1 Department of Nephrology and Renal Research Laboratory, Hospital of Bellvitge, L'Hospitalet, Medicine Department, University of Barcelona, CSUB and 2 Uriach Laboratories, Barcelona, Spain
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Abstract |
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Methods. Fifteen- to eighteen-month-old uninephrectomized male Sprague-Dawley rats were allocated into three groups and followed for 16 weeks: UNx, rats without ischaemia; UNxISC, ischaemic kidney (60 min), and UNxISC+UR, ischaemic kidney and UR-12670 from day 0 to the 16th week. Serum creatinine and proteinuria were monitored every 4 weeks. At the end of the study, conventional histology was performed and monocyte-macrophages were identified with the specific monoclonal antibody ED-1.
Results. The UNxISC group had severe acute renal failure with a high mortality rate, which was associated with incomplete restoration of renal function. Renal insufficiency in this group was sustained throughout the follow-up. Both UNx and UNxISC groups developed progressive proteinuria from the 12th week. Though UNxISC+UR group showed similar acute renal failure and mortality rate to the ischaemic non-treated group, serum creatinine decreased to levels similar to UNx group, which were maintained until the end of the study. Treatment of ischaemic kidneys with UR-12670 produced a slight decrease in 24-h proteinuria and a reduction in glomerulosclerosis, the mean tubulointerstitial score and number of monocyte-macrophages to values similar to UNx group.
Conclusions. The chronic administration of the PAF antagonist UR-12670 attenuates the long-term effects of ischaemia-reperfusion injury in uninephrectomized ageing rats. The beneficial effect of this agent suggests that PAF contributes to the progression to late renal damage in this model.
Keywords: ageing; chronic nephropathy; warm ischaemia; PAF; PAF receptor antagonist; tubulo-interstitial damage
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Introduction |
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Platelet-activating factor is a potent phospholipid mediator involved in acute inflammatory and immune responses [9]. Our group has shown that treatment with PAF receptor antagonists protects the kidney from experimental renal warm [10] or cold [11] ischaemia-reperfusion injury, as well as from delayed graft function in cadaveric kidney transplantation [12]. Recently, in a model of warm renal ischaemia and mass reduction in young rats, we reported that PAF is involved in the progression to late renal damage [13]. Experimental studies have also reported that PAF induces the synthesis of extracellular matrix proteins by mesangial [14] and tubular epithelial cells [15], suggesting a role of PAF in glomerulosclerosis and interstitial fibrosis.
Dual renal transplantation is one of the strategies proposed for kidneys from older donors to overcome the deleterious effect of grafting insufficient renal mass [16]. Indeed, this approach may also overcome the long-term consequences of transplanting only one ischaemic kidney, as our group has shown in a model of warm bilateral ischaemia but in young animals [17]. Unfortunately, because of the lack of donors, most centres only transplant a single older kidney, but attempt to minimize factors that may aggravate renal function and structure [1,2]. No study has evaluated in ageing rats the influence of warm renal ischaemia-reperfusion injury associated with nephron mass reduction on events that lead to chronic renal dysfunction, glomerulosclerosis, and severe tubulointerstitial damage. Here we assess the contribution of warm ischaemia to the development of chronic nephropathy in native kidneys in older uninephrectomized rats, and to assess whether PAF is involved in the progression of this damage by blocking its receptor with the oral PAF antagonist UR-12670.
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Materials and methods |
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Rats were allocated into three groups, as follows. Two control groups: the UNx group (single kidney), uninephrectomized rats with a left non-ischaemic kidney (n=12) and the UNxISC group (single ischaemic kidney), uninephrectomized rats with left renal warm ischaemia (n=20); and a long-term treated group: the UNxISC+UR, uninephrectomized rats with left renal warm ischaemia and UR-12670 administration from day 0 until the 16th week (n=24).
Before the experiment and every 4 weeks after the surgical procedure all animals were placed in metabolic cages in order to collect a 24-h urine sample. Animals were weighed at the same intervals and 0.5 ml of blood was obtained from the tail vein.
PAF antagonist administration
UR-12670 (Uriach Laboratories, Barcelona, Spain) is a potent orally active and highly selective receptor PAF antagonist that has been tested in several in vivo models [18,19]. The chemical name of UR-12670 is 1-[[1-(3,3-diphenylpropanoyl)-4-piperidyl]methyl]-1-H-2-methylimidazo [4,5-c] pyridine. It has a molecular weight of 438.57 and is a hydrosoluble compound with a 100% oral bioavailability [18]. UR-12670 was administered intravenously (i.v.) at 10 mg/kg, 5 min before reperfusion, and later given orally at 20 mg/kg by daily gavage.
