The role of the renin-angiotensin system in chronic kidney disease involves multiple peptides and receptors. Exerting antipodal pathophysiological mechanisms, renin inhibition and AT1 antagonism ameliorate renal damage. However, it is unclear which mechanism exerts better nephroprotection. We compared the renin inhibitor aliskiren with the AT1 antagonist losartan in mice with chronic kidney disease due to renal ablation. Doses were adjusted to equipotent inhibition of the renin-angiotensin system, determined via a dose-response quantifying plasma and renal renin expression. Six-week treatment with either 500 mg/l drinking water losartan or 50 mg·kg−1·day−1 aliskiren significantly decreased albuminuria, glomerular damage, and transcription rates of renal injury markers to a similar extent. An array analysis comparing renal gene expression of losartan- and aliskiren-treated mice evaluating >34,000 transcripts demonstrated regulation for 14 genes only, with small differences. No superior nephroprotection was found by combining losartan and aliskiren. Compared with plasma concentrations, aliskiren accumulated ∼7- to 29-fold in the heart, liver, lung, and spleen and ∼156-fold in the kidney. After withdrawal, plasma concentrations dropped to zero within 24 h, whereas renal tissue concentrations declined slowly over days. Withdrawal of aliskiren in mice with chronic kidney disease revealed a significantly delayed re-increase in albuminuria compared with withdrawal of losartan. This study demonstrates equieffective nephroprotection of renin inhibition and AT1 antagonism in mice with chronic kidney disease without additional benefit of combination therapy. These observations underscore the pivotal role of targeting ANG II to reduce renal injury.
- 5/6 nephrectomy
- renal impairment
there is evolving complexity in the renin-angiotensin system (RAS) because of the identification of a spectrum of bioactive angiotensin peptides and receptors that interact with these fragments. ANG II has long been considered the major bioactive contributor to chronic kidney disease (CKD). Chronic ANG II infusion induces albuminuria and renal injury (23). Conversely, angiotensin-converting enzyme (ACE) inhibition and ANG II type 1 receptor (AT1) antagonism are nephroprotective (1, 4, 29). However, it is controversial whether this efficacy is due to inhibition of ANG II production, blockade of the AT1 receptor, a shift of ANG II toward AT2 receptor transduction, or the continued or even increased presence of other bioactive angiotensin peptides that interact with receptors beyond the AT1 and AT2 receptor.
Renin inhibition suppresses the production of all angiotensin peptides (27), providing a unique approach to define the role of angiotensin peptides in the progression of CKD (20). Due to the high species specificity of the renin-angiotensinogen reaction, animal data facilitating a more detailed insight into the nephroprotective effects of renin inhibition are scarce. In double transgenic rats (dTGR) expressing both the human renin and angiotensinogen gene (11), renin inhibition decreases blood pressure and albuminuria (33). However, given the exceptionally high ANG II levels in these animals (up to 20 times normal) causing death within 7 wk without treatment (33), as well as the absence of the RAS feedback mechanisms, they are ill-suited for studying normal physiological events.
Renin inhibition and AT1 antagonism have been shown to improve albuminuria and end-organ damage. Several reports suggest that the beneficial effects of aliskiren are due to a decrease in ANG II production, while others have suggested an additive effect of aliskiren to antagonize the increased plasma renin activity (PRA) due to AT1 blockade (9). Renin inhibition has been suggested to be superior to ACE inhibition or AT1 antagonism in previous studies (22). Moreover, the combination of AT1 antagonism and renin inhibition has shown superior protection against renal fibrosis in mice (8, 39–40). However, in none of these studies was RAS inhibition quantified as in the present study, nor did the monotherapy exceed low-dose treatment. Therefore, we performed for the first time extensive dose-response curves for aliskiren and losartan and asked three questions. 1) What is the nephroprotective efficacy of aliskiren compared with equipotent high-dose treatment with the AT1 antagonist losartan? 2) Does aliskiren on top of losartan exert more nephroprotection than the monotherapies by i.e., inhibiting the increased PRA in response to losartan? 3) Are there differences between aliskiren and losartan after withdrawal of treatment? Renal ablation, a mainstay in experimental kidney research, was used for CKD induction in FVB/N mice. This model features all clinical hallmarks of CKD, including substantial albuminuria, uremia, and glomerulosclerosis (3). Furthermore, mice serve as the ideal target to study renin inhibition, because the IC50 of aliskiren for mouse renin (4.5 nM) is considerably closer to human renin (0.6 nM) than is rat renin (80 nM) (10, 27, 38).
