Protective effect of UR-12670 on chronic nephropathy induced by warm ischaemia in ageing uninephrectomized rats

Núria Lloberas1, Josep M. Cruzado1, Joan Torras1, Immaculada Herrero-Fresneda1, Marta Riera1, Manel Merlos2 and Josep M. Grinyó,1

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



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. In young animals, renal ischaemia/reperfusion injury and mass reduction are associated with chronic lesions that mimic those found in chronic rejection. We have shown that the phospholipid platelet-activating factor (PAF) participates in young animals in such chronic nephropaty. Here we examine the long-term effects of the orally active PAF antagonist, UR-12670 in ageing uninephrectomized rats exposed to prolonged warm ischaemia.

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



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Donor age has increased due to the continuous increase in organ demand [1,2]. Ageing kidneys have long been considered apt for organ transplantation although they may be susceptible to functional and structural changes [3,4]. A decline in glomerular filtration rate (GFR) and a parallel reduction in renal blood flow from the cortex to the medulla have been observed with age [5]. Glomerulosclerosis is the classic structural finding, but tubulointerstitial fibrosis also contributes to damage in ageing kidney [6]. Tubulointerstitial injury is associated with interstitial inflammation and fibroblast activation, and it has been related to ischaemia secondary to peritubular capillary injury and altered endogenous nitric oxide synthase (eNOS) expression [6]. Several other inflammatory mediators and fibrogenic growth factors, such as PAF [7] and TGF-ß1 [8], are involved in renal interstitial fibrosis associated with ageing.

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.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental design
Fifteen- to eighteen-month-old male Sprague-Dawley rats (650–750 g body weight, survival time of this animal colony is within 24 and 30 months) were used in this study. Animal care and interventions were conducted in accordance with the guidelines of the European Community Committee on Care and Use of Laboratory Animals and Good Laboratory Practice. Anaesthesia was induced with an intramuscular (i.m.) mixture of ketamine (75 mg/kg), atropine (0.05 mg/kg) and diazepam (5 mg/kg). For renal ischaemia, a medial laparotomy was performed and the left renal pedicle was occluded for 60 min using a non-traumatic clamp. Five days after ischaemia, right nephrectomy was performed. Animals were housed in a room kept at constant temperature with a 12-h dark/12-h light cycle and free access to tap water and a standard diet (protein 14.14%, fat 2.65% and fibre 4.19%; PANLAB SL, Barcelona, Spain).

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 1–2 mm-thick slices of the kidneys were fixed in 4% formaldehyde and embedded in paraffin. For light microscopy 3–4-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 3–4-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 ({Delta}) 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 {chi}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.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of UR-12670 on ischaemic acute renal failure in ageing uninephrectomized rats
We first analysed the early consequences of ischaemic injury on renal function and mortality in uninephrectomized ageing rats and the effect of UR-12670. Five out of 20 animals (25%) from UNxISC group and five out of 24 (20.8%) from the ischaemic UR-12670-treated group died within the first week because of acute renal failure (P, non significant) (Figure 1Go). There was no early mortality in the UNx group.



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Fig. 1. Kaplan-Meier survival analysis of the experimental groups. There were not significant differences among the three experimental groups (P=0.16).

 
Severity of acute renal failure was similar in both UNxISC and UNxISC+UR groups (Figure 2Go) with a peak serum creatinine of 309±49 and 277±40 µmol/l within the first week, respectively. Despite this similar extent of acute renal dysfunction the recovery of renal function was improved by UR-12670 treatment. Thus, 4 weeks after renal ischaemia, serum creatinine was similar in ischaemic UR-12670 treated and non-ischaemic animals whereas it was higher in the UNxISC group.



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Fig. 2. Serum creatinine profile after 60 min of warm renal ischaemia in older rats. The UNxISC group had a severe acute renal failure that was associated with an incomplete restoration of renal function. Though UNxISC+UR group showed similar acute renal failure than ischaemic non-treated group, serum creatinine decreased to levels similar to UNx group. *P=0.01; P<0.05 UNxISC vs UNxISC+UR and UNx.

 

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 1Go). No animal died from renal failure. Body weight remained stable and no differences were between the three groups throughout the follow-up (Table 1Go).