Biochemistry data
Every 4 weeks, serum creatinine (µmol/l) was determined by Jaffe's reaction on an autoanalyzer (Beckman Instruments, Palo Alto, CA, USA). Serum creatinine has been shown to be representative of kidney function in ageing rats [8]. At the same intervals, proteinuria (mg/24-h) was determined by the Ponceau method (Bayer Diagnósticos, Madrid, Spain). Finally, creatinine clearance was calculated by the standard formula (ml/min).
Histological studies
For histological studies, coronal 12 mm-thick slices of the kidneys were fixed in 4% formaldehyde and embedded in paraffin. For light microscopy 34-µm-thick tissue sections were stained with haematoxylin-eosin, periodic acid-Schiff and silver methenamine. Sections were reviewed by a pathologist blinded to the treatment groups, and examined for glomerular, vascular and tubulointerstitial lesions. The percentage of glomerulosclerosis was calculated. Tubular atrophy, tubular dilatation, interstitial cellular infiltrate, interstitial fibrosis, and vasculopathy were graded from 0 to 3+ as follows: 0, no abnormalities; 1+, abnormalities affecting less than one-third of the sample; 2+, abnormalities affecting between one- and two-thirds of the sample; 3+, abnormalities affecting more than two-thirds of the sample. The total tubulointerstitial score was the sum of the four individual tubulointerstitial parameters.
For immunohistochemical study, representative 34-µm-thick tissue sections were stained with a monoclonal mouse anti-rat ED-1 antibody for monocyte/macrophage (Oxford Biomarketing, Oxford, England). The sections were then incubated with purified rabbit anti-mouse IgG (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) by the avidine-biotine method and counterstained with haematoxylin. Positive ED-1 stained cells infiltrating kidneys were counted and expressed as mean±SEM of cells per field of view (ED-1+ cells/FV, 400x, 20 counted fields/kidney).
Glomerular morphometry
In periodic acid-Schiff sections, the glomerular tuft area was calculated by point counting, using a 20x20 point eyepiece reticular grid and a magnification of x200. In each case, 30 glomeruli were counted. Mean glomerular volume (GV, µ3/106) was estimated from its area (PxA) and corrected to account for the tissue shrinkage associated with paraffin embedding (f), according to the Weibel and Gómez formula (20): GV=[(PxA)3/2xB/K]/f, where P=average of points per glomerulus; A=point area (µ2); B=1.38, shape coefficient for a sphere; K=1.01, size distribution coefficient for a 10% coefficient of variance; and f=0.69, shrinkage coefficient.
The increment () of mean glomerular volume was calculated with the formula: [(GV study kidney-GV contralateral kidney)/GV contralateral kidney]x100, where the contralateral kidney is the right kidney excised 5 days after ischaemia.
Statistical analysis
To compare the mortality ratio from acute renal failure between treated and non-treated groups the 2 test was used. To analyse the overall survival, the Kaplan-Meier and log-rank methods were used. To compare more than two groups for proteinuria, serum creatinine, and creatinine clearance throughout the follow-up statistical analysis was performed by one-way analysis of variance for two-factor repeated measures. To determine the origin of the differences at any time point for those parameters and to compare ED-1+ cells the one-way analysis of variance followed by Scheffe's test were used. To compare histological data the non-parametric Kruskall-Wallis test and subsequent Connover's test was used. All P values were two tailed, and a P value of <0.05 was considered statistically significant. Data are presented as mean±SEM.
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Results |
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Long-term effect of UR-12670 on renal functional parameters after renal warm ischaemia in ageing uninephrectomized rats
Additional animals died in the three groups during the follow-up because of experimental stress or old age (Figure 1). No animal died from renal failure. Body weight remained stable and no differences were between the three groups throughout the follow-up (Table 1
).
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Effect of UR-12670 on long-term renal morphology after renal warm ischaemia in ageing uninephrectomized rats
At the end of the study, in all groups kidney weight was significantly higher than contralateral kidney at the time of ischaemia, suggesting compensatory hypertrophy after nephrectomy (Table 1) but there were no differences between the three groups. Accordingly, the increase in mean glomerular volume after nephrectomy was similar in all three groups (Table 2
). However, percentage of glomerulosclerosis in UNxISC group was higher than in UNx group. Treatment with UR-12670 was associated with a decrease in glomerulosclerosis to a value similar to that of the UNx group.
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Discussion |
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Ischaemic acute renal failure in young animals has been treated with several compounds with success [21,22]. However, not all the drugs or the therapeutic schedules are equally effective in young and ageing animals [23]. Indeed ischaemic ageing kidney is relatively resistant to protective drugs [23]. The group of Andreucci [24] reported that older rats presented a higher and more prolonged decrease in GFR than young rats after normothermic ischaemia. They also showed that dimethylthiourea protects young animals from ischaemic acute renal failure, although it had no effect on older animals. In contrast to dimethylthiourea, superoxide dismutase restored renal dysfunction in older rats, while it induced only slight changes in GFR in young rats. We have previously shown [11,13] that BN 52021 and UR 12670, both platelet-activating factor receptor antagonists, protected the kidney from ischaemic acute renal failure in young animals. In the present study, the lack of effect of UR-12670 in the early phase of warm ischaemic acute renal failure in ageing rats may be related to the particular resistance of ageing kidney to protective drugs. Old kidneys show alterations in the balance of vasoconstrictor and vasodilator factors [3], which reduces renal blood flow. Thus, although the PAF receptor antagonist has blocked the PAF-renal vasoconstriction action, the effect of the unbalance of other vasoactive systems, such as nitric oxide or prostaglandins, may predominate [9].
