Angiotensin type 1 and type 2 receptor blockade in chronic allograft nephropathy
Authors: J. Lutz, K. Risch, S. Liu, B. Antus, C. Schmaderer, M. Roos, N. Ouyang, M. Lehmann, U. Heemann
Journal: Kidney International (2006)

Angiotensin-II (Ang-II) type 1 (AT1) receptor blockers may delay the progression of chronic allograft nephropathy (CAN). However, neither the optimal time for initiating AT1 receptor blockade in order to delay CAN potentially nor the role of Ang-II type 2 (AT2) receptors under AT1 receptor blockade is known. Both AT receptors can regulate p53 expression and apoptosis. We investigated what time of initiation with AT1 blockers most effectively delayed CAN as well as the role of the AT2 receptor, and how angiotensin receptor blockade affected apoptosis and its regulating factors in this context in a rat model. Kidneys of Fisher (F344) rats were transplanted into Lewis rats. Animals were treated with AT1 (candesartan) and/or AT2 (PD123319) receptor antagonists, a calcium channel blocker, or vehicle (treatment periods: day -7 before to week 24 after transplantation (long term), week 12 to week 24 (late), day -7 to day +5 (early)) and observed the animals for 24 weeks. Reduction of proteinuria, grade of CAN, and number of apoptotic cells was most pronounced in animals receiving long-term AT1 receptor blockade. A combined AT1/AT2 blocker treatment reduced CAN similarly to AT1 blocker treatment alone. The number of apoptotic cells and the level of p53 mRNA were significantly lower in long-term AT1 blocker-treated animals. In summary, AT1 receptor blockade delayed the progression of CAN, particularly in animals treated long term. Reduction of apoptosis could be related to these beneficial effects. The AT2 receptor does not appear to play an important role in CAN. Angiotensin-II (Ang-II) type 1 (AT1) receptor blockers may delay the progression of chronic allograft nephropathy (CAN). However, neither the optimal time for initiating AT1 receptor blockade in order to delay CAN potentially nor the role of Ang-II type 2 (AT2) receptors under AT1 receptor blockade is known. Both AT receptors can regulate p53 expression and apoptosis. We investigated what time of initiation with AT1 blockers most effectively delayed CAN as well as the role of the AT2 receptor, and how angiotensin receptor blockade affected apoptosis and its regulating factors in this context in a rat model. Kidneys of Fisher (F344) rats were transplanted into Lewis rats. Animals were treated with AT1 (candesartan) and/or AT2 (PD123319) receptor antagonists, a calcium channel blocker, or vehicle (treatment periods: day -7 before to week 24 after transplantation (long term), week 12 to week 24 (late), day -7 to day +5 (early)) and observed the animals for 24 weeks. Reduction of proteinuria, grade of CAN, and number of apoptotic cells was most pronounced in animals receiving long-term AT1 receptor blockade. A combined AT1/AT2 blocker treatment reduced CAN similarly to AT1 blocker treatment alone. The number of apoptotic cells and the level of p53 mRNA were significantly lower in long-term AT1 blocker-treated animals. In summary, AT1 receptor blockade delayed the progression of CAN, particularly in animals treated long term. Reduction of apoptosis could be related to these beneficial effects. The AT2 receptor does not appear to play an important role in CAN. Chronic allograft nephropathy (CAN), a major cause of late allograft failure, is characterized by glomerulopathy, tubular atrophy, interstitial fibrosis, and vasculopathy.1.Hariharan S. Johnson C.P. Bresnahan B.A. et al.Improved graft survival after renal transplantation in the United States, 1988–1996.N Engl J Med. 2000; 342: 605-612Crossref PubMed Scopus (1626) Google Scholar,2.Racusen L.C. Solez K. 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Angiotensin-converting enzyme inhibitors.Circulation. 1998; 97: 1411-1420Crossref PubMed Scopus (609) Google Scholar and Angiotensin-II (Ang-II) type 1 (AT1) receptor blockers6.Amuchastegui S.C. Azzollini N. Mister M. et al.Chronic allograft nephropathy in the rat is improved by angiotensin II receptor blockade but not by calcium channel antagonism.J Am Soc Nephrol. 