Olive leaf extract counteracts epithelial to mesenchymal transition process induced by peritoneal dialysis, through the inhibition of TGFß1 signaling.

The mesothelial cells (MCs) play an important role in the morpho-functional alterations of the peritoneal membrane (PM) undergoing peritoneal dialysis (PD). MCs, through the epithelial-mesenchymal transition process (EMT), progressively acquire a myofibroblast-like phenotype, promoting peritoneal fibrosis (PF) and failure of peritoneal membrane function. Transforming growth factor ß1 (TGFß1), through canonical and non-canonical pathways, promotes the epithelial-mesenchymal transition (EMT) process leading to PF. To investigate the therapeutic potential of an olive leaf extract (OLE) on preserving peritoneal membrane function, we evaluated the effect of OLE on the TGFß1-induced EMT in mesothelial cells, Met5A, and elucidated the underlying molecular mechanisms. As assessed by changes in the expression of epithelial, mesenchymal, and fibrotic cell markers (such as E-cadherin, N-cadherin, α-SMA, fibronectin, vimentin), levels of matrix metalloproteinases (MMP2 and MMP9), and cell migration, OLE inhibited the TGFß1-induced EMT. Importantly, the beneficial effect of OLE was mediated by reduction of the TGFß1-induced activation of Smad2/3 signaling and the mitigation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Smad/non-Smad signaling pathways, activated by TGFß1, both reduce expression of epithelial marker E-cadherin which has a crucial role in EMT initiation. Interestingly, we observed that in presence of OLE activity of the E-cadherin, promoter was increased and concomitantly OLE reduced the nuclear content of its co-repressor SNAIL. Our results suggest the potential therapeutic of OLE to counteract fibrotic process in peritoneal dialysis patients.

Olive leaf extract counteracts cell proliferation and cyst growth in an in vitro model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of kidney cysts, leading to chronic kidney disease. Since the available treatment for ADPKD is limited, there is emerging interest for natural compounds as potential therapeutic candidates. The aim of our study was to investigate whether an olive leaf extract may be able to counteract the cyst growth in an in vitro model of ADPKD. We treated WT9-12 cells with an olive leaf extract (OLE). In monolayer culture we evaluated cell viability by the MTT assay, protein expression by western-blot analysis and apoptosis by DNA laddering and TUNEL assays. For functional studies we used transient transfection and ChIP assays. Intracellular calcium measurement was performed with a spectrofluorimeter using a fluorescent probe. 3D-cell-culture was used for cyst growth studies. OLE reduced the WT9-12 cell growth rate and affected intracellular signaling due to high c-AMP levels, as OLE reduced PKA levels, enhanced p-AKT, restored B-Raf-inactivation and down-regulated p-ERK. We elucidated the molecular mechanism by which OLE, via Sp1, transactivates the p21WAF1/Cip1 promoter, whose levels are down-regulated by mutated PKD1. We demonstrated that p-AKT up-regulation also played a crucial role in the OLE-induced anti-apoptotic effect and that OLE ameliorated intracellular calcium levels, the primary cause of ADPKD. Finally, using a 3D-cell-culture model we observed that OLE reduced the cyst size. Therefore, multifaceted OLE may be considered a new therapeutic approach for ADPKD treatment.

Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR.

Experimental evidence demonstrated that macroautophagy/autophagy exerts a crucial role in maintain renal cellular homeostasis and represents a protective mechanism against renal injuries. Interestingly, it has been demonstrated that in the human proximal tubular renal cell line, HK-2, the MTOR inhibitor rapamycin enhanced autophagy and mitigated the apoptosis damage induced by urinary protein overload. However, the underlying molecular mechanism has not yet been elucidated. In our study we demonstrated, for the first time, that in HK-2 cells, the exposure to low doses of rapamycin transactivated the NGFR promoter, leading to autophagic activation. Indeed, we observed that in HK-2 cells silenced for the NGFR gene, the rapamycin-induced autophagic process was prevented, as the upregulation of the proautophagic markers, BECN1, as well as LC3-II, and the autophagic vacuoles evaluated by transmission electron microscopy, were not found. Concomitantly, using a series of deletion constructs of the NGFR promoter we found that the EGR1 transcription factor was responsible for the rapamycin-mediated transactivation of the NGFR promoter. Finally, our results provided evidence that the cotreatment with rapamycin plus albumin further enhanced autophagy via NGFR activation, reducing the proapoptotic events promoted by albumin alone. This effect was prevented in HK-2 cells silenced for the NGFR gene or pretreated with the MTOR activator, MHY1485. Taken together, our results describe a novel molecular mechanism by which rapamycin-induced autophagy, mitigates the tubular renal damage caused by proteinuria, suggesting that the use of low doses of rapamycin could represent a new therapeutic strategy to counteract the tubule-interstitial injury observed in patients affected by proteinuric nephropathies, avoiding the side effects of high doses of rapamycin.