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Table 1. Body weight, kidney weight and creatinine clearance per 100 g body weight at the end of the experiment at 16 weeks

 
In the UNxISC group, the degree of renal dysfunction achieved in the fourth week was sustained throughout the follow-up, with a serum creatinine level significantly higher than UNx group (Figure 3Go). In the 16th week, UNxISC group showed moderate renal failure with a mean serum creatinine of 122±17 µmol/l. Conversely, continuous long-term treatment of ischaemic uninephrectomized rats with UR-12670 improved renal function, achieving serum creatinine values similar to those of UNx group (UNxISC+UR, 81±8; UNx, 73±4 µmol/l, P=0.01). Differences in creatinine clearance throughout the study paralleled those of serum creatinine (Table 1Go).



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Fig. 3. Serum creatinine profile throughout the follow-up. In the UNxISC group, the degree of renal dysfunction in the fourth week was sustained throughout the follow-up with a serum creatinine level significantly higher than UNx group. Conversely, continuous long-term treatment of ischaemic uninephrectomized rats with UR-12670 ameliorated renal function achieving serum creatinine values similar to those of UNx group. *P<0.05 UNxISC vs UNxISC+UR and UNx; $P<UNxISC vs UNx.

 
As shown in the Figure 4Go, uninephrectomy in ageing rats induced proteinuria that was not further enhanced by warm ischaemia. The continuous administration of UR-12670 had an anti-proteinuric effect. At the end of the study, proteinuria was 123±35 in UNx, 145±22 in UNxISC and 69±13 mg/24 h in UNxISC+UR group (P=0.01).



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Fig. 4. Twenty-four-hour proteinuria profile throughout the follow-up. Treatment with UR-12670 produced an anti-proteinuric effect. *P<0.05 UNxISC+UR vs UNxISC and UNx

 

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 1Go) 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 2Go). 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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Table 2. Histological parameters

 
Vascular lesions were diffusely mild in all groups. Myointimal hyperplasia was slightly, but not significantly, higher in UNxISC group than in the other two groups (Table 2Go). Tubular atrophy, tubular dilation and fibrosis were uniform within each group, and the UNxISC group showed the highest values (Table 2Go). This tubular injury in the UNxISC group was associated with higher generalized and focal inflammatory cell infiltration in the interstitium than in the UNx group. Large concentrations of inflammatory cells were seen in association with tubular dilation, tubular atrophy and interstitial fibrosis. Concerning the monocyte-macrophage composition of cell infiltrate, as determined with inmunostaining with the specific monoclonal antibody ED-1, UNxISC kidneys showed more ED-1 positive cells than UNx kidneys. Conversely, after long-term treatment with UR-12670 both the semiquantitative value and the number of ED-1 positive cells were similar to that of the UNx group. Finally, as shown in Table 2Go, the total tubulointerstitial score was significantly higher in UNxISC than in the other two groups.



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Here we explore the possibility that the administration of the PAF receptor antagonist UR-12670 could attenuate the long-term effects of ischaemia/reperfusion injury in uninephrectomized ageing rats. Our results indicate that warm ischaemia on kidneys from such rats induced chronic renal failure, and aggravated the glomerulosclerosis and tubulointerstitial lesions present in non-ischaemic kidneys. Animals chronically receiving UR-12670 did not develop chronic renal failure, and showed a marked reduction in tubulointerstitial injury and glomerulosclerosis. The beneficial effect of UR-12670 strongly suggests that PAF contribute to the progression of chronic renal damage in this model.

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.



   Acknowledgments
 
This study was supported by a grant from Uriach Laboratories, Barcelona, Spain, and by grants from Fondo de Investigaciones Sanitarias (FIS 98/0756 and 98/0029–02). Núria Lloberas is a fellow from ‘Fundació August Pi Sunyer’, Marta Riera from Fundació Catalana de Trasplantament and Immaculada Herrero-Fresneda from Instituto de Salud Carlos III (BISCIII 99/4262). Uriach Laboratories, Barcelona, Spain, kindly provided the UR-12670.



   Notes
 
Correspondence and offprint requests to: Josep M. Grinyó, Nephrology Department, Renal Research Laboratory, Pabello de Govern, Hospital de Bellvitge, CSUB, Feixa Llarga s/n, E-08907 L'Hospitalet, Barcelona, Spain. Back



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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 7.00
Revision received 13.11.00.