[Corrigendum] Establishment of hypoxia induction in an in vivo animal replacement model for experimental evaluation of pancreatic cancer.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that, on p. 156, the data panels shown to represent the 'CoCl2' and 'TRIP' data panels in Fig. 3 for the DAPI experiments were apparently the same, even though different experiments were being depicted here.  The authors were able to re­examine their original data files, and realized that this figure had been assembled incorrectly: there was an inadvertent mix­up of a pair of the DAPI control images. The revised version of Fig. 3, containing the correct DAPI data for the 'TRIP' experiment, is shown opposite. Note that the revisions made to this figure do not affect the overall conclusions reported in the paper. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [Oncology Reports 32: 153­158, 2014; DOI: 10.3892/or.2014.3196].

Factors influencing post-exercise proteinuria after marathon and ultramarathon races.

Post-exercise proteinuria is one of the most common findings observed after short and intensive physical activity, but is observed also after long runs with low intensity. The aim of this study was to analyze factors influencing proteinuria after marathon runs. Two groups of male amateur runners were studied. The results of 20 marathon finishers (42.195 m), with a mean age of 49.3 ± 6.85 years; and 17 finishers of a 100-km ultramarathon with a mean age of 40.18±4.57 years were studied. Urine albumin to creatinine ratio (ACR) was calculated before and after both races. The relationship between ACR and run pace, metabolites (lactate, beta hydroxybutyrate), markers of inflammation (CRP, IL-6) and insulin was studied. The significant increase in ACR was observed after both marathon races. ACR increased from 6.41 to 21.96 mg/g after the marathon and from 5.37 to 49.64 mg/g after the ultramarathon (p<0.05). The increase in ACR was higher after the ultramarathon that after the marathon. There was no correlation between run pace and proteinuria. There was no correlation between ACR and glucose, free fatty acids, lactate, beta-hydroxybutyrate and insulin levels. There was significant negative correlation between ACR and interleukin 6 (IL-6) (r =-0.59, p< 0.05) after ultramarathon. Proteinuria is a common finding after physical exercise. After very long exercises it is related to duration but not to intensity. There is no association between metabolic and hormonal changes and ACR after marathon runs. The role on inflammatory cytokines in albuminuria is unclear.

Intraductal papillary mucinous neoplasm of the pancreas rapidly xenografts in chicken eggs and predicts aggressiveness.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas has a high risk of progressing to invasive pancreatic ductal adenocarcinoma (PDA), but experimental models for IPMN are largely missing. New experimental systems for the molecular characterization of IPMN and for personalized prognosis and treatment options for IPMN are urgently needed. We analyzed the potential use of fertilized chicken eggs for the culture of freshly resected IPMN tissue. We transplanted 49 freshly resected IPMN tissues into eggs and compared the growth characteristics to IPMN tissues transplanted into mice; this was followed by an analysis of histology, morphology, and marker expression. Of the IPMN tissues transplanted into eggs, 63% formed tumor xenografts within 4 days, while none of the 12 IPMN tissues transplanted into immunodeficient mice engrafted. In the eggs, the grafting efficiency of high-grade (n = 14) and intermediate-grade (n = 17) dysplasia was 77% and was significantly higher than the 39% grafting efficiency of low-grade dysplasia (n = 18). According to mucinous expression, 46 IPMN tissues were classified into gastric (n = 6), intestinal (n = 3), oncocytic (n = 23), and pancreatobiliary (n = 14) subtypes. The grafting efficiency was highest for the pancreatobiliary subtype (86%), followed by the oncocytic (70%), gastric (33%) and intestinal (33%) subtypes. The morphology and expression patterns of mucins, progression markers and pancreatic ductal markers were comparable between the primary IPMN tissues and their xenograft copies. The individual tumor environment was largely maintained during subtransplantation, as evaluated upon passage 6. This new IPMN model may facilitate experimental studies and treatment decisions for the optimal personalized management of IPMN.

Dexamethasone mediates pancreatic cancer progression by glucocorticoid receptor, TGFß and JNK/AP-1.