Active compounds extracted from extra virgin olive oil counteract mesothelial-to-mesenchymal transition of peritoneal mesothelium cells exposed to conventional peritoneal dialysate: in vitro and in vivo evidences.

During peritoneal dialysis (PD), peritoneal mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype that, together with the inflammatory process, promotes tissue fibrosis and a failure of peritoneal membrane function. To date, there is no definitive treatment for the progressive thickening and angiogenesis of the peritoneal membrane associated with PD. In this study we tested, in vitro and in vivo, the ability of active compounds extracted from extra virgin olive oil (AC-EVOO) to counteract the mesothelial-to-mesenchymal transition process (MMT) observed in mesothelial cells chronically exposed to the conventional peritoneal dialysate (DL). In particular, we used a cultivar from southern Italy known to have a high polyphenol content. Our results showed that, in mesothelial cells exposed to DL, the combined treatment with AC-EVOO prevented the genic and protein upregulation of key mesenchymal and inflammatory markers, as well as the MCs' migratory capacity. Concomitantly, we tested the antifibrotic efficacy of AC-EVOO in mesothelial cells obtained from effluents of patients undergoing PD, whose "fibroblast-like" phenotype was defined by flow-cytometry assay. We observed that in these cells AC-EVOO significantly mitigated, but did not reverse, the MMT process. In conclusion, our preliminary results suggest that AC-EVOO can interfere with critical factors in the process of differentiation, preventing myofibroblast formation, but once fibrosis has already progressed it is unable to promote the redifferentiation to the epithelial phenotype. Further studies are needed to establish whether AC-EVOO could represent a new therapeutic target to prevent peritoneal fibrosis.

High doses of hydroxytyrosol induce apoptosis in papillary and follicular thyroid cancer cells.

PURPOSE: Recent evidences indicates that hydroxytyrosol, one of the main olive oil phenols, possess antitumor effects because of its pro-oxidant properties and the capacity to inhibit proliferation and to promote apoptosis in several tumor cell lines, although most of the results were obtained for breast and digestive systems cancers. METHODS: In this study, we evaluated the activities of hydroxytyrosol against papillary (TPC-1, FB-2) and follicular (WRO) thyroid cancer cell lines. RESULTS: Cellular viability revealed that high doses of hydroxytyrosol reduced cancer cells viability concomitantly with a reduction of cyclin D1 expression and an up-regulation of cell cycle key modulator p21 levels. In the same experimental conditions, Annexin V-PI staining and DNA laddering revealed that hydroxytyrosol exerts proapoptotic effects on papillary and follicular cancer cells. Furthermore, by Western blot analysis, we observed that hydroxytyrosol treatment reduced thyroid cancer cells viability by promoting apoptotic cell death via intrinsic pathway. CONCLUSIONS: Collectively, our results demonstrated for the first time that in thyroid cancer cells hydroxytyrosol promoted apoptosis at higher doses with respect to other cancer cells lines. Therefore, further studies will reveal the mechanisms by which thyroid cancer cells are more resistant to the proapoptotic effect exerted by hydroxytyrosol as well as the potential application as novel target therapeutic in thyroid cancer.

HeartDrive: A Broader Concept of Interoperability to Implement Care Processes for Heart Failure.

This paper originates from the HeartDrive project, a platform of services for a more effective, efficient and integrated management of heart failure and comorbidities. HeartDrive establishes a cooperative approach based on the concepts of continuity of care and extreme, patient oriented, customization of diagnostic, therapeutic and follow-up procedures. Definition and development of evidence based processes, migration from parceled and episode based healthcare provisioning to a workflow oriented model and increased awareness and responsibility of citizens towards their own health and wellness are key objectives of HeartDrive. In two scenarios for rehabilitation and home monitoring we show how the results are achieved by providing a solution that highlights a broader concept of cooperation that goes beyond technical interoperability towards semantic interoperability explicitly sharing process definitions, decision support strategies and information semantics.

Early cytomegalovirus-specific T-cell response and estimated glomerular filtration rate identify patients at high risk of infection after renal transplantation.

