Regular paper Vol. 57, No 4/2010 547–552

BACKGROUND
There is evidence that dyslipidemia is associated with chronic kidney disease (CKD) and it has been implicated in the progression of renal damage. Optimal management of dyslipidemia should therefore lead to renal benefits. A number of experimental models demonstrate a beneficial effect of statins in ameliorating renal damage. However, the exact mechanism by which statins protect against renal damage remains unclear.


METHODS
In a placebo-controlled, randomized, cross-over study we evaluated the influence of atorvastatin (ATO) 40 mg/day added to the renin-angiotensin-aldosterone systeme (RAAS) blockade on proteinuria and surrogate biomarkers of tubular damage or injury in 14 non-diabetic patients with proteinuria (0.4-1.8 g per 24 h) with normal or declined kidney function (eGFR 55-153 ml/min). In the eight-week run-in period, therapy using angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin II subtype 1 receptor antagonists (ARB) was adjusted to achieve a blood pressure below 130/80 mm Hg. Next, patients were randomly assigned to one of two treatment sequences: ATO/washout/placebo or placebo/washout/ATO. Clinical evaluation and laboratory tests were performed at the randomization point and after each period of the study. The primary end point of this study was a change in proteinuria measured as 24-h urine protein excretion (DPE). Secondary end points included urine N-acetyl-β-D-glucosaminidase (NAG) and α1-microglobulin (α1m) excretion.


RESULTS
The ATO therapy significantly reduced urine excretion of α₁m (p=0.033) and NAG (p=0.038) as compared to placebo. There were no differences in proteinuria, blood pressure, eGFR and serum creatinine between the ATO and placebo groups.


CONCLUSION
Atorvastatin treatment is safe and improves biomarkers of tubular damage or injury in non-diabetic patients with CKD.


INTRODUCTION
Despite recent progress, there is still no optimal therapy that stops progression of renal disease.Therefore, it is necessary to search for alternative therapeu-tic strategies which can further improve renal outcome (Renke et al., 2010).There is evidence that dyslipidemia is associated with chronic kidney disease (CKD) (Guijarro & Keane, 1993;Samuelsson et al., 1997).Experimental studies have established that lipids are damaging to the kidney (Keane et al., 1988;Rutkowski et al., 2003).The administration of various statins has been reported to exhibit beneficial effects in a number of experimental models of chronic kidney diseases suggesting that lipids may represent important therapeutic targets to halt or attenuate renal injury (Tylicki et al., 2003).The benefits of statins can be explained not only by their lipid-lowering potential but also by non-lipid related mechanisms, the so called "pleiotropic effects".Several studies have evaluated the effects of statins on the progression of CKD in human subjects but the results are controversial (Chan et al., 1992;Fuiano et al., 1996;Bianchi et al., 2003;Strippoli et al., 2008;Banach et al., 2009).Considering the prognostic impact of proteinuria reduction on long-term renal outcome, in the present study we evaluated the effects of addition of atorvastatin (ATO), a 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to background nephroprotective therapy consisting of angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin II subtype 1 receptor antagonists (ARB).ATO, in contrast to many other statins, does not require dosage modification at any level of renal function (K/DOQI 2003).The patients were then evaluated for proteinuria, inflammation, renal function, and surrogate biomarkers of tubular injury.The primary end point of this study was a change in proteinuria measured as 24-h urine protein excretion (DPE), in measurements available for each patient.Secondary end points included urine N-acetyl-β-d-glucosaminidase (NAG) and α 1microglobulin (a1m) excretion.