Glucocorticoids such as dexamethasone are widely co-prescribed with cytotoxic therapy because of their proapoptotic effects in lymphoid cancer, reduction of inflammation and edema and additional benefits. Concerns about glucocorticoid-induced therapy resistance, enhanced metastasis and reduced survival of patients are largely not considered. We analyzed dexamethasone-induced tumor progression in three established and one primary human pancreatic ductal adenocarcinoma (PDA) cell lines and in PDA tissue from patients and xenografts by FACS and western blot analysis, immunohistochemistry, MTT and wound assay, colony and spheroid formation, EMSA and in vivo tumor growth and metastasis of tumor xenografts on chicken eggs and mice. Dexamethasone in concentrations observed in plasma of patients favored epithelial-mesenchymal transition, self-renewal potential and cancer progression. Ras/JNK signaling, enhanced expression of TGFß, vimentin, Notch-1 and SOX-2 and the inhibition of E-cadherin occurred. This was confirmed in patient and xenograft tissue, where dexamethasone induced tumor proliferation, gemcitabine resistance and metastasis. Inhibition of each TGFß receptor-I, glucocorticoid receptor or JNK signaling partially reversed the dexamethasone-mediated effects, suggesting a complex signaling network. These data reveal that dexamethasone mediates progression by membrane effects and binding to glucocorticoid receptor.

Ethical euthanasia and short-term anesthesia of the chick embryo.

Fertilized chicken eggs are suggested as an alternative to mammalian models. The chorioallantoic membrane (CAM) of the chick embryo is widely used for examination of angiogenesis, xenotransplants and for virus production. Unfortunately, it is mostly not taken into account, that the chick embryo's ability to experience pain starts to develop at day 7 of breeding. In our view, this model is only in accordance with the 3 R principles, if an appropriate anesthesia of the chick embryo in potentially painful procedures is provided. Although many experimental approaches are performed on the none-innervated CAM, the euthanasia of the embryo strongly requires a more human technique than the usually used freezing at -20°C, decapitation or in ovo fixation with paraformaldehyde without prior anesthesia. However, protocols regarding feasible and ethical methods for anesthesia and euthanasia of avian embryos are currently not available. Therefore, we established an easy and reliable method for the euthanasia and short-term anesthesia of the chick embryo.

Establishment of hypoxia induction in an in vivo animal replacement model for experimental evaluation of pancreatic cancer.

Transplantation of tumor xenografts to fertilized chicken eggs is a promising animal replacement method, which has successfully been used for xenotransplantation of pancreatic ductal adenocarcinoma (PDA) cells. PDA is characterized by a pronounced tumor hypoxia, which mediates aggressive growth, therapy resistance and cancer stem cell (CSC) features. For in vivo experimental evaluation of hypoxia-targeting therapeutic strategies, the xenografting of tumors to chicken eggs combined with the induction of hypoxia is necessary. However, the chicken embryos do not survive the conventional method of hypoxia induction by a gas mixture of 1% O2, 5% CO2, 94% N2, not even when hypoxia is applied for only 30 min. Therefore, we employed chemical induction of hypoxia by the hypoxia mimetic agent cobalt chloride (CoCl2). Whereas CoCl2 did not further increase tumor growth, it mediated the induction of carbonic anhydrase IX (CAIX) in the tumor xenografts and led to enhanced expression of the human CSC markers CD133, Sox2 and CD44. Side-effects in chicken embryos were not observed as evaluated by H&E staining of embryo-derived liver sections and the determination of the embryo weight. These results suggest the successful induction of hypoxia in chicken eggs and xenografted tumors by CoCl2. For therapeutic intervention and as a control, we treated the eggs with the plant-derived anti-inflammatory agent triptolide, which recently showed promising effects toward hypoxia-induced tumor progression in experimental PDA. Triptolide abolished tumor growth and the CoCl2-induced hypoxic effects, without inducing obvious side-effects. Collectively, our data present a new in vivo animal replacement method for the successful induction of tumor hypoxia in PDA.

Triptolide reverses hypoxia-induced epithelial-mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions, a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as "thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation, aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity, migratory potential and a switch in morphology to a fibroblastoid phenotype, as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines, whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors, triptolide downregulated markers of CSCs, proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA.

Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells.