1998; 9: 1948-1955PubMed Google Scholar ameliorates the progression of chronic renal diseases including CAN.7.Mulec H. Johnsen S.A. Björck S. Long-term enalapril treatment in diabetic nephropathy.Kidney Int. 1994; 45: S141-S144Google Scholar However, the optimal time point for the initiation of AT1 receptor blockade to achieve the best amelioration of long-term graft function is as unknown as the contribution of the AT1 and the Ang-II type 2 (AT2) receptors in this context. Among other effects, signalling via the AT1 receptor increases blood pressure, and promotes cellular proliferation as well as extracellular matrix production, thus contributing to the progression of fibrosis. The role of the AT2 receptor on the other hand, which could be involved in antiproliferative, antiangiogenic, and blood pressure lowering effects during the development of CAN, is not yet clear. Thus, the amelioration of fibrosis in CAN by AT1 receptor blockade may also depend on signalling via non-blocked AT2 receptors. Furthermore, the mechanisms involved in the protective effects of AT1 receptor blockade are not fully understood. Apart from reduced proteinuria and proliferation, a reduced number of apoptotic cells in the graft may be related to the amelioration of CAN by AT1 receptor antagonists. Apoptosis is an intrinsically controlled process of cellular suicide8.Majno G. Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death.Am J Pathol. 1995; 146: 13-15Google Scholar and can significantly contribute to tissue damage by a reduction of functioning kidney cells.9.Ortiz A. Lorz C. Justo P. et al.Contribution of apoptotic cell death to renal injury.J Cell Mol Med. 2001; 5: 18-32Crossref PubMed Scopus (62) Google Scholar An increased number of apoptotic cells has been described in human graft biopsies with CAN,10.Laine J. Etelämäki P. Holmberg C. Dunkel L. Apoptotic cell death in human renal allograft rejection.Transplantation. 1997; 63: 101-105Crossref PubMed Scopus (59) Google Scholar whereas a reduced expression of cytoprotective factors together with an increased number of apoptotic cells has been observed in a rat model of CAN.11.Djamali A. Reese S. 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Cox A. et al.Angiotensin type 2 receptor is expressed in the adult rat kidney and promotes cellular proliferation and apoptosis.Kidney Int. 2000; 58: 2437-2451Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar These divergent results could be related to different cell types and treatment conditions. How Ang-II influences apoptosis is not fully understood. A potential factor involved in Ang-II-mediated apoptosis is p53, which can result in cell cycle arrest and/or apoptosis.19.Schuler M. Green D.R. Mechanisms of p53-dependent apoptosis.Biochem Soc Trans. 2001; 29: 684-688Crossref PubMed Google Scholar Stimulation of AT1 receptors increased p53 in parallel to the number of apoptotic cells in a model of stretch-induced stress in myocytes.20.Leri A. Liu Y. 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Effect of AT2 receptor blockade on the pathogenesis of renal fibrosis.Am J Physiol. 1999; 276: F39-F45PubMed Google Scholar Here, we analyzed the effects of AT1 and AT2 receptor blockade over different periods after kidney transplantation on the progression of CAN, the role of the AT2 receptor during CAN, and how AT receptor blockade influences apoptosis and the expression of p53 as a possible link between Ang-II and apoptosis regulation. Animals receiving long-term AT1- (AT1) and combined AT1/AT2 antagonist treatment (AT1+AT2) developed a significantly lower proteinuria than animals receiving AT2 antagonist- (AT2) and vehicle (VEH) treatment (P≤0.03; Figure 1a), whereas proteinuria tended to be lower as compared to animals treated with a calcium channel blocker (CCB) (P=0.06 and 0.11). Animals receiving late and early treatment developed no statistically significant differences (P>0.05; Figure 1b and c), although animals receiving late AT1- (AT1) and combined AT1-/AT2 antagonist treatment (AT1+AT2) had lower proteinuria as animals of groups AT2, CCB, and VEH. Creatinine clearances as well as mean arterial pressure and body weights did not differ significantly between the groups at week 24 (Table 1).Table 1Body weight (g; mean±s.e.m.), MAP (mm Hg; mean±s.e.m.), and creatinine clearance (ml/min; mean±s.e.m.)GroupAT1AT2AT1+AT2CCBVEHLong-term treatment (n)77886 Body weight (g)480.6±19.9474.0±12.6488.8±9.6488.0±9.7497.5±9.8 MAP (mm Hg)92.6±3.995.2±6.686.7±4.189.8±6.492.5±6.6 Creatinine clearance (ml/min/kg)4.01±0.374.09±0.364.02±0.434.66±0.553.78±0.24 Glomerulosclerosis (%)11.5±1.3°P=0.006 vs AT2, P=0.006 vs CCB, and P=0.012 vs VEH24.5±1.816.5±1.4P=0.012 vs AT2, P=0.012 vs CCB27.5±2.027.7±1.6 Grade of macrophage infiltration♦♦♦ 05—4—— Grade 121441 Grade 2—4—22 Grade 3—2—23 PCNA (cells/field of view)4.9±0.65.5±0.85.7±1.14.1±0.54.5±0.5Late treatment (n)67788 Body weight (g)467.0±20.2472.5±9.4474.3±19.4479.3±6.3479.1±11.9 MAP (mm Hg)93.7±8.191.3±7.188.8±5.894.8±8.595.4±5.9 Creatinine clearance (ml/min/kg)4.02±0.344.17±0.413.67±0.363.67±0.954.18±0.22 Glomerulosclerosis (%)18.5±2.423.4±3.118.1±1.721.1±2.122.1±1.5 Grade of macrophage infiltration 03—311 Grade 122333 Grade 213132 Grade 3—2—12 PCNA (cells/field of view)4.2±2.24.1±1.84.9±0.84.4±0.74.0±2.4Early treatment (n)64666 Body weight (g)472.0±13.5482.5±18.3484.7±8.5486.7±9.0460.5±16.6 MAP (mm Hg)94.7±4.294.0±10.191.6±4.891.0±8.893.6±6.9 