Emerging evidence indicates sustained effects of aliskiren beyond withdrawal (31, 35). Besides its long plasma half-life in humans, speculations about any putative renal accumulation of aliskiren were held to explain this phenomenon (10). We compared plasma concentrations with various tissue levels of aliskiren and analyzed its effect compared with losartan on albuminuria after withdrawal.
Using renal ablation in FVB/N mice as a model for CKD, this is the first study comparing renin inhibition and AT1 antagonism based on equipotent RAS inhibition in nontransgenic animals. This facilitates a meaningful evaluation of the nephroprotective efficacy of renin inhibition during treatment as well as of prolonged effects after its withdrawal.
MATERIALS AND METHODS
All experiments started in 10-wk-old male FVB/N mice. Aliskiren (Novartis) was administered via osmotic minipump infusion (model 2002, Alzet Osmotic Pumps; DURECT), and losartan (Sandoz) was dissolved in the drinking water. All animal procedures were approved by the local animal committee and were in accord with national and institutional animal care guidelines.
Dose-response curve and effect on angiotensin fragments.
To identify dosages for losartan and aliskiren yielding equipotent RAS inhibition, healthy FVB/N mice were treated with increasing dosages of both drugs for 14 days before renal renin was quantified with real-time PCR for RNA and immunostaining for protein levels. Plasma renin concentration (PRC) was measured as described elsewhere (25). PRC was determined by measuring ANG I formation in the presence of excess angiotensinogen after diluting of the samples to get rid of the aliskiren. Plasma angiotensin peptides were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry as previously described (19). The detection limit was 4 × 10−16 mol. In addition, plasma ANG II levels were also quantified with a conventional enzymatic immunoassay by an ANG II enzyme immunoassay kit from SpiBio (Massy, France). ANG II from the plasma was concentrated by using phenyl cartridges (SpiBio) and water and was eluated by methanol. The eluate was lyophilized by vacuum centrifugation at 4°C. The kit was used according to the manufacturer's protocol. For blood collection, an inhibitor cocktail (Protease Inhibitor Cocktail, Sigma) was used to prevent generation and/or degradation of ANG II.
Comparison of aliskiren and losartan in CKD.
Two weeks before the start of the experiment, two-thirds of the left kidney was removed as described (3). At day 0, renal ablation was completed by contralateral uninephrectomy. Four weeks thereafter, albuminuria and blood urea nitrogen (BUN) were measured and mice were randomized into three groups: untreated, losartan, and aliskiren. In the subsequent 6 wk, 500 mg/l drinking water losartan or 50 mg·kg−1·day−1 aliskiren were administered. The losartan dose for a 30-g mouse drinking 10 ml/day averages ∼167 mg·kg−1·day−1. Mice were euthanized after week 10 to collect organs and blood for further analyses. In a second set of experiments, double blockade with losartan plus aliskiren was performed concomitantly to both monotherapies.
Half-life of aliskiren in the kidney, heart, and plasma.
To determine the tissue and plasma half-life of aliskiren, healthy FVB/N mice were treated with 50 mg·kg−1·day−1 aliskiren for 14 days. Aliskiren was withdrawn, and five mice each were euthanized immediately (day 0) or 1, 3, or 7 days thereafter.
Plasma levels of ANG II after withdrawal of losartan and aliskiren.
In another set of experiments, healthy FVB/N mice were treated with aliskiren and losartan for 14 days. Aliskiren and losartan were withdrawn, and five mice each were euthanized immediately (day 0) or 1 or 3 days thereafter. ANG II plasma levels were measured by ELISA.
Mice were placed into metabolic cages for a 6-h urine collection. Urine albumin was measured using a commercially available mouse-specific ELISA (E90–134; Bethyl Laboratories) and urine creatinine by an autoanalyzer (Hitachi 717; Roche). Albuminuria was calculated as milligrams albumin per milligram creatinine.