Advanced androgen-independent prostate cancer (AIPC) is an aggressive malignancy with a poor prognosis. Apoptosis-resistant cancer stem cells (CSCs) have been identified in AIPC and are not eliminated by current therapeutics. Novel therapeutic options, which are currently being evaluated in patient studies, include TRAIL and the broccoli-derived isothiocyanate sulforaphane. Although neither agent targets normal cells, TRAIL induces apoptosis in most cancer cells, and sulforaphane eliminates CSCs. In this study, the established AIPC cell lines DU145 and PC3, with enriched CSC features, and primary patient-derived prostate CSCs were treated with sulforaphane and recombinant soluble TRAIL. We examined the effects of these drugs on NF-κB activity, self-renewal and differentiation potential, and stem cell signaling via spheroid- and colony-forming assays, FACS and western blot analyses, immunohistochemistry, and an antibody protein array in vitro and after xenotransplantation. We largely found a stronger effect of sulforaphane on CSC properties compared to TRAIL, though the agents acted synergistically when applied in combination. This was associated with the inhibition of TRAIL-induced NF-κB binding; CXCR4, Jagged1, Notch 1, SOX 2, and Nanog expression; ALDH1 activity inhibition; and the elimination of differentiation and self-renewal potential. In vivo, tumor engraftment and tumor growth were strongly inhibited, without the induction of liver necrosis or other obvious side effects. These findings suggest that sulforaphane shifts the balance from TRAIL-induced survival signals to apoptosis and thus explains the observed synergistic effect. A nutritional strategy for high sulforaphane intake may target the cancer-specific activity of TRAIL in CSCs.

Cytokine profile of mature milk from smoking and nonsmoking mothers.

We verified whether smoking during lactation influences breast milk cytokine (interleukin [IL]-1α, IL-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor-α) levels 30 to 32 days after delivery. The study group comprised 24 postpartum women who declared smoking >5 cigarettes per day. The control group included 45 nonsmoking postpartum women. Compared with nonsmoking women, smokers were characterized by significantly higher breast milk concentrations of IL-1α (P = 0.04), whereas no significant intergroup differences were observed in terms of remaining analyzed cytokines. Moreover, both groups were characterized by a similar fraction of women with detectable cytokine levels.

Renal function and low-molecular-weight proteins (cystatin C, ß2-microglobulin, neutrophil gelatinase-associated lipocalin) in child and young adult cancer survivors.

BACKGROUND: We sought to verify the hypothesis that children and young adults with cancer who have completed treatment differ according to the type and degree of renal damage. PROCEDURE: This study included 144 children and young adults (73 female) who had completed treatment for leukemias and lymphomas (group L, n=45), Wilms tumor (group W, n=52) and other solid tumors (group S, n=47). The following parameters were evaluated: serum concentrations of creatinine, cystatin C, ß2-microglobulin, neutrophil gelatinase-associated lipocalin and urine excretion of albumin, and urinalysis with sediment. Glomerular filtration rate (eGFR) was estimated using the classic Schwartz (eGFRSch), Schwartz redux (eGFRSchred), and Filler (eGFRFiller) formulas and with the new Schwartz equation for patients with chronic kidney disease (eGFRSchCKD). RESULTS: Group S had the lowest eGFRSchCKD and eGFRFiller, the highest serum cystatin C and the highest albumin excretion compared with groups L and W. Groups S and W had lower eGFRSch and eGFRSchred and higher serum ß2-microglobulin and neutrophil gelatinase-associated lipocalin compared with group L. Group W had lower eGFRSchCKD than group L. CONCLUSIONS: Children and young adults with cancer who have completed treatment differ in the type and degree of renal damage they sustain.

Effect of aliskiren on proteinuria in non-diabetic chronic kidney disease: a double-blind, crossover, randomised, controlled trial.

