Time course study of oxidative and nitrosative stress and antioxidant enzymes in K2Cr2O7-induced nephrotoxicity

Background

It has been established that hypothyroidism protects rats against renal ischemia and reperfusion (IR) oxidative damage. However, it is not clear if hypothyroidism is able to prevent protein tyrosine nitration, an index of nitrosative stress, induced by IR or if antioxidant enzymes have involved in this protective effect. In this work it was explored if hypothyroidism is able to prevent the increase in nitrosative and oxidative stress induced by IR. In addition the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was studied. Control and thyroidectomized (HTX) rats were studied 24 h of reperfusion after 60 min ischemia.

Methods

Male Wistar rats weighing 380 ± 22 g were subjected to surgical thyroidectomy. Rats were studied 15 days after surgery. Euthyroid sham-operated rats were used as controls (CT). Both groups of rats underwent a right kidney nephrectomy and suffered a 60 min left renal ischemia with 24 h of reperfusion. Rats were divided in four groups: CT, HTX, IR and HTX+IR. Rats were sacrificed and samples of plasma and kidney were obtained. Blood urea nitrogen (BUN) and creatinine were measured in blood plasma. Kidney damage was evaluated by histological analysis. Oxidative stress was measured by immunohistochemical localization of protein carbonyls and 4-hydroxy-2-nonenal modified proteins. The protein carbonyl content was measured using antibodies against dinitrophenol (DNP)-modified proteins. Nitrosative stress was measured by immunohistochemical analysis of 3-nitrotyrosine modified proteins. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was measured by spectrophotometric methods. Multiple comparisons were performed with ANOVA followed by Bonferroni t test.

Results

The histological damage and the rise in plasma creatinine and BUN induced by IR were significantly lower in HTX+IR group. The increase in protein carbonyls and in 3-nitrotyrosine and 4-hydroxy-2-nonenal modified proteins was prevented in HTX+IR group. IR-induced decrease in renal antioxidant enzymes was essentially not prevented by HTX in HTX+IR group.

Conclusion

Hypothyroidism was able to prevent not only oxidative but also nitrosative stress induced by IR. In addition, the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase seem not to play a protective role in this experimental model.     

Fibrogenesis in Kidney Transplant: Dysfunction Progress Biomarkers

Introduction

Fibrogenesis markers, such as alpha-actin (AA), CD163 (macrophages), and E-cadherin, have been studied as chronic kidney allograft injury (CAI) predictors, a major cause of allograft failure.

Polyomavirus Nephropathy: Ten-Year Experience

Background

Polyomavirus nephropathy (BKVN) is an important cause of chronic allograft dysfunction (CAD). Recipient determinants (male sex, white race, and older age), deceased donation, high-dose immunosuppression, diabetes, delayed graft function (DGF), cytomegalovirus infection, and acute rejection (AR) are risk factors. Reducing immunosuppression is the best strategy in BKVN. The objective of our study was to evaluate CAD progression after therapeutic strategies in BKVN and risk factors for graft loss (GL).

Three-dimensional reconstructions from non-deparaffinized tissue sections

Three-dimensional (3-D) reconstruction from microscopic images represents a useful tool for the study of biological structures in embryology and developmental biology. However, it is usually necessary to cope with many difficulties connected with the preparation of specimens. In order to minimize mutual displacement of structures in successive sections, the applicability of non-deparaffinized tissue sections for 3-D reconstruction was tested. Chicken embryos were fixed and stained in toto with eosin and then embedded in paraffin. About 30-μm-thick non-deparaffinized serial sections were used for obtaining initial data for 3-D reconstruction of larger stacks of embryonic bodies using either fluorescence or confocal microscope. The same sections served for both collecting optical serial sections of mesonephros as source images for its 3-D reconstruction, and immunohistochemical detection of fibronectin, laminin and vimentin. It was found that sections with retained paraffin preserve the mutual spatial relationships of tissue components as well as provide an excellent differentiation of structure. It makes the process of 3-D reconstruction easier. The localization of the products of immunohistochemical reactions demonstrated the co-localization of fibronectin and laminin in basal laminas and the presence of vimentin in glomeruli and mesenchymal tissue. The use of non-deparaffinized sections represents a less time consuming and more effective alternative to thin histological sections for the purpose of 3-D reconstruction, and enables further application of material.     

