Chronic allograft nephropathy, recently reclassified as interstitial fibrosis and tubular atrophy with unknown cause leading to reduced renal function and proteinuria still represents an important cause of graft loss after kidney transplantation (1).
Despite significant achievements in transplantation medicine, no specific therapy is available for chronic allograft dysfunction. The renin-angiotensin system (RAS) has been demonstrated to contribute to the progression of chronic kidney damage in various experimental models and might have relevance in kidney transplantation as well.
Angiotensin II (Ang II) type 1 receptor blockers (ARB) and angiotensin-converting enzyme (ACE) inhibition exert antifibrotic effects (2–4), attenuate podocyte injury (5), have antioxidative effects (6) and protect tubular cells from apoptosis (7) as previously shown in various experimental models.
Furthermore, we have previously demonstrated that both ARBs and ACE inhibitors delay the progression of chronic allograft nephropathy in experimental rat models (8). In humans, Artz et al. (9) reported that in patients with biopsy proven chronic allograft nephropathy, blockade of RAS resulted in increased graft survival and reduced proteinuria (10). Furthermore, the use of ACEI inhibitors and ARBs in patients experiencing delayed allograft function was associated with longer actual and functional transplant survival (11). The protective effect of RAS inhibition on graft dysfunction is mainly mediated by reduced cell proliferation, and thus, reduced extracellular matrix production in the glomeruli and interstitium, leading to ameliorated proteinuria and preserved overall graft function (8, 12).
At present, blockade of RAS is achieved mainly by ACE inhibitors and ARBs. More recently, direct renin inhibitors have also emerged as a potential therapeutic strategy to block the RAS (13).
Aliskiren is the first available direct renin inhibitor. It binds to the active site of renin, preventing angiotensinogen from binding and being cleaved to Ang I, inhibiting thereby the activation of the RAS at the rate-limiting step. It has a half-life of 40 hr and pharmacokinetic properties that place it as a promising drug in the focus of preclinical and clinical research (11).
Beside its blood pressure lowering effects, aliskiren was reported to reduce albuminuria and structural injury in experimental diabetic nephropathy (DN) (14). Furthermore, aliskiren presented favorably in reversing renal organ damage in renin and angiotensinogen double transgenic rats too (15).
Direct renin inhibitors might have several additional effects over ACE inhibitors and ARBs that might result in altered end effects of aliskiren. Theoretically, inhibiting the renin system can provide Ang II suppression, and down-regulation of Ang II breakdown products such as Ang (1–7), Ang (3–8) and a series of other components that are generated by the so-called ACE2. These breakdown products have been implicated in the regulation of vascular physiology and fibrosis, and their protective effect reducing kidney damage has been reported as well (16–19).
Because aliskiren has been shown to protect from kidney injury and might have potential additional effects over other RAS inhibitors, we examined aliskiren administration in an experimental model of chronic allograft dysfunction where no aliskiren study has yet been reported. The effects of aliskiren were compared with both vehicle treatment and well-described ARB candesartan.
Body Weight, Blood Pressure, Proteinuria, and Creatinine Clearance
Body weights did not differ significantly among the experimental groups (Table 1). Mean arterial blood pressure was significantly lower in both aliskiren and candesartan groups compared with VEH animals (both P<0.05) (Table 1). There was no difference between the two treatment groups (P>0.05).
The rate of proteinuria was measured every 4 weeks, and kidney function was assessed by calculating creatinine clearance at the time of harvesting 24 weeks after transplantation. In animals receiving aliskiren treatment, proteinuria did not differ significantly, whereas the candesartan-treated group developed significantly lower proteinuria from the 12th week after transplantation compared with the VEH group (P>0.05 and P<0.05, respectively) (Fig. 1).
In creatinine clearances, there was no significant difference between the experimental groups, however, there was a higher tendency in candesartan-treated animals versus VEH animals (P=0.056) (Table 1).