Before treatment, mice were slightly anesthetized with carbon dioxide and the retroorbital sinus was punctured with a glass capillary tube for heparinized blood collection. At death, blood was drawn intracardially. BUN and plasma cholesterol were determined by an autoanalyzer (Hitachi 717).
Systolic blood pressure was measured in conscious mice using computerized tail-cuff plethysmography (Process Control Blood Pressure 2900 series; TSE Systems) as described elsewhere (23). Mice were trained to get used to this procedure in advance, and initial measurements were not incorporated.
Kidney tissue was fixed with 4% neutral buffered formaldehyde, embedded in paraffin, and sectioned. Sections were stained for light microscopy with periodic acid-Schiff reagent. Glomerular injury was evaluated using a semiquantitative scale between 0 and 3. Proteinaceous casts were counted in the whole cortex. For assessment of interstitial injury, nonoverlapping cortical areas were analyzed by overlaying a grid containing 40 points. For renin immunohistochemistry, a polyclonal antibody was used as described elsewhere (24). Juxtaglomerular renin was quantified using a semiquantitative scale from 0 to 3. The size of the juxtaglomerular apparatus was found by computer-aided assessment.
Real-time PCR analyses.
Total RNA of the renal cortex was prepared according to standard laboratory methods. Quantitative real-time PCR analysis (StepOnePlus, Applied Biosystems) was performed using SYBR Green dye (Qiagen).
The following primers were used: 18S: forward 5′-CAC GGC CGG TAC AGT GAA AC-3′, reverse 5′-AGA GGA GCG AGC GAC CAA A-3′; fibronectin: forward 5′-AGA CCA TAC CTG CCG AAT GTA G-3′, reverse 5′-GAG AGC TTC CTG TCC TGT AGA G-3′; plasminogen activator inhibitor (PAI)-1: forward 5′-GGA CAC CCT CAG CAT GTT CA-3′, reverse 5′-TCT GAT GAG TTC AGC ATC CAA GAT-3′; renin: forward 5′-GCT CTG GAG TCC TTG CAC CTT-3′, reverse 5′-CTT GAG CGG GAT TCG TTC AA-3′; and renin receptor: forward 5′-TCA TTC GAC ACA TCC CTT GTG-3′, reverse 5′-AGG TTA TAG GGA CTT TGG GTG TTC T-3′.
Determination of aliskiren in plasma and tissue.
Aliskiren in plasma (25 μl) and tissue (2–10 mg wet wt) samples was determined using compound 21 as an internal standard (IS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to Wan et al. (37). Analytes were chromatographed on a Synergy Polar RP column [30 × 2-mm ID, Phenomenex, Torrance, CA, at a flow rate of 0.4 ml/min; 0- 0:30 min 10% B, 0:30–3 min increase to 90% B, 3–3:30 min 90% B, reequilibration at 10% B until 8 min, 10 mM ammonium acetate (A), acetonitril (B)]. The following transitions were used for the quantitative determination of analytes (ESI+): m/z 552.4 -> 418.4 and m/z 552.4 -> 436.4 for aliskiren, and m/z 476.4 -> 293.2 and m/z 476.4 -> 402.2 for IS.
Renal tissue from renal-ablated mice after 6-wk treatment with either 500 mg/l losartan or 50 mg·kg−1·day−1 aliskiren (n = 5 each) were used for the experiments. Affymetrix GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix) containing ∼34,000 genes and expressed sequence tags (ESTs) were used. Target preparation and hybridization with Gene Chip microarrays were performed as described by the manufacturer. Microarrays were scanned with Affymetrix GeneChip Scanner 7G. Expression signals were RMA background corrected and quantile normalized using Affymetrix Expression Console Software (version 1). Significance of gene regulation was determined by application of a Welch's unpaired t-test (R statistical platform, ver. 2.11.0), followed by a sample permutation correction for multiple testing. Genes were declared as significant when exhibiting a permutation-derived P value <0.05 and an absolute signal-log-ratio >0.8 (fold-change of 1.75). The data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Seris accession number GSE 34552. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE34552).
