Aliskiren reduces albuminuria after kidney transplantation *

Background: The renoprotective effects of the direct renin inhibitor, aliskiren, in renal transplant recipients have been supposed, but not finally proven. We performed an exploratory double-blind, losartan controlled, cross-over study to evaluate the influence of aliskiren, direct renin inhibitor, on albuminuria and other surrogate markers of kidney injury in patients after renal transplantation. The safety of this therapy was also evaluated. Method: 16 of 18 patients (12 M, 4 F), 48.3 ± 9.0 years, 57.7 ± 9.1 months after kidney transplantation, with hypertension and stable serum creatinine 1.4 ± 0.08 mg/dl without proteinuria, completed the protocol. Each patient underwent two 8-week treatment periods (one with 150 mg of aliskiren, and one with 50 mg of losartan) in random order, allowing an 8-week placebo washout between them. Results: There were no differences in albuminuria, transforming growth factor β-1 and 15-F2t-isoprostanes urine excretion between aliskiren and losartan. Creatinine serum level, eGFR, 24 h systolic and 24 h diastolic blood pressure were stable through the study. There were no differences in haemoglobin and potassium serum concentration between studied drugs. Conclusion: Aliskiren decreases albuminuria in renal transplant recipients with clinically minimal side effects. The effect does not differ from that of losartan.


INTRODUCTION
Interstitial fibrosis and tubular atrophy (IF/TA) in renal allografts, previously termed chronic allograft nephropathy (CAN), is a major cause of long-term renal graft dysfunction and loss (Solez et al., 1998).Although the functional and morphological findings in this pathology are well characterised, no effective therapy has been developed so far to prevent, limit, or reverse graft lesions (Paul, 1999).Agents inhibiting renin-angiotensinaldosteron system (RAAS) prevent and retard the progression of both diabetic and non-diabetic native kidney disease (Tylicki et al., 2005).Although the fundamental role of angiotensin II in the processes associated with the development of IF/TA, such as hypertension, arteriosclerosis, atherosclerosis and calcineurin inhibitor agents (cyclosporine, tacrolimus) toxicity, has been proven, the nephroprotective benefits of drugs inhibiting RAAS in renal transplant recipients remain unclear (Tylicki et al., 2003).Indeed, analyses of large databases and meta-analyses of small prospective clinical studies have provided conflicting results showing improved outcomes, detrimental effects or null effects of post-transplantation angiotensin converting enzyme inhibitors (ACEI) or angiotensin II receptor antagonists (ARA) therapy compared with other antihypertensive agents (Heinze et al., 2006;Opelz et al., 2006).Undoubtedly, ACEI and ARA are effective in reducing albuminuria or proteinuria in a similar manner as in the population with chronic native kidney disease (Tylicki et al., 2006).Consequently, according to the recent KDIGO recommendations, drugs that block the RAAS should be considered for use in all kidney transplant subjects with albuminuria after stable graft function is obtained (Roberts, 2014).Given the fact that microalbuminuria and macroalbuminuria are considered to be powerful independent predictors of end stage renal disease and death in renal transplant recipients, their use may offer both kidney and cardiovascular disease protection.
Direct renin inhibitor (DRI), aliskiren, is a representative of the newest class of agents inhibiting RAAS axis with a (similar as placebo) adverse reaction profile in monotherapy (Liu et al., 2013;Lizakowski et al., 2013b).Data on its renal effects in transplant patients is very limited.To shed more light on this issue we performed a randomized, controlled study to check influence of aliskiren on albuminuria and some other surrogate markers of kidney injury in this population.Losartan, ARA which was earlier evidenced to decrease albuminuria in renal transplant recipients, served as the control.

Patients.