Renal Morphological Changes
The degree of glomerulosclerosis remained unchanged in the aliskiren-treated animals (P>0.05 vs. VEH and P>0.05 vs. candesartan), whereas it was significantly reduced in the candesartan group compared with the VEH group and aliskiren-treated group (P<0.05, respectively) (Table 1, Fig. 2A). Animals treated with aliskiren developed significantly attenuated tubular atrophy compared with the VEH group (P<0.05). Also the candesartan-treated group had significantly reduced degree of tubular atrophy when compared with the VEH animals (P<0.05) (Table 1, Fig. 2A). There was no difference between the aliskiren and candesartan-treated groups (P>0.05). These results were underlined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining, demonstrating significantly lower number of apoptotic tubular cells in the aliskiren and in the candesartan-treated animals when compared with the VEH group (P<0.05, respectively) (Table 1, Fig. 2B) with no significant difference between the aliskiren and candesartan groups (P>0.05).
The degree of collagen deposition did not change in the aliskiren-treated animals versus VEH group, whereas it was significantly reduced in the candesartan-treated group when compared with the VEH and aliskiren-treated animals (P>0.05 and P<0.05, respectively) (Table 1, Fig. 2C). α-SMA staining was used to detect interstitial myofibroblast accumulation. This remained unchanged in the aliskiren group versus VEH group and versus candesartan group (P>0.05 vs. VEH and P>0.05 vs. candesartan), whereas it was significantly lower in the candesartan-treated group when compared with the VEH and aliskiren-treated groups (P<0.05, respectively) (Table 1, Fig. 2D). Macrophage infiltration (CD68-positive cells) did not differ significantly between the treatment groups, however there was a tendency toward lower infiltration in the candesartan-treated group when compared with the VEH-treated animals (P=0.062).
Urine Angiotensinogen Level
Urine angiotensinogen level did not differ in the aliskiren group when compared with VEH or candesartan group (P>0.05). Urine angiotensinogen was significantly decreased in the candesartan group versus VEH (P<0.01) (Fig. 3).
Serum Angiotensin II Level
Serum Ang II level was significantly lower in the renin inhibitor aliskiren group (P<0.01), whereas it was significantly higher in the candesartan group compared with VEH- and aliskiren-treated animals (both P<0.01) (see Figure, SDC 1,http://links.lww.com/TP/A509).
ACE2 activity in the serum was decreased both in the aliskiren- and in candesartan-treated animals when compared with VEH-treated rats (both P<0.05) (see Figure, SDC 2,http://links.lww.com/TP/A510). There was no difference between the candesartan and the aliskiren group.
Ang (1–7) Plasma Level
In the aliskiren-treated group, there was a lower plasma level of Ang (1–7) compared with both VEH- and candesartan-treated rats, however versus VEH group, it was demonstrated to be only a tendency (P=0.086). There was no difference between the candesartan and VEH group (P>0.05) (Fig. 4).
In our experimental model for chronic allograft dysfunction, we treated animals with the renin inhibitor aliskiren. To compare its impact not only with placebo but also with another type of RAS inhibition, a group of rats was treated with the ARB candesartan.
Our results clearly demonstrate that although aliskiren treatment was as well effective in the reduction of blood pressure as candesartan, it had no effect on renal function compared with vehicle-treated rats. Furthermore, glomerulosclerosis and deposition of collagen in the kidney remained unchanged as well, although a tendency toward reduction in glomerulosclerosis could be observed.
Both aliskiren and candesartan lowered the blood pressure, suggesting that the applied doses were pharmaceutically effective in both groups. The observation that there was a significant reduction in proteinuria in the candesartan but not in the aliskiren group indicate that in this model, changes in blood pressure itself do not impact the outcome of chronic kidney graft injury.
ACE inhibitors and ARBs have already been proven to have a beneficial effect on the long-term kidney graft function after transplantation (8–10). In contrast to ACE inhibitors and ARBs, direct renin inhibitors block the RAS more upstream.
Through blocking the RAS upstream of Ang I, aliskiren might reduce not only the ACE but also the ACE2-dependent signaling. Blocking ACE2 decreases not only the Ang II levels but also the formation of the so-called Ang II breakdown products that might lead to different end effects of renin blockers on organ injuries (20).