Creatinine clearance (ml/min/kg)4.23±0.34.12±0.333.78±0.223.85±0.384.01±0.24 Glomerulosclerosis (%)23.6±3.125.8±3.327.4±3.225.6±3.922.9±2.1 Grade of macrophage infiltration 01—211 Grade 121232 Grade 212222 Grade 321——1 PCNA (cells/field of view)4.9±1.05.9±1.45.0±0.95.7±0.75.0±0.4AT1, Angiotensin-II type 1; AT2, Angiotensin-II type 2; CCB, calcium channel blocker; MAP, mean arterial pressure; PCNA, proliferating cell nuclear antigen; VEH, vehicle.Glomerulosclerosis of graft tissue of the experimental groups: long-term treatment: P=0.024 vs VEH. Late and early treatment: P>0.05 AT1 and AT1+AT2 vs AT2, CCB, and VEH.Grade of infiltration by macrophages (CD68 positive cells): 0=<5 cells/field of view; grade 1=6–25 cells/field of view; grade 2=26–50 cells/field of view; and grade 3=>50 cells/field of view. Numbers represent number of cases with the respective grade of infiltration: Long-term treatment: ♦P=0.01 vs AT2, P=0.028 vs CCB, and P=0.016 vs VEH; ♦♦P=0.008 vs AT2, P=0.006 vs CCB, and P=0.013 vs VEH. Late and early treatment: P>0.05 AT1 and AT1+AT2 vs AT2, CCB, and VEH.Number of proliferating cells (PCNA) (cells/field of view; mean±s.e.m.).Long-term treatment (day -7 to week 24), late treatment (week 12 to week 24), and early treatment (day -7 to day +5); n=number of animals analyzed 24 weeks after transplantation.° P=0.006 vs AT2, P=0.006 vs CCB, and P=0.012 vs VEH°° P=0.012 vs AT2, P=0.012 vs CCB Open table in a new tab AT1, Angiotensin-II type 1; AT2, Angiotensin-II type 2; CCB, calcium channel blocker; MAP, mean arterial pressure; PCNA, proliferating cell nuclear antigen; VEH, vehicle. Glomerulosclerosis of graft tissue of the experimental groups: long-term treatment: P=0.024 vs VEH. Late and early treatment: P>0.05 AT1 and AT1+AT2 vs AT2, CCB, and VEH. Grade of infiltration by macrophages (CD68 positive cells): 0=<5 cells/field of view; grade 1=6–25 cells/field of view; grade 2=26–50 cells/field of view; and grade 3=>50 cells/field of view. Numbers represent number of cases with the respective grade of infiltration: Long-term treatment: ♦P=0.01 vs AT2, P=0.028 vs CCB, and P=0.016 vs VEH; ♦♦P=0.008 vs AT2, P=0.006 vs CCB, and P=0.013 vs VEH. Late and early treatment: P>0.05 AT1 and AT1+AT2 vs AT2, CCB, and VEH. Number of proliferating cells (PCNA) (cells/field of view; mean±s.e.m.). Long-term treatment (day -7 to week 24), late treatment (week 12 to week 24), and early treatment (day -7 to day +5); n=number of animals analyzed 24 weeks after transplantation. One animal of each group AT1, AT2, and VEH receiving long-term treatment died owing to surgical complications; one animal of group VEH died after week 12. One animal of group AT1 receiving late treatment died after surgery. One animal from each group AT1, AT2, and AT1+AT2 died after 20 weeks. One animal of group AT2 receiving early treatment died after 12 and 20 weeks, respectively. Animals of long-term treatment group AT1 or group AT1+AT2 developed a significantly lower grade of CAN as compared to the groups AT2, CCB, and VEH (P<0.05; Figure 2a). Animals treated late or early did not significantly differ in the grade of CAN. However, animals receiving late treatment of groups AT1 as well as group AT1+AT2 had, although not significant, lower grades of CAN (P>0.05; Figure 2b and c). Glomerulosclerosis was significantly lower in animals treated long term with the AT1 receptor antagonist as compared to groups AT2, CCB, and VEH (P≤0.012; Table 1, Figure 3a). Animals of group AT1+AT2 had a lower glomerulosclerosis as compared to the groups AT2, CCB, and VEH (P≤0.025). Glomerulosclerosis in animals receiving late and early treatment did not significantly differ, although, again, late treatment in groups AT1 and AT1+AT2 resulted in a lower glomerulosclerosis as compared to the groups AT2, CCB, and VEH (P>0.05; Table 1). Animals of long-term treatment group AT1 had a significantly reduced number of macrophages in graft tissues as compared to group AT2 (P=0.01). Macrophage infiltration was also lower as compared to groups CCB and VEH (P<0.03; Table 1). Animals of group AT1+AT2 had a significantly reduced macrophage infiltration as compared to groups AT2, CCB, and VEH (P<0.01). Late and early treatment resulted in no significant differences. However, animals of late treatment group AT1 and AT1+AT2 had a reduced number of graft-infiltrating macrophages as compared to groups AT2, CCB, and VEH, (P>0.05; Table 1). The number of proliferating cells did not differ significantly between the experimental groups at the end of the observation period 24 weeks after transplantation (Table 1). Animals of long-term treatment groups AT1 and AT1+AT2 had a significantly lower number of apoptotic cells in their grafts as compared to animals of group AT2 and VEH (P≤0.03; Figures 3b and 4a). The number of apoptotic cells in grafts