The ameliorative effects of Nigella sativa, thymoquinone, and bentonite against aflatoxicosis in broilers via AFAR and Nrf2 signalling pathways, and down-regulation of caspase-3.

1. Aflatoxins (AFs) are metabolites which especially have toxic effects on proteins, and are detoxified by the aflatoxin-B1 aldehyde reductase (AFAR) pathway. In this pathway, the aldo-keto reductase family 7, member A2 (AKR7A2) enzyme, which is controlled by nucleic-related erythroid factor 2 (Nrf2), plays an active role. However, data on the efficacy of this critical pathway in broilers is limited.2. The aim of the following study was to investigate the changes in the expression levels of AKR7A2, Nrf2, and caspase-3, and the effects of Nigella sativa seeds (NS), thymoquinone (TMQ), and bentonite (BNT) in broilers exposed to AFs.3. One-hundred broilers were divided into ten groups (control (CNT); AF; NS; TMQ; BNT; AF+TMQ; AF+NS; AF+BNT; AF+BNT+NS; AF+BNT+TMQ) and fed for 28 d. AF, TMQ, NS and BNT were added to diets at levels of 2 mg/kg, 300 mg/kg, 50 g/kg and 10 g/kg respectively.4. The addition of AF to the diet decreased AKR7A2 and Nrf2 levels dramatically, but increased caspase-3 (P < 0.01). TMQ, NS and BNT additions to the diet eliminated all negative effects caused by AF (P < 0.01); and AKR7A2 and Nrf2 were further raised in TMQ and NS groups when compared to the control group. TMQ and NS showed a positive effect on detoxification parameters when given together with BNT.5. Supplementation with NS and TMQ enhanced AF detoxification via the AFAR pathway, by increasing AKR7A2 and Nrf2 levels, in addition to reducing hepatocyte apoptosis.

An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics.

Comparative, quantitative mass spectrometry of proteins provides great insight to protein abundance and function, but some molecular characteristics related to protein dynamics are not so easily obtained. Because the metabolic incorporation of stable amino acid isotopes allows the extraction of distinct temporal and spatial aspects of protein dynamics, the SILAC methodology is uniquely suited to be adapted for advanced labeling strategies. New SILAC strategies have emerged that allow deeper foraging into the complexity of cellular proteomes. Here, we review a few advanced SILAC-labeling strategies that have been published during last the years. Among them, different subsaturating-labeling as well as dual-labeling schemes are most prominent for a range of analyses including those of neuronal proteomes, secretion, or cell-cell-induced stimulations. These recent developments suggest that much more information can be gained from proteomic analyses if the labeling strategies are specifically tailored toward the experimental design.

Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.

Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA.

Early Low Urinary CXCL9 and CXCL10 Might Predict Immunological Quiescence in Clinically and Histologically Stable Kidney Recipients.

We monitored the urinary C-X-C motif chemokine (CXCL)9 and CXCL10 levels in 1722 urine samples from 300 consecutive kidney recipients collected during the first posttransplantation year and assessed their predictive value for subsequent acute rejection (AR). The trajectories of urinary CXCL10 showed an early increase at 1 month (p = 0.0005) and 3 months (p = 0.0009) in patients who subsequently developed AR. At 1 year, the AR-free allograft survival rates were 90% and 54% in patients with CXCL10:creatinine (CXCL10:Cr) levels <2.79 ng/mmoL and >2.79 ng/mmoL at 1 month, respectively (p < 0.0001), and 88% and 56% in patients with CXCL10:Cr levels <5.32 ng/mmoL and >5.32 ng/mmoL at 3 months (p < 0.0001), respectively. CXCL9:Cr levels also associate, albeit less robustly, with AR-free allograft survival. Early CXCL10:Cr levels predicted clinical and subclinical rejection and both T cell- and antibody-mediated rejection. In 222 stable patients, CXCL10:Cr at 3 months predicted AR independent of concomitant protocol biopsy results (p = 0.009). Although its positive predictive value was low, a high negative predictive value suggests that early CXCL10:Cr might predict immunological quiescence on a triple-drug calcineurin inhibitor-based immunosuppressive regimen in the first posttransplantation year, even in clinically and histologically stable patients. The clinical utility of this test will need to be addressed by dedicated prospective clinical trials.

Polythiophenes and polythiophene-based composites in amperometric sensing.

