serotec, UK). This antibody is directed to a cytoplasmic antigen of tissue macrophages and monocytes [25]. As control, a peripheral blood smear and a spleen section of the rat were stained. T-lymphocytes (Helper T-cells: W3/25, suppressor T-cells: OX-8), and B-lymphocytes (OX-33) were demonstrated on 5 µm renal tissue cryosections prefixed in 4% formaldehyde (BDH Chemical Ltd, Poole, UK) buffered with 0.1 mol/l Na-cacodylate pH 7.4 containing 1% CaCl2. Sections were incubated overnight with W3/25 (1/800), OX-8 (1/400) or OX-33 (1/100) (Serotec, Oxford, UK). Appropriate antibody dilutions were determined in preliminary experiments. The W3/25 monoclonal antibody recognizes the rat CD4-equivalent present on T-helper cells [26], and, in lower density, on some macrophages [27]. The OX-8 monoclonal antibody reacts with T-suppressor/cytotoxic and natural killer cells, and is directed to the rat CD8-equivalent [28]. The OX-33 monoclonal antibody reacts with only peripheral B-cells [29]. Positively stained cells were quantified in 10 randomly chosen OSOM microscope fields (magnification 125) in each animal and expressed as positive cells per square millimetre. Endogenous tissue peroxidase activity was inhibited by immersion in methanol for 15 min, followed by 30 min incubation with 0.03% H2O2 in TSB. After washing, subsequent incubations were performed with the avidin biotin peroxidase complex (Vectastain, Vector Laboratories Inc., Burlingame, USA) and 9-amino-ethylcarbazole as the chromogen supplemented with the H2O2 as substrate. As controls, a spleen section of the rat was also stained. Nuclei were counterstained with methyl green.
Myeloperoxidase (MPO) assessment of rat post-ischaemic kidney
This colorimetric method measures the activity of MPO found in the azurophilic granules present in PMNs [30]. Normal and post-ischaemic kidney at 1, 2, 6, 12, and 24 h post-reperfusion was homogenized in 5 mmol/l potassium phosphate buffer first and then centrifuged at 30 000 g for 30 min at 4°C prior to extraction. To avoid interference with supernatants from control and post-ischaemic kidney the pellets were washed twice [31]. The resulting pellets expressed MPO activity after suspending them in 50 mmol/l phosphate buffer containing 0.5% hexadecyltrimethyl ammonium bromide (HTAB). The pellets were centrifuged at 40 000 g for 15 min; 0.2 ml specimen was added to 0.8 ml 50 mmol/l potassium phosphate buffer (pH 6.0) containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% H2O2. Absorbency was measured at 460 nm for 2 min, and assay linearity was confirmed. MPO activity, normalized to protein content of the supernatant, was expressed as the percentage of levels in kidneys subjected to sham surgery.
Identification/quantification methods of PMNs
H&E staining
Histochemical detection of polymorphonuclear cells was performed on methacarn-fixed, paraffin-embedded renal-tissue sections. The H&E staining was used to identify and to quantify the infiltration of polymorphonuclear cells, based upon the localization of the cell and morphology of the nucleus of the cell. Positively stained cells were quantified in 75 randomly chosen microscope fields (magnification 1000) in cortex, outer stripe of outer medulla (+ medullary rays), and inner medulla, expressed as positive cells per square millimetre.
ED-1 staining on cryosections in combination with MPO staining
In brief, after incubation with the mAb ED-1 against macrophages (step 1), MPO was stained using the chromogen benzidine dihydrochloride (step 2). Thereafter, MPO was inactivated (step 3) and the ED-1 staining itself was developed (step 4) (Figure 1). More in detail, 5 µm renal tissue cryosections were used, prefixed in formalin calcium (4% formaldehyde (BDH Chemical Ltd., Poole, UK)) buffered with 0.1 M Na-cacodylate pH 7.4 containing 1% CaCl2. In a first step, the sections were mounted on poly-L-lysine coated microscope slides and treated for 5 min with 0.003% trypsin III (Sigma Chemical Co., St Louis, USA) in 10 mmol/l TrisHCl buffer pH 7.3. After washing in TSB with 1% bovine serum albumin and treatment with normal horse serum (1/5), the sections were incubated overnight with the primary mouse anti-rat antibody ED-1 (1/15 000). This very specific antibody is directed to a cytoplasmic antigen of tissue macrophages, monocytes and dendritic cells [25]. Sections were then incubated for 30 min with biotinylated horse anti-mouse IgG. As second step, staining for the MPO was performed, using the chromogen benzidine dihydrochloride (30% ethanol, containing 1.5 g benzidine, pH 6.0) and hydrogen peroxide 0.03% as substrate. This shows the yellow-coloured intra-cytoplasmic granules. The sections were mounted with gelatine and photographed with a Leica microscope using the program KS 400 v. 2.0. In a third step, the sections were demounted to further develop the ED-1 mAb: after inhibition of endogenous peroxidase using methanol for 15 min and H2O2 0.03% in TSB for 30 min, sections were incubated with avidinbiotin labelled with peroxidase (Vectastain, Vector Laboratories Inc., Burlingame, USA) and developed with 9-amino-ethylcarbazole as chromogen and H2O2 as substrate, which finally shows the ED-1 positive red-brownish-coloured cytoplasm of the macrophage. The sections were mounted with gelatine/glycerine and photographed again.