of animals of late and early treatment groups AT1 or AT1+AT2 were not significantly different as compared to groups AT2, CCB, and VEH (P>0.05; Figure 4b and c). The lower number of apoptotic cells in animals of long-term treatment group AT1 was paralleled by a significant shift of the mRNA ratio of bcl-2/bax towards bcl-2 mRNA (coding for apoptosis-inhibiting Bcl-2) in group AT1 as compared to groups AT2 (P=0.045) and VEH (P=0.012), as well as group AT1+AT2 as compared to group VEH (P=0.018; Figure 5a). A significant shift of the mRNA ratio of bcl-2/bax towards bcl-2 mRNA was observed in animals of late treatment groups AT1 and AT1+AT2 (P<0.01; Figure 5b), whereas early treatment resulted in no significant differences as compared to controls (P>0.05; Figure 5c). Animals of long-term treatment group AT1 had significantly lower p53 levels as compared to animals of groups AT2 (P=0.03) and VEH (P=0.06; Figure 6a). Late and early treatment resulted in no significant differences in the expression of p53 (P>0.05; Figure 6b and c). Animals of long-term treatment group AT1 had significantly lower levels of caspase-1 and caspase-3 mRNA as compared to animals of groups AT2 and VEH (P≤0.03; Figures 7a and 8a). Animals of group AT1+AT2 had significantly reduced caspase-3 mRNA levels as compared to animals of groups CCB and VEH (P=0.03; Figure 7a), whereas caspase-1 mRNA levels were significantly lower as compared to animals of group VEH (P=0.012; Figure 8a).Figure 8Caspase-1 mRNA. (a) Animals receiving long-term (day -7 before transplantation to week 24 after transplantation) AT1 antagonist treatment (group AT1) had significantly lower levels of caspase-1 mRNA as compared to groups AT2, CCB, and VEH (*AT1 vs AT2: P=0.027; vs CCB: P=0.024; vs VEH: P=0.012. **AT1+AT2 vs AT2: P=0.14; vs CCB: P=0.09; vs VEH: P=0.012). (b) Animals receiving late (week 12 to week 24 after transplantation) AT1 and AT1+AT2 antagonist treatment (groups AT1 and AT1+AT2) had significantly lower levels of caspase-1 mRNA as compared to groups AT2, and VEH (*AT1 vs AT2: P=0.03; vs CCB: P=0.15; vs VEH: P=0.03. **AT1+AT2 vs AT2: P=0.03; vs CCB: P=0.09; vs VEH: P=0.03). (c) Animals receiving early treatment (day -7 before transplantation to day 5 after transplantation) had no significant differences, although levels were higher in animals of group VEH as compared to the other groups (P>0.05). □=AT1: treatment with AT1 receptor antagonist; =AT2: treatment with AT2 receptor antagonist; =AT1+AT2: treatment with AT1 and AT2 receptor antagonist; =CCB: treatment with CCB; and ■=VEH: treatment with vehicle.View Large Image Figure ViewerDownload (PPT) Animals of late treatment group AT1 had significantly lower caspase-1 and caspase-3 mRNA levels as compared to animals of group VEH (P<0.01; Figures 7b and 8b). Late combined AT1 and AT2 receptor antagonist treatment resulted in significantly lower caspase-1 mRNA levels as compared to animals of groups AT2 and VEH (P=0.03; Figure 8b). Animals receiving early treatment developed no significant differences (Figures 7c and 8c). In our model of CAN, AT1 receptor blockade reduced proteinuria, grade of CAN, and glomerulosclerosis as compared to animals receiving AT2 receptor blockade and controls receiving VEH treatment. The beneficial effects of AT1 receptor blockade were most obvious in animals treated for the entire period as compared to animals receiving late AT1 antagonist treatment. Although creatinine clearances did not differ between the experimental groups, after a longer follow-up, a significant difference could be expected between the groups in this experimental model as development of proteinuria usually precedes a drop in glomerular filtration rate. These results are in congruence with observations that inhibition of the renin–angiotensin system by angiotensin converting enzyme inhibition limits CAN even when treatment was delayed.23.Noris M. Mister M. Pezzotta A. et al.ACE inhibition limits chronic injury of kidney transplant even with treatment started when lesions are established.Kidney Int. 2003; 64: 2253-2261Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Although we also observed a reduction in proteinuria and grade of CAN in animals with delayed treatment, this effect was less pronounced in our experiment. The differences can be explained by use of an AT1 receptor antagonist, the shorter follow-up time, and a lower cyclosporin A dose in our series. Whether AT2 receptors, whose expression increases under pathological conditions,24.Ruiz-Ortega R. Esteban V. Suzuki Y. et al.Renal expression of angiotensin type 2 (AT2) receptors during kidney damage.Kidney Int. 2003; 64: S21-S26Abstract Full Text Full Text PDF Scopus (65) Google Scholar contribute to the beneficial effects after the blockade of AT1 receptors in delaying CAN is not known. Some