1 Department of Pediatric Nephrology and Rheumatology, 2 Institute of Neurological Sciences, Department of Neurology, Faculty of Medicine, 3 Department of Pharmacology Faculty of Pharmacy, Hacettepe University, 4 Department of Pathology, University of Ankara, Ankara, Turkey and 5 Sheffield Kidney Institute, Sheffield, UK
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Abstract |
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Methods. The alteration in the NO pathway was assessed by measuring nitrite levels in serum/urine and by evaluating the changes in vascular reactivity of the isolated perfused rat kidney (IPRK) system. Rats were stratified into control groups and ADR-induced nephropathy groups. These two groups were then divided into: group 1, animals receiving saline; and group 2, animals receiving aminoguanidine (AG) which is a specific inhibitor of inducible-NO synthase. On day 21, rats were sacrificed after obtaining material for biochemical analysis.
Results. Histopathological examination of the kidneys of rats treated with ADR revealed focal areas of mesangial proliferation and mild tubulointerstitial inflammation. They also had significantly higher levels of proteinuria compared with control and treatment groups (P<0.05). Urine nitrite levels were significantly increased in the ADR-nephropathy group (P<0.05). In the IPRK phenylephrine and acetylcholine related responses were significantly impaired in the ADR-nephropathy group. Apoptosis was not detected in controls. However, in the ADR-nephropathy group, numerous apoptotic cells were identified in the tubulointerstitial areas. Double staining revealed numerous interstitial apoptotic cells to stain for ED1, a marker for monocytes/macrophages. Treatment with AG prevented the impairment of renal vascular bed responses and reduced both urine nitrite levels and apoptosis to control levels.
Conclusion. We suggest that interactions between NO and apoptosis are important in the pathogenesis of the ADR-induced nephrosis.
Keywords: adriamycin-induced nephropathy; aminoguanidine; apoptosis; isolated perfused rat kidney; nitric oxide
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Introduction |
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Apoptosis is a programmed form of cell death. Its role in kidney disease is complex [9]. In certain experimental models of proliferative glomerulonephritis, apoptosis has been suggested to be a necessary process in the regulation of endothelial or mesangial cells in the repair process [10,11]. On the other hand, apoptosis has been implicated in the sclerotic process within the kidney; apoptotic bodies have been demonstrated in the sclerotic areas of experimental and human renal diseases [11,12].
We have set up a model of nephrotic syndrome in rats induced by adriamycin (ADR), to study the role of NO and its association with apoptosis. The alteration in the NO pathway in this experimental model was assessed by measuring nitrite levels in serum/urine and by evaluating the changes in vascular reactivity of the isolated perfused rat kidney. The isolated perfused kidney system enables us to study the role of NO, on the renal vascular tone. Since increased NO synthesis by iNOS is expected to be associated with the inflammatory response, we have then examined the effect of aminoguanidine (AG) treatment on the observed changes in NO-mediated responses and the induction of apoptosis. AG was chosen because it is a relatively selective inhibitor of iNOS, and not constitutive NOS [13]. We, thus, wished to confirm the effect of NO produced by iNOS by a specific inhibitor.
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Materials and methods |
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Isolated perfused kidney experiments
Rats were anaesthetized with pentobarbital sodium (30 mg/kg) by i.p. injection. After laparotomy with a midline incision, heparin (200 U/kg) was injected into the aorta. The aorta was ligated distal to the right renal artery, and a polypropylene cannula was inserted into the left renal artery through an incision. The left kidney was removed and placed into the perfusion system with the renal artery cannula to study the vascular reactivity of renal vascular bed. In the meantime a blood sample was collected from the inferior vena cava for analysis of serum nitrite levels, and the right kidney was resected for histopathological and immunohistochemical studies.
The Ethics Committee of Ankara University, Faculty of Veterinary Medicine, Ankara, Turkey, approved the animal experimentation.