AIM: To evaluate the proteinuria-lowering effect of a renin inhibitor (aliskiren), compared to placebo and to an angiotensin-converting enzyme inhibitor (perindopril), in patients with non-diabetic chronic kidney disease. METHODS: A randomised, double-blind, crossover trial was performed in 14 patients with nondiabetic chronic kidney disease with 24-h mean proteinuria of 2.01 g (95% CI, 1.36­2.66) and estimated creatinine clearance of 93±6.8 ml/min. The study consisted of five treatment periods. The patients were randomly assigned to receive aliskiren (150 mg), aliskiren (300 mg), perindopril (5 mg), perindopril (10 mg) or placebo. RESULTS: Aliskiren and perindopril reduced proteinuria. These effects were dose-dependent. Furthermore, 24-h proteinuria was reduced by 23% (mean 95% CI; 2­44) by treatment with aliskiren (150 mg), by 36% (95% CI, 17­55; P<0.001) with aliskiren (300 mg), by 7.1% (95% CI, 11­26) with perindopril (5 mg) and by 25% (95% CI, 11­39; P<0.05) with perindopril (10 mg), compared to placebo. No significant difference was found between the effects of aliskiren and perindopril. CONCLUSIONS: Aliskiren significantly reduced proteinuria. The antiproteinuric effect is probably similar to that of perindopril, for equivalent hypotensive dosages. The renin inhibitor provides a promising alternative approach for the treatment of patients with chronic proteinuric non-diabetic kidney disease.

Renal function and solitary kidney disease: Wilms tumour survivors versus patients with unilateral renal agenesis.

AIMS: To test the hypothesis that Wilms tumour survivors (WTs) experience increased disturbance in renal function, even after prompt treatment, compared to patients with unilateral renal agenesis (URA). METHODS: To assess the renal function of 30 WTs and 17 individuals with URA, the estimated glomerular filtration rate (eGFR) was calculated using the Schwartz and Filler formulas as well as the new Schwartz equation for chronic kidney disease. To measure kidney damage, serum levels and urine excretion of ß(2)-microglobulin (B2M), cystatin C (Cys C), neutrophil gelatinase-associated lipocalin (NGAL) were tested, N-acetyl-ß-glucosaminidase (NAG), and albumin urine excretion and urine sediment were examined. Blood pressure was measured. RESULTS: No differences were found between the groups in terms of eGFR, serum Cys C, B2M and NGAL concentrations. The urine excretion of Cys C, NGAL and NAG was similar in both groups. URA patients had higher B2M excretion than WTs. Arterial hypertension was present in 7/30 (23%) WTs and 1/17 (6%) patients with URA. CONCLUSIONS: WTs have similar eGFR to individuals with URA and are more likely to have arterial hypertension. The patients with URA have signs of tubular damage. This study demonstrates the need for nephrological monitoring of individuals with a single kidney.

Atorvastatin improves tubular status in non-diabetic patients with chronic kidney disease - placebo controlled, randomized, cross-over study.

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-ß-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.

The effect of N-acetylcysteine on blood pressure and markers of cardiovascular risk in non-diabetic patients with chronic kidney disease: a placebo-controlled, randomized, cross-over study.

BACKGROUND: Cardiovascular complications in patients with chronic kidney disease (CKD) are frequent. They show increased cardiovascular mortality and morbidity attributable to accumulation of several risk factors; e.g., hypertension, oxidative stress and elevated plasma homocysteine concentration. Despite recent progress in their management, there is still no optimal therapy that can stop progression of CKD and decrease cardiovascular outcome in these patients. Antioxidants, e.g., N-acetylcysteine (NAC), have been suggested as a promising medicament in this field. MATERIAL/METHODS: In a placebo-controlled, randomized, two-period cross-over study we evaluated the influence of eight weeks of NAC therapy (1200 mg/day) added to pharmacological renin-angiotensin system blockade on ambulatory blood pressure and surrogate markers of cardiovascular risk and injury in 20 non-diabetic patients with albuminuria [30-915 mg per creatinine mg] and normal or slightly decreased kidney function [eGFR 61-163 ml/min]. After eight weeks run-in period during which the therapy using angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers was settled, patients were randomly assigned to one of two treatment sequences: NAC/washout/placebo or placebo/washout/NAC. RESULTS: No significant changes in blood pressure, albuminuria and homocysteine plasma level were observed. CONCLUSIONS: NAC had no effect on blood pressure and surrogate markers of cardiovascular injury in non-diabetic patients with CKD.

Effect of pentoxifylline on proteinuria, markers of tubular injury and oxidative stress in non-diabetic patients with chronic kidney disease - placebo controlled, randomized, cross-over study.