groups suggested that signalling via the AT2 receptor mediates the beneficial effects associated with the blockade of AT1 receptors as AT2 receptor knockout mice developed a more severe fibrosis as compared to AT1 receptor knockout and wild-type mice in a model of interstitial fibrosis.25.Ma J. Nishimura H. Fogo A. et al.Accelerated fibrosis and collagen deposition develop in the renal interstitium of angiotensin type 2 receptor null mutant mice during ureteral obstruction.Kidney Int. 1998; 53: 937-944Abstract Full Text Full Text PDF PubMed Google Scholar Similarly, it has been suggested that high levels of Ang-II following AT1 receptor blockade stimulate non-engaged AT2 receptors, and thereby influence extracellular matrix turnover, which may explain the decreased fibrosis observed in a rat model of ischemic myocardial fibrosis.26.Liu Y.H. Yang X.P. Sharov V.G. et al.Effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists in rats with heart failure. Role of kinins and angiotensin II type 2 receptors.J Clin Invest. 1997; 99: 1926-1935Crossref PubMed Scopus (545) Google Scholar Contrary to these observations, in our experiment the protective effect of long-term AT1 receptor blockade even in the presence of additional AT2 receptor blockade suggested that signalling via the AT2 receptor is not of crucial importance for the delay of CAN. The differences between the models may be explained by variations in the expression of the two receptor subtypes with regard to tissue type, cell type, disease stage as well as the quality of receptor cross-talk. In accordance with our results are observations that AT1 receptor-activating antibodies can promote vascular rejections that may eventually contribute to the progression of CAN.27.Dragun D. Muller D.N. Brasen J.H. et al.Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection.N Engl J Med. 2005; 352: 558-569Crossref PubMed Scopus (679) Google Scholar How did AT1 receptor blockade delay the progression of CAN, particularly in the long-term-treated animals? Lowering blood pressure alone was not responsible for the favorable results of AT1 receptor blockade as blood pressure did not significantly differ between the groups, which is in congruence with previous reports.28.Benediktsson H. Chea R. Davidoff A. Paul L.C. Antihypertensive drug treatment in chronic renal allograft rejection in the rat: effect on structure and function.Transplantation. 1996; 82: 1634-1642Crossref Scopus (89) Google Scholar,29.Perna A. Ruggenenti P. Testa A. et al.ACE genotype and ACE inhibitors induced renoprotection in chronic proteinuric nephropathies.Kidney Int. 2000; 57: 274-281Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Apoptosis might contribute to a decrease of graft function by a reduction of functioning nephrons. Interestingly, Ang-II can induce apoptosis in tubular epithelial cells through the AT130.Bhaskaran M. Reddy K. Radhakrishanan N. et al.Angiotensin II induces apoptosis in renal proximal tubular cells.Am J Physiol Renal Physiol. 2003; 284: F955-F965Crossref PubMed Scopus (137) Google Scholar as well as the AT2 receptor.31.Weidekamm C. Hauser P. Hansmann C. et al.Effects of AT1 and AT2 receptor blockade on angiotensin II induced apoptosis of human renal proximal tubular epithelial cells.Wien Klin Wochenschr. 2002; 114: 725-729PubMed Google Scholar Thus, angiotensin receptor blockers could exert their protective effects also via an inhibition of apoptosis. Interestingly, Cao et al.32.Cao Z. Bonnet F. Candido R. et al.Angiotensin type 2 receptor antagonism confers renal protection in a rat model of progressive renal injury.J Am Soc Nephrol. 2002; 13: 1773-1787Crossref PubMed Scopus (118) Google Scholar observed an inhibition of apoptosis, together with a protective effect, by the AT2 receptor antagonist PD123319 in a model of subtotal nephrectomy. In another experiment, apoptosis of tubular cells was inhibited by an AT2 receptor blocker in a short-term model of obstructive nephropathy.22.Morrissey J.J. Klahr S. Effect of AT2 receptor blockade on the pathogenesis of renal fibrosis.Am J Physiol. 1999; 276: F39-F45PubMed Google Scholar In our experiments, the lower proteinuria and grade of CAN was paralleled by a lower number of apoptotic tubular cells and a shift of the ratio of bcl-2/bax mRNA levels toward the antiapoptotic Bcl-2 in parallel to reduced caspase-3 mRNA levels (coding for caspase-3 that belongs to the effector molecules of apoptosis) in animals treated with the AT1 receptor antagonist over the long term as compared to AT2 receptor antagonist-treated animals and VEH-treated controls. Although mRNA levels should be cautiously interpreted with respect to protein expression, the mRNA data in our model paralleled the number of apoptotic cells in the grafts. Thus, in our model, the AT1 receptor seems to influence apoptosis more as compared to the AT2 receptor. However, whether apoptosis is involved in the pathogenesis of CAN is not clear so far. Data supporting a pathogenic role of apoptosis for renal tissue damage is derived from ischemia/reperfusion experiments, where inhibition of apoptosis protected kidneys from acute ischemia/reperfusion injury.33.Daemen M.A. van’t Veer C. Denecker G. et al.Inhibition of apoptosis induced by ischemia–reperfusion prevents inflammation.J Clin Invest. 