Biochemical evaluation
Serum creatinine (by standard autoanalyser technique), urine protein levels (by the Biuret method), plasma and urine nitrite levels were measured in each group of rats by standard autoanalyser techniques. Nitrite was assayed colorimetrically after reaction with Griess reagent as described previously [15].
Histopathological examination
After removal of the kidney, all tissues were fixed in 10% formalin, embedded in paraffin and examined in multiple consequent sections. The histological study was carried out using haematoxylin eosin (H&E) and periodic acid-Schiff (PAS). Glomerular and tubular cells were counted at a magnification of x200. For quantification of glomerular cells and tubular cells, 20 glomeruli and 20 fields were counted, respectively, to take an average attributed to the animal study. For the quantitation of the interstitial cells 10 fields with x400 magnification were counted by one person blinded to the specimen experimental code (D.O.)
Immunohistochemistry
Sections 5-µm thick were cut from paraffin blocks and mounted on poly-L-lysine coated slides. After deparaffinization, slides were put in a microwave oven in 10 mM sodium citrate at 700 W (4x5 min) for antigen retrieval. Immunohistochemical staining using the streptavidinbiotin peroxidase method was performed with monoclonal primary antibody against proliferating cell nuclear antigen (PCNA, Biogenex, 1/80 dilution). Negative control sections were incubated with phosphate-buffered saline (PBS) or non-immune globulin instead of the primary antibody. Human tonsil sections served as positive control. PCNA positive tubular and interstitial cells were counted in 10 fields at a microscopic magnification of x400 (D.O.).
Detection of apoptotic cells with TUNEL staining
DNA fragmentation was detected in situ by 3'-end labelling using a kit containing terminal deoxynucleotidyl transferase (TdT) and digoxigenin-11-dUTP according to the manufacturer's recommendations (ApopTagR, Intergen, NY, USA). Briefly, formalin-fixed and paraffin-embedded 5-µm thick tissue sections were deparafinized and dehydrated. Nuclear proteins were stripped from the DNA by incubating in proteinase K for 30 min, and endogenous peroxidase was blocked with H2O2. Sections were incubated in a buffer containing TdT and digoxigenin-labelled dUTP, followed by digoxigenin conjugated peroxidase treatment. Diaminobenzidine (DAB) was used as chromogen and the background was stained with methyl green. Positive and negative controls were included in every set of experiments. Brown labelled TUNEL positive apoptotic cells in the glomeruli and tubulointerstitium were counted independently by two investigators under x400 magnification. Apoptotic cells were identified by the presence of various types of chromatin condensation (perinuclear ring formation, patches or apoptotic bodies), while cells showing diffuse cytoplasmic labelling considered as necrotic cells were not counted.
Double staining for both apoptosis and ED1 or -SMA
In order to identify the type of apoptotic cells, double immunohistochemical staining was used on paraffin sections for both ISEL (in situ end labelling) and ED1 (detecting the antigen on rat monocytes /macrophages; 1 : 50, Serotec, UK) or -SMA (smooth muscle actin) (detecting myofibroblasts; 1 : 250, DAKO, Denmark). Briefly, 4 mm sections of tissue were dewaxed, and hydrated according to standard protocols. Sections were then digested with proteinase K, endogenous peroxidase was blocked with 3% H2O2, and then taken through the ApopTag protocol from equilibration buffer to stop/wash as in the ApopTag method. Sections were pre-incubated with blocking horse serum for 30 min, labelled with the anti-ED1 or
-SMA antibody at 40°C overnight. Sections were labelled with biotinylated secondary anti-mouse IgG at 37°C for 30 min with alkaline phosphatase streptavidin for another 30 min and developed with Fast Red TR/Naphthol AS-MX solution to produce the bright pink colour. Subsequently, anti-digoxigenin peroxidase antibody was applied on the sections and the development was achieved by the solution of DAB, in which a yellow-brown colour indicates a positive staining.