Expression and functional activity of the very late activation antigen-4 molecule on human natural killer cells in different states of activation

In the present study we describe the expression and functional activity of the alpha4beta1 heterodimer molecule on human natural killer (NK) cells. Flow cytometric analyses showed that fresh and activated NK cells expressed high levels of very late activation antigen-4 (VLA-4) molecules. These cells bound to fibronectin (FN) and to its 38 000-MW proteolytic fragment through the VLA-4 integrin that was blocked with HP2/1 anti-alpha4 monoclonal antibodies (mAbs) and with the FN peptide fragment CS1. No inhibitory effects were observed in the presence of anti-alpha5 mAb, FN peptide fragment CS2 or other irrelevant mAb. Fresh NK cells were unable to aggregate, despite their expression of VLA-4, and only activated (cultured and lymphocyte-activated killer cells) NK cells showed homotypic aggregation with HP1/7 and HP2/4 anti-alpha4 mAb related to cellular activation. These results underline new evidence of how NK cells in different states of activation maintain different constitutive levels of alpha4beta1 integrin activity, and highlight the possibility of a different functional regulation by the cells bearing VLA-4, in the expression of these epitopes and their ability to interact with their ligands.     

Investigation of the role of signal transduction in attachment of ocular melanoma cells to matrix proteins: inhibition of attachment by calmodulin antagonists including tamoxifen

We investigated the role of signal transduction systems in the attachment of human uveal melanoma cells to matrix proteins. Ocular melanoma cells established from primary tumours attached rapidly to all substrates examined. Preferred substrates of attachment were collagens type I, III and IV and fibronectin rather than laminin, gelatin, arginine-glycine-aspartine, vitronectin, poly-I -lysine or plastic. All cells showed rapid attachment to the preferred substrates (80% within 10 min). Manipulation of intracellular cyclic AMP or protein kinase C activity had relatively little effect on cell attachment. In contrast, attachment was significantly reduced by manipulating either intracellular calcium or calmodulin. After 15 min at 37°C, the calcium ionophore ionomycin (5 m) reduced attachment to 25%, and TMB8 (50 M), which can reduce intracellular calcium, reduced attachment to 60%. The experimental calmodulin antagonist J8 (25 m), a substituted naphthalene sulphonamide, reduced attachment to 40%. Similarly tamoxifen (25 M), which has calmodulin antagonist activity in vitro,reduced attachment to 55%. Both J8 and tamoxifen inhibited cell attachment to a wide range of matrix proteins, suggesting that this effect on attachment is not dependent on the presence of specific adhesion receptors. Reduction of ocular melanoma tumour cell/matrix interactions through manipulation of intracellular calcium or calmodulin may therefore merit further investigation as a possible approach to reducing metastatic spread.     

Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland,an area of low incidence of infection

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Definition of the diversity and antimicrobial susceptibility of S. sonnei isolates may be helpful in the management of individual cases and outbreaks. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of S. sonnei predominantly (n = 59) from three counties in the west of Ireland. Phage typing (n = 17), plasmid profiling (n = 28), and integron analysis (n = 24) were performed with subsets of strains. PFGE typing permitted recognition of two major clusters: PFGE type A (n = 53) and PFGE type B (n = 14). PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed (n = 6) were phage type 3. PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates). Fifteen different plasmid profiles were identified among the 28 isolates analyzed. A class 2 integron was present in all 14 PFGE type B isolates. One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern. Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin. Our data demonstrate two predominant PFGE types among S. sonnei isolates circulating in this region. The limited diversity of the S. sonnei isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak. Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.     