1999; 104: 541-549Crossref PubMed Scopus (487) Google Scholar In kidney transplantation, observational studies have related apoptosis to CAN.10.Laine J. Etelämäki P. Holmberg C. Dunkel L. Apoptotic cell death in human renal allograft rejection.Transplantation. 1997; 63: 101-105Crossref PubMed Scopus (59) Google Scholar In models of unilateral ureteral obstruction, cell compartment-specific as well as time-dependent effects seem to influence apoptosis with different effects regarding interstitial volume or tubular atrophy for example.34.Bascands J.L. Schanstra J.P. Obstructive nephropathy: insights from genetically engineered animals.Kidney Int. 2005; 68: 925-937Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar In our series, long-term AT1 receptor blockade was associated with a reduced number of apoptotic cells and an amelioration of CAN, whereas the effects of late AT1 receptor blockade on apoptosis and CAN were not significant. On the other hand, our group previously showed that late administration of the mammalian target of rapamycin inhibitor everolimus, known to induce apoptosis in immune cells and activated non-immune cells, also ameliorated CAN, which was associated with an increased number of apoptotic cells.35.Lutz J. Zou H. Liu S. et al.Apoptosis and treatment of chronic allograft nephropathy with everolimus.Transplantation. 2003; 76: 508-515Crossref PubMed Scopus (49) Google Scholar Conversely in a model of renal ischemia/reperfusion, early administration of the mammalian target of rapamycin inhibitor sirolimus also increased the number of apoptotic cells, but augmented renal damage.36.Lieberthal W. Fuhro R. Andry C.C. et al.Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells.Am J Physiol Renal Physiol. 2001; 281: F693-F706PubMed Google Scholar It is difficult to compare the results of different studies as observation times, dosing of the drugs, and potential different effects of the drugs on apoptosis related to the type of the cell and its proliferation status might influence the overall effect. Further studies are needed to clarify what role apoptosis plays in progressive renal diseases and whether apoptosis could also be associated with varying effects at different periods during their progression. In our study, the differences in the number of apoptotic cells were probably not related to cellular proliferation in the graft tissues, as no significant differences in the number of proliferating cells were present. Interestingly, animals receiving long-term AT1 receptor antagonist treatment had lower caspase-1 mRNA levels together with a reduced macrophage infiltration of the graft tissue as compared to controls. As caspase-1 has been associated with apoptosis37.Syed F.M. Hahn H.S. Odley A. et al.Proapoptotic effects of caspase-1/interleukin-converting enzyme dominate in myocardial ischemia.Circ Res. 2005; 96: 1103-1109Crossref PubMed Scopus (66) Google Scholar and inflammation,38.Wong W.W. ICE family proteases in inflammation and apoptosis. Therapeutic strategies for modulating the inflammatory diseases.Agents Actions Suppl. 1998; 49: 5-13PubMed Google Scholar a link between those processes may exist. Thus, hypothetically, the function of caspase-1 (apoptotic/inflammatory) could depend on the surrounding environment. What could be the mechanisms responsible for a reduction of apoptosis by inhibition of the AT1 receptor? One of the pathways by which Ang-II can induce apoptosis is an upregulation of p53 that in turn can increase the number of AT1 receptor molecules, and, moreover, shifts the Bcl-2/Bax ratio toward the apoptosis-promoting Bax.21.Pierzchalski P. Reiss K. Cheng W. et al.p53 induces myocyte apoptosis via the activation of the rennin–angiotensin system.Exp Cell Res. 1997; 234: 57-65Crossref PubMed Scopus (123) Google Scholar,39.Leri A. Claudio P.P. Li Q. et al.Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin–angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar. This is supported by our findings in animals receiving long-term AT1 receptor blockade, as these animals had a reduced expression of p53 as compared to AT2 receptor antagonist-treated animals. Apart from a direct effect on the reduction of apoptosis in the grafts, indirect effects such as a decrease in proteinuria may also have accounted for the beneficial effects of AT1 receptor blockade, as proteinuria is known to induce apoptosis.40.Erkan E. De Leon M. Devarajan P. et al.Albumin overload induces apoptosis in LLC-PK(1) cells.Am J Physiol Renal Physiol. 