Renal transplant recipients were recruited from a cohort that successively attended our outpatient department.The inclusion criteria were as follows: posttransplantation period above 6 months, calcineurin inhibitor (cyclosporine or tacrolimus) based immunosuppression, stable cyclosporine or tacrolimus trough level in the last three months (no variations above 25%), stable renal function defined as eGFR > 30 ml/min (no variations above 5 ml/min/1.73m 2 in the last 3 months), arterial hypertension treated with one or two antihypertensive agents or blood pressure (BP) > 130/80 mmHg in patients not treated yet, albuminuria > 30 mg/g creati-nine.In none of the patients was a graft biopsy available.Patients with graft artery stenosis, unstable coronary heart disease, or decompensated congestive heart failure in the previous 6 months, subjects with an episode of malignant hypertension or stroke in the history, and diabetics, were excluded.
General protocol.The study was an exploratory randomised, double-blind, controlled cross-over trial in which the renal effects of therapy with aliskiren (A) and losartan (L) were compared.It consisted of an 8-week run-in period, 8 weeks of active treatment with aliskiren or losartan (period 1), 8 weeks of active treatment with the alternative medication (period 2), and an 8-week placebo-washout (W) period between them (Fig. 1).At the beginning, subjects who met the inclusion criteria entered the 8-week run-in screening period.All the hypotensive group of drugs were allowed with the exception of ACEI, ARA, DRI and mineralocorticoid receptor antagonists.The target BP was an office trough BP of 140/90 mmHg or less.At the end of the run-in period patients were randomly allocated to one of the two treatment sequences: L/W/A (sequence 1) or A/W/L (sequence 2).Allocation was performed independently of the research team member, according to a computer-generated randomization list.The study medications were introduced as single hypotensive drugs, or added to the current hypotensive agents, the dosage of which, once adjusted in the run-in period, was left unchanged throughout the study.Losartan was used at a dose of 50 mg, and aliskiren was administered at a dose of 150 mg.Drug compliance was assessed by tablet counts.Placebo preparation and both drugs blinding were performed at the Department of Pharmaceutical Technology, Medical University of Gdansk.Patients were recommended not to change their usual daily protein and sodium intake during the study period (there were no differences in sodium urine excretion between aliskiren and losartan).Dosage of cyclosporine or tacrolimus was not allowed to be modified either.Before and after each of the treatment periods, office trough BP, 24-h ambulatory BP, albuminuria, serum creatinine and potassium, haemoglobin, urine excretion of N-acetyl-β-D-glucosaminidase (NAG), transforming growth factor β-1 (TGF-β-1) and 15-F2t-isoprostanes (isoprostanes) were determined.eGFR was calculated.Patients were also asked to fill in a questionnaire for assessment of patient-reported side effects.The study was approved by the local ethical committee and the investigated patients all gave their informed consent.
Methods.The office trough BP was measured by mercury sphygmomanometer in a sitting position after 10 minutes of rest, and expressed as a mean value of two consecutive measurements taken 2 minutes apart.Ambulatory BP was measured continuously for 24 h using the Mobil-o-graph (version 12) monitoring system.BP was measured every 15 minutes during the day (7.00 a.m. to 10.00 p.m.) and every 30 minutes during the night (10.00 p.m. to 7.00 a.m.).Results of office BP measurements were analysed for systolic (SBP) and diastolic (DBP) values; those of ambulatory BP measurements for 24 h SBP, 24 h DBP.Albumin excretion was measured in the first morning spot urine sample, as commonly recommended (Bailie et al., 2005).The first morning urine specimen is preferred, because it correlates best with 24hour protein excretion, and is required to avoid postural albuminuria.The authors calculated the ratio of albumin to creatinine (UACR) to correct for variations in urinary concentration due to hydration.The concentration of albumin was measured by enzyme-linked immunosorbent assay (ELISA) using an Albumin (Immunodiagnostic AG, Bensheim, Germany) kit in accordance with the manufacturer's recommendations.The intra-assay and interassay coefficients of variations for this assay were 5.0% and 8.0%, respectively.Haemoglobin, serum creatinine and potassium levels were measured by the standard laboratory techniques.eGFR was calculated according to the CKD-EPI formula.Adverse effects were recorded at each visit in response to questionnaires.