BACKGROUND: Assessing the risk of cytomegalovirus (CMV) viremia in kidney transplant recipients (KTR) may be helpful to indicate in which patient it is worth starting antiviral treatment during preemptive strategy. METHODS: In 40 CMV-seropositive KTR preemptively treated with ganciclovir, we used interferon (IFN)-γ ELISpot test to evaluate whether monitoring T cells directed against phosphoprotein (pp) 65 and immediate early (IE)-1 antigens could predict the onset of viremia. RESULTS: CMV viremia occurred in 24 patients (60%) within 120 days after transplantation. Non-viremic patients had higher anti-pp65, anti-IE-1 T cells, and estimated glomerular filtration rate (eGFR) in the first 90 days after transplantation. At logistic regression, anti-pp65, anti-IE-1 T cells, and eGFR measured at day 30 were significantly associated with CMV infection. Cutoff values of 15 spot-forming cells (SFCs)/200,000 peripheral blood mononuclear cells (PBMCs) for anti-IE, 40 SFCs/200,000 PBMCs for anti-pp65, and 46.6 mL/min/1.73 m(2) for eGFR, respectively, predicted the risk of CMV infection with high sensitivity and specificity (area under the receiver operating characteristic curve >0.75). Using a classification tree model, we identified as high-risk patients those showing anti-pp65 <42 SFCs/200,000 PBMCs and eGFR <62 mL/min/1.73 m(2) , as well as anti-pp65 ≥42 and anti-IE-1 <6.5 SFCs/200,000 PBMCs. CONCLUSION: Monitoring CMV-specific T-cell responses and eGFR in the first month post transplant can identify patients at high risk of CMV infection, for whom preemptive antiviral therapy is recommended.

High nerve growth factor blood concentration in renal transplantation: a new prognostic marker?

BACKGROUND: Nerve growth factor (NGF) belongs to the family of neurotropic proteins NGF is markedly expressed in proteinuric renal diseases and in end-stage renal disease; it might be involved in kidney physiopathology. To date, little is known about NGF concentrations in kidney transplant recipients (KTRs). Because NGF exerts its action on cell survival and differentiation, tissue repair, and inflammatory responses, it may also be implicated in the pathogenesis of chronic allograft nephropathy. The aim of this study was to determine circulating NGF concentrations in KTRs and to ascertain their use as a prognostic marker for kidney transplant outcomes. METHODS: Using enzyme-linked immunosorbent assay, we performed quantification of NGF in the serum of 40 prevalent KTRs at baseline and at 6 months. RESULTS: NGF concentrations in KTRs averaged 1.16 ± 0.67 ng/mL. They negative-linearly correlated with recipient age. Logistic multivariate regression analysis showed NGF to be independently associated with increased proteinuria over the 6-month follow-up. CONCLUSIONS: Our data demonstrated that serum concentrations of NGF in KTRs were elevated and that they could be considered to be a prognostic marker in kidney transplantation.

Increasing levels of hemoglobin improve renal transplantation outcomes.

INTRODUCTION: Several studies have reported various data on prevalence of posttransplant anemia (PTA). We have little information about its impact on long-term graft outcomes and few studies of the optimal hemoglobin (Hb) target in kidney transplantation. METHODS: We examined retrospectively 144 kidney transplant recipients of mean age 44.4 ± 12.3 years and follow-up of 40.5 ± 4.6 months. Exclusion criteria were age below 18 years, multiorgan transplantation, and graft failure in the first year. Using simple and multiple linear regression models, we evaluated the potential prediction of a serum concentration of Hb at 1 year after renal transplantation on allograft outcome as measured by Δ% estimated glomerular filtration rate (eGFR), the difference between eGFR, measured with the Modification of Diet in Renal Disease (MDRD) formula, at the end of follow-up, and at 1 year. Multiple models were adjusted for recipient sex, recipient age, donor age, ESA therapy, acute rejection episodes (ARE), days of delayed graft function, human leukocyte antigen mismatches and cold ischemia time. RESULTS: At 1 year after transplantation, the mean Hb level was 13.77 ± 1.87 g/dL in males and 12.52 ± 1.53 g/dL in females. The average eGFR at 1 year was 63.07 ± 25.88 mL/min. At the end of follow-up, the mean Δ% eGFR was -5.73% ± 27.30%. Blood concentration of Hb correlated with donor, recipient sex, ARE, and eGFR at 1 year. There was a close correlation between the Δ% Hb and eGFR upon univariate analysis and the multiple linear regression model. Hb was the only predictor of transplant outcome. CONCLUSIONS: Many factors are involved in kidney allograft function. Among these, Hb is important. In this work we demonstrated that increasing levels of Hb at 1 year after transplantation seemed to predict better preservation of graft function, representing a marker of a good quality graft.