Restriction of HIV-1 replication in promonocytic cells: a role for IFN-alpha

A comparative study of the replication kinetics of human immunodeficiency virus type 1 (HIV-1) was performed in the promonocytic U937 cells and in the T lymphoblastoid H9 cells. If a productive HIV-1 infection of both cell types could be established, the time which elapses before most of the cells could express viral proteins is always proportionally longer for U937 cells than for H9 cells. Indeed, when U937 cells are infected with HIV-1, this nonproductive phase is followed by a lag phase during which the percentage of virus-producing cells is slowly increasing when compared to H9 cells. The restriction of HIV-1 replication in U937 cells might be consecutive to the lower adsorption of viral particles to these cells, even though the same percentage of U937 and H9 cells was expressing the CD4 molecule. Furthermore, we demonstrate that HIV-1 replication in U937 cells is mainly restricted by endogenous IFN-alpha. Indeed, addition of anti-IFN-alpha antibodies at the time of infection, during the nonproductive phase of the viral replication cycle, or during the lag phase leads to an earlier expression of viral proteins and/or to a rapid increase in the percentage of virus-producing cells. Likewise, the treatment of cultures of HIV-1 chronically infected U937 cells with the same antibodies induces an increased production of viral particles. Thus, IFN-alpha appears to be involved in the persistence of HIV-1 in the monocytes/macrophages of infected individuals.     

Diastrophic dysplasia and atelosteogenesis type II as expression of compound heterozygosis: first report of a Mexican patient and genotype-phenotype correlation

The osteochondrodysplasias represent a heterogeneous group of cartilage and bone diseases. Among these, achondrogenesis 1B, atelosteogenesis type II, diastrophic dysplasia, and autosomal recessive multiple epiphyseal dysplasia are caused by mutations in the solute carrier family 26 (sulfate transporter), member 2 gene (SLC26A2). This group of osteochondrodysplasias shows a continuous spectrum of clinical variability and shares many features in common. Usually, it is difficult to distinguish clinically among these patients. To date, several efforts have been made to correlate mutations in the SLC26A2 gene with phenotypic severity in the patients. We report on a Mexican girl with diastrophic dysplasia presenting some unusual clinical and radiographic features that are usually observed in atelosteogenesis type II. Molecular analysis of the SLC26A2 gene in this patient showed compound heterozygosity for the R178X and R279W mutations. In this patient, the combination of a mild and a severe mutation has apparently led to an intermediate or transitional clinical picture, showing an apparent genotype-phenotype correlation.     

Fluorescence in situ hybridisation (FISH) analysis of chromosome segregation and interchromosomal effect in spermatozoa of a reciprocal translocation t(9,10)(q11;p11.1) carrier

A couple was referred for exploration of repetitive abortions. The man was found to be a carrier of a balanced reciprocal translocation t(9;10)(q11;p11.1). The meiotic segregation of chromosomes 9 and 10 was analysed in 5,157 spermatozoa from this translocation carrier and in 15,255 spermatozoa from three control donors using three-colour fluorescence in situ hybridisation (FISH). The theoretical viability of the different segregation patterns was performed using the computer system HC Forum developed by the Department of Cytogenetics at the Grenoble University Medical School, La Tronche, France. A normal or balanced constitution was found in 56.25% of the analysed spermatozoa. The tertiary 3:1 segregation mode was the most frequently observed (14.37%). The frequencies of adjacent-1, adjacent-2 and 3:1 interchange modes were 12.85, 9.38 and 7.14% respectively. The cumulative frequency of non-viable imbalance was estimated at 20.91% according to the theorical viability of the different segregation patterns. Spermatozoa aneuploidy frequency was also evaluated for chromosomes X, Y and 18, and there was no evidence of interchromosomal effect in spermatozoa from the translocation carrier. FISH analysis of spermatozoa in combination with the viability theorical estimation of the different segregation patterns could be considered a useful tool for genetic counselling in carriers of reciprocal translocation.The experiments performed in this study comply with the current French laws.