The first morning urine sample was collected for the determination of NAG, TGF-β-1 and isoprostanes.The samples were stored at -75°C until assayed.TGF-β-1 urine concentration was analyzed using a commercially available solid-phase enzyme-linked immunosorbent assay (Quntykine; R&D Systems, Minneapolis, MN).The minimum level of TGF-β-1 detectable with the test was 7 pg/ml.NAG was determined by the spectrophotometric method according to Maruhn (8).Incubation medium contained, in a final volume of 0.4 ml, 5 mmol/l p-nitrophenyl-2-acetamido-2deoksy-β-d-glucopyranoside as a substrate in 50 mmol/l citrate buffer (pH 4.14).The reaction was started by the addition of 0.2 ml of undialysed urine, carried out for 15 min at 37°C, and then terminated with 1 ml of glycine buffer, pH 10.5.Absorbance was measured at 405 nm against a sample terminated at time zero.The calculation of the NAG level was done from the molar extinction coefficient of the product of the reaction, p-nitrophenol, equal to 18.5 cm 2 /µmol.From the preliminary experiments, it was clear that the dialysis of urine did not affect NAG level in urine.A commercial ELISA kit (Cayman Chemical Co.) was then used to measure the urinary excretion of 15-F2t-isoprostanes.Urinary NAG, TGF-β-1 and isoprostanes were reported per mg of urine creatinine to correct the variation in urine concentration.
Statistics.The primary end point of the study was a difference in UACR in the measurements available for each patient.The secondary end point was the presence of differences in NAG, TGF-β-1 and isoprostanes urine excretion in the measurements available for each patient.In addition, a safety analysis of the therapy with aliskiren, involving its influence on serum creatinine, potassium, and haemoglobin level, was performed.
The normality and homogeneity of the variances were verified by the Shapiro-Wilk test and the Levene test, respectively.Because of their skewed distribution, values of UACR, NAG, TGF-β-1 were logarithmically transformed before statistical analysis, and expressed as geometric means and 95% confidence intervals.Other results were expressed as means ± S.D. In the per-protocol design, the variable differences between aliskiren and losartan were assessed by t-test.A P < 0.05 (two-tailed) was considered statistically significant.Data was evaluated using STATISTICA (version 6.0 Stat Soft Inc.) software package.
To prevent or limit the possibility of a "period effect", we introduced a degree of balance into the study design, with a scheme of randomisation allowing every treatment sequence to be represented in every period with the same frequency.Overall, we had two different therapy sequences with the three treatment periods (Fig. 1).Equal number of patients (n=9) per sequence was randomised.Since 2 patients were prematurely withdrawn, this balance was fully respected at the study end.To prevent or limit the risk of "carryover" effect, we planned each treatment period for 8 weeks, and placebo treatment between them.Previous studies demonstrated that the effects of RAAS blocking agents on albuminuria and glomerular permselectivity are fully reversible within 4 weeks (Gansevoort et al., 1994).Thus, prolonging each treatment period to 8 weeks and introducing washoutplacebo therapy between them, allowed us to completely rule out any residual effect of previous treatment at the end of the eighth week of second period, when UACR was measured.

RESULTS
Of the 18 patients who entered the study, 16 (88.8%)completed the protocol.Their characteristics are presented in Table 1A and 1B.Two patients were withdrawn from the study, one subject from each of the 2 treatment sequences.One person was excluded due to important deviation from the study protocol.Another one resigned from participation in the study for personal reasons.Before data analysis, the "period effect" and "carryover effect" were tested and found to be not significant.