Data are expressed as the median [25th percentile; 75th percentile], unless otherwise indicated. In the case of an uneven distribution, laboratory parameters were logarithmically transformed for statistical analyses. Univariate ANOVA was performed using SPSS Statistics 18.0 (SPSS). All intergroup comparisons were performed pairwise using a least significant difference posttest. For the comparison between study groups having received renal ablation, the albuminuria before the start of therapy was used as covariate (ANCOVA). For the experiment of withdrawal of aliskiren and losartan in CKD, albuminuria before the start of therapy and at the day of withdrawal of therapy were used as covariates. Significance was considered for an error of α <5% (P < 0.05).
Interfering with the RAS stimulates renal renin expression due to inhibition of the ANG II-mediated negative-feedback loop. Thus quantifying renin allows one to estimate the magnitude of the RAS blockade (27). Equieffective RAS inhibition with losartan and aliskiren was identified via a dose-response measuring the reactive renin rise in healthy control mice. Renin RNA levels, plasma renin concentration, and renin immunohistochemistry revealed a dose-dependent increase for both drugs (Fig. 1A). Plasma concentrations and RNA levels of renin correlated well. Equipotent RAS inhibition was approximately observed for 500 mg/l losartan and 50 mg·kg−1·day−1 aliskiren. In this regard, the presumable overestimation of renin protein measurements in the immunoassay, promoted by conformational changes in prorenin during aliskiren therapy and the technical challenge to measure PRC in mice, was taken into account (6). Figure 1B depicts representative renin immunohistochemistry. Furthermore, renin inhibition decreased plasma concentrations of angiotensin fragments sufficiently (Fig. 1C). ANG I, II, and 1–9 were undetectable after aliskiren, but unaltered after losartan treatment.
Similar nephroprotective properties of aliskiren and losartan.
Equipotent losartan and aliskiren treatment in mice with CKD was begun 4 wk succeeding renal ablation. Before therapy, no difference was found in the removed kidney weight, BUN, and albuminuria between all groups, indicating similar induction of CKD (Table 1). Throughout the subsequent 6 wk, albuminuria increased to 16.22 [9.86; 67.40] in untreated renal-ablated mice and progressively decreased to 0.95 [0.45; 2.64] in losartan- and 0.59 [0.42; 1.47] in aliskiren-treated mice (Fig. 2A). This highly significant reduction was of comparable magnitude in both treatments (Fig. 2B). Intratubular proteinaceous casts, the morphological correlate of proteinuria, were observed in renal-ablated mice (Fig. 2D). Quantification revealed a significant reduction of casts for both treatments (Fig. 2C). Elevated plasma cholesterol levels indicated the nephrotic range of albuminuria (Fig. 2E). Both treatments reduced cholesterol levels significantly.
Glomerular and interstitial damage.
The abundant glomerular damage in untreated renal-ablated mice was significantly attenuated by losartan and aliskiren to a comparable extent (Fig. 3A). The interstitial area (Fig. 3B) was increased after renal ablation and not reduced by any therapy. Figure 3C depicts representative micrographs.
Renal ablation downregulated renin RNA (0.16 [0.10; 0.33]) compared with controls (Fig. 4A). Treatment with losartan (1.36 [0.96; 2.45]) or aliskiren (1.35 [0.54; 1.57]) leads to an approximately eightfold increase in renin compared with untreated renal-ablated mice, confirming equipotent RAS inhibition in CKD mice for the doses evaluated before in healthy mice. The RNA expression of the (pro)renin receptor was unaltered (Fig. 4B). Both treatments significantly decreased profibrotic markers such as PAI-1 (Fig. 4C) and fibronectin (Fig. 4D).
Renal function and systolic blood pressure.
Mortality did not differ between untreated and losartan- or aliskiren-treated mice (Table 2). Renal ablation decreased renal function, illustrated by increased BUN levels. Both drugs had no significant effect on BUN levels. Systolic blood pressure increased moderately in renal-ablated mice. This was attenuated by both treatments. We cannot differentiate from our data whether the observed nephroprotection is due to the small decrease in blood pressure or blood pressure-independent effects of renin inhibition and AT1 receptor blockade since we did not include a non-RAS inhibitor treatment group. However, there is overwhelming evidence from experimental and clinical data that, especially in CKD with proteinuria and the renal ablation model, RAS inhibition is superior to non-RAS inhibition. Analogous to blood pressure, the relative heart weight was increased in untreated, but reduced in treated renal-ablated mice.