METHODS
Patients were selected from a cohort that attended our renal outpatients' department.The inclusion criteria were as follows: age 18-65 years, chronic non-diabetic proteinuric nephropathy without dyslipidemia, normal or slightly impaired stable renal function expressed as estimated glomerular filtration rate (eGFR) above 45 ml/min, stable proteinuria above 300 mg/24 h, and no steroids or other immunosuppressive treatment for a minimum of six months before the study.Stable renal function and proteinuria were defined as a variability of serum creatinine and proteinuria less than 25 % during six months before the start of the study.Patients with total cholesterol less than 200 mg/dl, low-density lipoprotein (LDL) cholesterol < 130 mg/dl, and triglycerides < 150 mg/ dl were included.Exclusion criteria were as follows: nephritic syndrome, diabetes mellitus, cardiovascular disease (CVD), potassium serum level > 5.1 mmol/l, history of malignancy including leukemia and lymphoma, fertile women who were not taking oral contraceptives, pregnant or lactating women, patients with active liver disease, i.e., aspartate aminotransferase (ASAT) or alanine aminotransferase (ALAT) values more than three times the upper reference values, and known or suspected contraindications to the study medications, including a history of adverse reactions to statins, ACEI or ARB.
General protocol.The study was a prospective, placebo-controlled, randomized, two-period cross-over trial in which the renal effects of adding ATO (Sortis; Parke-Davis, Pfizer Polska) to background nephroprotective therapy with ACEI and/or ARB (Xartan; Adamed Polska) were evaluated.At the beginning, subjects entered an eight week run-in period during which the background nephroprotective therapy using pharmacological blockade of RAAS was adjusted to give a target blood pressure (BP) below 130/80 mm Hg (Table 1).At the end of the run-in period, patients were randomly assigned to one of two treatment sequences: twelve-week ATO (40 mg/day)/12week washout -background therapy/12-week placebo (sequence 1) or 12-week placebo/12-week washout -background therapy/12-week ATO (40 mg/day) (sequence 2) (Fig. 1).Allocation was performed by a person that was independent of the research team according to a computer generated randomized list.The patients received 40 mg of ATO as tablets (Sortis 40, Pfizer) once a day.The target BP during the whole study was an office visit BP of 130/80 mm Hg or less.The dosages of ACEI, ARB and diuretics, once established in the run-in period, were left unchanged throughout the study and in the washout period.At the randomization point and after the end of each treatment periods, office trough BP, serum creatinine, potassium, proteinuria measured as 24-h urine protein excretion (DPE), sodium excretion (Na ex), urea excretion, and surrogate markers of tubular injury (urine excretion of N-acetyl-β-d-glucosaminidase (NAG), α-1 -microglobulin (α1m)) were determined.The study was approved by the local ethical committee (NKE-BN/749/2003) and all the patients gave informed consent.The study was registered at www.clinicaltrials.gov and received a positive opinion (NCT00572312).
Procedures and laboratory analyses.The office trough BP was measured with Speidel+Keller sphyngomanometer in a sitting position after 10 min of rest and expressed as a mean value of two consecutive measurements taken 2 min apart.DPE, Na ex and urea excretion were evaluated on the basis of 24-h urine collection.All patients were equipped with a scaled container and were strictly informed how to collect 24-h urine.They collected two 24-h urinesof those the mean value of DPE was calculated for data evaluation.Patients were asked not to perform heavy physical activity on the urine collection days and were recommended not to change their usual daily protein and sodium intake during the study period.The excretion of urea was used to calculate the protein intake according to Maroni equation: protein intake normalized to weight (g/kg per day)=6.25×([urea-N-excretionurine 24 h (g/day)]+[0.0031×bodyweight (kg)])/ body weight (kg) (Maroni et al., 1985).eGFR was calculated according to Cockcroft-Gault formula (Cockcroft & Gault, 1976).NAG and α1m were analyzed in the second morning spot urine sample.NAG was determined by the spectrophotometric method according to Maruhn (1976).Incubation medium contained in a final volume of 0.4 ml, 5 nmol/l P-nitrophenyl-2-acetamido-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 made from the molar absorbance coefficient of the product of the reaction, P-nitrophenol, equal to 18.5 cm 2 /μmol.From preliminary experiments it was clear that the dialysis of urine did not affect NAG level in urine.Immunoturbidimetric test (Tina-quant α 1 -microglobulin, Roche, Basel, Switzerland) was used for quantification of α1m in urine.The detection limit of the method was 2 mg/l.Urinary NAG and α1m were reported per mg or g of urine creatinine to correct for the variation in urine concentration.We measured high sensitive C-reactive protein (hsCRP) with a commercial ELISA Kit (DRG, EIA-3954) and reported it in mg/l.Total cholesterol, LDL cholesterol, HDL cholesterol, serum triglyceride, ASAT, ALAT, creatine phosphokinase (CK), potassium, sodium, urea, protein and creatinine levels were measured in fresh blood samples drawn after fasting overnight for at least 12 h.These parameters were measured by standard laboratory techniques.Body mass index (BMI) was calculated asmass (kilograms) divided by height (meters) squared.Adverse effects were recorded at each visit in response to questionnaires or as observed by the investigators.
Statistics.The primary end point of this study was a change in DPE in measurements available for each patient.A sample size of 12 patients adequately allowed a power of 80 % to detect a difference in variables equal to within patient standard deviation, that is a standardized effect size of 1.0 at a significance level of 0.05 (two-tailed).Secondary end points included urine NAG and a1m excretion.Normality and homogeneity of the variances were verified by means of the Shapiro-Wilk test and Levene test, respectively.Because of their skewed distribution, diastolic BP, DPE, NAG excretion, hsCRP, serum creatinine and daily protein intake were logarithmically transformed before statistical analysis and expressed as geometric means and 95 % confidence intervals.Other results are presented as means ± S.E.M. Differences in variable changes between treatment with ATO and placebo were assessed using Student's t-test (Table 2).Differences in variables measured more than twice (Table 3) were assessed using ANOVA.P values less than 0.05 (2-tailed) were considered statistically significant.Data were evaluated using Statistica (version 7.1; StatSoft Inc., Tulsa, OK) software package.