Isolated perfused kidney
Perfusion of the isolated left kidney was carried out with oxygenated (95% O2, 5% CO2) and warmed (37°C) KrebsHenseleit solution, using a Harvard peristaltic pump (model 1203a) (Newport Beach, CA, USA) at a constant flow of 6 ml/min. KrebsHenseleit solution had the following composition (mM): NaCl, 95; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.6. Mean perfusion pressure was measured via a pressure transducer and monitored continuously on an IBM-compatible personal computer by transducer data acquisition system (MAY 95) (Commat, Ankara, Turkey).
The basal perfusion pressure of the isolated kidneys were stabilized within 30 min. Each kidney preparation from the control and ADR-nephropathy group of rats was used to study the following parameters. (i) Phenylephrine-induced pressor response: phenylephrine from 10-7 to 10-5 M concentration was added into the perfusion reservoir in a cumulative manner. The increase in perfusion pressure was determined and expressed as mmHg. (ii) Acetylcholine-induced vasodilator response: perfusion pressure was increased to 7080% of the maximum pressor response elicited by phenylephrine and acetylcholine at the dose of 0.05 and 0.1 µg was added by injection to a volume of 0.01 ml, through a rubber tube distal to the perfusion pump. The involvement of NO in the acetylcholine-induced vasodilator response was also tested. For this purpose, N-nitro-L-arginine methyl ester (L-NAME) (3x10-5 M) was added into the perfusion reservoir and acetylcholine response was re-evaluated in the control group. (iii) Papaverine and sodium nitroprusside-induced vasodilator response: the vasodilator response elicited by papaverine (2.5 and 5 µg) and sodium nitroprusside (2.5 and 5 µg) were evaluated as described above for acetylcholine. The decrease in perfusion pressure by acetylcholine, papaverine and sodium nitroprusside was expressed as percentage of phenylephrine-induced pressor response.
Statistical analysis
Data are expressed as mean±SEM. The statistical analysis of data was performed by analysis of variance followed by Tukey HSD test. P values <0.05 were considered significant. All the statistical analysis were made using SPSS software.
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Results |
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Biochemical analysis
Rats treated with ADR had significantly higher levels of proteinuria compared with control groups (P<0.05). Treatment with AG significantly reduced proteinuria of rats with ADR-induced nephropathy (Table 1). Serum creatinine levels remained normal in all.
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Renal vascular bed responses
In isolated rat kidneys baseline perfusion pressure was 85.2±5.4 mmHg in the control group (n=5). Perfusion pressure in the ADR-nephropathy group was 91.4±6.3 mmHg (n=6) and was not significantly different from that in the control group (P>0.05).
Phenylephrine (from 10-7 to 10-5 M) evoked a concentration-dependent increase in the perfusion pressure with a maximal pressor response of 172.4±17.1 mmHg in the control group. This response was significantly attenuated in the ADR-nephropathy group and the maximum pressor response reduced to 130.6±16.1 mmHg (P<0.05) (Fig. 1). Treatment with AG did not significantly alter either the baseline perfusion pressure or the pressor response elicited by phenylephrine in the control group; the maximum response was 189.8±16.4 mmHg. On the other hand, in the ADR-nephropathy group AG treatment completely reversed the attenuated phenylephrine response (P<0.05) (Fig. 1
).
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Histopathological examination
Histopathological examinations of the kidneys isolated from the ADR-nephropathy group of rats showed focal areas of mesangial proliferation and mild tubulointerstitial inflammation at the time of sacrifice. The number of tubulo-interstitial cells was significantly increased in the ADR-nephropathy group (90.18±5.86/field) when compared with all the other groups (P<0.01). the mean number of interstitial cells in the control group who received saline was 48.60±4.25/field. The mean number of interstitial cells in animals who received AG following ADR-nephropathy had decreased to 65.74±2.36/field. In the control group animals that received AG this number was 64.65±7.07/field.