To examine this hypothesis, first Ang II and ACE2 serum levels were measured. In accordance with our presumptions, the level of Ang II was significantly lower in the aliskiren-treated animals, whereas it was higher in the candesartan group compared with vehicle-treated rats. ACE2 activity was decreased to the same level in both treatment groups resulting therefore in a significantly lower Ang II/ACE2 ratio in the aliskiren-treated rats. Marked reduction in the Ang II levels might have several consequences. On one hand, Ang II signaling through the AT2 receptor might be ameliorated. Activation of AT2 receptor by Ang II seems to have vasodilative, antihypertrophic, antiproliferative, antioxidative, and antifibrotic effects helping to preserve tissue structure counterbalancing thereby the effects of AT1 receptor stimulation (21). On the other hand, the lower level of Ang II might result in lower production of Ang II breakdown products in the aliskiren animals. In our experiments, ACE2 was reduced in both groups to the same level compared with the vehicle group, suggesting that chronic administration of both agents decreases ACE2. There is only little known about the impact of RAS inhibitors on ACE2, however, contradicting our findings ARBs seem to rather upregulate ACE2 (22). Nevertheless, it is important to note that in our study, candesartan was administered for a markedly longer time, and ACE2 was measured in the plasma, not in the tissue.
ACE2 protects from chronic kidney injury, possibly by the regulation of Ang II breakdown products, in a rat model of DN (23). Of these breakdown products, Ang (1–7) and Ang (3–8) have gained the most attention as they have various favorable properties including vasomodulatory and antifibrotic effects.
Therefore, to further investigate our hypothesis, serum levels of Ang (1–7) were assessed. Ang (1-7) level was significantly lower in the aliskiren group compared with the vehicle-treated rats which itself could underline our hypothesis. However, Ang (1–7) levels were the same in the vehicle- and in the candesartan-treated animals, and yet, only candesartan-treated animals were protected from chronic allograft injury indicating the potential role of other factors as well.
An alternative mechanism for the different outcome in aliskiren-treated rats might be the different changes in the intrarenal RAS activity in the two groups. Urinary angiotensinogen level has been recently suggested to be a useful index of intrarenal RAS activity (24, 25), therefore, we assessed the level of angiotensinogen in the urine. In line with previous studies reporting that ARBs reduce urinary angiotensinogen level correlating well with proteinuria (26, 27), we have also demonstrated that the candesartan-treated rats had a significant decrease in the level of urinary angiotensinogen. On the other hand, aliskiren had no significant influence on urinary angiotensinogen levels; however, there was a lower tendency, hence indicating a major difference between aliskiren and candesartan effects in this model. This unchanged intrarenal RAS activity in the aliskiren animals could therefore also explain why functional and most histological signs of chronic allograft injury were not significantly influenced by aliskiren. Summarizing, we suggest that reduced Ang (1–7) formation or unchanged intrarenal RAS activity might be the cause of the lack of the protective aliskiren effects.
Interestingly, tubular atrophy, in particular, apoptosis of tubular cells was significantly reduced in both candesartan and aliskiren groups. We can only speculate through which mechanism tubular apoptosis was ameliorated also in the aliskiren-treated animals because all other parameters of chronic allograft injury remained unchanged. We propose that the lower tendency that was observed in urinary angiotensinogen level in the aliskiren animals might have been sufficient to reduce apoptosis of tubular cells, but insufficient to reduce proteinuria, glomerulosclerosis, and interstitial fibrosis.
To date, there have been no experimental/clinical data published comparing the renoprotective/antiproteinuric effects of ARBs and renin inhibitors. Data from the AVOID trial suggest that the addition of aliskiren to the ARB losartan provides an antiproteinuric effect additional to that of losartan alone; however, no comparison between ARB and aliskiren given in monotherapy has been made. Therefore, these data can be discussed here only with limitations (28).
As mentioned previously, in contrast to the results of our present study, aliskiren has been reported to protect from DN both in experimental models and in the clinical settings. However, it is important to emphasize that even though the outcome and renal morphology of DN (induced even in double transgenic renin-angiotensinogen mice) and chronic allograft injury show several similarities; there are basic differences in their pathomechanisms.