2001; 280: F1107-F1114PubMed Google Scholar Thus, it cannot be excluded that the number of apoptotic cells has additionally been affected through a reduction of proteinuria as a result of AT1 receptor antagonist treatment. In summary, long-term AT1 receptor blockade delayed CAN more effectively as late AT1 receptor blockade, while AT1 receptor signalling seems to be more important than signalling via the AT2 receptor. Clinical trials are needed to analyze whether an early initiation of AT1 receptor blockade could delay CAN in humans more effectively than delayed blockade. The protective effects of AT1 receptor blockade may be related to a lower number of apoptotic tubular epithelial cells in the graft related to the lower expression of p53. Kidneys of male Fisher rats (F344, RT1lvl) (170–210 g) (Charles River, Sulzfeld, Germany) were transplanted into male Lewis rats (LEW, RT11) (170–210 g) (Charles River, Sulzfeld, Germany).41.Szabo A. Lutz J. Schleimer K. et al.Effect of angiotensin-converting enzyme inhibition on growth factor mRNA in chronic renal allograft rejection in the rat.Kidney Int. 2000; 57: 982-991Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar The left native recipient kidney was removed and the left donor kidney was orthotopically transplanted by end-to-end anastomosis of renal artery, vein, and ureter under inhalative anesthesia with isoflurane. All animals received cyclosporin A subcutaneously (1.5 mg/kg/day; Sigma-Aldrich, Taufkirchen, Germany) from day 0 to 10 after transplantation, when the remaining right kidney was removed. Animals received no other immunosuppressive drugs (e.g. steroids). We assigned animals to five groups (Table 1): AT1 (AT1 receptor antagonist candesartan, 5 mg/kg/day); AT2 (AT2 receptor antagonist PD123319, 1.5 mg/kg/day); AT1+AT2 (candesartan and PD123319); the control groups received either vehicle (VEH) or the calcium channel blocker (CCB) lacidipine (1 mg/kg/day) to control for the potential blood pressure lowering effect of candesartan. The different treatments were applied for various periods: long-term treatment (day -7 to week 24 after transplantation; n=8/group); late treatment (week 12–24 after transplantation; n=8/group), and early treatment (day -7 to day 5 after transplantation; n=6/group). The observation ended after 24 weeks. In the respective groups, administration of the substances was initiated 7 days before transplantation in donors and recipients to achieve the maximum pharmacodynamic effect at transplantation. We determined 24-h urine protein excretion every 4 weeks.41.Szabo A. Lutz J. Schleimer K. et al.Effect of angiotensin-converting enzyme inhibition on growth factor mRNA in chronic renal allograft rejection in the rat.Kidney Int. 2000; 57: 982-991Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Serum creatinine levels were analyzed by an automatic laboratory analyzer (Synchron CX5, Beckman Coulter, Krefeld, Germany). Creatinine clearance was calculated according to the formula (CrCl=Ucrea (mg/dl)*diuresis (ml)/Screa (mg/dl)*1440 (min)/weight (kg)). Mean arterial blood pressure was measured intra-arterially before the removal of the graft by a digital analyzer using a pressure transducer (Siemens, Munich, Germany). Measurement was performed under standard conditions at 24°C room temperature between 0900 and 1200 hours. Paraffin-embedded tissue sections were stained with hematoxylin and eosin to evaluate tubulointerstitial fibrosis, vasculopathy, and tubular atrophy. CAN was graded according to parameters adapted from the BANFF 97 classification:2.Racusen L.C. Solez K. Colvin R.B. et al.The BANFF 97 working classification of renal allograft pathology.Kidney Int. 1999; 55: 713-723Abstract Full Text Full Text PDF PubMed Scopus (2771) Google Scholar 0=no signs of CAN; grade 1=mild CAN (mild fibrosis and tubular atrophy, 5–15% of section); grade 2=moderate CAN (moderate fibrosis and tubular atrophy, 16–50% of section); and grade 3=severe CAN (severe fibrosis and tubular atrophy, >50% of section). Grading also included vasculopathy with mild vasculopathy (intimal proliferation with luminal obstruction ≤25%), moderate vasculopathy (luminal obstruction >25–50%), and severe vasculopathy (luminal obstruction >50% of section). Glomerulosclerosis was analyzed by counting sclerotic and normal glomeruli of the section and given as the percentage of sclerotic glomeruli. Macrophage infiltration was evaluated with the alkaline phosphatase antialkaline phosphatase complex (Dako, Glostrup, Denmark) after incubation of cryostat sections (4 μm) with mouse anti-CD 68 (Serotec, Wiesbaden, Germany) and secondary