Microarray analysis of renal gene expression.
Microarray analysis of renal gene expression comparing 6-wk losartan or aliskiren treatment in mice with CKD revealed significant regulation with a signal log ratio (SLR) >0.8 of 14 genes only with a maximum of a 2.58-fold change (Fig. 5). No major pathway differentially expressed by renin inhibition or AT1 antagonism can be identified since the genes are a mixture of matrix, transporter cytokine, receptor, and transduction proteins as well as unidentified genes. In contrast, significant regulation was found of 157 genes for aliskiren and 127 genes for losartan treatment both compared with untreated mice (data not shown).
Due to the high variability of the renal damage caused by renal ablation, mice of the double blockade were only compared with mice treated with aliskiren or losartan monotherapy in the same set of experiments. Double blockade was not superior, as assessed for albuminuria (Fig. 6), BUN, systolic blood pressure, gene expression of renin and fibronectin, and glomerular sclerosis (Table 3). Although plasma potassium levels were slightly higher in ablated mice with and without RAS blockade, no significant difference was found between the different groups (controls 5.3 ± 0.1, ablation 6.3 ± 0.5, ablation+losartan 6.4 ± 0.4, ablation+aliskiren 6.1 ± 0.4, ablation+losartan+aliskiren 6.6 ± 0.2 mmol/l).
Plasma and tissue levels of aliskiren.
In healthy FVB/N mice, 50 mg·kg−1·day−1 treatment yielded aliskiren plasma levels of 0.60 μg/ml [0.50; 0.71] (Fig. 7). In contrast, aliskiren tissue levels were between 4.00 and 17.50 μg/g wet weight for the heart, lung, liver, and spleen. This revealed ∼7- to 29-fold higher levels in these tissues compared with plasma. Interestingly, renal tissue levels [93.85 μg/g (67.70; 122.25)] were ∼156-fold higher than plasma levels. Comparing micrograms per milliliter and per gram is reasonable since 1 gram matches roughly 1 ml (7).
Aliskiren was given to healthy FVB/N mice, the pumps were removed after 14 days, and mice (n = 5 each) were immediately euthanized or at day 1, 3, or 7 thereafter. Aliskiren plasma levels almost decreased to below detection within 24 h (<0.01 μg/ml) (Fig. 8A), whereas tissue concentrations remained detectable for several days in the kidney (Fig. 8B) and heart (Fig. 8C). ANG II reappeared already on day 1 after cessation of therapy (Fig. 8D).
Withdrawal of aliskiren and losartan in mice with CKD.
Six-week therapy with losartan and aliskiren reduced albuminuria in mice with CKD. Withdrawal of aliskiren and losartan revealed a re-increase in albuminuria. This re-increase was significantly lower following cessation of aliskiren than of losartan (Fig. 9A), suggesting the renal accumulation of aliskiren to be of pathophysiological relevance. As plasma ANG II in healthy mice reappeared already on the first day subsequent to withdrawal of aliskiren (Fig. 8D), the observed delayed re-increase in albuminuria in mice with CKD may not relate to plasma angiotensin levels. Since ANG II was measured by ELISA (Fig. 8D) and by mass spectrometry (Fig. 1), we confirm with two different methods that plasma levels of ANG II are not increased in losartan-treated mice.
The present study is the first to compare head-to-head aliskiren and losartan based on equipotent RAS blockade in mice with CKD. High-dose renin inhibition ameliorates CKD to at least the same degree as does high-dose AT1 antagonism. Administration on top of the AT1 antagonist did not further improve CKD. We cannot confirm that other bioactive angiotensin peptides beyond ANG II play an essential role in renal injury, at least not in the renal ablation model in mice. We cannot rule out from our data that intrarenal angiotensin levels are differently regulated compared with plasma fragments. However, van Esch et al. (36) recently showed the same downregulation of renal and plasma levels of angiotensin fragments in aliskiren-treated rats. Finally, this is the first study demonstrating accumulation of aliskiren in the murine kidney and to a lower degree in the heart, lung, liver, and spleen. After withdrawal of treatment, plasma levels of aliskiren ceased within 24 h, whereas aliskiren was eliminated from both the kidney and heart slowly over several days.