Renal function assessed by means of serum creatinine and eGFR remained stable during the study periods.There was no difference in potassium concentration be-   tween the treatments (Table 2).In none of the patients was a potassium level above 5.5 mmol/L observed.Haemoglobin level decreased numerically after aliskiren and losartan.This, however, was not associated with anaemia symptoms, and none of the patients needed treatment for anaemia.There were no differences between aliskiren and losartan in this regard (Table 2).Side-effects reported de novo during treatments are showed in Table 3.There were no significant differences in their frequency between the three treatments.

DISCUSSION
Some years ago, DRI, a new class of drugs that selectively inhibits angiotensin II formation at the first step of the RAAS cascade, were introduced to clinical practice.Aliskiren was the first orally bioavailable DRI approved for the treatment of hypertension.Once-daily oral treatment with aliskiren lowers BP effectively, with a safety and tolerability profile comparable with that of placebo (Liu et al., 2013;Lizakowski et al., 2013b).In our previous papers, we demonstrated that aliskiren significantly reduced proteinuria in nondiabetic chronic kidney disease, and this antiproteinuric effect was similar to that of ACEI in equivalent hypotensive dosages (Lizakowski et al., 2013a).In a double-blind, randomised, crossover study Persson et al. demonstrated the same in diabetics (Persson et al., 2009).Szeto et al. found that aliskiren has anti-proteinuric effect in patients with IgA nephropathy and persistent proteinuria despite ACEI or ARA (Szeto et al., 2013).Enthusiasm for the use of aliskiren declined after the ALTITUDE study that prompted the FDA to issue the recommendation not to use aliskiren, combined with other RAAS inhibiting agents, in patients with diabetes or moderate to severe renal impairment.The reason of such statement was the risk of hypotension, acute renal impairment, and hyperkalaemia during such combination therapy (Parving et al., 2012).Nevertheless, much evidence of the effectiveness and safety of aliskiren in monotherapy for the treatment of hypertension and proteinuria in different populations leads to the conclusion that aliskiren may be considered as an equivalent alternative to ACEI or ARA in certain conditions.
There are no controlled studies analysing the effects of aliskiren in renal transplant recipients so far.In experimental models of renal transplantation aliskiren had no effect on renal function (i.e.proteinuria, creatinine clearance), or on renal morphological changes (i.e.collagen deposition in glomerulosclerosis, myofibroblast accumulation and macrophage infiltration) determined 24 weeks after transplantation (Rusai et al., 2011).On the contrary, histopathological and ultrastructural studies showed that aliskiren may attenuate tacrolimus-induced renal damage in rats, implying that aliskiren may counteract nephrotoxicity associated with calcineurin inhibitors applied after renal transplantation (Al-Harbi et al., 2014).
In our previous report, we showed that angiotensin receptor antagonist, losartan, decreases albuminuria in renal transplant recipients (Tylicki et al., 2006).In this exploratory short-term study, we demonstrated, for the first time to our knowledge, no differences between losartan and aliskiren in this regard.The measurement of urinary albumin excretion provides a sensitive marker of glomerular injury with impaired permselectivity (Komenda et al., 2014).In renal transplant recipients, proteinuria is regarded as an excellent marker of poor long-term allograft prognosis (Fernandez-Fresnedo et al., 2004; Ibra-  et al., 2014).Albuminuria is also considered as an important risk factor for cardiovascular complications (Komenda et al., 2014).
Considering the fact that the extent of tubulointersitial damage is a fundamental predictor of kidney outcome, tubular cells have become a renal site of particular interest.To evaluate tubulointerstitial effects of our interventions, urine excretion of NAG, enzyme of the hydrolase class which is abundant in the kidney, predominantly in the lysosomes of the proximal tubular cells, was analysed as a secondary end point.It is physiologically excreted in low amounts in urine as a consequence of the normal exocytosis process (Price, 1982).The increased excretion of NAG is thought to be a specific marker of tubular injury in many renal pathologies (Sherman et al., 1983).Considering non-hemodynamic mechanisms of possible benefits of aliskiren i.e. antagonizing the proinflammatory and profibrotic effects of angiotensin II, we also evaluated the influence of aliskiren on urine excretion of TGF-β1 and isoprostanes.TGF-β1 is a key profibrotic cytokine involved in the pathogenesis of IF/TA in renal allografts.A significant correlation between intragraft TGF-β1 mRNA and renal allograft interstitial fibrosis was reported previously.Furthermore, TGF-β1 urine excretion is increased in overt IF/TA (Djamali et al., 2009;Sharma et al., 1996).