BACKGROUND: Inhibition of the renin-angiotensin-aldosterone system (RAAS) with angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin II subtype 1 receptor antagonists (ARB) is a common strategy used in the management of patients with chronic kidney disease (CKD). However, there is no universal therapy that can stop progression of CKD. Pentoxifylline (PTE) is a non-specific phosphodiesterase inhibitor with anti-inflammatory properties. It has been reported to have promising effects in CKD treatment. METHODS: In a placebo-controlled, randomized, cross-over study we evaluated the influence of PTE (1200 mg/day) added to RAAS blockade on proteinuria, surrogate markers of tubular injury and oxidative stress-dependent products in 22 non-diabetic patients with proteinuria (0.4-4.3 g per 24 h) with normal or declined kidney function [eGFR 37-178 mL/min]. In an eight-week run-in period, therapy using ACEI and/or ARB was adjusted to achieve a blood pressure below 130/80 mm Hg. Next, patients were randomly assigned to one of two treatment sequences: PTE/washout/placebo or placebo/washout/PTE. Clinical evaluation and laboratory tests were performed at the randomization point and after each period of the study. RESULTS: The PTE therapy reduced proteinuria (by 26%) as compared to placebo. There were no differences in alpha(1)-microglobulin, urine excretion of N-acetyl-beta-d-glucosaminidase (NAG), hsCRP, the urinary excretion of 15-F(2)t-isoprostane, blood pressure (BP), eGFR and serum creatinine between the PTE and placebo groups. CONCLUSION: Pentoxifylline may decrease proteinuria in non-diabetic patients with CKD.

Influence of megestrol acetate on nutrition and inflammation in dialysis patients - preliminary results.

Malnutrition is a common clinical problem in dialysis patients. So far the management of malnutrition in this population has not been fully successful. The aim of the study was to evaluate the efficacy and safety of use of megestrol acetate suspension in malnourished dialysis patients. Twenty-six hypoalbuminemic (albumin < or = 3.8 g/dl) dialysis patients took 160 mg of megestrol acetate daily for a period of two months. Anthropometry (dry weight, body mass index) and biochemical measurements of nutrition (serum albumin, triglycerides, total cholesterol) and inflammation (hsCRP, IL-1beta, IL-6) were performed on a monthly basis. The treatment led to a statistically significant increase (P < 0.05) in anthropometry and albumin concentration, with no statistically significant changes in total cholesterol, triglycerides and indices of inflammation. Side effects included overhydration, diarrhoea and hyperglycaemia. Thus, megestrol acetate may be an effective therapeutic agent in improving the nutritional status of carefully selected dialysis patients, while it might not mitigate inflammation. Because of the prevalent side effects it must be monitored closely.

TGF-beta(1), IL-10 and IL-4 in colostrum of allergic and nonallergic mothers.

OBJECTIVE: To determine transforming growth factor (TGF) beta(1), interleukin (IL) 4, and IL-10 concentrations in human milk and to assess the relationship between allergic disorders in mothers and the content of the interleukins in their milk. MATERIAL AND METHODS: Thirty allergic and 46 healthy mothers were included in the study. Colostrum was collected 2-3 days after delivery. Cytokine concentrations were determined with commercial enzyme-linked immunosorbent systems. RESULTS: TGF-beta(1)was found in milk from 23 women in the control group (53.49%) and 11 in the allergy group (37.93%). When TGF-beta(1) was present, the median concentration was higher in the allergy group than in the control (61.5 and 30.4 pg/mL, respectively; P < 0.004). IL-10 was present in the colostrum of all the women and the median IL-10 concentration did not differ between the allergy (50.5 pg/mL) and control (51.5 pg/mL) groups. The probability of occurrence of a positive IL-4 value in the allergy group was greater than in the control group (chi-squared [df=1] = 2.60, P < 0.053). Median IL-4 level did not differ significantly between the two groups (0.5 and 0.5 pg/mL respectively). CONCLUSIONS: TGF-beta(1) was detected less often in the colostrum of allergic mothers than in that of mothers without allergy (but the difference was not statistically significant). IL-4 was found more often in the colostrum of allergic mothers than nonallergic ones. The allergy status did not correlate with IL-10 concentration.

The effect of N-acetylcysteine on proteinuria and markers of tubular injury in non-diabetic patients with chronic kidney disease. A placebo-controlled, randomized, open, cross-over study.