RESULTS
Of the 14 patients who entered the study, 12 (86 %) completed the protocol.Two of them were dropped out because of withdrawal of informed consent.This decision was not due a side effect of therapy.Clinical characteristics of patients are listed in Table 1.

Twenty-four-hour urine protein excretion (DPE)
There was no significant change in DPE after ATO as compared to placebo (Table 2).

Serum lipid levels
Total cholesterol (P=0.001) and LDL cholesterol (P=0.001)decreased significantly after ATO as compared to placebo.There were no significant changes in triglyceride and HDL cholesterol during the study (Table 2).

Blood pressure, renal function, hsCRP, sodium and protein intake
The control of BP was adequate in all study periods; all patients reached the target office trough BP below 130/80 mm Hg.There were no differences in office trough systolic and diastolic BP between the treatment periods.Renal function assessed by means of serum creatinine and eGFR remained stable throughout the study.hsCRP levels had a tendency to decrease in ATO treatment but it was not statistically significant (P=0.47).There were no differences in sodium and protein intake between treatment periods (Table 3).

Safety
ATO therapy was well tolerated by all patients.Adverse effects were not reported.ASAT and CK were unchanged during the study period.ALAT statistically increased after ATO (P=0.049) but it was still in the normal range.

DISCUSSION
To the best of our knowledge, the present study was the first to evaluate the influence of atorvastatin, an HMG-CoA reductase inhibitor, on the markers of renal outcome in proteinuric CKD patients without dyslipidemia and CVD.We analysed the effects of ATO (40 mg/day) on proteinuria, the fundamental marker of glomerular injury and impaired glomerular permselectivity.Proteinuria is also a marker of long-term renal outcome.In the present study, the administration of ATO provided no change in proteinuria level (P=0.98) in non-diabetic CKD patients.Only a few randomized controlled trials directly addressing the effect of statins on renal function and proteinuria have been reported.Most of those studies were of small size or short duration, used a variety of statins, and many did not include a placebo arm.Some of them suggest that statins reduce proteinuria and the rate of decline of GFR (Bianchi et al., 2003;Tonelli, 2006).These positive effects have been summarized in published meta-analyses (Fried et al., 2001;Sandhu et al., 2006;Strippoli et al., 2008).Interestingly, there are also studies suggesting that statins, particularly at high doses, may increase proteinuria (Deslypere et al., 1990;Verhulst et al., 2004).Finally, the National Lipid Association Statin Safety Task Force recently reported that statin-induced proteinuria is not associated with renal impairment or renal failure (McKenney et al., 2006).
Considering that tubular epithelial cell injury may initiate the fibrosis process in kidneys and the fact that the extent of tubulointerstitial damage is a crucial predictor of renal outcome, tubular cells have become a renal site of particular interest.To evaluate tubulointerstitial effects of our interventions, the tubular involvement markers NAG and α1m were analysed (Bazzi et al., 2002).
An increased excretion of NAG is thought to be a specific marker of tubular injury in many renal pathologies including non-diabetic CKD (Bazzi et al., 2002).Increased urinary excretion of α1m, a low-weight protein physiologically filtered and reabsorbed by tubular cells, might indicate a reduced capacity of its reabsorption by such cells and it might be a marker of established tubular damage, with greater urinary concentrations pointing to greater severity of damage (Holdt-Lehmann et al., 2000).Our results show that treatment with ATO reduces markers of tubular injury.Similar results (although in experimental models) were described by Tsujihata and co-workers (2008).That group reported that ATO had inhibitory effects on renal tubular cell injury.In human subjects Nakamura and co-workers (2006) presented data suggesting that pitavastatin ameliorated tubulointerstitial damage in CKD patients.That effect was independent of the lipid-lowering effect (Nakamura et al., 2006).