Immunohistochemistry
There were no apoptotic cells in the kidney sections of animals in the control groups. In animals with ADR-nephropathy, apoptosis was not noted in the glomeruli. However, in this group, numerous apoptotic cells were identified in the tubulo-interstitial area (Table 1) (Fig. 3
). The ratio of apoptotic cells to the tubular cells was at a median of 0.0212. On the other hand, animals with ADR-nephropathy that received AG treatment, did not show any apoptosis (Table 1
). Apoptotic cells were to a large extent localized in areas of interstitial inflammation. Double immunostaining identified a large number of apoptotic monocytes/macropahages (ED1+ cells) in the animals with ADR-nephropathy (Fig. 4
). On the other hand in animals treated with AG, there were hardly any double-stained cells. Very few cells double-stained for
-SMA (myofibroblasts) and apoptosis in all groups.
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Discussion |
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On the other hand proteinuria per se, has a direct effect on cellular infiltration, as mentioned above. Protein overload has been suggested to induce functional alterations of tubular cells, overexpressing proinflammatory mediators [16]. In the presented study, the tubulointerstitial inflammation of the rats corresponded to the time of overt proteinuria. Thus, one may speculate that the attenuation of interstitial inflammation is all secondary to the inhibition of proteinuria by the medication. However, AG is a well-known inhibitor of iNOS. Thus, we may suggest that the inhibition of NO has suppressed the inflammatory response. In fact in animals treated with AG both the number of cells and the double-stained cells representing the macrophage/monocytes, have significantly decreased. This finding may imply that AG has inhibited infiltration of the macrophage series. On the other hand, AG also has NO-independent effects which could contribute to the inhibition of inflammation, such as inhibition of AGE formation [17].
Our histopathological findings showed mild but clear tubulointerstitial changes with cellular infiltrates, by light microscopical examination. Interstitial inflammation maybe an important determinant of the outcome of glomerular inflammation [18,19]. In a number of studies interstitial inflammatory cells were considered to be more important to renal function than cells in the glomeruli. Goumenos et al. [19] have suggested that myofibroblasts of the interstitium may be playing a crucial role in the pathogenesis of fibrosis in glomerular diseases. The tubulointerstitial cellular response and the localization of apoptotic changes in our rats with nephrosis emphasizes the importance of the tubulointerstitium in nephropathies [18]. It has been suggested that the interstitial changes are mainly produced by downstream diffusion of certain inflammatory mediators [18]. NO may well be one of the mediators concerned. An important feature of this study was the significant increase in interstitial cells and PCNA staining, along with the increased urine nitrite levels in the diseased animals, and there was a decrease in these cells after AG was administered. Furthermore, ED1 stained cells have also decreased after AG treatment. AG is a relatively specific inhibitor of iNOS, which is responsible of production of pathological amounts of NO. The alterations in the renal vascular responses further confirm the increased production of NO in this model. We suggest that these findings may be indicating the role of NO in the process.
The inhibition of NO production through AG has led to a decrease in the number of the cells in the interstitial area, along with a decline in urine nitrite levels and change in renal vascular bed responses. Cattel [6] has suggested that blood borne macrophages were the major source of glomerular NO. However, the role of intrinsic glomerular cells cannot be excluded, either acting subsequently or concomitantly. A limitation of our study was that we are unable to comment on whether the source of iNOS was resident mesangial cells or just infiltrating macrophages. Further immunohistochemical studies with iNOS staining would be required to clarify this point.
Physiological levels of NO, produced by constitutive NOS have a crucial role in the maintenance of renal haemodynamics [5]. NO has beneficial effects relating to its vasodilator and antithrombogenic properties. However, large amounts of NO, produced by iNOS, have been associated with inflammation and NO levels have correlated with the presence of infiltrating macrophages [6]. Weinberg et al. [20] have shown greater NO production and urine nitrite excretion to be associated with glomerulonephritis in their experimental model. On the other hand contradicting reports are present, assigning NO a beneficial role in the pathophysiology of glomerular diseases as well [6].