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ED-1 staining on cryosections in combination with naphthol AS-D chloroacetate esterase staining
In brief, after incubation with the mAb ED-1 against macrophages, the naphthol AS-D chloroacetate esterase reaction was performed. Thereafter, the ED-1 staining itself was developed. More in detail, ED-1 positive cells were demonstrated as described above; 4 µm renal tissue cryosections were used, prefixed in formalin calcium (4% formaldehyde (BDH Chemical Ltd, Poole, UK) buffered with 0.1 M Na-cacodylate pH 7.4 containing 1% CaCl2). In a first step, the sections were mounted on poly-L-lysine coated microscope slides and treated for 5 min with 0.003% trypsin III (Sigma Chemical Co., St Louis, USA) in 10 mM TrisHCl buffer pH 7.3. After washing in TSB with 1% bovine serum albumin and treatment with normal horse serum (1/5), the sections were incubated overnight with the primary mouse anti-rat antibody ED-1 (1/15 000). Sections were then incubated with biotinylated horse anti-mouse IgG. After 30 min incubation, as a second step, the chloroacetate staining was performed as described by Leder [32]: slides were incubated with naphthol AS-D chloroacetate esterase in 4% pararosaniline and 4% sodium nitrate in 0.1 M acetate buffer for 30 min. The sections were mounted with gelatine and photographed: cells containing red-brownish granules were regarded as positive. In a third step, the sections were demounted. After inhibition of endogenous peroxidase using methanol for 15 min and H2O2 0.03% in TSB for 30 min, sections were incubated with avidinbiotin horseradish peroxidase (Vectastain, Vector Laboratories Inc., Burlingame, USA) and developed as described above, to show the ED-1-positive cells. Control stainings were similar as described for MPO (see above).
HIS-48 (granulocytes) in combination with ED-1
In brief, first the primary antibody HIS-48 was demonstrated, photographs were taken, decoloured, and thereafter ED-1 staining was performed. More in detail, 5-µm renal tissue cryosections were prefixed and mounted as described above, and incubated overnight with HIS-48 (1/10) (monoclonal antibody against polymorphonuclear cells) [14]. As controls sections were also incubated with the primary mAb ED-1 (1/150 000) and mAb against -smooth muscle actin (
-SMA)(1/30 000). After inhibition of endogenous peroxidase using methanol for 15 min and H2O2 0.03% in TSB for 30 min, sections were incubated with biotinylated horse anti-mouse IgG for 30 min. Sections were then incubated with avidinbiotin horseradish peroxidase (Vectastain, Vector Laboratories Inc., Burlingame, USA) and developed with 9-amino-ethylcarbazole as chromogen and H2O2 as substrate. The sections were mounted with gelatine/glycerine and photographed. The sections were demounted, decoloured with isopropanol and again photographed. Then the sections were heated in a microwave [14] for 10 min in order to destroy the horse anti-mouse binding on HIS-48 and were allowed to cool down. The sections were blocked with polyclonal rabbit antiserum for 20 min and then incubated overnight with ED-1, which was a FITC (fluoresceine isothiocyanate)-labelled antibody. Then the sections were incubated with a rabbit polyclonal anti-FITC antibody, which was peroxidase labelled and thereafter, developed with 9-amino-ethylcarbazole as chromogen and H2O2 as substrate.