The long-term impact of the initial warm ischaemia injury was analysed by following up the animals for 16 weeks after the insult. Our group [13] has shown that a single kidney subjected to warm ischaemia in uninephrectomized young rats develops chronic renal insufficiency and morphological damage that mimics chronic transplant nephropathy, and that this damage was prevented by the long-term administration of the PAF receptor antagonist UR-12670. Using a similar experimental model, here we extend those observations to ageing animals. We found that kidneys from uninephrectomized ageing animals developed glomerular hypertrophy and proteinuria, but maintained stable renal function. These findings were similar to young rats but proteinuria in ageing uniphrectomized animals appeared earlier and became more severe [13]. When these older kidneys suffered from warm ischaemia, further abnormalities were seen. Thus, these animals developed severe renal insufficiency, tubulointerstitial damage and glomerulosclerosis with only slight vascular myointimal hyperplasia. In our previous work [13], renal function in young animals completely recovered within 4 and 8 weeks after ischaemia, and histology at 16 weeks showed minimal tubulointerstial abnormalities. The onset of renal failure in these rats delayed until 40th week and severe glomerulosclerosis and interstitial fibrosis appeared at 52th week. By the contrary, in UNxISC ageing animals renal function never recovered after ischaemia and, at the time of sacrifice at 16 weeks, severe chronic renal damage was observed. Those findings strongly suggest that ageing rats are more sensitive to the injury induced by warm ischaemia and nephron mass reduction, resulting in accelerated loss of renal function.
PAF is a key mediator in both the vasomotor and the inflammatory responses occurring early in post-renal ischaemia [10,11,25]. It has also been associated with the regulation of mechanisms responsible for renal changes in ageing [4]. The improvement of renal failure and the reduction in tubulointerstitial damage and glomerulosclerosis with UR-12670 treatment in the present study suggest that PAF could also be considered in ageing animals as an important mediator of renal tissue matrix remodelling following ischaemia in nephron mass ablation. The pathogenic mechanisms that lead to this chronic organ damage remain partially unknown and usually only studied in young animals. In this setting, however, it is known that post-ischaemic organs are progressively infiltrated by T cells and macrophages, mainly located in glomeruli and around vessels [26]. In our model, the ischaemic injury chronically damaged the tubulointerstitial compartment. The presence of a high number of ED-1+ cells in those ischaemic kidneys compared with the scarce number of those cells in non-ischaemic kidneys suggests that macrophages may act as effectors of this post-ischaemic chronic nephropathy. In various models of chronic nephropathy it has been demonstrated that infiltrating T-cells and macrophages are sources of PAF [15,27] and that treatment with a PAF antagonist reduces the number of interstitial cell infiltrates [28]. Moreover, human macrophages constitutively express PAF receptor mRNA transcripts as well as specific binding sites for this mediator [29]. In the present study, UR-12670 treatment dramatically reduced the number of infiltrating macrophages in the interstitium of ischaemic kidneys, suggesting that the PAF antagonist blocked the binding of PAF to its receptor in macrophages, thus inhibiting the activation and further recruitment of more effector cells.
Another mechanisms may explain the beneficial effect of UR-12670 in our chronic model. PAF alters the physicochemical properties of the glomerular capillary wall by both a direct effect [30] and through the action of inflammatory cells [31], inducing proteinuria. In fact, the increased synthesis of PAF from isolated glomeruli of aged rats may account for this local action [32]. In some proteinuric nephropathies [31] an enhanced glomerular PAF production has been documented and, in these studies, PAF receptor antagonists had an anti-proteinuric effect. Therefore, the lowering of proteinuria in UR-12670 treated rats compared with both ischaemic and non-ischaemic uninephrectomized animals, as seen in our model, could be related to the anti-proteinuric effect of PAF receptor blockade.
In summary, our results provide evidence that warm renal ischaemia and mass reduction in ageing animals induces chronic nephropathy mainly manifested as renal failure and tubulointerstitial damage. The protective effect of long-term treatment with the PAF receptor antagonist, UR-12670, suggests that PAF may be actively involved in the progression to this chronic nephropathy. The study of compounds that could be incorporated to the treatment of chronic nephropathies, such as that following single kidney transplantation from suboptimal donors, is needed.
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Acknowledgments |
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Notes |
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References |
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