This overview of polythiophene-based materials provides a critical examination of meaningful examples of applications of similar electrode materials in electroanalysis. The advantages arising from the use of polythiophene derivatives in such an applicative context is discussed by considering the organic conductive material as such, and as one of the components of hybrid materials. The rationale at the basis of the combination of two or even more individual components into a hybrid material is discussed with reference to the active electrode processes and the consequent possible improvements of the electroanalytical performance. In this respect, study cases are presented considering different analytes chosen among those that are most frequently reported within the classes of organics and inorganics. The use of a polythiophene matrix to stably fix biological elements at the electrode surface for the development of catalytic biosensors and genosensors is also discussed. Finally, a few possible lines along which the next research in the field could be fruitfully pursued are outlined. Furthermore, the work still to be done to exploit the possibilities offered by novel products of organic synthesis, even along paths already traced in other fields of electrochemistry, is discussed.

Therapeutic mTOR inhibition in autosomal dominant polycystic kidney disease: What is the appropriate serum level?

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease, and sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to significantly retard cyst expansion in animal models. The optimal therapeutic dose of sirolimus is not yet defined. Here, we report the history of a previously unknown ADPKD deceased donor whose kidneys were engrafted in two different recipients. One of the two received an immunosuppressive regimen based on sirolimus for 5 years while the other did not. After transplantation, both patients developed severe transplant cystic disease. Donor DNA sequence identified a new hypomorphic mutation in PKD1. The rate of cyst growth was identical in the two patients regardless of the treatment. While sirolimus treatment reduced the activation of mTOR in peripheral blood mononuclear cells, it failed to prevent mTOR activation in kidney tubular cells, this could account for the inefficiency of treatment on cyst growth. Together, our results suggest that the dose of sirolimus required to inhibit mTOR varies according to the tissue.

Classification of red wines by chemometric analysis of voltammetric signals from PEDOT-modified electrodes.

Nine different types of Italian red wines of four different varieties were analysed, without any sample pre-treatments, by voltammetric techniques using a poly(3,4-ethylenedioxythiophene)-modified electrode. The data matrices consisting of the currents measured at different potentials, by repeated Cyclic Voltammetry or Differential Pulse Voltammetry, are submitted to chemometric analysis. After explorative tests based on Principal Component Analysis, Partial Least Squares-Discriminant Analysis classification models are built both for the training and for the test sets. To this aim, different classification strategies are adopted, considering the responses from the two techniques either separately or joined together to form a data matrix including the whole voltammetric information.

Adsorption geometry variation of 1,4-benzenedimethanethiol self-assembled monolayers on Au(111) grown from the vapor phase.

1,4-benzenedimethanethiol was chemisorbed from the vapor phase onto Au(111). The chemisorption geometry, molecular orientation, and bonding properties were studied at different degrees of surface coverage by photoelectron spectroscopy, metastable deexcitation spectroscopy, and near-edge x-ray absorption fine structure spectroscopy at the carbon K edge. Two main chemisorption regimes were identified: at low coverage the molecules adopt a flat configuration, then, as the molecular density of the first layer increases, the reduction of the available chemisorption sites induces the newly bonded molecules to assume a vertical alignment, with only one of the sulphur head groups interacting with the substrate. Experimental results were interpreted on the basis of theoretical calculations that we performed on the free molecule concerning the molecular orbitals' density of states and simulated x-ray absorption.

Amperometric sensors based on poly(3,4-ethylenedioxythiophene)-modified electrodes: discrimination of white wines.

The voltammetric responses on selected white wines of different vintages and origins have been systematically collected by three different modified electrodes, in order to check their effectiveness in performing blind analysis of similar matrices. The electrode modifiers consist of a conducting polymer, namely poly(3,4-ethylenedioxythiophene) (PEDOT) and of composite materials of Au and Pt nanoparticles embedded in a PEDOT layer. Wine samples have been tested, without any prior treatments, with differential pulse voltammetry technique. The subsequent chemometric analysis has been carried out both separately on the signals of each sensor, and on the signals of two or even three sensors as a unique set of data, in order to check the possible complementarity of the information brought by the different electrodes. After a preliminary inspection by principal component analysis, classification models have been built and validated by partial least squares-discriminant analysis. The discriminant capability has been evaluated in terms of sensitivity and specificity of classification; in all cases quite good results have been obtained.

Development of an electronic tongue based on a PEDOT-modified voltammetric sensor.

Three different electrodes were tested for use as nonspecific amperometric sensors for blind analysis on real matrices, namely different fruit juices from different fruits or different brands. The first two electrodes were traditional Pt and Au electrodes, while the third one was modified with poly(3,4-ethylenedioxythiophene) conducting polymer. The sensors were tested separately, tested coupled to each other, and also tested together. The responses of the electrode system(s) were first screened via PCA and then their discriminant capabilities were quantified in terms of the sensitivities and specificities of their corresponding PLS-DA multivariate classification models. Particular attention was paid to analyzing the evolution of the response over subsequent potential sweeps. The modified electrode demonstrated the most discriminating ability, and it was the only system capable of satisfactorily performing the most complex task attempted during the analysis: discriminating between juices from the same fruit but from different brands. Moreover, the electrode "cleaning" procedure required between two subsequent potential sweeps was much simpler for the modified electrode than for the others. This electrode system was therefore shown to be a good candidate for use as an informative element in an electronic tongue applied to the analysis of other food matrices.

Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells.

The effects of flow-induced mechanical strains on the phenotype of proximal tubular cells were addressed in vivo and in vitro by subjecting LLC-PK(1) and mouse proximal tubular cells to different levels of flow. Laminar flow (1 ml/min) induced a reorganization of the actin cytoskeleton and significantly inhibited the expression of plasminogen activators [tissue-type (tPA) activity: 25% of control cells; tPA mRNA: 70% of control cells; urokinase (uPA) mRNA: 56% of control LLC-PK(1) cells]. In vivo, subtotal nephrectomy (Nx) decreased renal fibrinolytic activity and uPA mRNA content detectable in proximal tubules. Nx also induced a reinforcement of the apical domain of the actin cytoskeleton analyzed by immunofluorescence. These effects of flow on tPA and uPA mRNA were prevented in vitro when reorganization of the actin cytoskeleton was blocked by cytochalasin D and were associated, in vitro and in vivo, with an increase in shear stress-responsive element binding activity detected by an electrophoretic mobility shift assay in proximal cell nuclear extracts. These results demonstrate that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.

Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.

Little is known about the serial changes that might occur in renal capillaries after reduction of renal mass. In the current study, our aim was to document potential alterations in the morphology and proliferation of the renal cortical peritubular microcirculation at specific time points (7 and 60 days) after experimental 75% surgical nephron reduction using two strains of mice that we here demonstrate react differently to the same initial insult: one strain (C57BL6xDBA2/F1 mice) undergoes compensatory growth alone, whereas the other (FVB/N mice) additionally develops severe tubulo-interstitial lesions. Our data demonstrate that significant remodeling and proliferation occur in renal cortical peritubular capillaries after experimental nephron reduction, as assessed by microangiography using infusion of fluorescein isothiocyanate-labeled dextran, expression of the endothelial markers CD34 and Tie-2, and co-expression of CD34 and proliferating cell nuclear antigen, a surrogate marker of cell proliferation. This was accompanied by an increase of renal vascular endothelial growth factor protein levels and a change in distribution of this protein within the kidney itself. Moreover, most of these responses were accentuated in FVB/N mice in the presence of progressive renal disease and positively correlated with tubular epithelial cell proliferation. Hence, we have made three significant novel observations that illuminate the complex pathophysiology of chronic kidney damage after nephron reduction: 1) cortical peritubular capillaries grow by proliferation and remodeling, 2) vascular endothelial growth factor expression is altered, and 3) the development of tubulo-interstitial disease is genetically determined.

Using transgenic mice to analyze the mechanisms of progression of chronic renal failure.

An understanding of the mechanisms underlying the formation of renal lesions is necessary for the development of strategies aiming to delay the progression of chronic renal failure. The generation of transgenic mice in the past 20 years has contributed significantly to the study of this phenomenon. Overexpression and/or inactivation of single factors in renal tissue demonstrated that molecules such as growth factors, proto-oncogenes, and renin-angiotensin system elements play major roles in renal deterioration. Several mouse models of renal injury have been developed in the past 10 yr. Transgenic mice that exhibit a normal phenotype under physiologic conditions allow analysis of the roles of single factors in the progression of chronic renal failure when renal injury models are used. Using this strategy, it was demonstrated that vascular adaptation, which is a process that involves the endothelin/nitric oxide balance, is essential for the survival of mice after nephron reduction and that the epidermal growth factor/activator protein-1/Bcl-2 pathway is involved in the development of renal lesions after renal injury, possibly via adjustment of the proliferation/apoptosis balance. Moreover, it was demonstrated that selective inhibition of epidermal growth factor signaling in the kidney successfully prevents the progression of chronic renal failure. These results indicate the power of transgenesis for elucidation of the pathogenesis of renal disease.

Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury.

The role of EGF in the evolution of renal lesions after injury is still controversial. To determine whether the EGF expression is beneficial or detrimental, we generated transgenic mice expressing a COOH-terminal-truncated EGF-R under the control of the kidney-specific type 1 gamma-glutamyl transpeptidase promoter. As expected, the transgene was expressed exclusively at the basolateral membrane of proximal tubular cells. Under basal conditions, transgenic mice showed normal renal morphology and function. Infusion of EGF to transgenic animals revealed that the mutant receptor behaved in a dominant-negative manner and prevented EGF-signaled EGF-R autophosphorylation. We next evaluated the impact of transgene expression on the development of renal lesions in two models of renal injury. After 75% reduction of renal mass, tubular dilations were less severe in transgenic mice than in wild-type animals. After prolonged renal ischemia, tubular atrophy and interstitial fibrosis were reduced in transgenic mice as compared with wild-type mice. The beneficial effect of the transgene included a reduction of tubular cell proliferation, interstitial collagen accumulation, and mononuclear cell infiltration. In conclusion, functional inactivation of the EGF-R in renal proximal tubular cells reduced tubulo-interstitial lesions after renal injury. These data suggest that blocking the EGF pathway may be a therapeutic strategy to reduce the progression of chronic renal failure.