Great care was taken to determine equipotent doses for aliskiren and losartan with respect to RAS inhibition to avoid any putative difference in nephroprotection to be merely attributed to incommensurable RAS blockade. Renin inhibition inhibits ANG II production whereas AT1 antagonism blocks binding to the AT1 receptor. Therefore, in the present study we used a downstream signal of the AT1 receptor, which is renin expression. Binding of ANG II to the AT1 receptor inhibits renin expression and inhibition of the RAS regardless of which position increases renin expression. As shown before, two different RAS inhibitors inhibit the RAS to the same extent if the increase in renin expression is of the same magnitude (27). Furthermore, RAS inhibition is almost maximal if a further dose increase does not increase renin expression any longer. In the present work, near-maximal doses were used to prevent additional effects of the double blockade to be simply due to underdosing of the monotherapies. In fact, in the recent AVOID trial, addition of aliskiren in patients with diabetic nephropathy pretreated with 100 mg losartan lowered proteinuria by an additional 18%, suggesting the superiority of a double blockade over monotherapy (32). However, 100 mg losartan/day is not a high-dose RAS blockade in patients. The present study, based on near-maximal doses, did not reveal superior nephroprotection by the double blockade. This clearly shows that inhibiting the increase in renin activity in response to AT1 antagonism does not result in additional nephroprotection.
Measuring angiotensin fragments and discovering new ones have been described by us (17–19). In the present study, we measured ANG II levels by mass spectrometry and by ELISA and were unable to detect increased levels of ANG II and other fragments in mice treated with high-dose losartan. It has been suggested that the levels of ANG II and other fragments increase in species treated with angiotensin receptor blockers. However, it should be taken into account that angiotensinogen levels in mice are low (as opposed to in humans), so that increases in renin result in depletion of angiotensinogen as shown previously (30). Consequently, a rise in renin (as will always occur during RAS blockade) will not necessarily result in a rise in ANG II (28).
Excellent nephroprotection by renin inhibition has been demonstrated in double transgenic rats carrying both the human renin and angiotensinogen gene as well as in diabetic rats overexpressing the mouse renin-2 gene (22, 33). However, since the overexpression of the renin gene causes end-organ damage, it is expected that aliskiren is protective. In fact, both transgenic models are based on high renin levels and are contrary to human CKD and renal ablation used in this study, where renin is downregulated.
Renin inhibition has been suggested to be superior to ACE inhibition or AT1 antagonism in previous, probably less well-controlled studies (22). Moreover, the combination of AT1 antagonism and renin inhibition has shown superior protection against renal fibrosis in models of diabetic nephropathy, unilateral ureter obstruction, and hypertensive renal injury in mice (8, 39–40). However, in none of these studies was RAS inhibition quantified as in the present study, nor did aliskiren monotherapy exceed low-dose treatment. Further studies also reported protective effects of renin inhibition. In some of those studies, however, the effects were compared vs. untreated animals (10, 12, 16, 21, 34).
Six-week administration of losartan and aliskiren significantly lowered albuminuria and glomerular injury. Both drugs did not reduce the increased interstitial volume caused by renal ablation. This is in line with our previous work demonstrating that RAS blockade decreases glomerular damage but is less efficient with regard to interstitial injury (13–14). Whether RAS blockade induced regression of renal injury or delayed progression of CKD in this study remains elusive. Long-term RAS inhibition beyond 6 wk may resolve this issue. Aliskiren has been shown to accumulate in the kidney of rats (10). Feldman et al. (10) found a ∼46-fold kidney-to-plasma ratio in rats treated with 10 mg·kg−1·day−1 aliskiren (10). The present study validates and expands these observations. This is the first report in mice that aliskiren accumulates ∼156-fold in the kidney compared with plasma. Moreover, we demonstrate for the first time a ∼7- to 29-fold accumulation in the heart, lung, liver, and spleen. After withdrawal, aliskiren plasma levels dropped within 24 h to nondetectable, whereas a delayed decline over several days was found in the heart and kidney. Interestingly, this contrasts with plasma half-life of aliskiren in humans (∼30–40 h after multiple dosing) (15), which was used to explain sustained reduced blood pressure after a missed dose and may counteract adverse effects due to imperfect adherence (5, 31). Interspecies variation of the pharmacokinetic entities of aliskiren thus need further evaluation.