To evaluate the role of NO in the renal vascular changes induced by ADR-nephropathy, isolated perfused rat kidney was used. Renal vascular bed responses have not been previously studied in nephrotic syndrome. We have observed clear changes in the renal vascular bed, which we have interpreted to be due to excess NO production. The endothelium-dependent vasodilatory response elicited by acetylcholine was significantly attenuated in the ADR-nephropathy group whereas papaverin and sodium nitroprusside-induced vasodilatory responses that are independent of endothelium were preserved. NO produced in large amounts has been suggested to cause the down-regulation of endothelial cNOS or has direct cytotoxic effects on the endothelium [7]. Thus, the induction of iNOS in renal vascular bed may explain the impairment of endothelial response in our experimental nephropathy. The protection of the acetylcholine response by AG treatment supports this proposal and reflects its selective inhibitory potency on iNOS. The attenuated phenylephrine-induced contractile response in the ADR-nephropathy group and its prevention by AG treatment further suggests the increased production of NO in the renal vascular bed. Thus, the renal vascular reactivity studies have validated the effect of NO produced by iNOS, in ADR-nephropathy.
Another adverse effect of NO maybe through inducing cytotoxicity and DNA damage. The interaction of NO with superoxide anion generates peroxinitrite, which induces lipid peroxidation and cytotoxicity [5]. iNOS mediated activation of endonuclease is suggested to play a role into nucleosomal fragments and apoptosis [7]. Again NO-induced inactivation of glutathione peroxidase and oxidative stress may also explain the cytotoxic and apoptotic effects of NO [7]. The association of NO production and apoptosis has been previously demonstrated in a number of cell types such as cortical neurons and vascular smooth muscle cells [8]. Wang et al. [7] have recently demonstrated this relationship in the model of human lupus nephritis as well. The authors have suggested that induction of iNOS, detected by immunohistochemical staining, played a role in the occurrence of apoptosis in the glomeruli of lupus nephritis. We have shown a relationship between iNOS and apoptosis in an experimental kidney disease as well. The association of NO pathway and apoptosis was strengthened by the disappearance of apoptosis in rats treated with AG.
Apoptosis has contradictory effects in kidney diseases. Apoptosis has been assigned a beneficial role in proliferative glomerulopathy induced by anti-Thy-1.1 antibody administration; apoptosis has been suggested to be necessary for regulating the number of intrinsic endothelial cells and for removing the unwanted cells of inflammation [10,11]. On the other hand, Makino et al. [12] have observed TUNEL-positive cells mainly in the sclerotic lesions. Again the role of apoptosis in sclerosis was evident in the rat remnant kidney model [12]. Thus, although apoptosis may initially be a regulatory mechanism, uncontrolled apoptosis seems to be involved in the sclerotic process. In our study model the late phase has not been studied since association with sclerosis was not an aim of this study. However, we have identified the significance of apoptosis in the acute phase of this experimental nephrosis and its association with the NO pathway. An important feature of apoptosis in this study was the tubulo-interstitial localization of the TUNEL-positive cells. Double staining revealed that these cells were negative for -SMA, which characterizes myofibroblasts. Thus, apoptosis was evident in the infiltrating cells in this disease model and may be expected to serve a beneficial role. On the other hand since apoptotic cells have outgrown the proliferative response, it may be speculated that fibrosis may be favoured. Thus, late phase assessments of adriamycin nephrosis are necessary to comment on the prognostic role of apoptosis in this disease model.
We suggest that marked NO production maybe important in the pathogenesis of ADR-nephropathy. Animals with ADR-nephropathy presented with increased nitrite levels and evidence of increased NO production in the renal vasculature along with (i) proteinuria, reflecting the clinical disease activity; (ii) interstitial cell increment, reflecting inflammation; and (iii) apoptosis. These effects including cellular infiltration, were suppressed with the administration of an iNOS inhibitor, AG, which is suggestive of the role of NO in the aforementioned features. The double staining of the inflammatory cells suggested to us that the increased cells undergoing apoptosis were mainly blood-borne. Further studies are needed to enlighten the localization of iNOS formation.
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Acknowledgments |
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Notes |
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References |
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