rabbit anti-mouse IgG followed by development with fast red chromogene solution (Dako, Glostrup, Denmark). Cells with positive staining were counted and infiltration was graded: 0=<5 cells/field of view; 1=6–25 cells/field of view; 2=26–50 cells/field of view; and 3=>50 cells/field of view. At least 20 fields of view per section and per specimen were evaluated at × 400 magnification. Two independent observers evaluated the samples in a blinded manner. The number of proliferating cells was analyzed after incubation of the sections with mouse anti-proliferating cell nuclear antigen antibody (Dako, Glostrup, Denmark) followed by incubation with a secondary horse anti-mouse antibody according to the Avidin Biotin Complex Method and development of the color signal by peroxidase (Vectastain elite Avidin Biotin Complex Kit; Vector Laboratories, Burlingame, CA, USA). Tubular epithelial cells with nuclear brown staining were counted per field of view at × 100 magnification. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assays were performed in frozen sections incubated with TUNEL solution42.Cursio R. Gugenheim J. Ricci J.E. et al.A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis.FASEB J. 1999; 13: 253-261Crossref PubMed Scopus (207) Google Scholar (Roche Diagnostics, Mannheim, Germany). After washing, sections were incubated with a rabbit anti-digoxygenine antibody (Roche Diagnostics, Mannheim, Germany). Binding was visualized using fast red chromogene solution. Tubular epithelial cells with positive staining (red nuclei) were counted per field of view. At least 20 fields of view per section and per specimen were evaluated at × 400 magnification. Two independent observers evaluated the samples in a blinded manner. Total RNA was prepared as previously described for the analysis of mRNA levels of Bcl-2, Bax, caspase-1, and caspase-3.41.Szabo A. Lutz J. Schleimer K. et al.Effect of angiotensin-converting enzyme inhibition on growth factor mRNA in chronic renal allograft rejection in the rat.Kidney Int. 2000; 57: 982-991Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar A substantial part of renal tissue consists of cortical and medullar tubular cells (63 and 34% of relative renal tissue volume).43.Clapp Wl. Abrahamson D.R. Development and gross anatomy of the kidney.in: Tisher C.C. Brenner B.M. Renal Pathology with Clinical and Functional Correlations. JB Lippincott Company, Philadelphia1994: 48Google Scholar Thus, a gross part of the isolated mRNA is derived from tubular cells. Radioactive antisense riboprobes (32P-UTP) were prepared by in vitro transcription (RiboQuant Multi-Probe Ribonuclease Protection Assay (RPA) System, Pharmingen, San Diego, CA, USA)35.Lutz J. Zou H. Liu S. et al.Apoptosis and treatment of chronic allograft nephropathy with everolimus.Transplantation. 2003; 76: 508-515Crossref PubMed Scopus (49) Google Scholar followed by hybridization of total RNA samples with their respective rat antisense riboprobes according to the manufacturer's protocol. Then, RNA and RNase digestion and separation on a 6% polyacrylamide gel were performed. Blots were scanned on a Fuji-BAS phosphor imager (Fuji, Düsseldorf, Germany), digitized, and density of bands was measured. The ratio between specific mRNA band densities and housekeeping gene mRNA band (glyceraldehyde-3-phosphate dehydrogenase) densities were calculated. Tissue samples were homogenized in lysis buffer and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples were transferred to Hybond-CExtra membranes (Amersham Pharmacia Biotech, Aylesbury, UK), blocked with 5% skimmed milk in Tris-buffered saline/0.5% Tween 20 overnight at 4°C. Membranes were incubated with the following antibodies after washing in Tris-buffered saline/Tween: primary antibodies – monoclonal mouse anti human p53 (1:5000; Pharmingen, San Diego, CA, USA), polyclonal rabbit anti β-actin (1:10 000; Sigma, St Louis, MO, USA); secondary antibodies – goat anti-mouse immunoglobulin G-horseradish peroxidase conjugated (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-rabbit immunoglobulin G-POD conjugated (1:5000; Vector Laboratories, Burlingame, CA, USA). Antibodies were diluted in 5% skimmed dry milk in Tris-buffered saline/Tween buffer. Blots were washed and incubated with LumiLight working solution (Roche Diagnostics GmbH, Mannheim, Germany). Density of bands was analyzed and the ratios between p53- and β-actin bands were calculated. Data are expressed as mean±s.e.m. Data were tested using the χ2 test or Mann–Whitney U-test. Bonferroni's method was applied to avoid inflation of type I error in multiple testing settings. A global P-value of <0.05 was considered significant. All tests were performed two-tailed. Data were analyzed using the SPSS statistical software package (v. 13.0, SPSS GmbH, Munich, Germany).