In DN, there is basically an overload of tubular cells with glucose leading to higher glucose reabsorption that results in the activation of various metabolic pathways which in turn, stimulate signaling pathways leading to extracellular matrix synthesis. Furthermore, tubular glucose hyperreabsorption contributes to glomerular hyperfiltration paralleled by hypertrophy of tubular cells. More importantly, in the recent years, activation of the RAS mainly through triggered renin release in the juxtaglomerular apparatus has been recognized as a major hallmark of DN pathology (29). On the other hand, the cause of chronic allograft dysfunction involves the additional complexity of both alloantigen-dependent and -independent factors well reflected in this rat model too (30). Despite being similar to those in DN, renal morphological changes in chronic allograft injury are the endpoints of even more complex multifactorial, final pathways after more types of injury. Furthermore, renin release which is typical in DN has not yet been observed in chronic allograft injury. Cyclosporine, for example, has been shown to induce Ang II production in the kidney given after transplantation (31) probably contributing thereby significantly to the calcineurin inhibitors nephrotoxicity. This provides further proof of evidence why ARBs might protect from chronic allograft injury. It is therefore plausible that due to these major differences in their pathomechanisms and the prominent role of enhanced renin production in DN, renin inhibition does not have the same, protective effect in chronic renal changes after transplantation as in DN.
In summary, our experimental study demonstrated that the renin inhibitor aliskiren did not reduce the progression of chronic allograft dysfunction in a rat model of kidney transplantation. This interesting finding might be related to reduced formation of the protective Ang II breakdown products such as Ang (1–7) or to unchanged intrarenal RAS activity in aliskiren-treated rats demonstrated by urinary angiotensinogen levels.
MATERIALS AND METHODS
Animals, Surgery, and Treatment
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) as described previously (32) (see SDC 3,http://links.lww.com/TP/A511).
We assigned animals to three groups (Table 1): the control group received vehicle (VEH) treatment (saline, n=9), candesartan group (AT1 receptor antagonist, n=6) candesartan (5 mg/kg/day); aliskiren (renin inhibitor, n=7) (10 mg/kg/day). The drugs were administered through oral gavage.
The observation ended 24 weeks after transplantation. 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.
Paraffin-embedded tissue sections were stained with periodic acid-Schiff (PAS) reaction, original magnification to evaluate tubular atrophy. Glomerulosclerosis was analyzed by counting sclerotic and normal glomeruli of the section and given as the percentage of sclerotic glomeruli. Glomerulosclerosis was present when collapse of capillary lumens, mesangial matrix expansion, hyalinosis, and thickening of the glomerular basement membrane could be observed. A minimum of 50 glomeruli were counted.
Tubular atrophy (presence of tubules with thick redundant basement membranes or a reduction of greater than 50% in tubular diameter compared with surrounding nonatrophic tubules) was graded on a scale from 0 to 3: grade 0, no signs of tubular atrophy; grade 1, mild tubular atrophy (5%–15% of section); grade 2, moderate tubular atrophy (16%–50% of section); and grade 3, severe tubular atrophy (>50% of section).
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling
TUNEL analysis was performed on frozen sections fixed in paraformaldehyde 4% as described previously (33). All positive tubular epithelial cells in each section were counted at a magnification ×40 and related to the number of view fields per section.
Immunohistochemical studies were performed on frozen sections fixed in acetone (4°C) as described previously (34). Infiltration of CD68 immunoreactive cells was scored on a scale of 0 to 3.
Data are expressed as mean±SD. Data were analyzed by χ2 test (tubular atrophy scoring, parametrical data) or by Kruskal-Wallis analysis (nonparametrical data) with Dunn's post hoc test using the Sigma plot statistical analysis software (version 11.0, Systat Software GmbH, Erkrath, Germany). A global P value less than 0.05 was considered significant.
The authors thank Sandra Haderer and Marcel Konhäuser for their technical assistance.
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