Thyroid status and kidney transplantation outcomes.

INTRODUCTION: It is known that end-stage renal disease patients can display abnormal thyroid gland function, which may cause autoimmune hypothyroidism or subclinical alterations. The impact of thyroid function on graft outcomes is not completely clear among renal transplant patients. The aim of this study was to evaluate thyroid function among a cohort of 136 consecutive renal recipients in correlation with clinical parameters of graft function. MATERIALS AND METHODS: We performed a cross-sectional study on 136 subjects including 84 males and 52 females of overall mean age of 49.71 ± 10.98 years who underwent renal transplantations between 2005 and 2009 and had a mean follow-up of 28.3 ± 15.7 months. All patients were treated with a calcineurin inhibitor, steroids, and mycophenolate mofetil. The exclusion criteria were age below 18 years, multiorgan transplantation, graft failure in the first 6 months, or presence of a thyroid neoplasm. We evaluated levels of serum FT3, FT4, and thyroid-stimulating hormone (TSH) in relation to the following parameters: body mass index (BMI), serum creatinine, estimated glomerular filtration rate estimated glomerular filtration rate (eGFR) by Modification of Diet in Renal Disease (MDRD) formula, proteinuria/24 hours, serum sodium, potassium, calcium, phosphorus, cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides, and hemoglobin (Hb). RESULTS: Only 6.4% of our transplant recipients were treated with levothyroxine sodium. The patients showed an average FT3 of 3.24 ± 0.5 mg/dL; average FT4 of 0.84 ± 0.1 mg/dL, and mean TSH of 1.29 ± 0.8 mg/dL. The study showed no relationship between thyroid hormones and age of the transplant, while there was a significant difference in FT3 levels between men and women. We also observed a significant correlation between FT3 and serum creatinine, eGFR, serum sodium, BMI, and Hb; whereas there was no correlation with other variables. The correlations between FT4 and TSH and all examined variables were not significant. CONCLUSIONS: The interactions between the thyroid and the kidney have been incompletely studied among patients with renal transplants. Our data showed that the presence of low serum FT3 levels correlated with worse graft function, anemia, BMI, and serum sodium. Thus low FT3 levels could be predictive of graft function, especially in the 5 years posttransplantation.

Proteinuria is a predictor of posttransplant anemia.

INTRODUCTION: Posttransplant anemia (PTA) involves many factors. Although the link between the hemoglobin (Hb) levels and renal function is known, the relationship between proteinuria and PTA hemoglobin has not been widely explored. The aim of this study was to evaluate whether proteinuria was a predictor of anemia and whether erythropoietin-stimulating agent therapy was a protective factor for kidney damage among transplantation patients. METHODS: We retrospectively examined 144 kidney transplant recipients of mean age 44.4 ± 12.3 years and a mean follow-up period of 40.5 ± 4.6 months. Exclusion criteria were age under 18 years, multiorgan transplantation, proteinuria at 6 months over 1.5 g/d, and transplant failure within the first year. Using regression models, we evaluated the potential predictive power of proteinuria at 6 months after renal transplantation for anemia as expressed by Hb levels at 1 year. RESULTS: The frequency of patients with PTA was 38.89% at 1 year, 35.21% at 2 years, and 31.43% at 3 years. Variables with significant correlations with anemia upon univariate analysis were: proteinuria, donor age, acute rejection, estimated glomerular filtration rate, s-creatinine, and salbumin. Upon multivariate regression analysis 24-hour proteinuria and s-albumin remained independent predictors of 1-year PTA. Univariate analysis among the entire cohort showed a significant correlation between 1-year Hb and proteinuria/24 hours at 6 months (P=.007), an observation that was confirmed in the adjusted model along with recipient sex. Patients were then divided into two groups regarding treatment with erythropoiesis stimulating agents (ESA). Multivariate analysis showed that proteinuria (P=.005) was a predictor of Hb only among the group of patients who did no receive erythropoietin, whereas this relationship disappeared among the group treated with ESA. CONCLUSIONS: These results showed that proteinuria at 6 months was a predictor of Hb levels at 1 year. Treatment of transplant patients with ESA may be a protective factor for renal endothelial damage expressed as proteinuria.

Impact of continuous value of body mass index on graft loss in overweight patients.