The pleiotropic effects of statins have important clinical implications, independent of their lipid-lowering effects (Fathi et al., 2004;Tonelli et al., 2004;Epstein and Campese, 2005;Nissen et al., 2005;Ridker et al., 2005;Goicoechea et al., 2006;Panichi et al., 2006;Renke et al., 2010).In our previous pilot study we confirmed that ATO therapy attenuated oxidative stress in patients with CKD (Renke et al., 2010).They are at an increased risk for CVD, and recent reviews suggested that inflammation and oxidative stress could be the primary mediators explaining the burden of CVD in CKD patients (Arici & Walls, 2001).Moreover, inflammation plays a central role in the progression of CKD (Tonelli et al., 2005;Zoja et al., 2006).Our study used hsCRP, a protein found in the blood, as a marker of inflammation.Interestingly, patients with elevated basal levels of CRP are at an increased risk of diabetes, hypertension and cardiovascular disease (Pradhan et al., 2001;Dehghan et al., 2007).In our study this parameter had a tendency to decrease with ATO treatment, but the result was not statistically significant (P=0.47).The fact that most of the patients had serum hsCRP levels in the normal range at the beginning of the study is probably the main reason why our results are different from those of some other authors (Chang et al., 2002;Ichihara et al., 2002;Vernaglione et al., 2004).Our study confirms the findings of others (Newman et al., 2006;Shurraw & Tonelli 2006;Newman et al., 2008) that ATO therapy is well tolerated by CKD patients.Adverse effects were not reported during the study period.It is unlikely that confounding factors might have influenced the outcome of the present study.The treatment periods did not differ with respect to blood pressure, patients` sodium and protein intake as well as renal function.We believe that the nephroprotective properties of ATO need to be addressed further in future controlled long term studies.
A potential limitation of the study is the relatively small sample size, although it was sufficiently powered to detect a significant difference equal to the S.D. value between treatment periods.A further limitation would be the fact that the participants were selected on the basis of their stability.The 24-h urine collections used to assess proteinuria may be associated with significant collection errors, largely because of improper timing and missed samples, leading to over-and under-collection.In addition, one should realize that the potential benefits for tubules and interstitium were extrapolated from presumptive early surrogates.Such evidence should be confirmed by histological examination.
In conclusion, the study results suggest that treatment with ATO (40 mg/day) for 12 weeks in nondialysis patients with CKD induced, in addition to its lipid-lowering effect, a significant decrease in biomarkers of tubular injury and damage without change in proteinuria.The treatment was safe and well tolerated by patients.

Table 1 . Patient characteristics at baseline
Note: To convert serum creatinine in mg/dl to µmol/l, multiply by 88.4; eGFR in ml/min/1.73m 2 to ml/s/1.73 m 2 , multiply by 0.01667; Abbreviations: BMI, Body mass index; hsCRP, high sensitive C-reactive protein; eGFR, estimated glomerular filtration rate