This rabbit anti-ED-1 FITC-labelled antibody was used in order to exclude aspecific staining with the previous secondary polyclonal horse anti-mouse antibody and avidinbiotin complexes. In another control experiment aspecific binding of ED-1 with the horse anti-mouse antibody, used during first staining step, was excluded using -SMA in the first staining.
Labelling of rat peripheral blood cells with mAb HIS48
Erythrocytes from 20 µl of heparinized blood were lysed (hypotonic buffer, 10 min, 20°C) and leukocytes were then incubated (30 min, 4°C) with mAb HIS-48. A second incubation step (30 min, 4°C) with FITC-conjugated rabbit-anti-mouse IgG (RAM-FITC) followed washing of the cells (PBS+0.1% BSA+0.05% NaN3). Cells were resuspended in washing buffer prior to analysis on a FACStar plus (Becton & Dickinson, Immunocytometry Systems). To identify positive cells, these were sorted onto poly-L-Lysine-coated glass slides and stained by MayGrünwaldGiemsa standard procedure.
Statistics
Data are presented as means±standard deviation. They were compared with an one-way ANOVA analysis, and a StudentNewmanKeuls test was used to prove qualitative differences by using the software package SPSS. Significant differences were anticipated when P<0.05.
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Results |
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Kidney function (Figure 2C)
Unilateral nephrectomy, without any ischaemia, caused a slight increase in the serum creatinine (S-crea) levels on day 2 (S-creanephrectomy day 0=0.75 mg/dl±0.05 vs S-creanephrectomy day 2=1.22 mg/dl±0.13; P<0.05), and returned to normal on day 3. A period of 60 min of warm ischaemia caused a severe acute renal failure on the second day (S-creaischaemia day 0=0.75 mg/dl±0.05 vs S-creaischaemia day 2=4.0 mg/dl±1.9; P<0.05). Functional recovery of the kidney started after day 2 and was almost complete at day 7 (S-creaischaemia day 7 =1.06 mg/dl±0.11; not significant).
Damage and regeneration/PCNA positivity (Figures 3, 4
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The zones of most severe injury (PAS staining) and proliferation (PCNA staining), which are the OSOM and medullary rays, are exactly the same zones of most important leukocyte infiltration (OX-1 pan leukocyte staining) (Figure 3). The evolution of damage and regeneration of the kidney tubules was scored semi-quantitatively (Figure 4
). In cases of a unilateral nephrectomy without ischaemia of the unique kidney, no signs of damage or regeneration were seen in the remaining kidney during the study period of 10 days. No PCNA positivity or infiltrating leukocytes was noticed. In contrast to the non-ischaemic groups, 60 min of warm ischaemia resulted in remarkable damage of the proximal S3 segment and the thick ascending limb of the nephron in the OSOM and medullary rays. The first day after the ischaemia, almost half of the tubules showed a complete detachment (score 4) of their epithelial cells from their basement membrane. On day 2, the first signs of regeneration appeared and they reached a maximum at day 3, together with a maximum of PCNA positivity. The characteristics of this early regeneration consisted of small non-differentiated cells with strong PCNA-positive nuclei, localized on the basement membrane of the proximal tubule and the thick ascending limbs (score 5). The following days, the volume of the cytoplasm of these cells increased (score 6), until finally the brush border recovered (back to score 0). The amount of PCNA-positive cells decreased in time and returned to baseline from the 5th day onwards. At the end of the investigated period (day 10), half of the proximal tubules were regenerated completely.
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Interstitial leukocytes (Figure 5)
In the non-ischaemic group, no increase in number of interstitial leukocytes (OX-1 staining) could be seen within the study period of 10 days. Ischaemia of a unique kidney resulted in a gradually increasing interstitial leukocyte infiltrate in the OSOM during the 10 days investigation period. At day 10, the interstitial infiltrate was quite prominent (OX1ischaemia=1733± 10 cells/mm2).
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W3/25 staining (CD4+ molecule)
A lymphocytic infiltration occurred from day 5 on in the ischaemic group, and persisted after 10 days.
OX-8 staining (CD8+ molecule)
The outer stripe of the outer medulla contained only a small but significant number of CD8+ cells, entering the ischaemic kidney at the 3rd day (35±6.76 cells/mm2 vs controls=6.8±8.05 cells/mm2 (P<0.05)) and persisting until day 10.
OX-33 staining (B-cells)
The spleen section was clearly positive for B-cells. Neither the control kidney nor the ischaemic kidney contained B-cells.