BACKGROUND: Inhibition of the renin-angiotensin-aldosterone system with angiotensin-converting enzyme inhibitors (ACEI) and/or angiotensin II subtype 1 receptor antagonists (ARB) constitutes a strategy in the management of patients with chronic kidney disease. There is still no optimal therapy which can stop the progression of chronic kidney disease. Antioxidants such as N-acetylcysteine (NAC) have been reported as a promising strategy in this field. METHODS: In a placebo-controlled, randomized, open, 2-period cross-over study, we evaluated the influence of NAC (1,200 mg/day) added to renin-angiotensin-aldosterone system blockade on proteinuria and surrogate markers of tubular injury and renal fibrosis in 20 non-diabetic patients with proteinuria (0.4-6.36 g/24 h) with normal or decreased kidney function (estimated glomerular filtration rate 61-163 ml/min). Subjects entered the 8-week run-in period during which the therapy using ACEI and/or ARB was established with blood pressure below 130/80 mm Hg. Next, patients were randomly assigned to 1 of 2 treatment sequences: NAC/washout/placebo or placebo/washout/NAC. Clinical evaluation and laboratory tests were performed at the randomization point and after each period of the study. RESULTS: No significant changes in laboratory tests were observed. CONCLUSION: NAC had no effect on proteinuria, surrogate markers of tubular injury or renal fibrosis in non-diabetic patients with chronic kidney disease.

Triple pharmacological blockade of the renin-angiotensin-aldosterone system in nondiabetic CKD: an open-label crossover randomized controlled trial.

BACKGROUND: Agents inhibiting the renin-angiotensin-aldosterone (RAAS) system have an important role in slowing the progression of chronic kidney disease. We evaluated the hypothesis that the addition of an aldosterone receptor antagonist to an angiotensin-converting enzyme (ACE) inhibitor and angiotensin II type 1 (AT-1) receptor blocker (ARB) (triple RAAS blockade) may provide an additional benefit compared with an ACE inhibitor and ARB (double RAAS blockade). DESIGN: Randomized open controlled crossover study. SETTING & PARTICIPANTS: 18 whites (7 women, 11 men) from the Outpatient Department of Nephrology with chronic nondiabetic proteinuric kidney diseases, mean age 42.4 +/- 1.9 years (SEM). INTERVENTIONS: In the 8-week run-in period, all participants received the ACE inhibitor cilazapril (5 mg), the ARB telmisartan (80 mg), and the diuretic hydrochlorothiazide (12.5 mg) as double RAAS blockade to achieve the target blood pressure of less than 130/80 mm Hg. Participants were then randomly assigned to 2 treatment sequences, either the addition of spironolactone (25 mg) (triple RAAS blockade) through 8 weeks followed by double RAAS blockade through 8 weeks (sequence 1) or double RAAS blockade followed by triple RAAS blockade (sequence 2). MAIN OUTCOME MEASURES: 24-hour urine protein excretion (primary end point) and markers of tubular injury and fibrosis (secondary end points). Analysis was performed using analysis of variance for repeated measurements. RESULTS: At baseline, mean serum creatinine level was 1.16 +/- 0.09 mg/dL (103 +/- 8 micromol/L), estimated glomerular filtration rate was 107.8 mL/min (95% confidence interval, 93 to 140.9 [1.8 mL/s; 95% confidence interval, 1.55 to 2.35; Cockcroft-Gault formula), and 24-hour mean proteinuria was 0.97 +/- 0.18 g. Mean urine protein excretion was 0.7 g/24 h (95% confidence interval, 0.48 to 0.92) less after triple RAAS blockade than after double RAAS blockade (P = 0.01), without change in blood pressure. Urine excretion of N-acetyl-beta-d-glucosaminidase (P = 0.02) and amino-terminal propeptide of type III procollagen (P = 0.05) also significantly decreased. Potassium levels increased significantly after triple therapy (P = 0.02). However, no patient was withdrawn because of adverse effects. LIMITATIONS: Absence of blinding, small sample size, short treatment period, absence of histological assessment. CONCLUSIONS: Administration of an aldosterone receptor antagonist in addition to double RAAS blockade with an ACE inhibitor and ARB may slow the progression of chronic kidney disease. Additional studies are necessary to confirm this result.