The mechanisms for the accumulation of aliskiren are yet unknown. There is evidence that aliskiren accumulates in renin secretory granules, and, after exposure to renin-secreting cells, aliskiren-bound renin is released (26). In addition, Batenburg et al. (2) reported increased prorenin half-life in rat vascular smooth muscle cells after aliskiren binding. It is therefore likely that a part of the renal-accumulated aliskiren observed in our study is bound to renin within the juxtaglomerular cells. We propose that aliskiren accumulates ∼7- to 29-fold in the tissue by a yet unknown mechanism possibly involving the (pro)renin receptor or the putative clearance receptor of renin, the M6P/IGF2-receptor (2). The higher renal accumulation may relate to the renin content of the kidney.
Although renal accumulation of aliskiren was only measured in the present study in healthy mice, we speculate that the renal aliskiren accumulation may cause prolonged antiproteinuric effects after treatment cessation in CKD mice, emphasizing its putative pathophysiological significance. The re-increase in albuminuria was significantly lower after withdrawal of aliskiren than of losartan. Due to the short plasma half-life of aliskiren we observed in mice, this effect was most likely independent of plasma concentrations. This is in accordance with the recent observation of a prolonged antiproteinuric effect of aliskiren in rats (35). However, renal accumulation and prolonged efficacy after withdrawal may also be disadvantageous. Recently, the ALTITUDE trial, in which aliskiren was given on top of ACE inhibition and AT1 antagonism to high-risk patients with diabetes and renal impairment, was prematurely terminated due to increased morbidity and side effects like hyperkalemia in the double blockade group. In cases of volume depletion by i.e., diarrhea, complete and long-lasting RAS blockade may aggravate acute renal failure. A similar observation was made in the double blockade group in the ONTARGET study (41).
In summary, RAS blockade at the level of renin and the AT1 receptor, adjusted for equipotent dosages, offers similar nephroprotection in mice with CKD caused by renal ablation. This underscores the importance to target ANG II for nephroprotection. Combining renin inhibition and AT1 antagonism did not exert additional effects. Finally, aliskiren accumulated ∼156-fold in the kidney and ∼7- to 29-fold in other organs compared with plasma, providing prolonged antiproteinuric effects.
This study was supported by a grant from Novartis and by a grant of the German Research Foundation to U. Wenzel.
This study was supported in part by Novartis. A. H. J. Danser and U. Wenzel have received funding and a speaker's fee from Novartis.
Author contributions: C.F., C.K., R.A.S., and U.O.W. provided conception and design of research; C.F., S.L., C.K., A.H., J.B., E.S., I.G., A.J.D., A.-R.F., H.v.G., V.J., and U.O.W. performed experiments; C.F., S.L., C.K., N.D., E.S., T.S., J.V., I.G., A.J.D., A.-R.F., H.v.G., V.J., G.N., and U.O.W. analyzed data; C.F., S.L., C.K., E.S., T.S., A.J.D., V.J., G.N., and U.O.W. interpreted results of experiments; C.F., J.V., and U.O.W. prepared figures; C.F. and U.O.W. drafted manuscript; C.F., S.L., C.K., E.S., T.S., J.V., A.J.D., and U.O.W. edited and revised manuscript; C.F., S.L., C.K., A.H., J.B., N.D., E.S., T.S., J.V., I.G., A.J.D., A.-R.F., H.v.G., V.J., R.A.S., G.N., and U.O.W. approved final version of manuscript.
We thank M. Reszka, S. Schröder, and S. Gatzemeier for excellent technical assistance.
- Copyright © 2012 the American Physiological Society