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Early staining of PMNs vs monocytes/macrophages: analysis of 024 h (Figure 6![]() |
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MPO assessment of rat post-ischaemic kidney during the first 24 h post-I/R (Figure 6![]() |
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Identification of infiltrating cells (Figure 7![]() |
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ED-1 staining on cryosections in combination with MPO-staining
Figure 7A shows MPO staining of ED-1-positive cells, indicating that macrophages, like neutrophils, stain for MPO. By performing the staining procedure as described above but without incubation with the primary ED-1 mAb, no positive staining was observed, indicating no aspecific binding of the secondary mAbs and the avidinbiotin complexes, or residual MPO activity.
ED-1 staining on cryosections in combination with chloroacetate staining
Figure 7B shows naphthol AS-D chloroacetate esterase staining of ED-1-positive cells, which shows that macrophages, like neutrophils, stain with chloroacetate. By performing the staining procedure as described above but without incubation with the primary ED-1 mAb, no positive staining was observed, indicating no aspecific binding of the secondary mAbs and the avidinbiotin complexes, no residual activity of the endogenous peroxidase activity.
HIS-48 (granulocytes) in combination with ED-1
Figure 7C shows that ED-1-positive cells (macrophages) also stain for HIS-48. Different control experiments were performed (data not shown): first, by performing the double-staining procedure as described above but without incubation with the anti-ED-1 FITC mAb, no positivity was observed, indicating no aspecific binding of the second staining procedure. However, the ED-1 itself might bind with parts of the primary staining procedure. Therefore, a second control staining was performed by a double-immunohistochemical staining combining mAb against
-SMA in the first staining with ED-1 FITC in the second staining. The typical pattern of peritubular and periglomerular
-SMA staining and also that in the walls of large blood vessels is different from the typical interstitial pattern of macrophage staining. If in our control staining ED-1 should bind non-specifically to the secondary biotinylated horse anti-mouse antibody of the first staining, the ED-1 staining would have had the staining pattern of
-SMA, which was not the case. Hence the secondary staining for ED-1 was specific for the monocyte/macrophage cell type. In order to exclude aspecificity or decreased activity of a FITC-labelled antibody, a control staining was performed by combining the ED-1 in the first staining with the ED-1 FITC-labelled antibody in the second staining. Not surprisingly, although less intensive, all cells positive for ED-1 were also positive for the FITC-labelled antibody.
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Labelling of rat peripheral blood cells with mAb HIS48 (Figure 8![]() |
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Discussion |
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The presence of these leukocytes, particularly of neutrophils, is generally considered as a damaging event, exacerbating I/R damage [17]. Recent experiments that interfere with leukocyte activation and adhesion/infiltration (mAbs to ICAM-1 and/or LFA-1, ICAM-1 antisense oligonucleotides, etc.) have shown that the kidney can be functionally protected against post-I/R injury [7,34]. On the other hand, phagocytes are also regarded as important scavengers of apoptotic cells or necrotic debris, and their presence in the kidney following I/R injury could alternatively represent a repair process [1]. Yet these infiltrating inflammatory cells may be a source of growth-stimulating substances [35], implying a role in the repair process after ARF [3638].
Although many data are available concerning the overall cellular infiltrate after injury, only scarce information is available on the careful identification and time course of the different subsets of these adhering/infiltrating and proliferating leukocytes [5]. Recently, a mixed mononuclear cell (macrophage and CD4+ cell) infiltration of the kidney, within a few days after I/R, was described after 45 min warm ischaemia [39] and after experimental cold ischaemia [40], with limited information, however, as to its exact topographical localization and kinetics. In the latter and many other studies, no important neutrophilic infiltration was mentioned, while even very recently, others, using enzymatic tests and membrane markers described mainly neutrophilic adhesion/infiltration [12,13,21,34]. These findings underscore discussion in the literature concerning the number and role of neutrophils after renal I/R. Our results contribute to the unravelling of this controversy.