INTRODUCTION: High body mass index (BMI) is associated with increased cardiovascular mortality and risk of progression to end-stage renal disease both among the general population and among renal transplant patients. However, in the latter condition no unequivocal studies have been reported in the literature. The aim of our study was to investigate continuous versus categorical values of BMI (World Health Organization classification) as an independent risk factor in renal transplantation. PATIENTS AND METHODS: We retrospectively studied 194 renal transplant patients (128 males and 66 females) whose mean age at transplant was 43.9 years. They had 5 years follow-up. To investigate the association between BMI and graft survival, we performed univariate and multivariate analyses using the Cox regression model. This model was adjusted both for classical covariates (age, gender, time on dialysis, HLA mismatches, donor status) and other covariates as delayed graft function (DGF), acute rejection episodes (AR), and chronic allograft nephropathy (CAN), which are universally recognized to be predictors of graft loss as evidenced by a need for dialysis treatments. RESULTS: At the time of transplantation, the BMI averaged 24.4 +/- 2.65 kg/m(2). Upon univariate analysis, age (P = .049), BMI (P = .005), DGF (P = .009), ARE (P < .0001), and CAN (P = .001) were significantly related to poor transplant outcomes. Upon multivariate analysis, only the BMI value, considered as continuous value (P = .013), DGF (P = .030), and ARE (P < .0001) were significantly related to graft loss. CONCLUSIONS: BMI as a continuous value represented an independent risk factor for renal transplant loss at 5 years. Correction of pretransplant body weight both in overweight (25

Prediction of chronic allograft nephropathy using classification trees.

INTRODUCTION: For its intrinsic potential to mine causal relations, machine learning techniques are useful to identify new risk indicators. In this work, we have shown two classification trees to predict chronic allograft nephropathy (CAN), through an evaluation of routine blood and urine tests. METHODS: We retrospectively analyzed 80 renal transplant patients with 60-month follow-up (mean = 55.20 +/- 12.74) including 52 males and 28 females of overall average age of 41.65 +/- 12.52 years. The primary endpoint was biopsy-proven CAN within 5 years from transplantation (n = 16). Exclusion criteria were multiorgan transplantations, patients aged less than 18 years, graft failure, or patient death in the first 6 months posttransplantation. Classification trees based on the C 4.8 algorithm were used to predict CAN development starting from patient features at transplantation and biochemical test at 6-month follow-up. Model performance was showed as sensitivity (S), false-positive rate (FPR), and area under the receiver operating characteristic curve (AUC). RESULTS: The two class of patients (no CAN versus CAN) showed significant differences in serum creatinine, estimated Glomerular Filtration Rate with Modification of Diet in Renal Disease study formula (MDRD), serum hemoglobin, hematocrit, blood urea nitrogen, and 24-hour urine protein excretion. Among the 23 evaluated variables, the first model selected six predictors of CAN, showing S = 62.5%, TFP = 7.2%, and AUC = 0.847 (confidence interval [CI] 0.749-0.945). The second model selected four variables, showing S = 81.3%, TFP = 25%, and AUC = 0.824 (CI 0.713-0.934). CONCLUSIONS: Identification models have predicted the onset of multifactorial, complex pathology, like CAN. The use of classification trees represent a valid alternative to traditional statistical models, especially for the evaluation of interactions of risk factors.

Decisional trees in renal transplant follow-up.

INTRODUCTION: The predictive potentialities of application of data mining algorithms to medical research are well known. In this article, we have applied to a transplant population classification trees to build predictive models of graft failure, evaluating the interactions between body mass index (BMI) and other risk factors. The decision trees have been widely used to represent classification rules in a population by a hierarchical sequential structure. PATIENTS AND METHODS: We retrospectively studied 194 renal transplant patients with 5 years of follow-up (128 males, 66 females, mean age at time of transplant of 43.9 +/- 12.5 years). Exclusion criteria were: age < 18 years, multiorgan transplant, and retransplant. The BMI was calculated at the time of transplantation. In the classification algorithm, we considered the following parameters: age, sex, time on dialysis, donor type, donor age, HLA mismatches, delayed graft function (DGF), acute rejection episode (ARE), and chronic allograft nephropathy (CAN). The primary endpoint was graft loss within 5-years follow-up. RESULTS: The classification algorithm produced a decision tree that allowed us to evaluate the interactions between ARE, DGF, CAN, and BMI on graft outcomes, producing a validation set with 88.2% sensitivity and 73.8% specificity. Our model was able to highlight that subjects at risk of graft loss experienced one or more events of ARE, developed DGF and CAN, or has a BMI > 24.8 kg/m(2) and CAN. CONCLUSIONS: The use of decision trees in clinical practice may be a suitable alternative to the traditional statistical methods, since it may allow one to analyze interactions between various risk factors beyond the previous knowledge.