[Growth factors. Role in the progression of renal lesions]. / Facteurs de croissance. Rôle dans la progression des lésions rénales.

FROM PATHOPHYSIOLOGY TO THERAPEUTICS: Nephrologists are faced with the continuing problem of helping patients avoid the onset or retard the development of end-stage renal failure. Despite the treatments available, the risk is still high for patients and the cost a heavy burden for the public health budget. These facts underline the importance of a detailed understanding of the mechanisms leading to the destruction of renal parenchyma in order to develop therapeutic strategies capable of slowing the inevitable progression of kidney lesions. GROWTH FACTORS: It is currently recognized that a major reduction in the number of functional nephrons, whatever the initial cause, leads in itself to a progressive deterioration of healthy nephrons and finally to complete destruction of the kidney. The underlying mechanisms remain largely unknown. One possible mechanism would involve an overexpression of several growth factors in the damaged renal parenchyma. We present in this review experimental data obtained with various approaches, including pharmacological and/or dietetic modulations and the establishment of transgenic mouse lines, to demonstrate the key role played by growth factors in the progression of renal lesions. The pathways followed by these growth factors in the process of renal destruction as well as certain elements leading to their overexpression are also discussed.

Sodium restriction decreases AP-1 activation after nephron reduction in the rat: role in the progression of renal lesions.

Renal hyperplasia and hypertrophy are early events after nephron reduction which precede progressive destruction of the remnant kidney. Restriction of dietary sodium content was shown to reduce renal lesions following nephron reduction. AP-1 is a transcription factor, resulting from heterodimerization of fos and jun proteins, which mediates the effects of mitogenic growth factors. To elucidate the role of AP-1 in growth processes involved in renal deterioration, we evaluated whether restriction of dietary sodium content (0.25 vs. 0.50% sodium w/w) affected AP-1-DNA binding and hyperplasia in the remnant kidney after nephron reduction (70% nephrectomy). Cell proliferation, evaluated by PCNA immunostaining, increased progressively from day 7 to day 60 in glomeruli, proximal and distal tubules and loops of Henle of nephrectomized (Nx) rats compared to control sham-operated (C) animals. AP-1-DNA binding activity increased 7 and 14 days after surgery, but it was reduced below C values at day 60. c-fos and c-jun expression were also reduced in Nx rats at day 60. Sodium restriction significantly reduced the number of PCNA-stained cells in glomeruli and tubules at days 14 and 60, but not at day 7, whereas it decreased AP-1 activation at all times of the study. This effect was associated to a marked reduction of renal lesions in Nx rats. In conclusion, we showed that, after nephron reduction, the beneficial effect of sodium restriction was associated with a reduction of hyperplasia and AP-1 activation, but that the latter did not parallel delayed cell proliferation rate in remaining nephrons. Thus, we propose that different transduction pathways are involved in cell proliferation after nephron reduction, according to the time of evolution of renal lesions.

Renal tubular cells cultured from genetically modified animals.

The culture of renal tubular cells from genetically modified animals opens the opportunity of biochemical, cell biology and physiological studies under strictly controlled conditions. Either primary cultures or cell lines can be used. Through two examples of primary cultures of proximal tubular cells obtained from knock-out mice, important information about the function of proteins were obtained. Mice lacking vimentin, an intermediate filament normally reexpressed in tubular cells during regeneration and culture, have a normal tubular function under basal conditions. Proximal cells grown from these animals exhibit a defect in sodium-glucose cotransport activity, most likely related to alterations in the dimer/monomer ratio of the transporter in the apical membranes. These alterations may be important in terms of tubular function during the recovery phase following acute tubular necrosis. The situation is strikingly different with regard to mice lacking HNF-1, a transactivator involved in the transcription of multiple genes. These animals suffer from severe Fanconi syndrome related to decreased expression of proximal transporters including isoforms of sodium-glucose (SGLT2) and sodium-phosphate (NPT1) cotransporters. Whereas transport defects are observed in isolated tubules, they are no longer apparent in cultured proximal cells because the expression of these isoforms is suppressed under culture conditions. These observations illustrate the interest and limits of the in vitro models for studying renal function in transgenic animals.