First, during the first 2448 h the detailed identification and quantification of the interstitial leukocytes in the rat kidney after severe warm I/R injury showed a pronounced acute tubular necrosis, with only a moderate monocyte/macrophage infiltration and proliferation. It is important to stress that the decrease in body temperature from 37°C to 28°C during anaesthesia and surgery might change kinetics and other properties of the cellular infiltrate, especially at early time points after ischaemia. Despite there being no significant increase of PMNs (identified by routine H&E staining) and the slightly increased monocytes/macrophages (ED-1 staining) during the first 12 h, the MPO activity, generally related to presence of PMNs [793031], was significantly increased in the first hours post-ischaemia. This early (1 h) to maximal (12 h) MPO activity of the post-ischaemic kidney remains to be explained in the absence of significant increased number of inflammatory cells. It could be argued that PMNs and monocytes/macrophages do not infiltrate early after reperfusion but only adhere long enough to the activated endothelium to release their enzymes (like MPO) and to interfere with microcirculation with blockage of the vascular flow in the capillary network (no reflow) [4142], followed by re-entry to the circulation (hit-and-run phenomenon). In this respect, MPO activity reflects in fact more the activation of the adhering inflammatory cells and eventual of residential interstitial macrophages, resulting from post-ischaemic chemotactic and other cytokine activation, rather than infiltration itself. After this first critical period of 24 h, the number monocytes/macrophages was 1225 times higher than the scarcely present PMNs, which remained comparable to control values. This indicates that firm adhesion and diapedesis has taken place, generating the classical infiltration and proliferation observed in many conditions (ischaemia, HgCl2, obstruction, etc.). It could be suggested therefore that the immediate beneficial functional effect of anti-adhesion therapy with for example antibodies to ICAM-1 and LFA-1, has to be explained at the level of early intravascular (microcirculation) leukocyte activation, and adhesion, but not at the level of the interstitial infiltration, which appears slightly later, at a time when the functional recovery is already operational.
Secondly, we could demonstrate that MPO activity is not a specific measure of PMN presence, since MPO content of post-ischaemic tissue reflects not only presence of PMNs but at least also of monocytes/macrophages and/or of activated residential interstitial macrophages [304344]. In addition, by combined histochemical and immunohistochemical methods, we could demonstrate naphthol AS-D chloroacetate esterase activity in macrophages, present in the renal interstitium after I/R. Again, the latter staining is extensively used as specific for PMNs [101221]. Finally, staining of ED-1 (specific for macrophages) followed by HIS-48, a monoclonal antibody considered to be specific for polymorphonuclear cells, showed that macrophages also react with mAb HIS-48. Flow cytometric analysis of circulating white blood cells demonstrating the high affinity of macrophages for the anti-HIS-48 antibody, supported this result. Hence, none of the so-called cell-specific stainings for PMNs have proved to be specific for this particular cell type, and cross-react with monocytes/macrophages. The H&E staining (morphology of cell nucleus) remains the gold standard for identification of PMNs. MPO assays or chloroacetate esterase stainings should be regarded as tools to quantitate both PMNs and monocytes/macrophages. These observations explain the long lasting and frequently observed dissociation between the use of H&E staining vs enzymatic and/or membrane markers in the identification in the number of neutrophils [145].
Three days post-I/R, functional recovery starts, at the moment of maximum tubular cell proliferation. Only later, after 510 days, the mononuclear infiltrate becomes quite prominent, consisting of a sequential accumulation and proliferation of monocytes/macrophages and helper T-cells, a few suppressor/cytotoxic T-cells and no B-cells. These cells are most prominent at the site of maximal damage/regeneration, which is the OSOM and medullary rays. The total number of adhering/infiltrating PMNs in the different nephron segments remained unchanged up to 10 days post-I/R, their absolute number in the interstitium remaining far beyond that of other inflammatory cells.
In conclusion, the non-specificity of the so-called neutrophil-specific identification methods (MPO- activity, naphthol AS-D chloroacetate esterase or mAb HIS-48 staining), cross-reacting with monocytes/macrophages, explains the controversy in the literature concerning the number of PMNs in post-ischaemic injury. The only reliable method to quantitate PMNs remains H&E staining. Twelve to 24 h after severe warm I/R renal injury, a pronounced acute tubular necrosis occurs in the absence of a marked cellular infiltrate, along with a striking increase of MPO activity. This important MPO activity in this very early critical post-ischaemia period most probably reflects the activation of the adhering inflammatory cells (PMNs and monocytes/macrophages). Only at the later time of regeneration a sequential infiltration/proliferation of monocytes/macrophages and T-cells becomes prominent. The total number of PMNs remained low during the first 10 days post-I/R. These observations underscore an early effect of the few activated neutrophils, associated with the higher number of activated monocytes/macrophages.
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Acknowledgments |
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Notes |
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References |
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