The urinary excretion of 15-F2t-isoprostanes is a reliable and sensitive marker of intrarenal oxidative stress, the process known to be involved in the inflammation and kidney fibrosis (Renke et al., 2013).Losartan decreased NAG urine excretion to a greater extent than aliskiren in studied patients.One may assume that the greater sodium intake observed during aliskiren therapy might affect the results.No differences between aliskiren and losartan were found in TGF-β1 and 15-F2t-isoprostanes excretion.Small sample size and relatively short term of observation do not allow drawing final conclusions from these observations.The question, whether potentially beneficial effects of aliskiren on albuminuria translate into the long-term renal graft outcome, remains open.Evidence may be provided only by histological examinations, or controlled studies focused on graft function.Until now, there has been no such report for aliskiren.Moreover, the nephroprotective benefits of either ACEI or ARA in renal transplant recipients remain unclear as well.Some years ago, we demonstrated that losartan decreases albuminuria and surrogates for tubular damage and graft fibrosis (Tylicki et al., 2007).Since then, the research on this issue has not progressed too far.The Study on Evaluation of Candesartan Cilexetil after Renal Transplantation (SE-CRET) was not able to evidence the beneficial influence of ARA, candesartan, on graft failure/patients all-cause mortality/cardiovascular morbidity, but confirmed that candesartan reduced proteinuria (Philipp et al., 2010).The recent study of Ibrahim et al. was also inconclusive.They conducted a double-blind, randomized, placebocontrolled trial, to determine whether losartan prevents the expansion of the cortical interstitial compartment, the precursor of fibrosis.Treatment with losartan did not lead to a statistically significant reduction in a composite of interstitial expansion or ESRD from IF/TA in kidney transplant recipients.On the other hand, in a secondary analysis, losartan seemed to reduce the risk of a composite of doubling of interstitial volume, or all-cause ESRD (Ibrahim et al., 2014).A retrospective analysis by Heinze et al. found evidence supporting the benefit of RAAS blockers in kidney transplant recipients (Heinze et al., 2006), whereas the Collaborative Transplant Study report developed by Opelz and coworkers (2006) revealed no improvement of patient or graft survival in transplant recipients treated with ACEI or ARA (Opelz et al., 2006).Quite recently, a large retrospective analysis showed that the rate of cardiovascular death in kidney transplantation recipients receiving ACEI/ARA, or other antihypertensive medications, is virtually identical (Opelz et al., 2014).
A potential limitation of our study was the small sample size.Lack of repeated measurements of UACR at each study visit may be also recognized as a limitation.Calculation of albumin/creatinine ratio in the urine does not completely eliminate the variability of the urine albumin excretion.Treatment periods may be too short, and potentially beneficial renal effects may not yet fully develop.The effects for tubules and interstitium in the study were extrapolated only from presumptive early surrogates.It should also be taken into account, that the authors chose for their study a series of stable renal transplant recipients with excellent graft function.Given these together, the study results allow a conclusion only of pilot character.
In conclusion, aliskiren decreases albuminuria in renal transplant recipients with clinically minimal side effects.The renal effects do not differ from that of ARA, losartan.Given the strong prognostic value of albuminuria for graft outcome (Fernandez-Fresnedo et al., 2004), and the fact that albuminuria is also considered as an important risk factor for cardiovascular complications (Fernandez-Fresnedo et al., 2002), the present study may support the hypothesis that aliskiren improves allograft and patient outcome.